Patent Grant
12138303
A sequence listing electronically submitted with the present application as an ASCII text file named 1101-016US02ReplacementSequenceListing_ST25.txt, created on Aug. 6, 2024 and having a size of 73,547 bytes, is incorporated herein by reference in its entirety.
The present disclosure relates to therapeutic compositions, vaccines and immunogenic compositions comprising one or more immunogenic, attenuated, and/or non-cytotoxic proteins derived from Staphylococcus pseudintermedius. The compositions comprise recombinant attenuated proteins expressed from synthetic genes altered to produce the attenuated proteins. In particular, the disclosure relates to vaccines and immunogenic compositions comprising one or more natural or recombinant immunogenic, attenuated, non-cytotoxic Staphylococcus pseudintermedius proteins capable of inducing the production of antibodies reactive with S. pseudintermedius proteins naturally secreted and/or displayed on the bacterial surface and involved in host immune dysfunction and/or evasion. The disclosure further relates to therapeutic compositions comprising one or more natural or recombinant immunogenic, attenuated, non-cytotoxic Staphylococcus pseudintermedius proteins capable of improving the host immune response independent of production of antibody.
Staphylococcus pseudintermedius is the primary cause of canine pyoderma (skin infection). Pyoderma is the most common canine dermatologic disease and the organism is also associated with urinary tract infections, wound and surgical site infections, external ear otitis, abscess formation, mastitis and endocarditis. As many as 30-40% of the S. pseudintermedius isolates tested in clinical laboratories in some geographical regions are methicillin-resistant (MRSP). These bacteria are resistant to all β-lactam antibiotics. The vast majority of MRSP are also resistant to other clinically useful antibiotics and an increasing number of MRSP are resistant to all antimicrobials available to veterinarians. Accordingly, there is a need for immunogenic compositions capable of inducing antibodies to, and/or a protective immune response to S. pseudintermedius.
Development of vaccines to control staphylococcal infections is a high priority, however there are no licensed S. pseudintermedius vaccines. This is likely due to a lack of information about S. pseudintermedius protein functions, surface accessibility and epitope conservation. Information about other species of bacteria, especially staphylococci, is useful in the development of a S. pseudintermedius vaccine, however, this species is unique with regard to its disease presentation, genetic structure and composition, host range, and the virulence proteins it produces. Whole bacterial staphylococcal vaccines have not been efficacious. With an estimated greater than 2,000 different proteins produced by each bacterium, it is essential to identify and direct the immune response against the most important antigen targets. However, many of these proteins are potentially harmful to animals and must be attenuated to make them safe without eliminating epitopes that induce a protective immune response. Effective staphylococcal defenses are rooted in the ability of the bacteria to neutralize and/or destroy important components of their host's defenses.
There accordingly remains a need in the art for novel immunogenic compositions suitable for preparing S. pseudintermedius vaccines and therapeutic compositions. To meet this identified need in the art, conserved S. pseudintermedius cell surface-bound and secreted immunogenic proteins have been identified and attenuated and/or non-cytotoxic immunogenic recombinant proteins produced. The attenuated and/or non-cytotoxic immunogenic recombinant S. pseudintermedius cell surface-bound and cell-expressed proteins are suitable for vaccine development, including multivalent vaccines, and for manufacture of therapeutic compositions.
In one aspect of the present disclosure, a multivalent immunogenic therapeutic composition is provided, comprising one or more recombinant proteins selected from the group consisting of an attenuated immunoevasive Staphylococcus pseudintermedius Protein A (SEQ ID NO:2) and an attenuated immunoevasive Staphylococcus pseudintermedius Leukotoxin S (SEQ ID NO:4) and optionally, one or more suitable pharmaceutical or veterinary excipients and carriers. The composition may further comprise one or more of an attenuated immunoevasive Staphylococcus pseudintermedius Nucleotidase adenosine synthase protein (AdsA) (SEQ ID NO:6), an attenuated immunoevasive Staphylococcus pseudintermedius coagulase (SEQ ID NO:8), an attenuated immunoevasive Staphylococcus pseudintermedius Leukotoxin F (SEQ ID NO:10), and an attenuated immunoevasive Staphylococcus pseudintermedius exotoxin 15 (SEQ ID NO:12).
In an embodiment, the multivalent immunogenic therapeutic composition consists essentially of a recombinant attenuated immunoevasive Staphylococcus pseudintermedius Protein A (SEQ ID NO:2), a recombinant attenuated immunoevasive Staphylococcus pseudintermedius Leukotoxin S (SEQ ID NO:4), a recombinant attenuated immunoevasive Staphylococcus pseudintermedius Nucleotidase adenosine synthase protein (AdsA) (SEQ ID NO:6), a recombinant attenuated immunoevasive Staphylococcus pseudintermedius coagulase (SEQ ID NO:8), a recombinant attenuated immunoevasive Staphylococcus pseudintermedius Leukotoxin F (SEQ ID NO:10), and a recombinant attenuated immunoevasive Staphylococcus pseudintermedius exotoxin 15 (SEQ ID NO:12), and optionally, one or more suitable pharmaceutical or veterinary excipients and carriers. In another embodiment, the multivalent immunogenic therapeutic composition consists essentially of a recombinant attenuated immunoevasive Staphylococcus pseudintermedius Protein A (SEQ ID NO:2), a recombinant attenuated immunoevasive Staphylococcus pseudintermedius Leukotoxin S (SEQ ID NO:4), a recombinant attenuated immunoevasive Staphylococcus pseudintermedius Nucleotidase adenosine synthase protein (AdsA) (SEQ ID NO:6), a recombinant attenuated immunoevasive Staphylococcus pseudintermedius coagulase (SEQ ID NO:8), a recombinant attenuated immunoevasive Staphylococcus pseudintermedius Leukotoxin F (SEQ ID NO:10), and a recombinant attenuated immunoevasive Staphylococcus pseudintermedius exotoxin 15 (SEQ ID NO:12); and optionally, one or more suitable pharmaceutical or veterinary excipients and carriers.
In another aspect of the disclosure, a vaccine composition is provided comprising one or more recombinant proteins selected from the group consisting of a recombinant attenuated Staphylococcus pseudintermedius Protein A (SEQ ID NO:2) and a recombinant attenuated Staphylococcus pseudintermedius Leukotoxin S (SEQ ID NO:4); and one or more of a suitable pharmaceutical or veterinary adjuvant, a suitable pharmaceutical or veterinary excipient, and a suitable pharmaceutical or veterinary carrier. The vaccine composition may further comprise one or more of an attenuated immunoevasive Staphylococcus pseudintermedius Nucleotidase adenosine synthase protein (AdsA) (SEQ ID NO:6), an attenuated immunoevasive Staphylococcus pseudintermedius coagulase (SEQ ID NO:8), an attenuated immunoevasive Staphylococcus pseudintermedius Leukotoxin F (SEQ ID NO:10), and an attenuated immunoevasive Staphylococcus pseudintermedius exotoxin 15 (SEQ ID NO:12).
In an embodiment, the vaccine composition consists essentially of a recombinant attenuated immunoevasive Staphylococcus pseudintermedius Protein A (SEQ ID NO:2), a recombinant attenuated immunoevasive Staphylococcus pseudintermedius Leukotoxin S (SEQ ID NO:4), a recombinant attenuated immunoevasive Staphylococcus pseudintermedius Nucleotidase adenosine synthase protein (AdsA) (SEQ ID NO:6), a recombinant attenuated immunoevasive Staphylococcus pseudintermedius coagulase (SEQ ID NO:8), a recombinant attenuated immunoevasive Staphylococcus pseudintermedius Leukotoxin F (SEQ ID NO:10), and a recombinant attenuated immunoevasive Staphylococcus pseudintermedius exotoxin 15 (SEQ ID NO:12), and optionally, one or more suitable pharmaceutical or veterinary excipients and carriers. In another embodiment, the vaccine composition consists essentially of a recombinant attenuated immunoevasive Staphylococcus pseudintermedius Protein A (SEQ ID NO:2), a recombinant attenuated immunoevasive Staphylococcus pseudintermedius Leukotoxin S (SEQ ID NO:4), a recombinant attenuated immunoevasive Staphylococcus pseudintermedius Nucleotidase adenosine synthase protein (AdsA) (SEQ ID NO:6), a recombinant attenuated immunoevasive Staphylococcus pseudintermedius coagulase (SEQ ID NO:8), a recombinant attenuated immunoevasive Staphylococcus pseudintermedius Leukotoxin F (SEQ ID NO:10), and a recombinant attenuated immunoevasive Staphylococcus pseudintermedius exotoxin 15 (SEQ ID NO:12); and optionally, one or more suitable pharmaceutical or veterinary excipients and carriers.
In another aspect of the disclosure, a multivalent chimeric protein is provided comprising one or more recombinant attenuated immunoevasive proteins from Staphylococcus pseudintermedius cell surface-bound and/or secreted proteins, and comprising the sequence set forth as SEQ ID NO: 14.
In another aspect of the disclosure, a Staphylococcus pseudintermedius Protein A synthetic gene (SEQ ID NO:1) codon optimized for expressing a recombinant attenuated immunoevasive Protein A in Escherichia coli is provided.
In another aspect of the disclosure, a Staphylococcus pseudintermedius Leukotoxin S synthetic gene (SEQ ID NO:3) codon optimized for expressing a recombinant attenuated immunoevasive leukotoxin in Escherichia coli is provided.
In another aspect of the disclosure, a Staphylococcus pseudintermedius Nucleotidase adenosine synthase protein (AdsA) synthetic gene (SEQ ID NO:5) codon optimized for expressing a recombinant attenuated immunoevasive Nucleotidase ADsA in Escherichia coli is provided.
In another aspect of the disclosure, a Staphylococcus pseudintermedius coagulase synthetic gene (SEQ ID NO: 7) codon optimized for expressing a recombinant attenuated immunoevasive coagulase in Escherichia coli is provided.
In another aspect of the disclosure, a Staphylococcus pseudintermedius Leukotoxin F synthetic gene (SEQ ID NO: 9) codon optimized for expressing a recombinant attenuated immunoevasive gamma-hemolysin in Escherichia coli is provided.
In another aspect of the disclosure, a Staphylococcus pseudintermedius exotoxin 15 synthetic gene (SEQ ID NO: 11) codon optimized for expressing a recombinant attenuated immunoevasive exotoxin 15 in Escherichia coli is provided.
In the following description, there are shown and described embodiments of the disclosed recombinant attenuated S. pseudintermedius cell-surface and secreted proteins, and multivalent immunogenic compositions and vaccines including same. As it should be realized, the disclosed proteins, multivalent immunogenic compositions, and vaccines are capable of other, different embodiments and their several details are capable of modification in various, obvious aspects all without departing from the devices and methods set forth and described in the following claims. Accordingly, the drawings and descriptions should be regarded as illustrative in nature and not as restrictive.
The accompanying drawing figures incorporated herein and forming a part of the specification, illustrate several aspects of the disclosed recombinant attenuated S. pseudintermedius cell-surface and secreted proteins, and multivalent immunogenic compositions and vaccines, and together with the description serve to explain certain principles thereof. In the drawings:
Reference will now be made in detail to embodiments of the disclosed recombinant attenuated S. pseudintermedius cell-surface and secreted proteins, and multivalent immunogenic compositions and vaccines, examples of which are illustrated in the accompanying drawing figures.
Any citations, gene sequences, accession numbers, and reference sequences included or referred to in this application form a part of the disclosure and are incorporated herein in their entirety by reference. It will be appreciated that the embodiments shown and described in this patent application are an illustration of one of the modes best suited to carry out the invention. The invention is capable of other different embodiments, and its several details are capable of modification in various, obvious aspects all without departing from the invention. Accordingly, the drawings and descriptions provided herein will be regarded as illustrative in nature and not as restrictive. Various embodiments of the methods and compositions of the present disclosure will now be described by way of the following Examples.
S. pseudintermedius produces cell surface-bound and secreted proteins that variously inhibit phagocytosis, neutralize complement, bind immunoglobulins via their Fc and VH3 regions, cause apoptosis of B cells and kill leukocytes. Thus, these proteins contribute to the evasion of both the innate and adaptive branches of the immune system. In accordance with the foregoing need identified in the art, the present investigators have identified a number of such proteins as likely candidates for vaccines (see Table 1). Secreted S. pseudintermedius protein A acts as a superantigen, destroys B cells and prevents antibody production. S. pseudintermedius protein A bound to the surface of bacteria neutralizes antibody and prevents it from agglutinating, opsonizing or fixing complement. S. pseudintermedius adenosine synthase protein (AdsA) present on the bacterial surface produces adenosine from host-derived ATP, ADP and AMP found in damaged host tissue. The adenosine inhibits phagocytosis of bacteria by neutrophils. S. pseudintermedius coagulase facilitates the formation of a fibrin coating on the bacterial surface and is a hallmark of virulence in staphylococci although it has characteristics that differ significantly from coagulase produced by other coagulase-positive staphylococci. S. pseudintermedius leukotoxins, including leukotoxin S, leukotoxin F, and exotoxin 15 destroy leukocytes disabling their host's immune defenses.
S. pseudintermedius Vaccine Antigens
The functions of the above proteins in vitro were identified and confirmed using recombinant S. pseudintermedius proteins and canine leukocyte targets. Their secretion and/or surface accessibility was determined using culture supernatant, surface labeling, pull down experiments and GC mass spectroscopy. For GC mass spectroscopy log phase bacterial cultures of S. pseudintermedius strains (shown with their sequence types) 06-3228 (ST68), 08-1661 (ST71) and NA45 (ST84) were analyzed. These strains are representative of the major S. pseudintermedius genotypes (clonal complexes) most commonly isolated from canine infections in the United States and Europe.
S. pseudintermedius is known to produce a wide variety of virulence factors. Some of these are surface-associated while others are secreted. The purpose of the studies described herein was to analyze the whole secretome (secreted proteins) and surface-associated proteins in S. pseudintermedius by Mass Spectrometry (MS) in an effort to identify potential vaccine candidates. The specific objectives of this study were: (a) to analyze the entire bacterial secretome (b) to identify secreted immunoglobulin-binding (Ig-binding) proteins and (c) to identify proteins accessible on the surface of the bacteria. The secretome, Ig-binding proteins and trypsin-treated proteins were compared among three S. pseudintermedius isolates using their respective genomes as reference databases. Orthologous proteins among the three isolates were identified and from this group, proteins common to all three isolates were further analyzed to determine if they were good vaccine candidates. Mass spectrometry was used to identify the surface proteins and secreted proteins from three strains of S. pseudintermedius; 06-3228 (ST 68), 08-1661 (ST71) and NA45 (ST 84) which are representative of the major clonal populations in the United States and Europe.
