Patent Grant
9409975
A novel interaction between Candida albicans (C. albicans) surface proteins and heparin, a drug commonly used in patients at risk for catheter-associated biofilms and candidemia, is disclosed with an antibody that reduced or prevented a microorganism surface protein from binding to heparin, and reduced catheter-associated biofilm formation, where the reduction in biofilm formation can reduce the microorganism's virulence in a patient, with reduction encompassing any level of decrease.
Heparin is a highly sulfated, non-branched, anionic disaccharide composed of uronic acid, predominantly iduronic acid, in 1,4 linkage with glucosamine. Heparin is a known anticoagulant that is often infused in indwelling catheters to prevent the blood flowing through the catheter from clotting. Heparan sulfate is composed of the same disaccharide but is less sulfated and has a more varied structure than heparin. Heparan sulfate linked to core proteins, such as syndecans or glypicans, forms heparan sulfate proteoglycans (HSPGs). HSPGs are widely expressed on mammalian cell surfaces and in the extracellular matrix. Heparin binding motifs can bind to both heparin and heparan sulfate.
Several microbial proteins bind heparin and heparan sulfate, e.g., HIV-1 gp120, CypA, and Tat; hepatitis C envelope glycoprotein E2; herpes simplex virus glycoproteins B, C, and D; dengue virus envelope protein; Plasmodium falciparum circumsporozoite (CS) protein, Listeria monocytogenes ActA, and LcI of Legionella pneumophila (1-11). Such microbial protein interactions with surface HSPGs typically promote binding and entry of the microbial organism into the mammalian cell (except for HIV-1 gp120 and Tat), but the mechanisms by which individual microbial proteins recognize heparin or heparan sulfates are largely undefined. Positively charged tripeptides containing Lys or Arg in the amino terminus of L. monocytogenes ActA (aa 40-230) and in L. pneumophila LcI (aa 69-349) were shared with tripeptides in a number of heparin-binding mammalian proteins such as EGF-like growth factor and vonWillebrand factor, but the precise amino acids that mediated binding to heparin were not defined for ActA or LcI (10-11).
Eukaryotic proteins that bind heparin express either linear or conformational heparin binding motifs (HBM). Linear heparin binding motifs are short, conserved peptides containing both basic (B) and hydropathic (X) amino acids in specific patterns first identified by Cardin (XBBXBX) and Weintraub (XBBBXXBX) and subsequently expanded by Sobel (XBBBXXBBBXXBBX) (12-14). Molecular modeling studies suggested that basic amino acids such as lysine, arginine, and histidine were critical for interaction with anionic sulfate or carboxylate groups in heparin through electrostatic and hydrogen bonds. For example, substituting alanine for basic amino acids in linear HBM in the morphogen sonic hedgehog abolished binding to heparin (15). However, because many heparin-binding proteins failed to exhibit linear motifs, the concept of spatial orientation of basic residues was propounded (16). The CPC clip motif, a structural signature in which two cationic residues surround one polar residue, was conserved in eukaryotic heparin binding proteins, as well as in vaccinia complement protein and papillomavirus 18 capsid protein (17)
Because of its propensity to form biofilms in implanted medical devices such as indwelling central venous catheters, Candida albicans (C. albicans) is a leading pathogen in infections of indwelling catheters. A biofilm is a multilayered structure of microbes embedded in a polysaccharide matrix that forms on central venous catheters as well as implanted prosthetic joints, contact lenses, and other such medical devices (18, 19). A biofilm is undesirable because antibiotics and host defenses cannot penetrate it, thus a biofilm prevents elimination of microorganisms that have attached or adhered to a surface of an implanted medical device. For example, because of the high flow-through in a central venous catheter, sessile projections from a biofilm break off and are carried into the bloodstream to cause infection that can be carried by the blood to other sites in the body.
Heparin is a known anticoagulant, and catheters are often infused with heparin to prevent blood flowing through the catheter from clotting (20). Because heparin inhibited attachment of C. albicans to extracellular matrix proteins (21), the inventors hypothesized that C. albicans interacted with heparin through linear heparin binding motifs (HBMs): conserved sequences of basic (Arg, Lys, H is) and hydropathic amino acids in prescribed patterns.
Thirty-four C. albicans surface proteins encoding at least one linear heparin binding motif (HBM) were identified from a sequence-based search. Twelve of these 34 proteins flanked recently described transcriptionally active regions that are involved in biofilm production. Thirty-three of these 34 proteins are known targets of master biofilm regulators (22).
C. albicans binding to 2.5 units (12.5 μg) solid-phase heparin in an ELISA assay was significantly decreased by desulfation of heparin at the 2-O or 2-N groups, and by preincubation of C. albicans with heparin. The protein Int1 contained the largest number of HBMs; Int1 is a surface protein that is involved in adhesion, filamentation, and antigenic recognition. In the Int1 sequence 804QKKHQIHK (SEQ ID NO: 1), alanine mutation of lysine residues at positions 805/806 significantly reduced binding of the Int1 mutant to heparin. Rabbit IgG recognizing the polypeptide 799HKQEKQKKHQIHKV812 (SEQ ID NO: 4) inhibited C. albicans binding to heparin by 19%; pre-immune rabbit IgG had no effect.