Bacterial Strains and Growth Conditions
S. pseudintermedius clinical isolates 06-3228 and 08-1661 were obtained from the Clinical Bacteriology Lab at the University of Tennessee, Knoxville. Strain NA45 was a gift of Faye Hartmann of the University of Wisconsin School Of Veterinary Medicine. Bacterial colonies from blood agar plates were inoculated into 5 ml of sterile Trypticase Soy Broth (TSB) (BD Biosciences, San Jose, CA) and incubated overnight at 37° C. with shaking at 225 rpm (Excella E24 Incubator Shaker, New Brunswick Scientific). Fifty microliters of overnight culture were inoculated into 5 ml of fresh, sterile TSB to initiate log-phase bacterial cultures. The bacteria were grown at 37° C. with shaking at 225 rpm until an optical density of OD600=0.4-0.6 was reached.
Preparation of Whole Secretome
Log phase bacterial cultures were centrifuged at 10,000 g for 30 minutes at 4° C. (Sorvall RC-5C Plus Superspeed Centrifuge). Without disturbing the bacterial pellet, the supernatant was collected and passed through a 0.45 μm filter (Whatman, GE Healthcare Lifesciences, Pittsburgh, PA). The filtrate was concentrated using an Amicon® Ultra-4 centrifugal filter (EMD Millipore Corp., Billerica, MA) and stored at −20° C. until further analysis.
Purification of Immunoglobulin-Binding Proteins from Bacterial Culture Supernatants
Log phase bacterial cultures were centrifuged at 10,000 g for 30 minutes at 4° C. The supernatant was passed through an IgG-sepharose column (GE Healthcare Lifesciences, Pittsburgh, PA). Briefly, the column was equilibrated with five bed volumes of binding buffer containing 50 mM Tris, 2.7 mM potassium chloride and 0.137 M NaCl pH 8.0. The bacterial supernatant was added to the column to allow attachment of IgG binding proteins. Unbound proteins were washed off with 15 bed volumes of binding buffer. The bound proteins were eluted in 0.1-0.2 M Glycine/HCl pH 2.5-3.0. The pH of the elution fraction was adjusted to 7.0 using 0.5M Tris and stored at −20° C. until further analysis.
Purification of Trypsin-Treated Surface Proteins
Log phase bacterial cultures were centrifuged at 4,200 g for 3 min and washed three times with PBS. The pellet was suspended in 1 ml digestion buffer (0.6M sucrose buffered with 50 mM Tris, pH 7.5). 200 μg of porcine trypsin (Sigma-Aldrich, St. Louis, MO) was added and the cells were digested for 1 hour at 37° C. Protease inhibitor cocktail set IV (Calbiochem, EMD Millipore Corp, Temecula, CA) was then added to the mixture at a final concentration of 0.1 μg/ml. The supernatant containing tryptic peptides was stored at −20° C. until further analysis.
Biotin Labeling of Surface Proteins
Log phase bacterial cells were harvested by centrifugation at 10,000 g for 1 min and washed twice with ice-cold phosphate buffered saline (PBS) pH 8.0.100 μl of 10 mM EZ-Link Sulfo-NHS-LC-Biotin reagent (Thermo Scientific, Waltham, MA) per 1 ml of cell suspension was added and incubated for 30 min at room temperature. The cells were washed three times with PBS and 100 mM glycine was added to quench and remove excess biotin. The biotin-labeled bacteria were stored in aliquots at −20° C. until further use. The biotin-labeled proteins were digested with trypsin as described previously and were stored at −20° C. until further use.
Purification of Biotin-Labeled Surface Proteins Using Monomeric Avidin
Biotin-labeled surface proteins were purified using a monomeric avidin column (Pierce Monomeric Avidin Kit, Thermo Scientific, Waltham, MA) as per manufacturer's protocol. Briefly, the monomeric avidin column was washed with 8 ml of PBS followed by the addition of 6 ml of biotin blocking buffer to block any non-reversible biotin binding sites. The excess biotin was removed from the reversible binding sites by addition of 12 ml of regeneration buffer. The biotin-labeled, trypsin-cleaved proteins were loaded onto the column. Unbound proteins were washed off with PBS. The bound proteins were eluted using elution buffer in six, 2 ml fractions. The eluates were stored at −20° C. until further use.
LC-MS/MS Analysis of S. pseudintermedius Secretome and Surface-Associated Proteins
Samples interrogating S. pseudintermedius supernatant and surface-associated proteins were prepared for shotgun LC-MS/MS analysis. Trypticase soy broth (media alone) and bacterial culture supernatant passed through control Sepahrose beads (without IgG) served as controls. Prior to protein denaturation with sodium dodecyl sulfate (SDS), samples were concentrated/solvent exchanged on molecular weight cut-off (MWCO) spin filters (Vivaspin, GE Healthcare Lifesciences, Pittsburgh, PA) that retain proteins/polypeptides>5 kDa in size (˜50 amino acids or larger). Retained proteins were then re-suspended in 4% SDS, 100 mM Tris-HCl, 5 mM dithiothreitol (DTT), transferred to an Eppendorf tube, and incubated at 95° C. for 5 min. Iodoacetamide was then added to a final concentration of 15 mM and samples incubated for 20 min in the dark at room temperature. Denatured and reduced proteins were then digested to peptides with 2 μg of proteomics-grade porcine trypsin (Sigma-Aldrich, St. Louis, MO) for 4 hours. Following digestion, peptide samples were adjusted with 200 mM NaCl-0.1% formic acid and passed through a 10 kDa MWCO Vivaspin2 spin filter to collect appropriately sized tryptic peptides for LC-MS/MS analysis.
With the aid of a pressure cell, peptides were loaded onto a biphasic MudPIT back column containing both strong-cation exchange and reversed-phase (RP) resins-5u Luna or 5u Kinetex, respectively (Phenomenex, Torrance, CA) and separated. Briefly, loaded samples were washed offline then placed in line with an in-house pulled nanospray emitter packed with 15 cm of RP resin. Peptides were then separated and analyzed with either a 2-step MudPIT LC-MS/MS protocol (salt cuts of 50 mM and 50 mM ammonium acetate) over a 4-hr period (supernatant samples) or a 1-step protocol (500 mM ammonium acetate) over a 2-hr period (surface proteins and pulldown samples). All samples were measured with a hybrid LTQ XL-Orbitrap (Thermo Scientific, Waltham, MA) mass spectrometer (MS) as follows: data-dependent acquisition, 1 full scan followed by 10 MS/MS scans. Orbitrap mass analyzer was set to 15K resolution, LTQ isolation window=2.2 m/z, monoisotopic precursor selection, dynamic exclusion window, duration, and max=3 m/z, 60s (30s for supernatant samples), and 500 respectively. The immunogenicity of peptides was predicted using the POPI v.2.0 web server.
Results
LC MS/MS Data Analysis
MS/MS spectra were searched against sample-specific proteome databases concatenated with common protein contaminants using MyriMatch v.2.2. Peptide spectrum matches (PSM) were filtered and assembled to proteins using IDPicker v.3.0 with false-discovery rates, as assessed by evaluating MS/MS spectra that matched to reversed decoy sequences, controlled at <1% at the peptide-level. Proteins calls required two distinct peptides at a minimum. Peptide intensities were assessed by chromatographic area-under-the-curve (AUC) using IDPicker's embedded spectra/label-free quantification option and protein abundances derived via summation of constituent peptides intensities. To qualitatively compare the proteins identified across each strain of S. pseudintermedius, OrthoMCL (OrthoMCL Database, Version 5.0) was employed to identify orthologous protein sequences. Identified protein data for each specific S. pseudintermedius sequence type were then re-cast into their orthologous groups and compared across all sample types to identify proteins common to all three pathogenic strains with special emphasis on those that are surface-associated and/or secreted. To help winnow down the list of potential secreted and surface-bound antigen targets, PSORTb v.3.0 was used to assign subcellular location to each identified protein.
Identification of Surface Proteins, IgG-Binding Proteins and Extracellular (Secreted) Proteins by Mass Spectrometry
Using the analysis method described above, a total of 580 proteins were identified in the isolate 06-3228, 596 in 08-1661 and 542 in NA45. In order to narrow down the spectrum of proteins for analysis across all three sequence types, ortholog protein prediction was performed using the web-based tool OrthoMCL. A total of 364 predicted orthologous proteins were identified across all three sequence types. Excluding proteins that were classified as cytoplasmic in origin, there were 72 proteins predicted to be either secreted, cell wall-associated or of unknown cellular location across all three sequence types. These proteins were evaluated for inclusion in a vaccine based on their predicted virulence characteristics.
Upon contact with host blood, the coagulation system prevents the dissemination of microbial invaders by fibrin deposition around microbes. Phagocytosis of bacteria by neutrophils is strongly enhanced by opsonization of bacteria with plasma factors such as antibodies and complement. Staphylococci use several mechanisms to generate a protective coat composed of polysaccharide capsule or fibrin that prevent uptake of staphylococci by neutrophils or macrophages. Coagulase (Coa) is an important virulence factor that interacts with fibrinogen (Fg) and induces the formation of fibrin clots through activation of prothrombin that enables the establishment of staphylococcal disease. Coagulase activity has been observed in S. pseudintermedius, however, the responsible protein has not been identified or studied.
A recombinant protein (prCoa) was produced from the S. pseudintermedius gene commonly annotated as von Willebrand factor binding protein but which shares only 40% similarity to the same protein in S. aureus. A chromogenic assay was used to measure the activity of the staphylocoagulase-prothrombin complex directly for comparing the affinity of S. pseudintermedius Coa to prothrombin from bovine or human origin. Weak specificity for human prothrombin compared to bovine prothrombin was observed. The ability of prCoa to facilitate the escape of S. pseudintermedius from phagocytosis by forming a bridge between opsonizing antibody, complement and fibrinogen was studied. Evidence from this work suggests S. pseudintermedius Coa is an important candidate for a novel therapeutic or a novel vaccine that may elicit antibodies that enhance phagocytic clearance. The aim of this study was to identify the coagulase protein of S. pseudintermedius, clone the gene and produce a recombinant protein to study its biological properties.
Material and Methods
Bacterial Strains
A total of 15 clinical isolates previously identified as S. pseudintermedius (Table 2) and submitted to the University of Tennessee, College of Veterinary Medicine Clinical Bacteriology Laboratory as well as from European and North American collaborators were used. They represent nine MLST lineages associated with methicillin resistance. Bacterial isolation and identification procedures were those routinely used in the laboratory substantially as described above. Isolates were grown and maintained on tryptic soy blood agar (BD). S. aureus strain (ATCC23529) and the S. epidermidis (ATCC12228) were used as controls.
S. pseudintermedius
DNA Extraction
All DNA manipulations were performed using standard methods (Sambrook et al., 1989). Isolates were grown on blood agar plates overnight at 37 C and bacteria derived from a single colony were suspended in 5 ml of trypticase soy broth (TSB) (Becton, Dickinson and Co., Sparks, MD) in a rotary shaker at 225 rpm. Bacteria were harvested from 1.8 ml of microbial culture and DNA extracted using the UltraClean® Microbial DNA Isolation Kit (MO BIO, No 12224-50).
PCR Primers and Conditions
Primers were designed for amplification and sequencing of the S. pseudintermedius putative coagulase and vWbp genes based upon published NA45, 063228, and 081661 our genomic data (GenBank accession no. CP016072.1, CP015626.1, and CP016073.1 respectively; Table 3) using the IDT SciTools application (Integrated DNA Technologies, Coralville, IA). PCR amplification of the full-length coa genes was carried out with oligonucleotide primers (Table 4). The reaction mixtures consisted of 25 μl total volume solutions containing 2.5 μl of genomic DNA, 20 pmol of each primer (1 μl), 12.5 μl rTaq polymerase enzyme, and 8 μl nuclease free water. Amplification conditions consisted of an initial denaturation (94 C for 1.5 min) followed by 35 cycles of denaturation (94c for 60s), annealing (55 C for 2 min), and extension (72 C for 60 s), with a single final extension (72 C for 5 min). PCR products were visualized on 1.4% agarose gels.
Restriction enzyme sites (underlined) for Nco I and BamH1 were designed in the forward primer/reverse primer.
Sequence Analysis
PCR products of expected sizes were treated to destroy single-stranded DNA using ExoSap-IT purification reagent (USB Corp., Cleveland, OH) and submitted to the University of Tennessee, Molecular Biology Resource Facility for DNA sequencing using the dideoxy chain-termination method. PCR primers were used for direct DNA sequencing of PCR amplification products. BLAST sequence alignment tool (ncbi.nlm.nih.gov/blast) and Geneious software were used to determine nucleotide sequence similarity between S. pseudintermedius isolates.
Cloning, Expression, and Purification of Coa Protein
Cloning of the coa gene was carried out according to the manufacturer's instructions using pETBlue™-2(Novagen) vector with an expanded multiple cloning site (MCS) and C-terminal HSV•Tag® and His•Tag® sequences, an ampicillin resistance gene and lacZ region for blue/white screening. PCR amplification of full-length coa was carried out with oligonucleotide primers which included NcoI and BamHI restriction sites to facilitate cloning. PCR products from S. pseudintermedius strain 081661 were digested with NcoI and BamHI and purified using Wizard® SV Gel and PCR Clean-Up System according to the manufacturer's instructions. They were ligated into the vector, transformed into DH5-alpha E. coli and plated on LB agar containing 50 μg/ml ampicillin, IPTG, 40 μg/ml X-gal. The recombinant plasmid was extracted using a GeneJET Plasmid Miniprep Kit according to the manufacturer's instructions. The presence of the cloned fragment was confirmed by PCR.
The coa gene construct was expressed in Tuner™ (DE3) pLacI E. coli competent cells (Novagen). They were plated on LB agar containing 50 μg/ml ampicillin and 34 μg/ml chlormphenicol (LB amp-cam). Exponential phase cultures were inoculated 1:100 from overnight starter cultures and grown to an optical density (600 nm) of 0.6. Isopropyl β-D-thiogalactopyranoside (SIGMA) was added to a final concentration of 1 mM and the cells were incubated at 30° C. for an additional 4 min in a shaking incubator at 225 rpm with aeration for induction of protein expression.
For the purification of recombinant protein, 100 ml of induced bacterial cultures were centrifuged at 10000×g for 15 min. Bacteria were suspended in 5 ml of Bug Buster master mix (Novagen) with 20 μl protease inhibitor (Cocktail Set III, EDTA-Free—Calbiochem) and incubated for 30 min at 37 C in a shaking incubator at 225 rpm. Samples were centrifuged at 16,000×g for 45 min at 4° C. to remove insoluble cell debris. Proteins were applied to an immobilized metal ion affinity chromatography according to the manufacturer's instructions (PrepEase Ni-TED column). Proteins were eluted using HIS-Select Elution Buffer (Sigma) containing successively higher concentrations of imidazole up to 250 mM. Protein concentrations were determined with the bicinchoninic acid (BCA) assay (Thermo Scientific).