Consequences of heparin binding in vitro included removal of C. albicans surface antigens such as Eno1, Pgk1, Tdh3, and Ssa1/2, which themselves contain putative HBMs; impairment of histatin-mediated killing; and modulation of gene expression. In vivo, substitution of alanine residues for lysines at positions 805/806 in 804QKKHQIHK (SEQ ID NO: 1) markedly attenuated biofilm formation in central venous catheters in rats. In addition, pre-incubation of C. albicans with rabbit IgG recognizing the polypeptide 799HKQEKQKKHQIHKV812 (SEQ ID NO: 4) inhibited biofilm formation in vivo; pre-immune IgG had no inhibitory effect. These results identify linear HBM in C. albicans surface proteins, characterize specific lysine residues that mediate heparin binding, and demonstrate relevance for innate and adaptive immunity in vitro and biofilm formation in vivo.
The inventive method and composition ameliorated this undesirable situation by using antibodies against a specific heparin-binding motif that is expressed by surface proteins on C. albicans; other microorganisms also express similar heparin binding motifs. Without being held to a single theory, the disclosed antibodies reduce or prevent the undesirable biofilm from forming on the surface of a medical device, particularly a plastic device, implanted in a patient. Microorganisms include bacteria, yeast, fungi, etc. as known to one skilled in the art. Medical devices include those implanted or implantable in a patient, particularly plastic medical devices, including but not limited to catheters and central lines but excluding current non-plastic implanted joints, pacemakers, pacemaker wires, and spinal rods.
In one embodiment of the inventive method, antibodies were produced against a linear heparin binding motif that was expressed by a surface protein of Candida albicans (C. albicans). Related heparin binding motifs occur in surface proteins from Staphylococcus epidermidis (S. epidermidis) and Staphylococcus aureus (S. aureus).
Linear heparin binding motifs are sequential amino acids that conform to one of three consensus motifs (Cardin, Sobel, or Weintraub motifs); they have been identified in multiple mammalian proteins that bind heparin. While conformational heparin binding motifs also occur in some proteins, they must be identified by crystal structure of the protein. Antibodies to these linear heparin binding motifs inhibited C. albicans from adhering to heparin bound to a plastic surface (a microtiter plate).
Of the six million patients who have a central line implanted each year, more than 75,000 will develop bloodstream infections (23). Central line-associated blood stream infections (CLABSI) are a major source of hospital-acquired infection; over 43,000 CLABSI occurred among patients hospitalized in U.S. intensive care units (ICUs) in 2001, which is 3.2 infections for every 1000 line days. With a protocol for their sterile insertion and daily maintenance, CLABSI in ICUs dropped to 18,000 in 2009, but even with these improvements in ICUs procedures, more than 23,000 CLABSI occurred in patients on inpatient wards and 37,000 CLABSI occurred in outpatient hemodialysis patients in 2009. Even with a 50% reduction in CLABSI in ICU patients, the annual cost of these infections across the U.S. is more than $13 billion (23)
Staphylococcus epidermidis is the most common cause of CLABSI in central lines, with gram-negative rods second, S. aureus third; and Candida species fourth (24). In peripherally inserted central catheters (PICC), i.e., central lines inserted through peripheral veins in the arm, Candida species are the second most common cause of line infection (25). Candida spp. are equivalent to S. aureus in infections in central lines in the ambulatory setting (26). Among Candida spp, C. albicans is the most common cause of CLABSI (24).
When Candida species infect a central venous catheter, the organism enters the bloodstream to cause candidemia. Hosts at highest risk of candidemia, include burn patients and patients on a pump during coronary artery bypass procedures (27). Neutropenic oncology patients, premature newborns, and patients with major abdominal surgery such as intestinal resections are also high-risk patients with 8- to 16-fold the number of infections of other patient groups (27).
A major risk factor in candidemia is the presence of central lines (28-33), whose lumens are a site for formation of microbial biofilms that are effectively shielded from antimicrobial agents and host defenses. A second commonality is the use of heparin as an anticoagulant in most catheters. The amount of heparin is often considerable; e.g., a premature newborn in a neonatal intensive care unit may receive 150 units/kg/day, compared to 2.8 units/kg for a 70 kg adult receiving a 100 unit flush twice a day (20).
The ability to bind heparin is a unifying feature among S. epidermidis, S. aureus, and, Candida albicans (34-36), three of the leading causes of catheter-associated bloodstream infections. In experiments with S. aureus, high concentrations of heparin have been shown to increase S. aureus biofilm formation in vitro (37).
A first step in biofilm formation is microorganism adhesion or adherence to a surface of the implanted device, such as a catheter (38). In
Heparin is associated with central line infections, particularly those due to Candida spp. In a randomized trial of 260 central lines, 128 were coated with chlorhexidine and 132 were coated with heparin; all Candida colonization and all candidemias occurred in patients whose central lines were coated with heparin (39). A second study in renal dialysis patients showed that those patients who received 5000 units of heparin in the middle of dialysis had considerably more catheter loss due to infection compared to patients not treated with heparin. Although staphylococcal infections were most common in this patient group, candidemia also occurred (40).
One mechanism by which heparin interacts with proteins is through linear heparin binding motifs, conserved sequences of basic amino acids such as lysine, arginine, and histidine interspersed with hydropathic amino acids (14). Microbial heparin-binding proteins are identified by short consensus motifs of basic (B) and hydropathic (X) amino acids, as defined by Cardin [XBBXBX], Weintraub [XBBBXXBX], or Sobel [XBBBXXBBBXBBX].