A chromogenic staphylocoagulase assay was used to detect the presence of S. pseudintermedius coagulase. Single colonies of bacterial isolates were cultured overnight in 2 ml of TSB at 37° C. Bacteria were centrifuged at 12000×g for 2 min then supernatants were concentrated using Amicon Ultra-0.5 centrifugal filters. Tests were performed in flat-bottom microtiter plates. The molar concentrations of staphylocoagulase (SC) and prothrombin (P) were calculated using molecular weights of 62,000 for coagulase and 72,000 for human and bovine prothrombin. An 18 μl aliquot of concentrated supernatant or an equimolar amount of purified prCoa was mixed with 1×10−16 M of human prothrombin or bovine prothrombin and incubated for 30 min at 37 C. Chromogenic thrombin tripeptide substrate H-D-Phe-Pip-Arg-pNA (S-2238) was added to a final concentration of 1 mM in a total reaction buffer of 100 μl PBS per each well. After an initial reading the reaction was allowed to proceed by incubating in the dark for 1, 4 or 8 h at 37 C. The absorbance was continuously measured at 405 nm and positive, negative, and reagent controls (prothrombin alone without the substrate) were included with each batch, with S. aureus ATCC25923 as the positive control and S. epidermidis ATCC12228 as the negative control. The change in absorbance was plotted and the slope of the curve (dA/dt) was interpreted to be the rate of S-2238 hydrolysis was measured as the increase in the absorbance, and thus reflective of enzymatic function (% Coa prothrombin complex activity). The generated thrombin activity in the supernatant was measured using chromogenic thrombin peptide.
Phagocytosis Assays
For fluorescent labeling of S. pseudintermedius 081661 strain with pHrodo dye (Invitrogen, Mulgrave, Australia), bacteria were suspended in PBS, pH 7.2 and incubated with 1 mM of pHrodo™ Green DMSO solution for 45 minutes at room temperature protected from light. Bacteria were washed twice with PBS, suspended in PBS and 10% glycerol, and stored at −80° C. until further use.
For whole blood phagocytosis, 10 μL of pHrodo green-labeled S. pseudintermedius 081661 strain (109 cell/ml) were mixed with 10% rabbit plasma for 30 min at 37 C in the presence of prCoa (50 or 100 μg/ml) or buffer. Then 200 μl of dog blood freshly collected in EDTA, was added and incubated for 30 min at 37 C. The reaction was stopped using red blood cell lysing buffer (Sigma); samples were washed with PBS and analyzed by flow cytometry. Gating of cells was done on the basis of forward and side scatter. For each sample fluorescence intensity of 10,000 gated neutrophils was determined. Phagocytosis was expressed as the percentage of neutrophils that became fluorescent.
Flow Cytometry Assays with S. pseudintermedius and S. Epidermidius
Bacteria grown to mid log phase were incubated with 50 μl dog serum for 30 min at 37 C, washed with PBS and incubated with prCoa (50 μg/ml) for 1 hour at 37 C with shaking. After another washing step, bacteria were incubated with FITC conjugated Fg from pooled human plasma (50 μg/ml, Zedira) for 1 hour at 37 C with shaking. Controls included heat inactivated serum with added prCoa, FITC-fibrinogen or dog serum only with FITC-fibrinogen. Washed bacteria were analyzed by flow cytometry. Bacteria were gated on the basis of forward and side scatter properties and 10,000 bacteria were analyzed.
Binding Assays
To detect the ability of prCoa to bind with complement factor C3, microtiter plates were coated overnight at 4° C. with affinity purified goat anti-dog C3 (Bethyl) (10 μg/ml). After washing the wells with PBST, dog serum (undiluted and two-fold serially diluted) were added and incubated for one hour at 37 C, then incubated with prCoa (5 μg/ml) for another hour at 37 C. prCoa binding was detected using al:3000 dilution of HRP-conjugated goat anti-HIS monoclonal antibody (Thermoscientific) and visualized using TMB (Pierce) substrate.
To detect the binding of IgG, IgG-Fc, and IgG-Fab to prCoa, canine IgG was digested with immobilized papain using a Fab preparation Kit (Thermo Scientific) according to the manufacturer's instructions using a 6 hr incubation for 1 mg/ml dog IgG whole molecule (Rockland) with papain. Digestion was ensured by diluting the sample in Laemmli sample buffer 2× without reducing agent (Biorad) and running the digested IgG molecule in 7.5% polyacrylamide gels. This was followed by purification and separation of Fab fragments from Fc fragments using prepacked, immobilized Thermo Scientific™ NAb™ Protein A Plus Spin Column. prCoa (2 μg/ml) coated plates were incubated with canine whole molecule IgG (Rockland), Fc, or Fab fragments (beginning concentration 10 μg/ml then two-fold serially diluted) for one hour at 37 C. After washing, HRP conjugated sheep anti-dog IgG heavy chain (1:4000) (Invitrogen) was added and detected through incubation with TMB substrate.
To measure the binding of prCoa to prothrombin human and bovine prothrombin (21 μg/ml) was adsorbed to a microtiter plate well at 4° C. for 16 h. After washing the wells with PBST, prCoa (beginning with 21 μg/ml then two-fold serially diluted) was added and the plates were incubated for 1 h. Following incubation with HRP-conjugated goat anti-His monoclonal antibodies (thermoscientific) (1:3,000 dilution) for 1 h, TMB substrate (Pierce) was added. Bound proteins were quantified by measuring the absorbance at 450 nm in a microplate reader. All experiments were performed twice for reproducibility.
Statistical Analysis
Repeated measures mixed effects analyses of variance were used to test within and between subject effects for binding ability of prCoa (human and bovine prothrombin, complement C3 and TgG, Fab, Fc), bacterial supernatant and the coagulase recombinant protein chromogenic assay and phagocytosis assay. A 2×4 analysis of variance was used to test between subject effects for fibrinogen FITC deposition. Post hoc analysis included simple contrasts when comparing to a control or pairwise comparisons. All post hoc tests used a Bonferonni adjustment.
Results
Amplification and Sequencing of the Coa Gene from Staphylococcus pseudintermedius Strains
PCR analysis showed that the putative S. pseudintermedius coagulase gene was found in all 15 clinical isolates of S. pseudintermedius tested at the expected size of 1500 bp (data not shown).
Time Course of the Hydrolysis of Chromogenic Substrate with Concentrated Culture Supernatant
The use of a chromogenic assay shows that supernatant of all S. pseudintermedius strains contains sufficient staphylocoagulase for detection within 1 h except E141. Fmv2999 was negative for coagulase reactivity with human or bovine prothrombin and NA12 only reacted with human prothrombin and not bovine prothrombin as shown in
The presence of coa and the production of staphylocoagulase by the S. pseudintermedius strains (n=15), were assayed by PCR, the plasma tube clotting method, and chromogenic assay as shown in Table 3 and
Binding of 081661, 063228, NA45 Concentrated Bacterial Supernatant with Human and Bovine Prothrombin
Binding of Prothrombin, Canine IgG, and Complement C3 to Coagulase Recombinant Protein of S. pseudintermedius Strain 081661
The ability of coagulase to bind to immobilized complement C3 is shown in
Detection of the Clotting Activity of Coagulase Recombinant Protein
Strong coagulation of rabbit plasma was detected with a single colony of S. pseudintermedius 081661. Both concentrations of prCoa tested yielded positive results within 5 hr after inoculation and remained at 24 hr.
The use of a chromogenic assay (
Coagulase Mediated Fibrinogen Covering of S. pseudintermedius and S. epidermedius
Using flow cytometry we observed that coa mediates Fg binding to opsonized bacteria (
Coa Inhibits Phagocytosis in the Presence of Rabbit Plasma
prCoa blocked phagocytosis in a reaction that depends on the interaction of coa with both prothrombin and fibrinogen to form a protective fibrin shield around the bacteria (
Discussion
Coagulase activity is considered a key virulence factor for S. pseudintermedius. The coagulase gene identified in S. aureus does not have significant similarity to any genes found in S. pseudintermedius. The S. pseudintermedius gene annotated previously as producing von Willebrand factor binding protein, however, is only 40% similar to the corresponding S. aureus gene. The predicted S. pseudintermedius protein has domains similar those of proteins with coagulase activity and for that reason was investigated for its coagulase function.
Although some non-staphylocoagulase-producing S. pseudintermedius strains test positive in the tube coagulase test, it was found by PCR analysis that the occurrence of the putative coa gene (often annotated previously as von Willibrand binding protein) examined in this study is common among clinical isolates of S. pseudintermedius.
The chromogenic assay showed that 063228 coagulase activity is not significantly different from S. aureus at all time points with bovine prothrombin indicating that this strain is able to form a complex with bovine prothrombin. It was significantly different from S. aureus with human prothrombin indicating that this strain is able to form a complex with human prothrombin but with less reactivity than S. aureus.
The coagulase recombinant protein was able to bind with canine IgG, Fc and Fab domains. Based on BLAST sequence alignment of IgG binding domain and the linker domain (L), the most likely region that binds with IgG is the linker domain.
Coa residues 467-491 showed 38% and 44% identity to the complement binding domain of sbi protein of S. pseudintermedius and S. aureus respectively with R470, N 478 and 7 linker amino acids in between consistent with the results of functional binding assays (ELISA and western blot) performed in this study. However, secreted Coa protein is not known to interact directly with the bacterial surface but can induce the formation of an Fg shield at some distance from the bacterial surface.
In this study, using flow cytometry, we showed another novel mechanism that allows secreted S. pseudintermedius Coa to localize at the cell surface of S. pseudintermedius by binding to surface-bound IgG and C3 and the attachment of soluble Fg to the bacterial surface creating a protective fibrinogen shield. It is likely that at least some of the Fg molecules are converted to fibrin through the action of Coa activated prothrombin. This effect is not only specific for S. pseudintermedius but also extends to protect other microorganisms, as demonstrated with S. epidermidius.
In this study we demonstrated that S. pseudintermedius coagulase effectively blocks phagocytosis of bacteria with addition of rabbit plasma and dog whole blood and the decrease of the percentage of phagocytosis is dose dependent on the concentration of the added coagulase. The gene targeted in this study encodes a novel S. pseudintermedius protein that displays coagulase activity, binds immunoglobulin and complement C3, and provides protection from phagocytosis. It is proposed as a good candidate for a vaccine to protect against disease caused by this organism.
S. pseudintermedius expresses protein A on its surface and secretes it during the exponential growth phase. Phagocytosis experiments with S. pseudintermedius showed that blocking of protein A enhanced phagocytosis in whole blood, neutrophils and in a DH82 canine macrophage-like cell line. Taken together, the results demonstrate that S. pseudintermedius produces protein A that binds the Fc region of immunoglobulins and may serve as a potential virulence factor by evading the host immune system.
The purpose of this study was to characterize the cytotoxic effect of recombinant S. pseudintermedius Protein A (SpsQ) on dog B cells, develop attenuated SpsQ (SpsQ-M), and evaluate antibody raised against SpsQ-M in clinically healthy dogs. Staphylococcus pseudintermedius SpsQ-M was tested for its antigenicity and B cell killing. The results from this study suggest that S. pseudintermedius SpsQ may serve as a key component in a vaccine or as part of an immunotherapeutic treatment.
Bioinformatics Analysis
MSA of spsQ from diverse isolates of S. pseudintermedius (n=100), was performed using Geneious, version 9.1.3. The bacterial localization prediction tool, PSORTb version 3.0.2 (psort.org/psortb), was used to determine the topology and domain structure of SpsQ and SpsP. SpsQ modeling and binding site prediction were performed using Protein Homology/analogY Recognition Engine V 2.0 (Phyre2) (sbg.bio.ic.ac.uk/phyre2), and the 3DLigandSite (sbg.bio.ic.ac.uk/3dligandsite), using SpA as a basis to predict the IgBDs in each domain. A pairwise sequence alignment of SpsQ and SpsP was used to identify conserved amino acids critical for IgG Fc and B cell receptor binding.
Identification of IgBDs was guided by S. aureus SpA secondary structure and based on S. aureus SpA residues responsible for dual reactivity of each domain in SpA with Fcγ and Fab and shared by S. pseudintermedius SpsQ.
Geneious, version 9.1.3 was used to select the locations for amino acids to be substituted in each IgBD and to design a full-length, four domain (SpsQ-M) attenuated S. pseudintermedius protein A construct (SpsQ-M). Glutamine (Q) 5 and 6, as well as aspartate (D) 32 and 33 in each domain of SpsQ were selected as critical amino acids for the association of SpsQ with immunoglobulin. To test this, substitutions of Q5K (lysine), Q6K, D32A (alanine), and D33A were introduced into each IgBDs of SpsQ.
Bacterial Strains and Growth Conditions
The S. pseudintermedius strain used in this study, strain 06-3228, was isolated at the University of Tennessee, College of Veterinary Medicine Bacteriology Laboratory. It represents the most common multilocus sequence type (ST) previously reported in the United States (ST68). Bacterial colonies grown on blood agar plates were inoculated into 5 ml of sterile trypticase soy broth (TSB) (BD Biosciences, San Jose, CA, USA Cat No. RS1-011-21) and incubated overnight at 37° C. with shaking at 225 rpm (Excella E24 Incubator Shaker, New Brunswick Scientific). Fifty microliters of overnight culture were inoculated into 5 ml of fresh, sterile TSB to initiate log-phase bacterial cultures. Bacteria were grown at 37° C. with shaking at 225 rpm until an optical density of OD600=0.4-0.6 was reached.
Cloning, Expression, and Purification of Recombinant Wild-Type and Non-Toxigenic S. pseudintermedius SpsQ
Bacteria from a single colony of S. pseudintermedius strain 06-3228 obtained from blood agar plates were grown in TSB at 37° C. with 225 rpm shaking. DNA was extracted using a MO BIO UltraClean® Microbial DNA Isolation Kit (QIAGEN Inc., USA Cat No. 12224-50) according to the manufacturer's instructions. Oligonucleotide primers (Integrated DNA Technology, Coralville, USA) (Table 5) were designed using a PrimerQuest Tool (idtdna.com/Primerquest/Home/Index) based on the whole genomic sequence of S. pseudintermedius strain 06-3228 that we determined.
Native full length spsQ forward
Staphylococcus pseudintermedius (NotI and BamHI
The spsQ open reading frame (ORF) without the regions encoding the predicted N-terminal signal peptide was amplified from S. pseudintermedius 06-3228 genomic DNA. The ORF of spsQ-M was amplified from a PMA-SpsQ-M plasmid (Table 6) (Life Technologies Corp., Carlsbad, CA, USA), containing a synthetic spsQ-M gene. PCR was performed using taq polymerase (rTaq, Takara, USA Cat No. R004) and the following cycling conditions were performed: initial denaturation at 95° C. for 90 seconds, 30 cycles of denaturation at 94° C. for 30 seconds, annealing at 55° C. for 30 seconds, and extension at 72° C. for 1 minute followed by a final extension at 72° C. for 5 minutes. All ORFs were amplified without a 6× histidine tag because pETBlue-2 (Table 6) allowed T7lac promoter-based expression of target genes with C-terminal histidine•Tag® sequences. PCR products were Sanger sequenced at The University of Tennessee Genomics Core facility.
S. pseudintermedius protein A (SpsQ-M)
To clone full length S. pseudintermedius spsQ and spsQ-M, the PCR products were digested with NotI and BamHI, then ligated into pETBlue-2, an expression vector with C-terminal HSV•Tag® and His•Tag® sequences (Novagen, USA Cat No 0.70674). The pETBlue-2 construct transformed into DH5-alpha E. coli chemically-competent cells (Table 6) (New England BioLabs Inc., USA Cat No. C2987I) by heat shock and DH5-alpha bacteria were plated on LB agar plates with 100 μg/mL ampicillin. The plasmid constructs were transformed into Tuner™ (DE3) pLacI E. coli chemically-competent cells (Table 6) (Novagen, USA Cat No 0.70623) by heat shock and the bacteria were plated on LB agar containing 50 μg/ml ampicillin and 20 μg/ml chloramphenicol.
To express recombinant protein, a single colony of Tuner™ (DE3) pLacI E. coli was inoculated into LB broth containing 50 μg/ml ampicillin and 20 μg/ml chloramphenicol and bacteria were grown overnight at 37° C. with 225 rpm shaking. LB broth containing 50 μg/ml ampicillin and 20 μg/ml chloramphenicol was inoculated with a 1:100 dilution of overnight culture and grown at 37° C. with 225 rpm shaking until a 600 nm optical density between 0.4 and 0.6 was reached. Protein expression was induced by addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Teknova, USA Cat. No. I3431) and bacteria were grown for 4 h at 30° C. with shaking at 225 rpm. Bacterial cultures were centrifuged at 12,000×g for 5 min in 5 ml of protein extraction reagent (BugBuster, Novagen, USA Cat No. 70584) and 20 μl of 100× protease inhibitor (Cocktail Set III, EDTA-Free Calbiochem, USA Cat No. 539134), and subsequently incubated for 30 min at 37° C. in a shaking incubator at 225 rpm. Bacteria were pelleted by centrifugation at 12,000×g for 45 min at 4° C. Recombinant protein was purified from the supernatant using affinity purification (HisPur™ Ni-NTA Spin Purification Kit, Thermo Scientific, USA Cat No. 88228). Protein concentrations were determined using a bicinchoninic acid (BCA) assay (Thermo Scientific, USA Cat No. 23227).
Production of Antibodies Against Recombinant Proteins
Recombinant SpsQ-M (SEQ ID NO:2) at 100 μg/0.5 cc in phosphate buffered saline (PBS) (pH 7.2) was injected in the lateral thorax by the subcutaneous route, into three clinically normal dogs. Injections were given once every 7 days for a total of three injections with a control dog receiving PBS (pH 7.2) only. Blood (6 cc) was collected from a jugular vein using a 20 g needle and 12 cc syringe 4 times, on days −7, 8, 15 and 29. The collected blood was left undisturbed at room temperature for 30 min followed by centrifugation at 2,000×g for 10 min in a refrigerated centrifuge.
SDS-PAGE and Western Blots
Protein samples were resolved by SDS-PAGE in 4-12% polyacrylamide gels (Invitrogen, USA Cat No. NP0322BOX) and electrophoretically transferred onto nitrocellulose membranes (Thermo Scientific, USA Cat No. 77010). The blots were blocked overnight in 5% (w/v) nonfat dried milk powder dissolved in phosphate buffered saline containing 0.05% polyethylene glycol sorbitan monolaurate (Tween 20) (PBS-T) at 4° C. The blocked membranes were incubated with a 1:2,000 dilution of horseradish peroxidase (HRP)-conjugated anti-6×his tag monoclonal antibody (Thermo Scientific, USA Cat No. MA1-21315-HRP) in 0.05% PBS-T for 1 h with 225 rpm shaking at room temperature. After five washes with 0.05% PBS-T, bound antibodies were detected using 1-Step™ chloronaphthol substrate solution (Thermo Scientific, USA Cat No. 34012).
Enzyme-Linked Immunosorbent Assay
For measurement of the antigenicity of recombinant proteins, affinity-purified SpsQ and SpsQ-M were coated on ELISA plates (Corning, USA Cat No. 3590) at 2 pg/ml in PBS (pH 7.2). The plates were washed with PBS-T and incubated with HRP-chicken anti-protein A antibody (Gallus Immunotech, USA Cat No. APA) for 1 h at 37° C., then washed. For this and all subsequent ELISA assays, plates were washed three times with PBS-T between all incubations, bound antibodies were detected using TMB substrate (Thermo Scientific, USA Cat No. N301), reactions were stopped with 0.18 M sulphuric acid and optical density read at 450 nm on a plate reader (Bio TEK, USA Cat No. EL800). The experiment was repeated at least three times and a p-value of <0.05 was considered significant, which was the same for all the experiments unless mentioned otherwise.
To test the reactivity of serum from a dog with chronic pyoderma, S. aureus SpA and S. pseudintermedius SpsQ and SpsQ-M were coated on ELISA plates (Corning, USA Cat No. 3590) at 2 μg/ml in PBS. They were incubated with serum at a dilution of 1:2000. Bound IgG, IgM, and IgA were detected by HRP-goat anti-dog IgG-heavy and light chain (Bethyl Laboratories, Inc., USA Cat No. A40-123-1), HRP-goat anti-dog IgM μ chain (Bethyl Laboratories, Inc., USA Cat No. A40-116-2), HRP-goat anti-dog IgA (Bethyl Laboratories, Inc., USA Cat No. A40-104P) at a dilution of 1:8000 in PBS-T. Dog whole IgG molecule (Rockland, USA Cat No. 004-0102-1000) was used for comparison to measure non-specific IgG binding.
To detect a specific antibody response against SpsQ-M in injected dogs, recombinant S. pseudintermedius proteins and commercial SpA were coated on ELISA plates as previously described and incubated with two-fold serially diluted serum from injected dogs (1/1000-1/32000). Bound IgG was detected using HRP-goat anti-dog IgG-heavy and light chain (Bethyl Laboratories, Inc., USA Cat No. A40-123-1) with serum from uninjected dogs used as negative controls. The experiment was run in duplicate and a p-value of <0.05 was considered significant.
The Ability of S. pseudintermedius SpsQ to Kill B Cells and Induce B Cell Apoptosis
A total of 100 μg of purified recombinant SpsQ or SpsQ-M was mixed with isolated peripheral blood mononuclear cells (PBMC) in 1 ml of RPMI medium supplemented with 10% fetal bovine serum and incubated for 1.5 h at 37° C. in a 5% CO2 incubator. To detect early phases of B cell apoptosis, phosphatidylserine was measured on the surface of cells using pacific blue-conjugated annexin (Thermo Scientific, USA Cat No. A35136). B cells were identified using phycoerythrin (PE) conjugated mouse anti-canine CD21 (clone: CA2.1D6) (BIO-RAD, USA Cat No. MCA1781PE) that recognizes canine CD21 (complement receptor type 2) on mature B lymphocytes. Stained B cells were analyzed using a flow cytometer (Attune acoustic focusing cytometer). B cells were also incubated with the same recombinant proteins as described above but for 3.5 h in order to detect B cell death. Cells were stained with Sytox green (Life Technologies, Inc., USA Cat No. 1776406) and PE-conjugated mouse anti-canine CD21. Gates were placed on cells positive for PE and these B cells were analyzed by flow cytometry.
To determine the protective effect of canine anti-SpsQ-M on B cells, recombinant S. pseudintermedius SpsQ was incubated for 30 minutes at 37° C. with serum from SpsQ-M injected dogs. The experiment was run in duplicate and a p-value of <0.05 was considered significant.
For flow cytometry analysis the cut-off for apoptosis or cell death was established using leukocytes incubated without SpsQ. Mean fluorescent intensity was determined from all B cells.
Statistical Analysis
A one-way ANOVA and Tukey-Kramer method were used to measure the significant differences between SpsQ, SpsQ-M, and SpA on inducing apoptosis and causing B cell death. However, two-way ANOVA and Tukey-Kramer methods were performed to test if there were significant differences in SpA, SpsQ or SpsQ-M binding with canine antibodies. All analyses were conducted using the GraphPad Prism software (Version 7, GraphPad Software Inc.).
Results
Bioinformatics Analysis and S. pseudintermedius Protein A Characteristics
S. pseudintermedius ED99, represents isolates that have genes encoding both SpsQ and SpsP. SpsQ contained four and SpsP had genes encoding a maximum of three Ig-binding domains (
Multiple sequence alignment (MSA) between each domain of SpsQ and domain C of SpA, the most similar domain to SpsQ IgBDs, showed that each domain in SpsQ has binding sites for the IgG Fcγ (Glutamine (Q) s and Q 6), and Fab region (aspartate (D) 32 and D33) of surface membrane-associated variable heavy 1 (VH1)-encoded B cell antigen receptors (
Cloning, Expression, and Purification of Recombinant SpsQ and SpsQ-M
Recombinant polyhistidine tagged SpsQ and SpsQ-M were produced in E. coli and purified using HisPur Ni-NTA resin under native conditions and eluted using an imidazole gradient. The molecular weights of SpsQ and SpsQ-M determined in western blots were of the expected sizes (47.2 and 47.6 kDa, respectively) (data not shown).
Antigenicity of SpsQ and SpsQ-M and their Reactivity with Serum from a Dog with Pyoderma
Chicken anti-protein A, chosen because protein A does not bind chicken immunoglobulin, had strong reactivity with affinity-purified SpsQ-M similar to that with S. aureus SpA and S. pseudintermedius SpsQ, indicating the attenuated protein retained its antigenicity (data not shown). Sera from a dog with a history of chronic pyoderma showed significantly lower reactivity against SpsQ-M (
S. pseudintermedius Protein A Induced Apoptosis of Canine B Cells
Affinity-purified SpsQ-M had a low apoptotic effect on B cells at 1.5 h in contrast to recombinant SpsQ at the same time point, with a mean+/−SEM fluorescent intensity (MFI) difference of 2052±285.2 between SpsQ and SpsQ-M (p-value of 0.0063) (
SpsQ-M Induces Specific Antibody Responses in Dogs that Reduce the Effect of SpsQ on B Cells In Vitro
ELISA analysis of sera obtained from dogs on days −7, 8, 15 and 29 (relative to injections) showed that antibodies against S. pseudintermedius SpsQ-M and SpsQ were detected on day 15 and reached the highest level on day 29 (P<0.0001) compared to pre-injection control sera (
Pre-incubation of SpsQ, at a concentration of 100 μg/ml in PBS with dog anti-SpsQ-M resulted in 105665±900.3 SEM, n=1 reduction in annexin mean fluorescent intensity (MFI) as compared with that of SpsQ treatment alone (
Discussion
Protective host immune responses against extracellular pathogens are typically antibody-mediated. Unfortunately, staphylococcal infection does not usually establish protective immunity and efficacious staphylococcal vaccines have proven difficult to develop. Most vaccine research has focused on S. aureus, which most commonly affects humans and is distinguished from S. pseudintermedius, which is primarily of veterinary concern.
S. pseudintermedius SpsQ binding with the Fab region of surface membrane-associated VH1-encoded B-cell antigen receptors induces apoptosis in canine B cells. We developed a non-toxigenic SpsQ (SpsQ-M; SEQ ID NO:2) by substitution of residues responsible for dual reactivity of each SpsQ domain with IgG Fcγ and Fab regions of surface membrane-associated VH1-encoded B-cell antigen receptors. Compared to staphylococcal protein A, SpsQ-M had much lower reactivity with IgG. Moreover, IgG, IgM, and IgA in sera from a dog with a history of chronic pyoderma had little immune-mediated binding to SpsQ-M indicating a lack of natural production of antibody elicited by infection with S. pseudintermedius. Comparing purified recombinant wild-type and mutant SpsQ, we found SpsQ-M had a significantly lower toxic effect on canine B lymphocytes.
It should be noted that specific antibody directed against native protein A cannot be measured due to the non-immune binding of protein A to immunoglobulin. After injecting a clinically healthy dog with SpsQ-M, we observed a high titer of SpsQ-specific antibodies. These results indicate that SpsQ, similar to SpA, suppresses adaptive immune responses during staphylococcal infection which may explain why previous S. pseudintermedius infections are not associated with protective immunity against recurrent infection. Dog anti-SpsQ-M abolished the superantigenic effect of SpsQ that triggers apoptotic cell death in canine B cells.
In this study is reported the identification of a novel cell wall-anchored 5′-nucleotidase in S. pseudintermedius (SpAdsA) that has no more than 52% identity with other bacterial adenosine synthase genes but which contains conserved functional domains. Furthermore we show that S. pseudintermedius uses adenosine synthase anchored on the cell wall to synthesize adenosine that suppresses the canine innate immune response and facilitates its survival.
Bacterial Strains and Plasmids
The S. pseudintermedius strains used in this study were as previously discussed, representing the most common sequence types (ST) in the United States, included 06-3228 (ST68), 08-1661 (ST71) and NA45 (ST84).
Plasmid construct pMA-RQ-Bs-M Ado 370-1868, containing a mutated synthetic S. pseudintermedius SpAdsA gene with BamHI/NotI cloning sites was obtained commercially (Invitrogen™ GeneArt™ Gene Synthesis—Thermo Fisher Scientific).
Bioinformatics Analysis and S. pseudintermedius 5′-Nucleotidase Characteristics
A rooted phylogenetic tree (UPGMA, unweighted pair group method with arithmetic mean) of 5′-nucleotidases was generated with Geneious version 9.1.3 (28) using complete protein sequences of SpAdsA (S. pseudintermedius 08-1661, ANS88668.1), S5 nA (S. pyogenes, AAK33792)(29), AdsA (S. aureus, ESR29110)(11), Ssads (S. suis, YP001197640)(14), 5′-nucleotidases (Staphylococcus delphini, WP_096556274.1), 5′-nucleotidases (Enterococcus faecalis, WP_010714156.1) and 5′-nucleotidases (Staphylococcus intermedius WP_086428047.1). Multiple sequence alignment of S. pseudintermedius 5′-nucleotidase proteins was performed using Geneious version 9.1.3. The bacterial localization prediction tool, PSORTb version 3.0.2 (available at psort.org/psortb) was used to predict the protein topology and domain structure of SpAdsA. Protein modeling and binding site prediction was performed using Phyre2 web portal (available at sbg.bio.ic.ac.uk/phyre2/) and 3DLigandSite (available at sbg.bio.ic.ac.uk/3dligandsite).
Polymerase Chain Reaction (PCR) Amplification of the Wild and Mutant SpAdsA Gene from S. pseudintermedius
Bacteria from a single colony of S. pseudintermedius strain 08-1661 obtained from blood agar plates were grown in tryptic soy broth (TSB) (Becton, Dickinson and Co., Sparks, MD) at 37° C. with 225 rpm shaking. DNA was extracted using MO BIO UltraClean® Microbial DNA Isolation Kit (QIAGEN Inc. Cat No. 12224-50) according to the manufacturer's instructions. Oligonucleotide primers with BamHI and NotI restriction sites (underlined) were designed using Geneious version 9.1.3 based on the complete genome sequence of S. pseudintermedius strain 08-1661.