In the invention, a computer-based algorithm examined all 400 C. albicans surface and cell wall proteins for heparin binding motifs (HBM). Of the 6,000 known Candida albicans proteins, about 400 are localized to the cell wall of yeast or hyphae, depending upon the extraction technique (41). A sequence-based search identified putative Cardin, Sobel, or Weintraub motifs in 159 C. albicans proteins. Table 1 shows HBM in cell surface proteins expressed by C. albicans. The 34 proteins in Table 1 include only those that have the attribution “cell surface”, “fungal cell wall”, “yeast cell wall”, or “hyphal cell wall” as the cellular component in the Candida Genome Database, where *=Weintraub motif; #=Cardin motif; ‡=Identified by molecular modeling studies; A=“cell wall” in protein description (as of January 2011); obtained from Candida Genome Database (CGD); B=“cell wall” in Gene Ontology (GO) annotation (as of January 2011); obtained from CGD; C=cell wall proteins as reviewed by Alberti-Segui (42) and D=cell wall proteins as reviewed by Chaffin (41).
The following procedure was used to identify a set of cell wall proteins in C. albicans with HBM. The first step combined evidence to identify possible cell wall proteins in C. albicans. Specifically, using “cell wall” as keywords, 154 putative cell wall proteins were identified from protein descriptions, and another 245 unique cell wall proteins were identified from Gene Ontology functional annotations from Candida Genome Database (www.candidagenome.org; Assembly 21). Also included were 125 non-redundant cell wall proteins identified by Alberti-Segui (50) and another 174 reported by Chaffin (22) and mapped annotations to Assembly 21. The second step screened for consensus HBM in protein ORF sequences of these cell wall proteins from Assembly 21. Three types of consensus motifs were included: Weintraub (XBBBXXBX), Sobel (XBBBXXBBBXXBBX), and Cardin (XBBXBX) motifs, where B is a basic amino acid and X is a hydropathic amino acid. In the algorithm, basic amino acids are H, K, and R. Hydropathic amino acids are W, F, Y, L, I, C, M, G, V, S, T, A, N, P, and Q. A total of 159 cell wall proteins were identified that have possible HBM, and some have multiple motifs. The 159 proteins that met the search criteria were then manually curated to select only those proteins whose cell wall localization was confirmed by manual or computational methods in the Candida Genome Database.
Proteins with GPI anchoring sequences within these 159 cell wall proteins with HBM were further identified. Three Web servers were used to perform GPI anchoring sequence prediction: SignalP (43), GPI-SOM (44), and PredGPI (45). Combined results of these three prediction methods indicated that 15 out of these 159 proteins had GPI anchoring sequences, four of which overlapped with reviewed GPI anchoring proteins (46).
The algorithm identified 34 cell wall proteins with potential linear heparin binding sequences that matched the Cardin, Weintraub, and Sobel motifs. The 34 identified cell wall proteins were Ahp1, Als7, Atcl, Bud2, Cat1, Cef3, Chs1, Crh12, Dot4, Not5, Pdi1, Pga4, Pgk1, Phr3, Rbt1, Rps6A, Sam2, Srb1, Ssa2, Ssb1, Tdh3, Eft2, Eno1, Gap1, Gph1, Gpm2, Hem13, Hsp104, Hsp70 (also called Ssa1), Ino1, Int1, Ipp1, Ugp1, Xyl2. Three of these proteins, Als7, Pga4, and Rbt1, have GPI anchors. The genes encoding eleven of these proteins, Als7, Cat1, Dot4, Eno1, Gph1, Ino1, Rbt1, Sam2, Srb1, Ssa2, and Ugp1, are located in newly defined transcriptionally active regions that are critically involved in biofilm formation (22). Moreover, many of these proteins are known to be regulated by at least one of six master biofilm regulators; by RNA-seq, where RNA-seq is defined as the use of high-throughput sequencing techniques to sequence cDNA in order to get sequence information about the transcriptome, the sample's RNA content; CAT1, GAP1, GPH1, and HSP104 are up-regulated in biofilm formation, while CHS1, EN01, GPM2, HEM13, INO1, IPP1, PGK1, RPS6A, SAM2, SRB1, SSA2, SSB1, and TDH3 are down-regulated (22)
Given the presence of putative HBM among C. albicans surface proteins and the possible interactions with heparin in central venous catheters or with heparin sulfate proteoglycans expressed on host tissues such as vascular endothelium, the biochemical determinants and immunologic consequences of this interaction were defined. The protein Int1 (SEQ ID NO: 5) (accession number P53705.2; GI 187608862) had the highest number of HBM (five). Int1 (SEQ ID NO: 5) spans 1711 amino acids and is localized to the cell wall of the bud neck in C. albicans. It mediates adhesion, hyphal formation, and virulence, defined as the ability to cause disease such as bloodstream infection (47). Using the same search technique, HBM were also detected in surface proteins of S. epidermidis and S. aureus.