Primers for the amplification of native SpAdsA were W-Ado-Forward: GCATGAGGATCCGAAACGACTGCAACGCATAC (SEQ ID NO:21) and W-Ado-Reverse: GCATGAGCGGCCGCTCCACCTGAAGCTGTAAAGTC (SEQ ID NO:22). Primers for amplification of mutant SpAdsA were M-AdoForward: GCATGAGGATCCATGTTAAGATTGTCGGCTAAAAAAG (SEQ ID NO:23) and M-AdoReverse: GCATGAGCGGCCGCTCCACCTGAAGCTGTAAAGTC (SEQ ID NO:24).
The native SpAdsA open reading frame (ORF) (1539 bp) without the regions encoding the predicted N-terminal signal and C-terminal sortase anchoring sequence was amplified from 60 ng of genomic DNA of S. pseudintermedius 08-1661 by 35 cycles of PCR with rtaq polymerase (Takara, Cat No. R004) at an annealing temperature of 55° C. using primer W-Ado-Forward and W-Ado-reverse. The ORF of mutant SpAdsA (1536 bp) was excised from pMA-RQ-Bs-M Ado plasmid using BamHI-NotI restriction enzymes.
Cloning, Expression and Purification of Recombinant Wild and Mutant SpAdsA of S. pseudintermedius 08-1661
The PCR products of wild and mutant SpAdsA were digested with the restriction enzymes BamHI and NotI and purified using QuickClean II Plasmid Miniprep Kit (Genscript, Cat No. L00420) according to the manufacturer's instructions. Purified product was ligated into pETBlue™-2 (Novagen, WI USA), transformed into DH5-alpha E. coli and plated on LB agar plates with 100 μg/mL ampicillin, 80 μg/mL X-gal and 50 μM IPTG (Teknova, Cat No. L1925). For screening the clones containing recombinant plasmid, X-gal chromogenic substrate was added to the agar plates. The recombinant plasmid was extracted using GeneJET Plasmid Miniprep Kit according to the manufacturer's instructions (ThermoFisher, Cat No. K0502). The presence of the cloned fragment was confirmed by PCR. pETBlue™-2 constructs were transformed in Tuner™ (DE3) pLacI E. coli chemically-competent cells (Novagen, Cat No 0.70623) by heat shock and plated on LB agar containing 50 μg/ml ampicillin (Sigma-Aldrich, Cat No. A5354) and 20 μg/ml chloramphenicol (Sigma-Aldrich, Cat No. C0378). A single colony was used to inoculate LB broth containing 50 μg/ml ampicillin and 20 μg/ml chloramphenicol and the broth was incubated overnight at 37° C. with shaking at 225 rpm. Fresh LB medium containing 50 μg/ml ampicillin and 20 μg/ml chloramphenicol was inoculated at a 1:100 dilution and grown to an optical density of 0.6 at wavelength 600 nm then protein expression was induced by addition of 1 mM isopropyl β-D-1-thiogalatopyranoside (IPTG) for 4 hr at 30° C. with shaking at 225 rpm.
A suspension of IPTG-induced Tuner cell bacteria (100 ml) was centrifuged at 12000×g for 5 min. Bacteria were suspended in 5 ml of protein extraction reagent (BugBuster, Novagen Cat No. 70584) and 20 μl 100× Protease Inhibitor Cocktail Set III, EDTA-Free (Calbiochem, Cat No. 539134), incubated for 30 min at 37° C. in a shaking incubator at 225 rpm, and centrifuged at 12,000×g for 45 min at 4° C.
Recombinant protein was purified using a HisPur™ Ni-NTA Spin Purification Kit (Thermo Scientific, Cat No. 88228) according to the manufacturer's instructions. Protein concentrations were determined with the Pierce™ BCA Protein Assay Kit (Thermo Scientific, Cat No. 23225).
SDS-PAGE and Western Blot
Protein samples were resolved by SDS-PAGE in 4-12% polyacrylamide gels (Invitrogen, Cat No. NP0322BOX) and electrophoretically transferred onto a nitrocellulose membrane (Thermo Scientific, Cat No. 77010). The blots were blocked overnight in 5% (wt/vol) nonfat dried milk powder in 0.05% polyethylene glycol sorbitan monolaurate (Tween 20) containing phosphate buffered saline (PBS-T) at 4° C. The blocked membranes were incubated with a 1:2,000 dilution of horseradish peroxidase (HRP)-conjugated 6×his tag monoclonal antibody (Thermo Scientific, Cat No. MA1-21315-HRP) in 0.05% PBS-T for 1 h with 225 rpm shaking at room temperature. After five washes with 0.05% PBS-T bound antibodies were detected using 1-Step™ chloronaphthol substrate solution (Thermo Scientific, Cat No. 34012).
Adenosine Synthase Activity
Overnight cultures of S. pseudintermedius 06-3228 and 08-1661 were centrifuged and washed twice with phosphate buffer saline (PBS) and then suspended in 1 ml digestion buffer (0.6 M sucrose buffered with 50 mM Tris, pH 7.5) containing 200 μg trypsin from porcine pancreas (Sigma-Aldrich, Cat NO. T4799). Bacteria were digested with trypsin for 1.5 h at 37° C., then the supernatant containing S. pseudintermedius SpAdsA was obtained after centrifugation at 10,000×g for 5 minutes at 4° C. Four ml of S. pseudintermedius tryptic digest and recombinant SpAdsA protein at 0.15 μg/ml were suspended in 500 μL TM buffer (50 mM Tris-HCL [pH 7.5], 5 mM MnCl2), then hydrolysis of ATP (Sigma-Aldrich, Cat NO. A26209) and AMP (Sigma-Aldrich, Cat NO. 01930) was carried out in the presence of 1 mM nucleotide at 37° C. for 15 minutes. Inorganic phosphate (IP) release was measured using a malachite green phosphate kit according to the manufacturer's protocol (Sigma-Aldrich, Cat NO. MAK307).
Bacterial Survival in Blood
Overnight cultures of S. pseudintermedius 06-3228 and 08-1661 were diluted 1:100 into fresh TSB and grown for 3 hours at 37° C. Staphylococci were centrifuged 9000×g, washed twice, and diluted in PBS to yield an O.D 600 of 0.5. Then 100 μl of 4×106 CFU per ml of bacteria were mixed with 500 μl of fresh canine blood, collected in EDTA. The samples were incubated at 37° C. with 100 rpm shaking and harvested at 60, 90 and 120 minutes. They were incubated on ice for 30 minutes in 1 ml of Red Blood Cell Lysing Buffer Hybri-Max™ (Sigma-Aldrich, Cat NO. R7757), then bacteria were pelleted at 9,000×g at 4° C. and plated on tryptic soy agar (TSA) for enumeration of viable bacteria.
Flow Cytometry Analysis
Bacteria for the phagocytosis assay were labeled using a pHrodo™ phagocytosis particle labeling kit (Life technologies, P35369) (10). Briefly, overnight cultures of S. pseudintermedius 06-3228 and 08-1661 were diluted 1:100 into fresh TSB and grown for 3 hours at 37° C. Bacteria were centrifuged, washed twice, and diluted to 20 mg/mL wet weight in 0.1 M sodium bicarbonate, pH 8.5, and pHrodo™ dye was dissolved in DMSO at 10 mM. The bacteria were suspended at a final dye concentration of 0.5 mM. The inhibitory effect of SpAdsA on canine neutrophils was evaluated in blood samples by exposing the EDTA-whole blood samples to pHrodo™ dye-labeled S. pseudintermedius 06-3228 and 08-1661 for 30 and 60 minutes at 37° C. The RBCs were lysed using Red Blood Cell Lysing Buffer Hybri-Max™ (Sigma-Aldrich, Cat NO. R7757) followed by centrifugation and washing. The final cell pellet, containing leukocytes, was suspended in wash buffer and analyzed using a flow cytometer equipped with a 488-nm argon ion laser (Attune acoustic focusing cytometry).
Statistical Analysis
Data were analyzed using general liner modes with treatment, protein and time as the independent variables while inorganic phosphate (IP) release, mean fluorescence intensity (MFI) and colony forming units per ml (CFU ml−1) were used as response variables, respectively. Multiple comparisons were performed with Tukey's adjustment. Statistical significance was identified at the significant level of 0.05. Rank transformation on response variables were conducted when the normality assumption was violated. All analyses were conducted using PROC mixed in the SAS system for Windows version 9.4 (SAS Institute Inc., Cary, NC).
Results
Bioinformatics Analysis and S. pseudintermedius 5′-Nucleotidase Characteristics
Bioinformatic analysis of the predicted 780-amino acid sequence showed significant homologies to members of the 5′-nucleotidase/apyrase protein family (Interpro accession number: IPRO006179) with two characteristic domains. Dimetal-containing phosphoesterases (DMPs) (Interpro accession number: IPRO29052) are between position 147 and 401 with two domain signature sequences D1 and D2 (
A BLAST search of the GenBank database revealed related putative nucleotidases in other pathogenic bacterial species within the Staphylococcus intermedius group including Staphylococcus delphini (91% amino acid identity) and Staphylococcus intermedius (88% amino acid identity). Only limited sequence identity was found with S. aureus AdsA (52.4%), Enterococcus fecalis 5′-nucleotidase (36.0%), S. pyogenes S5 nA (20.1%) and S. suis Ssads (20.0%). No other species of bacteria with homologous proteins were identified in the database.
The Phre2 web tool was used to analyze SpAdsA based on the X-ray structure of S. suis type 2 5′-nucleotidase. Aspartic acids (positions 154 and 189), histidine (positions 156, 318, 354 and 356) and asparagine (position 221) within the SpAdsA signature sequences were predicted to be essential for divalent metal cation binding (
Cloning, Expression and Purification of Recombinant S. pseudintermedius SpAdsA
SpAdsA is predicted to have an N-terminal signal sequence and C-terminal sortase anchoring domain. Recombinant SpAdsA proteins (SEQ ID NO:6) lacking those sites were produced to facilitate a study of the mature protein. The sizes of recombinant native and mutant SpAdsA were 55.8 kDa and 55.4 kDa respectively, as determined by western blot using monoclonal antibody directed against the HIS tag.
S. pseudintermedius 5′-Nucleotidase Synthesizes Adenosine
Tryptic digests of surface proteins of S. pseudintermedius 06-3228 showed hydrolytic activity that released adenosine from AMP and ATP (
Sp AdsA Facilitates Survival of S. pseudintermedius in Canine Blood
To investigate the potential immune evasive attributes of the 5′-nucleotidase activity of SpAdsA, the effect of adenosine on the survival of S. pseudintermedius strains 06-3228 and 08-1661 in canine blood was examined. As shown (
Adenosine Mediates Inhibitory Signals Through A2A Receptors on Canine PMNs
To determine the role of A2A receptors on canine PMNs in the signaling pathway of adenosine we performed pharmacological inactivation of A2AR using the A2A receptor antagonist ZM241385. With ZM241385, survival of S. pseudintermedius 06-3228 and 08-1661 was significantly decreased at all of time points (06-3228; p<0.0108); 08-1661; p≤0.0265) compared to the same bacterial strains without the antagonist (
Adenosine Inhibits Phagocytic Activity of Canine Blood PMNs for S. pseudintermedius
The effect of SpAdsA on PMN-mediated phagocytosis was determined with pHrodo™ labeled bacteria used for flow cytometry. PMN were gated using forward and side scatter characteristics. As shown (
The addition of 5′-(α, β-methylene) diphosphate (500 μM) significantly increased phagocytosis of S. pseudintermedius 06-3228 in dog blood after incubation periods of 30 minutes at 37° C. (
Addition of ZM241385 (1 μM), however, antagonized the ability of S. pseudintermedius strain 06-3228 incubated in dog blood at 37° C. for 30 minutes (
For S. pseudintermedius strain 08-1661, we found that A2A receptor antagonist diminished the S. pseudintermedius strain 06-3228 phagocytosis evasion when incubated in dog blood for 30 and 60 minutes at 37° C. (
Discussion
This study showed how S. pseudintermedius modulates neutrophil function by perturbing adenosine levels using SpAdsA residing on its cell wall. It demonstrates that recombinant SpAdsA and S. pseudintermedius cell wall proteins containing SpAdsA hydrolyze ATP and AMP generating inorganic phosphate and adenosine. Neutrophils play a crucial role as professional phagocytes in the host defense against pathogens. They release adenosine and can respond to it via G-protein coupled receptors expressed on their surface in a dose dependent manner. Although extracellular adenosine is essential for the suppression of inflammation, accumulation of high concentrations of adenosine is also harmful. Disturbance of the immune homeostasis maintained by adenosine through perturbing its level likely affects host immune responses during infection.
A search of the GenBank database for SpAdsA protein in other members of the Staphylococcus intermedius group (SIG) found that 5′-nucleotidase predicted to be produced by members of this group share over 81% amino acid identity. Conversely, SpAdsA shares no more than 52% amino acid identity with proteins produced by S. aureus, Enterococcus and streptococcal species.
SpAdsA was considered a good candidate for producing adenosine synthase due to the presence of conserved domains associated with this enzymatic activity in other species of bacteria. Native and mutant 5′ nucleotidase proteins excluding signal peptides and anchoring sequences were used for this study because the full length expressed proteins were not readily soluble in aqueous solution and would not represent mature proteins. Enzymatic inhibition using an ADP analogue or A2 receptor antagonist significantly reduced bacterial survival in dog blood suggesting that S. pseudintermedius SpAdsA plays an important role in canine pathogen immune evasion and may serve as an important candidate target for novel anti-infective drugs and vaccines against canine pyoderma. We used an A2 receptor inhibitor to determine if adenosine signaling occurs through the A2 receptor in canine cells and found that the addition of ZM241385 stimulated phagocytic cell activities against S. pseudintermedius.
The present study revealed the diminished ability of canine blood to kill S. pseudintermedius in the presence of exogenous adenosine. Conversely, addition of ADP analogue or ZM241385 prevented S. pseudintermedius escape from phagocytic killing. In summary, these results indicate that SpAdsA plays an important role in promoting S. pseudintermedius survival and inhibiting PMN phagocytosis and killing activity by synthesis of adenosine and is therefore a good vaccine candidate against S. pseudintermedius infection.
Through genome sequencing and analysis of the S. pseudintermedius secretome using liquid chromatography mass spectrometry (described above) we identified two proteins, “LukS” and “LukF”, encoded on a degenerate prophage contained in the genome of S. pseudintermedius isolates. No other leukocidins were found in any of the available S. pseudintermedius genome sequences. Phylogenetic analysis of LukFS components in comparison to the rest of the leukocidin family showed that LukS was most closely related to S. aureus LukE, LukP and gamma hemolysin subunit A, whereas LukR was most similar to S. aureus gamma hemolysin subunit B. The killing effect of recombinant S. pseudintermedius LukF and LukS on canine polymorphonuclear leukocytes was determined using a flow cytometry cell permeability assay. The cytotoxic effect occurred only when the two recombinant proteins were combined. Engineered mutant versions of the two-component pore-forming leukocidins, produced through amino acids substitutions at selected points, were not cytotoxic. Anti-LukFS produced in dogs against attenuated proteins reduced the cytotoxic effect of native canine leukotoxin which highlights the importance of LukFS as a promising component in a vaccine against canine S. pseudintermedius infections. It is noted that occasionally herein the nomenclature LukS is used interchangeably with LukN and LukF is used interchangeably with LukR.
Bacterial Strains, Plasmids and Growth Conditions
The S. pseudintermedius strains used in this study, representing the most common multilocus sequence types (ST) previously reported in the United States, included 06-3228 (ST68), 08-1661 (ST71) and NA45 (ST84). Strains 06-3228 and 08-1661 were isolated at the University of Tennessee, College of Veterinary Medicine Bacteriology Laboratory. Strain NA45 was a gift of Faye Hartmann of the University of Wisconsin, School of Veterinary Medicine.
Plasmid construct pMA-LukS-M and pMA-LukF-M, each containing a mutated, synthetic S. pseudintermedius gene (designed as described below) with BamHI/NotI cloning sites, was obtained commercially (Life Technologies Corp., Carlsbad, CA).
Bacterial colonies grown on blood agar plates were inoculated into 5 ml of sterile trypticase soy broth (TSB) (BD Biosciences, San Jose, CA Cat No. RS1-011-21) and incubated overnight at 37° C. with shaking at 225 rpm (Excella E24 Incubator Shaker, New Brunswick Scientific). Fifty microliters of overnight culture were inoculated into 5 ml of fresh, sterile TSB to initiate log-phase bacterial cultures. Bacteria were grown at 37° C. with shaking at 225 rpm until an optical density of OD600=0.4-0.6 was reached.
LC-MS/MS Analysis of S. pseudintermedius Supernatant
LC-MS/MS analysis was as previously described.
Bioinformatics Analysis
A rooted phylogenetic tree (UPGMA (unweighted pair group method with arithmetic mean) of LukFS was generated with Geneious version 11.0.3 using the complete protein sequences of each leukotoxin subunit with S. pseudintermedius protein A serving as an outgroup (Table 7). Multiple sequence alignment (MSA) of LukS and LukF proteins from diverse isolates of S. pseudintermedius was performed using Geneious, version 11.0.3. The bacterial localization prediction tool, PSORTb version 3.0.2 (psort.org/psortb) was used to determine the topology and domain structure of LukF and LukS.
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus aureus
Staphylococcus
pseudintermedius
Staphylococcus
pseudintermedius
S. pseudintermedius LukF and LukS modeling and binding site prediction was performed using Protein Homology/analogY Recognition Engine V 2.0 (Phyre2) (sbg.bio.ic.ac.uk/phyre2) and the 3DLigandSite (sbg.bio.ic.ac.uk/3dligandsite) using S. aureus LukS-PV, LukF-PV, LukP and LukQ as a basis to predict the critical amino acids for protein function. PHAST (phast.wishartlab.com/index.html) and PHASTER (phaster.ca) were used for prophage detection in a total of 22 S. pseudintermedius isolates. LukS-M was designed with the following substitutions: T57F, K128A and S239A and the LukF-M substitutions were S59D and N65A.
Polymerase Chain Reaction (PCR) Amplification of LukN and LukR
Bacteria from a single colony of S. pseudintermedius strain 06-3228 obtained from blood agar plates were grown in TSB at 37° C. with 225 rpm shaking. DNA was extracted using a MO BIO UltraClean® Microbial DNA Isolation Kit (QIAGEN Inc. Cat No. 12224-50) according to the manufacturer's instructions. Oligonucleotide primers (Integrated DNA Technology, Coralville, USA) (Table 8) were designed using a PrimerQuest Tool (idtdna.com/Primerquest/Home/Index) based on the genomic sequence of S. pseudintermedius strain 06-3228. The native LukF and LukS open reading frames (ORF) (933 and 981 bp, respectively) without the regions encoding the predicted N-terminal signal peptide were amplified from S. pseudintermedius 06-3228 genomic DNA and the ORF of mutant LukS and LukF were amplified from pMA-LukS-M and pMA-LukS-M plasmids (Life Technologies Corp., Carlsbad, CA), respectively (Table 9). PCR was performed using tact polymerase (rTaq, Takara, Cat No. R004) and the following cycling conditions were performed: initial denaturation at 95° C. for 90 seconds, 30 cycles of denaturation at 94° C. for 30 seconds, annealing at 55° C. for 30 seconds and extension at 72° C. for 1 minute followed by a final extension at 72° C. for 5 minutes. All ORFs were amplified without a histidine tag because pETBlue-2 allowed T7lac promoter-based expression of target genes with C-terminal histidine•Tag® sequences. PCR products were Sanger sequenced at The University of Tennessee Genomics Core facility.
NotI, XhoI and BamHI restriction sites are underlined.
pseudintermedius LukN
pseudintermedius LukR
Kluyveromyces
lactis
pseudintermedius LukR
Cloning, Expression and Purification of Recombinant Native and Attenuated LukNR
To clone S. pseudintermedius native and mutant LukS and mutant LukF, their PCR products were digested with NotI and BamHI, then ligated into pETBlue-2 (Novagen, Cat No 0.70674) and transformed into DH5-alpha E. coli chemically-competent cells (Table 9) (New England BioLabs Inc., Cat No. C2987I) by heat shock. The DH5-alpha E. coli were plated on LB agar plates with 100 μg/mL ampicillin. The plasmid constructs were transformed into Tuner™ (DE3) pLacI E. coli chemically-competent cells (Table 9) (Novagen, Cat No 0.70623) by heat shock and the Tuner™ (DE3) pLacI E. coli were plated on LB agar containing 50 μg/ml ampicillin and 20 μg/ml chloramphenicol.
To express recombinant S. pseudintermedius native and mutant LukS and mutant LukF, a single colony of Tuner™ (DE3) pLacI E. coli was inoculated into LB broth containing 50 μg/ml ampicillin and 2011 g/ml chloramphenicol and bacteria grown overnight at 37° C. with 225 rpm shaking. LB broth containing 50 μg/ml ampicillin and 20 μg/ml chloramphenicol was inoculated with a 1:100 dilution of overnight culture and grown at 37° C. with 225 rpm shaking until a 600 nm optical density of between 0.4 and 0.6 was reached. Protein expression was induced by addition of 1 mM Isopropyl β-D-1-thiogalatopyranoside (IPTG) (Teknova, Cat. No. 13431) and bacteria were grown for 4 hr at 30° C. with shaking at 225 rpm. Bacterial cultures were centrifuged at 12,000×g for 5 min in 5 ml of protein extraction reagent (BugBuster, Novagen Cat No. 70584) and 20 μl of 100× protease inhibitor (Cocktail Set III, EDTA-Free Calbiochem, Cat No. 539134) and incubated for 30 min at 37° C. in a shaking incubator at 225 rpm. Bacteria were pelleted by centrifugation at 12,000×g for 45 min at 4° C. Recombinant proteins were purified using affinity purification (HisPur™ Ni-NTA Spin Purification Kit, Thermo Scientific, Cat No. 88228).
Recombinant native LukF was cloned using an integrative expression vector (pKLAC2) and expressed in Kluyveromyces lactis (New England Biolabs, Cat No. E1000S). Recombinant protein was purified from K. lactis supernatant using affinity purification (HisPur™ Ni-NTA Spin Purification Kit, Thermo Scientific, Cat No. 88228). Protein concentrations were determined using a bicinchoninic acid (BCA) assay (Thermo Scientific, Cat No. 23227).
SDS-PAGE and Western Blot
Protein samples were resolved by SDS-PAGE in 4-12% polyacrylamide gels (Invitrogen, Cat No. NP0322BOX) and electrophoretically transferred onto nitrocellulose membranes (Thermo Scientific, Cat No. 77010). The blots were blocked overnight in 5% (wt/vol) nonfat dried milk powder in 0.05% polyethylene glycol sorbitan monolaurate (Tween 20) containing phosphate buffered saline (PBS-T) at 4° C. The blocked membranes were incubated with a 1:2,000 dilution of horseradish peroxidase (HRP)-conjugated anti-6×his tag monoclonal antibody (Thermo Scientific, Cat No. MA1-21315-HRP) in 0.05% PBS-T for 1 h with 225 rpm shaking at room temperature. After five washes with 0.05% PBS-T bound antibodies were detected using 1-Step™ chloronaphthol substrate solution (Thermo Scientific, Cat No. 34012).
Preparation of Canine Anti-S. pseudintermedius LukNR
Recombinant LukS-M and LukF-M produced in E. coli were purified using affinity chromatography (as above) and endotoxin concentrations were measured using a ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (Genscript, Cat. No. L00350). Recombinant LukS-M and LukF-M at 20 μg each/0.5 cc in phosphate buffered saline (PBS) (pH 7.2) were injected in the lateral thorax by the subcutaneous route, into three clinically normal dogs. Injections were given once every 7 days for a total of three injections with a control dog receiving PBS (pH 7.2) only. Blood (6 cc) was collected from a jugular vein 4 times, on days −7, 8, 15 and 29. The collected blood was left undisturbed at room temperature for 30 min followed by centrifugation at 2,000×g for 10 min in a refrigerated centrifuge.
Enzyme-Linked Immunosorbent Assay
For measurement of recombinant protein antigenicity, LukS-M and LukF-M were coated separately onto ELISA plates (Corning, Cat No. 3590) at 2 μg/ml in PBS (pH 7.2). The plates were washed with 0.05% PBS-T and incubated with two-fold serial diluted serum from dogs (injected with recombinant proteins) for 1 h at 37° C., then bound IgG was detected using HRP-conjugated goat anti-dog IgG heavy and light chain (Bethyl Laboratories, Inc. Cat No. A40-123-1). ELISA assays plates were washed three times with PBS-T between all incubations, bound antibodies were detected using TMB substrate (Thermo Scientific, Cat No. N301), reactions were stopped with 0.18 M sulphuric acid and optical density read at 450 nm on a plate reader (Bio TEK, EL800). The experiment was repeated a minimum of three times and a p-value of <0.05 was considered significant for all the experiments unless otherwise stated.
PMN Cell Permeability Assay
Canine blood was collected from healthy dogs using a sterile blood collection system with EDTA anti-coagulant (BD Vacutainer). Then, 600 μl of dog blood was added to 1 ml of red blood cell lysing buffer (Hybri-Max™, Sigma-Aldrich, Cat No. R7757-100 ML) for 30 min at 37° C. in 15 ml sterile plastic tube, centrifuged and re-suspended in 1 ml RPMI medium supplemented with 10% fetal bovine serum. PMNs were incubated with recombinant proteins (LukS and LukF, LukS alone, LukF alone, LukS-M and LukF-M) in a volume of 500 μl in RPMI medium supplemented with 10% fetal bovine serum in a 5% CO2 incubator for 30 min. Supernatant of S. pseudintermedius 06-3228 was harvested at log phase to test the toxic effect of secreted LukF S. PMNs were stained with 1 μl of Sytox Green (Life technologies, Inc. Cat No. 1776406) for 30 min, washed with PBS (pH 7.2) twice and analyzed using a flow cytometer (Attune acoustic focusing cytometer) by gating on PMNs based on their side and forward scatter properties. In order to measure the protective effect of anti-LukFS on canine PMNs, recombinant LukF and LukS were incubated with canine anti-LukS-M LukF-M at a dilution of 1:100 for 30 min at 37° C., then tested with the cell permeability assay as previously described.
Biotin Labeling of S. pseudintermedius Wild Type and Attenuated LukFS
Purified recombinant S. pseudintermedius wild type and attenuated LukS and LukF at 500 μg/ml in PBS (pH 7.2) were incubated with 50 μl of 10 mM EZ-Link Sulfo-NHS-LC-Biotin reagent (equal to 20-fold molar excess of biotin) (Thermo Scientific, Cat No. 21327) for 30 min at room temperature. Excess biotin was removed using an Amicon Ultra-0.5 Centrifugal Filter Unit with a 30 kDa molecular weight cut-off (Milipore sigma, Cat No. UFC5030). The biotin-labeled proteins were stored at −20° C. until further use.
To test the binding of wild type and attenuated LukS and LukF to canine PMNs, biotin labelled recombinant proteins were incubated with PMNs from a clinical healthy dog for 30 minutes at room temperature, the cells were washed, then PMNs were incubated with 1:500 dilution of avidin-FITC conjugate (Sigma-Aldrich, A2050) at room temperature for 30 minutes in the dark. Unbound conjugate was removed by washing and the amount of binding was determined using a flow cytometer (Attune acoustic focusing cytometer) by gating on PMNs based on their side and forward scatter properties.
Statistical Analysis
All analyses were conducted using the GraphPad Prism software (Version 7, GraphPad Software Inc.).
Results
LC MS/MS Data Analysis of S. pseudintermedius Culture Supernatant
We used LC-MS/MS to screen culture supernatants of three clinical strains of S. pseudintermedius (06-3228, 08-1661 and NA45) for putative leukocidins and identified proteins with homology to LukS and LukF subunits of previously described leukocidins. LukS and LukF proteins were detected in the supernatant fraction of 06-3228, 08-1661 and NA45, respectively (Table 10). The LukFS components were detected in the supernatants of the three isolates as two proteins secreted independently with 310 and 326-amino acid sequences for LukS and LukF, respectively.
LukS and LukF were secreted by S. pseudintermedius 06-3228, 08-1661 and NA45. Secretome proteins were compared among the three isolates using their respective genomes as reference databases. Percent coverage was calculated by dividing the number of amino acids in all found peptides by the total number of amino acids in the entire protein sequence.
LukFS: A New Phage Encoded Leucocidin
Multiple sequence alignment (MSA) analysis showed that LukFS is conserved among S. pseudintermedius strains including 06-3221, 08-1661 and NA45. The LukF and LukS genes contain open reading frames in S. pseudintermedius that are 933 and 981 bp in length respectively, with amino acid identities over 99.4% between strains.
A 14.9 kb incomplete prophage (similar to Φ Staphy_96_NC_007057) was identified in the genome of S. pseudintermedius 06-3228. A BLAST search of Φ Staphy_96_NC_007057 using complete genome sequences of S. pseudintermedius strains available in the GenBank database and others sequenced in our lab but not yet published, revealed that approximately 7 Kb of the phage are present in all of S. pseudintermedius isolates examined (a total of 22 isolates). They also contain the coding DNA sequences (CDS) of ascorbate-specific PTS system EII A, B and C components, probable L-ascorbate-6-phosphate lactonase UlaG (L-ascorbate utilization protein G), phosphoglycerate mutase family 2 and hypothetical protein.
Phylogenetic analysis of LukFS in comparison to the entire leukocidin family showed that LukS is most closely related to S. aureus LukE, LukP and gamma hemolysin subunit A. S. pseudintermedius LukF is most closely related to S. aureus gamma hemolysin subunit B (data not shown).