804QKKHQIHK‡
1383THKGRF#
1530MKRGKP#
1593FKKRFFKL*
1612SHKTRA#
176SHRIRV#
310IKKFKY#
368IRHFKG#
442NKKGKS#
519CHKCHN#
635FKRFKF#
136GKKVKA#
146VKKFRQ#
168AHRAHS#
185QRKRALKA*
192AKKVKN#
114QKRSRF#
333VKKLKP#
210NKKFKN#
301GKKYRG#
53VKRARY#
199ARRSKS#
258LRRLRT#
45IKKNHL#
121GKKVKQ#
171LKRIHN#
257IRRGRY#
74TKKGKL#
115PHHHLNRY*
141AKKGKF#
420TKHIHN#
259AKRPRP#
623LRKYKG#
72QRKLKT#
259LRRLRT#
126PKRQKT#
581NKHNRI#
123FHKAHG#
959PKRVKV#
82GKKTRI#
263LRRLRT#
380PKKLKF#
70GHKIKV#
656TKHHIPKA*
1482SKRNKN#
161MKRAKV#
330THRFKF#
716LRKGKS#
Heparin effect on Candida albicans growth and morphology was determined. A single colony of BWP17 wt was inoculated into 3 ml YPD medium and incubated overnight at 30° C. with shaking at 225 rpm. Overnight cultures were diluted to an OD600 of 0.1 in 5 ml of YPD (yeast) or RPMI-HEPES (hyphae) in a 50 ml conical polypropylene tube and incubated in the presence or absence of 100 units/ml preservative-free pharmaceutical heparin at 30° C. for six hours. Cells (1 ml) were fixed with an equal volume of 4% formaldehyde in FACS buffer at 4° C. for one hour, then pelleted (7,000 rpm for 3 minutes) and washed with PBS. After reconstitution in 0.5 ml PBS, filipin (50 mg/ml stock solution in DMSO) was added to a final concentration of 100 μg/ml and incubated with cells at room temperature for five minutes. Cells were pelleted, washed with PBS and mounted on a slide using Fluoromount G. For calcofluor white staining, after six hours incubation, 1 μl of 1 mg/ml stock solution of calcofluor white in 0.1 N NaOH was added to 100 μl cells to a final calcofluor white concentration of 10 μg/ml.
Microscopy was performed by imaging filipin, PKH26, DAPI, and heparin-Alexa Fluor 488 on a Nikon Ti-E inverted microscope with a 100×CFI APO oil NA 1.49 objective. Filipin was excited with a Prior Lumen 200 metal halide light source set at 10% light output. This light was further attenuated by ND4 and ND8 neutral density filters in series to reduce light output to approximately 3% of output from the liquid light guide. The filters used for imaging were EX 360/40, dichroic 400 nm Ip, EM 460/50. Images were acquired with an Andor iXon emccd camera. Exposure times were 391 ms. Excitation of PKH26/DAPI/Heparin-Alexa Fluor 488 triple staining was accomplished with a Nikon A1R si laser scanning confocal. DAPI was imaged with 405 nm excitation and a 450/50 filter with a laser power of 16.3 and a photomultiplier tube (pmt) voltage of 92. Alexa Fluor 488 was imaged with 488 nm excitation from an Argon-ion laser and a 525/50 filter with a laser power of 6.0 and a pmt voltage of 84. PKH26 was imaged with 561 nm excitation and a 595/50 filter with a laser power of 13.2 and a pmt voltage of 108. Gains were kept below 110 to eliminate contribution from C. albicans autofluorescence; lack of autofluorescence was confirmed by comparison to a control sample without heparin-Alexa Fluor 488. Images were processed with Nikon NIS-Elements AR 4.11.00 64-bit software. Calcofluor white slides were examined using a Zeiss Axiovert 200M fluorescent microscope equipped with a Plan-Apochromat 63×/1.40 oil objective lens with 1.6× optivar and DAPI filter at 350 nm excitation and 460 nm emission. Images were captured using a Zeiss Axiocam color camera and processed with Axiovision version 4.8.2.
Flow cytometry was performed by inoculating a single colony of BWP17 wt into 3 ml YPD medium and incubating overnight at 30° C. with shaking at 225 rpm. Overnight cultures were diluted to OD600 of 0.1 in 5 ml of YPD or RPMI-HEPES and incubated in the presence or absence of 100 units/ml preservative-free pharmaceutical heparin at 30° C. for one hour. One ml aliquots of each mixture were removed, pelleted (7,000 rpm for 3 min), washed twice with PBS, and reconstituted in 1 ml PBS. Flow cytometry analysis was performed using the ImagestreamX flow cytometer (Amnis, Seattle Wash.) equipped with a 405 nm, 488 nm, 653 nm, laser and multi-mag function. The 40× magnification, 10 mm/sec flow rate, and 488 nm laser were used to collect SSC and Brightfield parameters. The flow cell allows particles up to 100 μm wide (height unlimited) to be collected in the instrument. INSPIRE (v.6.0) software was used to acquire events. Software analysis using IDEAS (v5.0) identified percent hyphae using aspect ratio, height and width features using the side scatter (Channel 6) and brightfield (Channel 1) parameters. Objects were selected within each file and tagged to use as identification of truth sets within the population.
For growth curves in the presence or absence of soluble heparin, a single colony of BWP17 wt was inoculated into 3 ml YPD medium and incubated overnight at 30° C. with shaking at 225 rpm. Overnight cultures were diluted to an OD600 of 0.1 in 5 ml of YPD, RPMI-MOPS, RPMI-HEPES or CSM in a 50 ml conical polypropylene tube. Preservative-free pharmaceutical heparin (1000 units/ml) was added to yield a final concentration of 100 units/ml in the heparin-treated samples. Cultures were grown at 30° C. with shaking and OD600 measured periodically. Doubling times (td) were calculated based on the equation td=ln 2/μ (μ=specific growth rate (48). One hundred units/ml heparin, the concentration recommended to prevent clotting of central venous catheters (20), had no effect on doubling times of planktonic yeast cells grown in YPD, RPMI-HEPES, RPMI-MOPS or CSM, as shown in
Binding of heparin by C. albicans was examined in vitro. Heparin was labeled with Alexa Fluor 488 by a method modified from Osmond (49). Briefly, 5 mg heparin (Sigma) was dissolved in 0.5 ml MES buffer (0.1M 2-(N-morpholino)ethanesulfonic acid hydrate, pH 4.5, Sigma), then mixed with a solution of 1 mg Alexa Fluor 488 hydrazide (Life Technologies) in 0.4 ml MES buffer. After adding 0.2 ml of EDC solution (15 mg 1-ethyl-3-(3-dimethylaminopropyl) carbo-diimide/ml water, Thermo Scientific), the mixture was stirred at room temperature for 30 min. A second 0.2 ml aliquot of EDC solution was added and the mixture stirred at room temperature for an additional 30 min. After adding 1.3 ml NaOAc (1 M, pH 4.8) and stirring at room temperature for one hour, heparin-Alexa Fluor 488 was purified on a PD-10 desalting column (GE Healthcare) equilibrated in autoclaved nanopure water. Fractions containing the labeled material detected at 490 nm were combined and dried overnight on a SpeedVac concentrator (Savant) with heating. The resulting solid was dissolved in autoclaved nanopure water to 10 mg/ml and stored at 4° C.
Heparin-Alexa Fluor 488 bound to PKH26- and DAPI-labeled C. albicans. A single colony of BWP17 wt was inoculated into 3 ml YPD medium and incubated overnight at 30° C. with shaking at 225 rpm. Using a PKH26 Red Fluorescent Cell staining kit (including PKH26 dye and diluent C, Sigma), 2×107 cells were mixed with 1 ml diluent C. To this solution was added a mixture of 4 μl PKH26 dye in 1 ml diluent C, and the mixture allowed to stand at room temperature for 3 min. After adding 10 ml 3% BSA, the mixture was centrifuged at 3,000 rpm for 7 min. The resultant magenta-colored cells were washed with 10 ml each of 1% BSA and PBS, with centrifugation after each step. After supernatant removal, the cells were reconstituted in 10 ml fresh PBS. Two 1 ml (2×106 cells) aliquots were placed in separate 15 ml conical tubes, centrifuged, and supernatants removed. Each was reconstituted with 2 ml YPD, and heparin-Alexa Fluor 488 (0.1 ml of above 10 mg/ml solution) was added to the experimental sample. Control and experimental samples were incubated at 30° C. with shaking (225 rpm) for 30 min, at which time 0.4 ml aliquots of each were removed, pelleted (10,000 rpm for 3 min), and washed twice with PBS. After reconstitution with 0.5 ml PBS, DAPI (4′,6-diamidino-2-phenylindole dihydrochloride (Sigma, stock solution of 5 mg/ml) was added to a final concentration of 1 μg/ml and the solution allowed to stand at room temperature for 10 min. Cells were pelleted and washed twice with PBS, then mounted on a microscope slide using Fluoromount G.
To measure the binding of C. albicans to solid-phase heparin, a heparin-binding ELISA assay was developed, as follows. A single colony of each C. albicans wild type or mutant strain from a YPD plate was inoculated into 3 ml YPD medium and incubated overnight at 30° C. with shaking at 225 rpm. Sigma heparin (fresh solution made daily), diluted to 25 units/ml in autoclaved, sterile-filtered PBS was added as 0.1 ml aliquots to each well of an allyl amine-coated 96-well heparin binding microtiter plate (BD Biosciences). The plate was incubated at room temperature overnight in the dark (50). In the morning, the plate was washed with acetate buffer (100 mM NaCl, 50 μM NaOAc, 0.2% Tween 20, pH 7.2), incubated with 3% bovine serum albumin (BSA) in PBS at 30° C. for 1 hour, then washed with PBS. In the meantime, overnight cultures of C. albicans were subcultured by dilution to OD600 of 0.2 in 25 ml of YPD, and grown at 30° C. to mid-log phase (OD600 0.6-0.7). Cells were pelleted (3,000 rpm for 7 minutes), washed twice with PBS and reconstituted in RPMI-HEPES to 4×105, 2×105, and 1×105CFU/ml, respectively. One hundred μl of each C. albicans dilution was applied to the microtiter wells; experiments for each strain were performed in quadruplicate. The plate was incubated at 30° C. for one hour and washed with PBS to remove non-adherent C. albicans. One-tenth ml of a biotinylated polyclonal rabbit anti-C. albicans antibody raised against soluble proteins in a C. albicans lysate (Meridian Life Science), which had been diluted 1:2500 in FACS-Tween (0.05% Tween 20 in FACS buffer consisting of 0.3% BSA), was added to each well. The plate was incubated at 30° C. for one hour, then washed with PBS-Tween (0.05% Tween 20 in PBS). One-tenth ml streptavidin alkaline phosphatase (Biolegend) diluted 1:10,000 in FACS-Tween was then added to each well, and the plate was incubated at 30° C. for 30 minutes. After washing with PBS-Tween and AKP buffer (100 mM Tris base, 50 μM MgCl2, 100 mM NaCl, pH 9.5.), 0.1 ml alkaline phosphatase substrate (KPL) was added to each well. After 45 minutes, absorbance at 595 nM was read on a Beckman Coulter DTX 880. When desulfated heparin analogs, chondroitin sulfate, or dermatan sulfate were used, equimolar amounts of the analogs (with respect to heparin) were applied to the allyl amine-coated 96-well plate instead of heparin.