Multiple sequence alignment (MSA) analysis showed that LukFS in S. pseudintermedius is a unique leukotoxin and that each functional component shares considerable similarity with other staphylococcal leukotoxin fractions (Table 11).
S.
pseudintermedius
S.
pseudintermedius
A BLAST search of LukFS in the GenBank database revealed that it shares sequence similarity with other pathogenic bacterial species within the Staphylococcus intermedius group. LukS shares 98.7% identity with Staphylococcus intermedius and 90.6% identity with Staphylococcus delphini. LukF has 99% amino acid identity with S. intermedius and 85% amino acid identity with S. delphini.
The LukS and LukF model developed with the Phyre2 web portal, using S. aureus LukPQ as a basis to predict critical residues for protein function (
Cloning, Expression and Purification of Recombinant S. pseudintermedius LukS and LukF
Recombinant polyhistidine tagged wild type and mutant LukS and LukF were generated in E. coli and LukR was secreted in the culture supernatant of K. lactis using an integrative expression vector (pKLAC2). Recombinant proteins were purified using HisPur Ni-NTA resin under native conditions and eluted using an imidazole gradient. The molecular weights of LukS, LukS-M, LukF and LukF-M determined in western blots were of the expected sizes (39.43, 39.12, 37.27 and 37.59 kDa, respectively). The endotoxin levels of purified recombinant, attenuated LukS and LukF were below 0.5 endotoxin units/mg protein.
Attenuated LukFS Induces Specific Antibody Responses
Specific antibodies against S. pseudintermedius wild type and attenuated LukF and LukS were detected using an indirect ELISA, with sera collected on days −7, 8, 15 and 29, after the second injection of LukS-M and LukF-M (on day 15) and was higher on day 29 (P<0.0001) compared to pre-injection control sera (
Antibodies against S. pseudintermedius wild type LukS and LukF were detected using an indirect ELISA. Recombinant S. pseudintermedius LukS and LukF proteins were coated on ELISA plates, then incubated with two-fold serially diluted serum from dog vaccinated with the same proteins. High reactivity with LukS and LukF was seen from sera collected two weeks after the third injection of LukS-M and LukF-M (P=0.0001****) and was higher (P=0.0003***) compared to pre-injection sera. The values represent averages from two independent experiments.
LukFS Kills Canine PMNs
Canine PMNs were highly susceptible to LukFS with lysis induced within 30 minutes at a concentration of 200 ng of each leukotoxin component (P<0.0001) and a 1:2 S. pseudintermedius 06-3228 supernatant dilution (P<0.0001) (
Dog Anti-LukFS Reduced the Cytotoxic Effect of Canine Leukotoxin on PMNs
Dog anti-LukFS at a dilution of 1:100 preincubated with LukFS showed a 52645±3570, n=2 reduction in mean fluorescent intensity (MFI) compared with LukFS treatment alone (
Discussion
We identified a new member of the two-component leucocidin family. LukFS is highly conserved among S. pseudintermedius isolates and appears to be the only leucocidin produced by this species. It is associated with an incomplete prophage that occurs in degenerate form across all S. pseudintermedius isolates for which genomic sequence is available.
In accordance with its host distribution in a canine opportunistic pathogen, LukFS is cytotoxic against dog PMNs. This highlights the immune-evasive attribute of S. pseudintermedius LukFS in the dog, in line with the assumed function of other phage-encoded leukocidins that similarly have a host-specific function and distribution.
LukS-M and LukF-M, attenuated to reduce their toxicity, bound to canine leukocytes without causing any significant killing suggesting the mutations disrupted their ability to oligomerize, an essential function required for cytolysis. The low concentration of endotoxin in the recombinant proteins and the low toxicity exhibited by the attenuated protein produced in E. coli suggests that endotoxin did not play a role in neutrophil killing.
Analysis of the S. pseudintermedius secretome using liquid chromatography mass spectrometry (LC-MS) guided by genome sequences, we identified an S. pseudintermedius exotoxin provisionally named SpEX. This exotoxin binds to and kills polymorphonuclear leukocytes (PMNs) and peripheral blood mononuclear cells (PBMCs) and inhibits chemotaxis at sublethal concentrations. A mutant version of SpEX, produced through amino acids substitutions at selected points, had diminished cytotoxicity. Anti-SpEX produced in dogs reduced the inhibitory effect of native SpEX on canine PMNs chemotaxis and was statistically significant in protecting the dog immune cells from the toxic effect of the native recombinant protein. These results suggest that attenuated SpEX produced in this study is a good candidate for inclusion in a vaccine or a therapeutic for use in the treatment and prevention of S. pseudintermedius infections.
Bacterial Strains, Plasmids and Growth Conditions
The S. pseudintermedius strains used in this study, representing the most common multilocus sequence types (ST) reported in the United States, included 06-3228 (ST68) and 08-1661 (ST71) were isolated at the University of Tennessee, College of Veterinary Medicine Bacteriology Laboratory. Strain NA45 was a gift of Faye Hartmann of the University of Wisconsin, School of Veterinary Medicine.
A plasmid construct containing a mutated, synthetic S. pseudintermedius spEX (designed as described below) with BamHI/NotI cloning sites, was obtained commercially (Genscript Piscataway, NJ USA) (Table 12). Bacterial colonies grown on blood agar plates were inoculated into 5 ml of sterile trypticase soy broth (TSB) (BD Biosciences, San Jose, CA Cat No. RS1-011-21) and incubated overnight at 37° C. with shaking at 225 rpm (Excella E24 Incubator Shaker, New Brunswick Scientific). Fifty microliters of overnight culture were inoculated into 5 ml of fresh, sterile TSB to initiate log-phase bacterial cultures. Bacteria were grown at 37° C. with shaking at 225 rpm until an optical density (OD600) of 0.4-0.6 was reached.
LC-MS/MS Analysis of S. pseudintermedius Supernatant
LC-MS/MS analysis was as described above.
Bioinformatics Analysis
Multiple sequence alignment (MSA) of SpEX proteins from diverse isolates of S. pseudintermedius was performed using Geneious, version 11.0.3. S. pseudintermedius SpEX modeling and binding site prediction were performed using Protein Homology/analogY Recognition Engine V 2.0 (Phyre2) (sbg.bio.ic.ac.uk/phyre2) and the 3DLigandSite (sbg.bio.ic.ac.uk/3dligandsite). A pairwise sequence alignment of S. pseudintermedius SpEX and S. aureus SSL11 was used to identify conserved amino acids critical for SpEX activity. We designed a full-length, attenuated S. pseudintermedius SpEX construct (SpEX-M), with the following amino acid substitutions using Geneious, version 11.0.3: D 102A, T125P, T206P and R217A.
Polymerase Chain Reaction (PCR) Amplification of spEX
Bacteria from a single colony of S. pseudintermedius strain 06-3228 obtained from blood agar plates were grown in TSB at 37° C. with 225 rpm shaking. DNA was extracted using a MO BIO UltraClean® Microbial DNA Isolation Kit (QIAGEN Inc. Cat No. 12224-50) according to the manufacturer's instructions. Oligonucleotide primers (Integrated DNA Technology, Coralville, USA) (Table 13) were designed using a PrimerQuest Tool (idtdna.com/Primerquest/Home/Index) based on the genomic sequence of S. pseudintermedius strain 06-3228.
The native SpEX open reading frames (ORF) (705 bp) without the regions encoding the predicted N-terminal signal peptide were amplified from S. pseudintermedius 06-3228 genomic DNA and the ORF of mutant SpEX were amplified from a pUC19-spEX-M plasmid (Genscript Piscataway, NJ USA) (Table 12). PCR was performed using taq polymerase (rTaq, Takara, Cat No. R004) and the following cycling conditions were performed: initial denaturation at 95° C. for 90 seconds, 30 cycles of annealing at 55° C. for 30 seconds and extension at 72° C. for 1 minute followed by a final extension at 72° C. for 5 minutes. All ORFs were amplified without a histidine tag because pETBlue-2 allowed T7lac promoter-based expression of target genes with C-terminal histidine •Tag® sequences. PCR products were sequenced at The University of Tennessee Genomics Core facility.
Cloning, Expression, and Purification of Recombinant Native and Attenuated SpEX
To clone S. pseudintermedius native and mutant SpEX, their PCR products, amplified from synthetic genes, were digested with NotI and BamHI, then ligated into pETBlue-2 (Novagen, Cat No 0.70674) (Table 12) and transformed into DH5-alpha E. coli chemically-competent cells (Table 12) (New England BioLabs Inc., Cat No. C2987I) by heat shock. The DH5-alpha E. coli were plated on LB agar plates with 100 μg/mL ampicillin. The plasmid constructs were transformed into Tuner™ (DE3) pLacI E. coli chemically-competent cells (Table 12) (Novagen, Cat No 0.70623) by heat shock and the Tuner™ (DE3) pLacI E. coli were plated on LB agar containing 50 μg/ml ampicillin and 201 μg/ml chloramphenicol.
To express recombinant S. pseudintermedius native and mutant SpEX, a single colony of Tuner™ (DE3) pLacI E. coli was inoculated into LB broth containing 50 μg/ml ampicillin and 20 μg/ml chloramphenicol and the bacteria grown overnight at 37° C. with 225 rpm shaking. LB broth containing 50 μg/ml ampicillin and 20 μg/ml chloramphenicol was inoculated with a 1:100 dilution of overnight culture and grown at 37° C. with 225 rpm shaking until a 600 nm optical density of between 0.4 and 0.6 was reached. Protein expression was induced by addition of 1 mM Isopropyl β-D-1-thiogalatopyranoside (IPTG) (Teknova, Cat. No. I3431) and bacteria were grown for 4 hr at 30° C. with shaking at 225 rpm. Bacterial cultures were centrifuged at 12,000×g for 5 min. in 5 ml of protein extraction reagent (BugBuster, Novagen Cat No. 70584) and 20 μl of 100× protease inhibitor (Cocktail Set III, EDTA-Free Calbiochem, Cat No. 539134) and incubated for 30 min at 37° C. in a shaking incubator at 225 rpm. Bacteria were pelleted by centrifugation at 12,000×g for 45 min at 4° C. Recombinant proteins were purified using affinity purification (HisPur™ Ni-NTA Spin Purification Kit, Thermo Scientific, Cat No. 88228).
Protein concentrations were determined using a bicinchoninic acid (BCA) assay (Thermo Scientific, Cat No. 23227).
SDS-PAGE and Western Blot
Protein samples were resolved by SDS-PAGE in 4-12% polyacrylamide gels (Invitrogen, Cat No. NP0322BOX) and electrophoretically transferred onto nitrocellulose membranes (Thermo Scientific, Cat No. 77010). The blots were blocked overnight in 5% (wt/vol) nonfat dried milk powder in 0.05% polyethylene glycol sorbitan monolaurate (Tween 20) containing phosphate buffered saline (PBS-T) at 4° C. The blocked membranes were incubated with a 1:2,000 dilution of horseradish peroxidase (HRP)-conjugated anti-6×his tag monoclonal antibody (Thermo Scientific, Cat No. MA1-21315-HRP) in 0.05% PBS-T for 1 h with 225 rpm shaking at room temperature. After five washes with 0.05% PBS-T bound antibodies were detected using 1-Step™ chloronaphthol substrate solution (Thermo Scientific, Cat No. 34012).
Preparation of Canine Anti-S. pseudintermedius SpEX
Recombinant SpEX-M (SEQ ID NO:12) produced in E. coli was purified using affinity chromatography (as above) and endotoxin concentration was measured using a ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (Genscript, Cat. No. L00350). Recombinant SpEX-M at 20 μg each/0.5 cc in phosphate buffered saline (PBS) (pH 7.2) were injected in the lateral thorax by the subcutaneous route, into three clinically normal dogs. Injections were given once every 7 days for a total of three injections with a control dog receiving PBS (pH 7.2) only. Blood (6 cc) was collected from a jugular vein 4 times, on days −7, 8, 15 and 29 (relative to injections). The collected blood was left undisturbed at room temperature for 30 min followed by centrifugation at 2,000×g for 10 min in a refrigerated centrifuge.
Enzyme-Linked Immunosorbent Assay
For measurement of recombinant protein antigenicity, SpEX and SpEX-M were coated separately onto ELISA plates (Corning, Cat No. 3590) at 2 μg/ml in PBS. The plates were washed with 0.05% PBS-T and incubated with two-fold serial diluted serum from dogs (injected with recombinant proteins) for 1 h at 37° C., then bound IgG was detected using HRP-conjugated goat anti-dog IgG-heavy and light chain (Bethyl Laboratories, Inc. Cat No. A40-123-1). ELISA assays plates were washed three times with PBS-T between all incubations, bound antibodies were detected using TMB substrate (Thermo Scientific, Cat No. N301), reactions were stopped with 0.18 M sulphuric acid and optical density read at 450 nm on a plate reader (Bio TEK, EL800). The experiment has repeated a minimum of three times and a p-value of <0.05 was considered significant for all the experiments unless otherwise stated.
PMN Preparation
Canine blood was collected from healthy dogs using a sterile blood collection system with EDTA anticoagulant (BD Vacutainer). Then, 600 μl of dog blood was added to 1 ml of red blood cell lysing buffer (Hybri-Max™, Sigma-Aldrich, Cat No. R7757-100 ML) for 30 min at 37° C. in 15 ml sterile plastic tube, centrifuged and re-suspended in 1 ml RPMI medium supplemented with 10% fetal bovine serum.
PMN Transmigration Assay
A neutrophil transmigration assay protocol as previously described (24) was used with modifications. The assay was conducted in modified 24-well plates with polycarbonate membrane (3.011M pore size and 6.5 mm well diameter) chamber (Corning Incorporated, NLD Cat No. 3415). Six hundred microliters of DMEM medium was placed in each well and 10% of fetal bovine serum was used as a PMN chemoattractant. S. pseudintermedius 06-3228 culture supernatant harvested at log phase was used as positive control and DMEM medium alone was used as negative control. Subsequently, isolated canine PMNs (1×106) were added to the top (basolateral) chamber and incubated at room temperature for 6 h. PMNs that migrated across the bottom (apical) chamber were quantified using a Countess™ II FL Automated Cell Counter (Thermo Scientific, USA Cat No. AMQAF1000).
In order to measure the protective effect of anti-SpEX on neutrophil chemotaxis, recombinant SpEX was incubated with canine anti-SpEX at a dilution of 1:100 for 30 min at 37° C., then tested with PMN transmigration assay as previously described.
Complement C5 Binding Assay
Human complement component C5 was coated onto ELISA plates (Corning, Cat No. 3590) at 2 μg/ml in PBS as previously described. Recombinant SpEX and SpEX-M were added at 500 ng/ml in PBS (pH 7.2) for 1 h at 37° C., then bound recombinant proteins were detected using (HRP)-conjugated anti-6×his tag monoclonal antibody (Thermo Scientific, Cat No. MA1-21315-HRP) at dilution of 1:1000 in PBS-T (pH 7.2). Bound antibodies were detected using TMB substrate (Thermo Scientific, Cat No. N301), reactions were stopped with 0.18 M sulphuric acid and optical density read at 450 nm on a plate reader (Bio TEK, EL800). The experiment has repeated a minimum of three times and a p-value of <0.05 was considered significant for all the experiments unless otherwise stated.