The ability of soluble heparin to inhibit C. albicans binding to plate-bound heparin was demonstrated by pre-incubating 1×106 CFSE-labeled C. albicans with 200 units heparin at 37° C. for one hour in wells of a 96-well black plate. C. albicans was labeled with carboxyfluorescein succinimidyl ester (CFSE), succinimidyl ester (CFSE; Invitrogen), which is used to track cell division and therefore does not kill the organism (28), as follows. A single colony of C. albicans was suspended in CSM to an OD600 of 0.1, diluted 1:200 into 25 ml CSM and grown at 30° C. overnight to mid-log phase (OD600 0.6-0.7). Cells were washed twice in sterile PBS and suspended in PBS at 2×107 CFU/ml. Carboxyfluorescein succinimidyl ester (Life Technologies) was dissolved in DMSO, diluted in PBS to 50 μM, and 0.5 ml CFSE mixed with 0.5 ml C. albicans suspension. The mixture was incubated for 20 min at room temperature on a rotator, washed once with FACS buffer, once with PBS, then suspended in 1 ml FACS buffer. Intensity of labeling was determined as the mean fluorescence intensity using a BD Accuri C6 cytometer (San Jose, Calif.) with excitation at 488 nm and a 533/30 emission filter. Wells of a black 96-well microtiter plate were incubated with 0.1 mg poly-D-lysine for one hour at room temperature, washed three times with PBS, then incubated with 200 units of heparin in 100 μl RPMI-HEPES overnight at room temperature. The following afternoon, C. albicans wild type strain and mutants were labeled with CFSE as follows: after growth of C. albicans to mid-log phase in CSM medium, organisms were washed two times in PBS and suspended in PBS at a concentration of 2×107/ml. 0.5 ml cells was mixed with 0.5 ml CFSE, covered in foil, and rotated on a mixer for 20 minutes at room temperature. CFSE-labeled C. albicans cells were pelleted in a minifuge at 3000 rpm for four minutes, washed once with FACS buffer (0.3% bovine serum albumin in PBS), washed again with PBS, and then suspended in 1 ml FACS buffer at a concentration of 1×107/ml. 100 μl CFSE-labeled C. albicans (1×106) were deposited in test wells for 60 minutes at room temperature. At the end of the incubation period, wells were washed three times with PBS, and fluorescence in each well was measured (Beckman Coulter Multimode detector DTX880).
Pre-incubation of CFSE-labeled C. albicans with 100 units/ml heparin decreased binding to heparin by 27% (p=0.022; data not shown). C. albicans also recognizes the related structures of chondroitin sulfate and dermatan sulfate. Binding of C. albicans to heparin and to chondroitin sulfate was equivalent, but binding to dermatan sulfate was reduced (p=0.019; data not shown). These results confirmed that the binding of C. albicans to heparin and similar glycosaminoglycans (chondroitin sulfate and dermatan sulfate) could be reproducibly measured and, in the case of heparin, specifically inhibited by pre-incubating the organisms with heparin. These results also suggested that surface components of C. albicans may mediate C. albican binding to heparin.
Location of HBM in Int1 (SEQ ID NO: 5) were determined. Five overlapping polypeptides spanning amino acids 51-1711 of Int1 were expressed with a 6×His tag (SEQ ID NO: 54) in S. cerevisiae BJ3501 and purified by affinity chromatography (HisTrap column; GELifesciences). Fractions containing the His-tagged polypeptide were pooled, diluted with loading buffer (10 mM phosphate, pH 7.0 plus 250 mM NaCl) and applied to a heparin sepharose column (HiTrap Heparin HP; GE Lifesciences). Heparin-binding polypeptides were eluted with a step gradient of NaCl (0.5-2 M) in loading buffer. Eluted fractions were analyzed by SDS-PAGE and immunoblot using anti-His tagged antibody (Santa Cruz) and chemiluminescent detection (SuperSignal West Pico Mouse IgG Detection Kit, Pierce) according to manufacturers' instructions. Polypeptides spanning aa 656-1193 and aa 1548-1711 bound to heparin-Sepharose and were eluted with NaCl, indicating that the HBM in those domains, schematized as Motif 1 (SEQ ID NO: 1), Motif 4 (SEQ ID NO: 2), and Motif 5 (SEQ ID NO: 3) (
The linear polypeptide spanning aa 656-1193 contains one potential heparin binding site, 804QKKHQIHK (SEQ ID NO: 1) (basic residues shown in red) (Motif 1 in Table 1 and
To test whether 804QKKHQIHK (SEQ ID NO: 1) (Motif 1) and/or 1593FKKRFFKL (SEQ ID NO: 2) (Motif 4) mediated the binding of heparin to C. albicans, standard PCR-mediated mutagenesis (51) was used to construct a set of isogenic INT1 disruptants and mutants (Table 2).
Construction of mutants was as follows. C. albicans genomic DNA was isolated from saturated overnight cultures using glass beads as described (52). A lithium acetate method was used to transform C. albicans (41). Plasmids and PCR products were purified using kits (Fermentas/ThermoFisher, Pittsburg Pa.) or established methods (53). Pfu enzyme (New England Biolabs) with High Fidelity buffer was employed for all amplifications. Products were sequenced to affirm fidelity prior to use. Primers are described in Table 3.
A single copy of Int1, including 1450 bp upstream and 548 bp downstream from the Int1 open reading frame (www.candidagenome.org, Assembly 21), was integrated into the hisG locus of the int1−/− strain VBIDM2 (54) to produce the reconstituted strain KO509. Briefly, a copy of Int1 was generated by PCR, using primers 1 and 2 with BWP17 wt DNA as template and cloned into the SacI/MluI sites of pGEMHIS (51) to create pKO509. pKO509 was digested with SwaI and transformed into VBIDM2 to create the reconstituted strain KO509. PCR-mediated overlap extension mutagenesis (55) was used to produce copies of Int1 mutated at putative heparin binding domains. Briefly, primer pairs 1+3 and 2+4 (or 1+5 and 2+6) were used to produce two overlapping fragments of INT1 in which putative heparin binding domains were mutated (FKKRFFKL (SEQ ID NO: 2)→FKAAFFKL (SEQ ID NO: 53) or KQKKHQ (SEQ ID NO: 64)→KQAAHQ (SEQ ID NO: 63)), and a full length mutated sequence generated in a third per using primers 1+2 with the fragments as template. The mutated sequences were cloned into the SacI/MluI sites of pGEMHIS to create pKO503 and pKO507, respectively. A construct mutated at both sites (pKO508) was produced using primers 1+5 and 2+6 with plasmid DNA from pKO503 as template. Full length mutated products cloned into pGEMHIS and used to transform VBIDM2 as above, producing strains KO503, KO507, and KO508. The correct insertion and orientation of all constructs was confirmed by PCR. In growth curves performed with and without 100 units/ml heparin at 30° C. in RPMI-HEPES, there was no difference in the doubling times of the wild type, Δint1 mutant, INT1 reintegrant, or the reintegrants containing alanine substitutions in Motif 1 (804QKKHQIHK (SEQ ID NO: 1) to QAAHQIHK (SEQ ID NO: 52)), Motif 4 (1593FKKRFFKL (SEQ ID NO: 2) to FKAAFFKL (SEQ ID NO: 53)), or Motifs 1 and 4. Percent binding of each mutant to heparin in the ELISA assay was compared to the wild type according to the formula [Absorbance595 mutant]/[Absorbance595 wild type]×100.
The ELISA assay used a commercially available 96-well microtiter plate coated with polymerized allylamine (BD Biosciences) (27), in which wells were inoculated with 2.5 units (12.5 μg) heparin in 100 μl phosphate buffered saline (PBS) per well. The plate was covered and allowed to stand at room temperature overnight, then washed with acetate buffer (100 mM NaCl, 50 mM NaOAc, 0.2% Tween, pH 7.2). Wells were washed three times with PBS, then inoculated with 1×104 to 4×104 Candida albicans yeast cells that were grown overnight at 30° C. in yeast peptone dextrose (YPD) broth, washed, and resuspended in RPMI-HEPES at 1×107 organisms/ml. The plate was then incubated at 30° C. for one hour, then washed with PBS-Tween (PBST, PBS/0.05% Tween). A commercially available biotinylated anti-Candida antibody (Meridian Life Science, Memphis Tenn.) was diluted 1:1500 in FACS-TWEEN (0.3% BSA/0.05% Tween), added to the wells, incubated at 30° C. for one hour, then washed with PBST. Streptavidin-alkaline phosphatase (BioLegend, San Diego Calif.) was then added to the wells at a 1:10,000 dilution in FACS-TWEEN and incubated at 30° C. for 30 min, then washed with PBST and AKP buffer (100 mM Tris, 100 mM NaCl, 50 mM MgCl2*6H2O). BluePhos@ microwell substrate (Kirkegaard and Perry Laboratories, Gaithersburg Md.) was added to the wells and allowed to react for 45 min at room temperature. The color change was read spectrophotometrically at absorbance of 595 nm.
Because Motif 1 in Int1 appeared to mediate a considerable proportion of the binding of C. albicans to heparin, a peptide encompassing this motif HKQEKQKKHQIHKV (SEQ ID NO: 4) was used to immunize rabbits with a commercial protocol (Pacific Immunology). Affinity-purified immune IgG and IgG from pre-immune rabbit serum (both from Pacific Immunology) were compared for the ability to inhibit binding of C. albicans to heparin (
Polyclonal IgG to the HKQEKQKKHQIHKV (SEQ ID NO: 4) motif in domain 3 of Int1 blocked 19% of binding of wild type C. albicans (WT) to heparin.
In other systems, heparin has been reported to cleave surface proteins (56), change protein conformation (57), and bind trypsin-sensitive lysine and arginine residues in histone H1 (58). In order to understand whether incubation with heparin changed the conformation of C. albicans surface proteins, 1×107 C. albicans were incubated for one hour at 3° C. on a rotator with pharmacologic heparin (20,000 units/ml) diluted in RPMI-HEPES to a working concentration of 250 units/ml. C. albicans 1×107 C. albicans in an equal volume of RPMI-HEPES served as control. Supernatants were removed. 300 μl of Hep+ and Hep− supernatants were incubated with 100 μl avidin agarose beads (Thermo Scientific) for 60 min at room temperature on a rotator in the presence of 50 units heparin (Hep+); an equal volume of RPMI-HEPES was substituted for heparin in the Hep− supernatants. Beads were pelleted, and 100 μl beads were incubated with 100 μl 3.0 M NaCl for 30 mins at room temperature on a rotator. Beads were pelleted and the supernatants were withdrawn; 100 μl TCA was added to 100 μl Hep+ or 100 μl Hep− supernatants and incubated on ice overnight at 4° C. The TCA precipitates were stored at −80° C. until analysis by mass spectroscopy.