Hemolysis Assay
In order to detect the ability of recombinant SpEX to bind with complement component C5 and inhibit its function, a hemolysis assay was performed. Bovine erythrocytes were sensitized to complement by incubation with rabbit IgG fraction anti-bovine red blood cells (ICN Cappel, Cat No. 55271) diluted 1:25, for 30 min at 37° C. with gentle mixing. Dog serum, diluted 1:4, was pre-incubated with 500, 1000 and 2000 ng/ml of recombinant native SpEX for 30 min at 37° C. and gentle shaking (100 rpm). One hundred microliters of sensitized bovine RBCs were added and further incubated for 30 min at 37° C. with gentle shaking. After centrifugation at 4200×g for 5 min, the absorbance of the supernatant was measured at 450 nm. Heat-inactivated serum mixed with PBS (pH 7.2) was used as a negative control and 25% normal dog serum used as a positive control.
PMNs and PBMCs Permeability Assay
PMNs and PBMCs were separated as previously described. PMNs and PBMCs were incubated with recombinant proteins (SpEX and SpEX-M) at concentration of 100 μg in a volume of 500 μl in RPMI medium supplemented with 10% fetal bovine serum in a 5% CO2 incubator for 30 min. The supernatant of S. pseudintermedius 06-3228 was harvested at log phase to test the toxic effect of secreted SpEX. PMNs and PBMCs were stained with 1 Sytox green (Life Technologies, Inc. Cat No. 1776406) for 30 min, washed twice with PBS (pH 7.2) and analyzed using a flow cytometer (Attune acoustic focusing cytometer) by gating on PMNs and PBMCs based on their side and forward scatter properties.
To determine the protective effect of canine anti-SpEX-M on PMNs and PBMCs, recombinant S. pseudintermedius SpEX at concentration of 100 μg/ml in PBS (pH 7.2) was incubated for 30 minutes at 37° C. with serum from SpEX-M injected dogs. The experiment was run in duplicate and a p-value of <0.05 was considered significant.
For flow cytometry analysis the cut-off for cell death was established using leukocytes incubated without SpEX. Mean fluorescent intensity was determined from all gated cells.
Statistical Analysis
Each experiment was repeated at least three times and a p-value of <0.05 was considered significant. Experiments including ELISA and flow cytometry on serum from injected dogs were run in technical duplicates. All analyses were conducted using the GraphPad Prism software (Version 7, GraphPad Software Inc.).
Results
S. pseudintermedius SpEX Secreted in the Culture Supernatant
LC-MS/MS was used to screen culture supernatants of three clinical strains of S. pseudintermedius (06-3228, 08-1661 and NA45), representing the major sequence types occurring in the United States as determined by multilocus sequence typing. SpEX was detected in the supernatants of the three isolates with 234-amino acid sequences.
S. pseudintermedius 06-3228 secrete SpEX that shares 91.45, 88.89% amino acid identity with that of 08-1661 and NA45 strains, respectively with mean pairwise % identity of 92.9% and 208 identical amino acid. The mean molecular weight is 26.093 kDa and the mean isoelectric point is 6.30.
Bioinformatics Analysis and S. pseudintermedius SpEX Characteristics
A BLAST search of SpEX in the GenBank database revealed that it is highly conserved in S. pseudintermedius isolates and it shares sequence similarity with other pathogenic bacterial species within the Staphylococcus intermedius group. SpEX shares 71.8% amino acid identity with Staphylococcus intermedius and 73.5% amino acid identity with Staphylococcus delphini.
The SpEX protein sequence contains a signal peptide sequence from positions 1 through 35 detected by SignalP 4.1 server. By searching protein family and domain databases Interpro, Pfam and PROSITE, we found that SpEX has an N-terminal oligonucleotide/oligosaccharide binding (OB-fold) domain in residues 43-126 that folds into a five-stranded beta-barrel and a C-terminal β grasp domain in residues 150-217 (
SpEX has approximately 47% amino acid sequence identity with staphylococcal superantigen-like protein 11 (SSL11) and less than 30% amino acid identity with other SSL members.
Cloning, Expression, and Purification of Recombinant S. pseudintermedius SpEX
Recombinant polyhistidine tagged native and mutant SpEX were generated in E. coli and purified using HisPur Ni-NTA resin under native conditions and eluted using an imidazole gradient. The molecular weights of SpEX and SpEX-M determined in western blots were of the expected sizes (27.63 and 27.49 kDa, respectively).
SpEX Interferes with Complement Function
The HRP-conjugated anti-6×his tag monoclonal antibody detected recombinant SpEX bound to human C5. SpEX was significantly bound to human C5 compared to SpEX-M at the same dilution (500 ng/ml with P<0.0013**) (
Moreover, SpEX in hemolytic assays with bovine erythrocytes caused inhibition of hemolysis at a concentration dependent manner. Starting at concentration of 500 ng, we found that SpEX significantly reduced the hemolysis of sensitized bovine erythrocytes compared to positive control with P<0.0001****. SpEX at concentration of 4 showed no significant difference in hemolysis with the negative control (
SpEX Inhibits PMN Chemotaxis
The PMN transmigration assay was used to detect the inhibitory effect of recombinant SpEX on PMNs chemotaxis in vitro. Recombinant SpEX at concentration of 200 ng/ml significantly inhibited the migration of PMNs induced by fetal bovine serum compared to SpEX-M at the same dilution with P<0.0001****. The chemotaxis inhibition by culture supernatant of S. pseudintermedius 06-3228 was significantly higher than SpEX P<0.0060** (
Attenuated SpEX Induces Specific Antibody Responses
ELISA analysis of sera obtained from dogs on days −7, 8, 15 and 29 (relative to injections) showed that IgG against S. pseudintermedius SpEX-M (SEQ ID NO:12) and SpEX was detected on day 15 (P<0.0001****) and reached the highest level on day 29 (P<0.0001****) compared to pre-injection control sera (
SpEX Kills Canine PMNs and PBMCs
Canine PMNs and PBMCs were highly susceptible to SpEX with lysis induced within 30 minutes at a concentration of 100 μg/ml of PBS (pH 7.2) of recombinant SpEX and a 1:2 S. pseudintermedius 06-3228 supernatant dilution (
Canine Anti SpEX-M Reduce the Effect of SpEX on PMNS and PBMCs In Vitro
Canine anti-SpEX at dilution of 1:100 in PBS (pH 7.2) preincubated with recombinant SpEX at concentration of 200 ng/ml significantly diminished the chemotaxis inhibition of SpEX with P<0.0001**** (
Pre-incubation of Canine anti-SpEX with SpEX at a concentration of 100 μg/ml in PBS resulted in reduction in mean fluorescent intensity (MFI) as compared with that of SpEX treatment alone (
Discussion
Using mass spectrometry and genomic information, it was possible to identify an exotoxin secreted by all of the S. pseudintermedius isolates including 06-3221, 08-1661 and NA45, representing the three distinct sequence types that predominate in the United States. In this study, we identified S. pseudintermedius SpEX, an exotoxin secreted by S. pseudintermedius. Despite the sequence difference, SpEX has a typical SSLs tertiary structure consisting of an N-terminal oligonucleotide/oligosaccharide binding (OB-fold) domain that folds into a five-stranded beta-barrel and a C-terminal β grasp domain. S. aureus SSLs are a family of structurally related molecules that exert an entirely different function through a variety of structural mechanisms this explains why we found SpEX in addition to sharing the chemotaxis inhibitor property of SSL11, it has a cytotoxic effect against PBMCs and PMNs and complement activity inhibition through C5 binding. SpEX target components of innate immunity by binding complement factor C5, inhibits complement activation. SpEX is a chemotaxis inhibitor and cytotoxic against dog PMNs and PBMCs that has come into infection sites. The combination of these mechanisms effectively help the bacteria to survive and multiply in their hosts and increase the likelihood of transmission.
Canine antibodies to SpEX efficiently neutralize and diminish its chemotaxis inhibitory and cytotoxic effect on PMNs and PBMCs highlighting the likely immune-evasive attribute of S. pseudintermedius SpEX in the dog. Furthermore, SpEX-M (SEQ ID NO:12) with amino acid substitutions in its predicted functional domains showed diminished immune evasive properties compared to SpEX.
A vaccine composition was made comprising recombinant attenuated S. pseudintermedius immunosuppressive proteins as described above. The composition comprised recombinant attenuated S. pseudintermedius protein A (SEQ ID NO:2), recombinant attenuated S. pseudintermedius leukotoxin S (SEQ ID NO:4), recombinant attenuated S. pseudintermedius leukotoxin F (SEQ ID NO:10), recombinant attenuated S. pseudintermedius exotoxin 15 (SEQ ID NO:12), and recombinant attenuated S. pseudintermedius spdsa (SEQ ID NO:6).
The compositions were administered individually and as a mixture to healthy research dogs in a series of 3 vaccinations at one-week intervals. Serum was collected pre-vaccination and at weeks 1, 2, 3, and 5 post-vaccination. Canine IgG response to the vaccine was determined by an ELISA assay substantially as described above. The vaccine elicited significant increases in IgG to each of the vaccine components compared to a control comprising no antigen (
Dogs with pyoderma were cultured to confirm S. pseudintermedius infections. Blood was obtained prior to injection and dogs were vaccinated with a vaccine comprising 20 μg each of recombinant attenuated immunosuppressive proteins as set forth in Example 7. Antibody reactivity prior to and after injections was determined by ELISA using HRP conjugated anti-canine IgG. Infected dogs had low levels of antibody prior to injection and a strong antibody response to the vaccine (
Any citations and published gene sequences referred to in this application form a part of the disclosure and are incorporated herein in their entirety by reference. While the terms used herein are believed to be well-understood by one of ordinary skill in the art, definitions are set forth to facilitate explanation of certain of the presently-disclosed subject matter.
Following long-standing patent law convention, the terms “a,” “an,” and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a cell” includes a plurality of such cells, and so forth.
Unless otherwise indicated, all numbers expressing quantities of ingredients, properties such as reaction conditions, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless indicated to the contrary, the numerical parameters set forth in this specification and claims are approximations that can vary depending upon the desired properties sought to be obtained by the presently-disclosed subject matter.
As used herein, the term “about,” when referring to a value or to an amount of a composition, dose, sequence identity (e.g., when comparing two or more nucleotide or amino acid sequences), mass, weight, temperature, time, volume, concentration, percentage, etc., is meant to encompass variations of in some embodiments ±20%, in some embodiments ±10%, in some embodiments ±5%, in some embodiments ±1%, in some embodiments ±0.5%, and in some embodiments ±0.1% from the specified amount, as such variations are appropriate to perform the disclosed methods or employ the disclosed compositions.
The term “comprising”, which is synonymous with “including” “containing” or “characterized by” is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. “Comprising” is a term of art used in claim language which means that the named elements are essential, but other elements can be added and still form a construct within the scope of the claim.
As used herein, the phrase “consisting of” excludes any element, step, or ingredient not specified in the claim. When the phrase “consists of” appears in a clause of the body of a claim, rather than immediately following the preamble, it limits only the element set forth in that clause; other elements are not excluded from the claim as a whole.
As used herein, the phrase “consisting essentially of” limits the scope of a claim to the specified materials or steps, plus those that do not materially affect the basic and novel characteristic(s) of the claimed subject matter. With respect to the terms “comprising”, “consisting of”, and “consisting essentially of”, where one of these three terms is used herein, the presently disclosed and claimed subject matter can include the use of either of the other two terms.
As used herein, the term “and/or” when used in the context of a listing of entities, refers to the entities being present singly or in combination. Thus, for example, the phrase “A, S, C, and/or O” includes A, S, C, and O individually, but also includes any and all combinations and subcombinations of A, S, C, and O.
The term “antibody” (Ab) as used herein includes monoclonal antibodies, polyclonal antibodies, multispecific antibodies and antibody fragments, as long as they exhibit the desired biological activity. The term “polyclonal antibody” as used herein refers to an antibody obtained from a population of heterogeneous antibodies, i.e., they are secreted by different B cell lineages within the body. The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations. Monoclonal antibodies are highly specific, being directed against a single antigenic site.
The term “antibody” (Ab) as used herein also includes antibody fragments. An “antibody fragment” is a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include but are not limited to: Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.
The foregoing description of preferred embodiments has been presented for purposes of illustration and description. It is not intended to be exhaustive or to limit the invention to the precise form disclosed. Obvious modifications or variations are possible in light of the above teachings. The embodiments were chosen and described to provide the best illustration of the principles of the disclosed subject matter and its practical application to thereby enable one of ordinary skill in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. All such modifications and variations are within the scope of the invention as determined by the claims when interpreted in accordance with the breadth to which they are fairly, legally and equitably entitled.
This continuation patent application claims the benefit of priority in US patent application Ser. No. 16/636,682 filed on Feb. 5, 2020, which in turn is the national stage of international patent application no. PCT/US2018/046281 filed on Aug. 10, 2018 which in turn claims the benefit of priority in U.S. provisional patent application Ser. No. 62/543,676 filed on Aug. 10, 2017, the entire disclosures of which are incorporated herein by reference in their entirety.
| Number | Name | Date | Kind |
|---|---|---|---|
| 9879054 | Bannoehr | Jan 2018 | B2 |
| 11141473 | Kania | Oct 2021 | B2 |
| Number | Date | Country |
|---|---|---|
| WO 2011045573 | Apr 2011 | WO |
| Entry |
|---|
| Moodley et al., Veterinary Microbiology, 2009; 135:320-326 (Year: 2009). |
| Worthing et al., Veterinary Microbiology, 2018; 213:58-65. (Year: 2018). |
| International Search Report and Written Opinion for PCT/US2018/046281 dated Apr. 29, 2019. |
| NCBI, GenBank accession No. WP_065520714.1, Jul. 21, 2016. Available via URL https://www.ncbi.nlm.nih.gov/protein/WP_065520714.1/. |
| NCBI, GenBank accession No. WP_020220128.1, Jun. 30, 2013. Available via URL https://www.ncbi.nlm.nih.gov/protein/WP_020220128.1/. |
| Ruzauskas, M. et al., ‘Characterization of Staphylococcus pseudintermedius isolated from diseased dogs in Lithuania’, Pol. J. Vet. Sci., 2016, vol. 19, No. 1, pp. 7-14. |
| Garbacz, K. et al., ‘Pathogenicity potential of Staphylococcus pseudintermedius strains isolated from canine carriers and from dogs with infection signs’, Virulence, Jan. 17, 2013, vol. 4, No. 3, pp. 255-259. |
| Number | Date | Country | |
|---|---|---|---|
| 20210379173 A1 | Dec 2021 | US |
| Number | Date | Country | |
|---|---|---|---|
| 62543676 | Aug 2017 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 16636682 | US | |
| Child | 17389982 | US |