Six biological replicates of TCA precipitated protein pellets from equal cellular equivalence of heparin-treated (Hep+) and untreated (Hep−) conditioned medium from cultures of C. albicans were each solubilized in 50 μL Laemmli gel buffer. To remove any residual TCA and to further concentrate the samples, each was subjected to buffer exchange and concentration using an Amicon ultra 3 kDa microfuge filtration cartridge at 14,000×g for 15 min with five subsequent additions of 50 μL of 1× Laemmli gel buffer between spins. The resulting retained proteins (6 Hep+ and 6 Hep−) were subsequently prepared for SDS-PAGE by combining into two pools of three samples for the Hep+ and Hep− conditioned medium. The replicate sample pools were loaded onto two 4-12% mini gels and separated using the MOPS buffer system followed by silver staining to visualize the proteins using the Sigma Proteosilver system. The proteins were prepared for identification and quantitation by mass spectrometry by gridding the gel lanes from the replicates of Hep+ and Hep− samples into 11 equal regions followed by in gel trypsin digestion and extraction of peptides as described (59)
The recovered peptides from the gridded gel sections were analyzed by liquid chromatography coupled nano-electrospray mass spectrometry (nLC-MSMS) on a TripleTOF 5600 mass spectrometer (AB Sciex, Toronto Canada) attached to an Eksigent (Dublin Calif.) nanoLC ultra nanoflow system. Recovered peptides from each fraction were loaded on to IntegraFrit Trap Column (outer diameter of 360 μm, inner diameter of 100, and 25 μm packed bed) from New Objective, Inc. (Woburn Mass.) at 2 μl/min in FA/H2O 0.4/99.2 (v/v) for 10 min to desalt and concentrate the samples. For the chromatographic separation of peptides, the trap-column was switched to align with the analytical column, Acclaim PepMap100 (inner diameter of 75 μm, length of 15 cm, C18 particle sizes of 3 μm and pore sizes of 100 Å) from Dionex-Thermo Fisher Scientific (Sunnyvale Calif.). The peptides were eluted using a linear gradient from 95% phase A (FA/H2O 0.4/99.6, v/v) to 40% phase B (FA/ACN 0.4/99.6, v/v) from 5 min to 22.5 min (2% ACN/min) at a flow of 300 mL/min. As the peptides eluted from the column they were sprayed into the mass spectrometer using NANOSpray® III Source (AB Sciex, Toronto Canada). Ion source gas 1 (GS1), ion source gas 2 (GS2) and curtain gas (CUR) were respectively kept at 7, 0 and 25 vendor specified arbitrary units. Interface heater temperature and ion spray voltage was kept at 150 C, and at 2.3 kV respectively. Mass spectrometer method was operated in positive ion mode set to go through 25 minutes, where each cycle performing one TOF-MS scan type (0.25 sec accumulation time, in a 400 to 1600 m/z window) followed by twenty information dependent acquisition (IDA)-mode MS/MS-scans on the most intense candidate ions selected from initially performed TOF-MS scan during each cycle having a minimum 150 counts. Each product ion scan was operated under vender specified high-sensitivity mode with an accumulation time of 0.05 sec and a mass tolerance of 50 mDa. Former MS/MS-analyzed candidate ions were excluded for 10 sec after its first occurrence, and data were recorded using Analyst®-TF (1.5.1) software.
NanoLC-MSMS data collected from the Hep+ and Hep− samples were converted to Mascot generic files and searched against the SwissProt fungal database on an in house server running Mascot version 2.2.07 (Matrix Science, Ltd). Specific search parameters included up to two missed tryptic cleavages, carbamidomethylation of Cys, oxidation of Met, peptide and fragmentation mass tolerance of 0.1 Da. Only proteins with a minimum of two peptides with Mascot peptide score indicating a peptide identity and a false discovery rate (FDR) against and inverse database at less than 1% were reported. Semi-quantitative measurements between the Hep+ and Hep− proteins were generated using a minimum of two tryptic peptides from each protein as surrogates for the amount of proteins from the two groups. This was accomplished by capturing extracted ion profiles for each peptide and then comparing the mono-isotopic peak intensity at the apex of the signal for the M+2H or M+3H signal for each peptide. An average ratio of the maximal peak intensity between two independent peptides for each protein presented was used to determine semi-quantitative ratios of proteins between the Hep+ and Hep− samples.
To determine the levels of these proteins in control versus heparin-treated organisms, the relative level of several tryptic peptides were used as surrogates for the protein levels.
Compared to untreated organisms, heparin treatment led to a seven-fold increase in intensity of IDVVDQAK (SEQ ID NO: 65) from Eno1 (
Other peptides from Eno1, Pgk1, and Tdh3 also exhibited increased intensities, ranging from 10-16 fold the intensity of the corresponding peptides from untreated organisms (data not shown), further validating significant increases in the protein levels in the supernatants for heparin-treated cultures. Of the 12 proteins whose peptides were found in highest concentration in the supernatant (Table 4), all but one are known to be localized to the cell wall; cellular localization of Eft1 is not known. Eight of the twelve, including Eno1, Pgk1, Tdh3 and Ssa1/2, themselves contain putative heparin binding motifs (Table 1) and are considered critical antigens for innate and adaptive immune responses against C. albicans (31-36).
#cell wall proteins by Gene Ontology.
In order to evaluate the consequences of removal of Ssa1 and Ssa2 (Table 4), both targets of histatin, a histatin killing assay was performed as follows. C. albicans strains were grown to late log phase overnight in YPD. A single colony was suspended in 1 ml YPD, diluted 1:500 into 10 ml YPD, and incubated overnight at 30° C. and 225 rpm to OD600 1.0. Yeast cells were washed twice in PBS and 2×104 cells suspended in 250 μl RPMI+10 mM HEPES, pH 7.0, with or without 500 units/ml heparin (Sigma). The cells were incubated at 37° C. for one hour with shaking, washed twice with 10 mM phosphate buffer, pH 7.4, and suspended in 20 μl phosphate buffer. Histatin 5 (final concentration 15 μM; Peptides International) or 10 mM phosphate buffer was added to the preincubated cells (total volume 40 μl) and incubated further at 37° C. for 90 minutes with shaking. YPD (360 ul) was added to each tube, a 40 μl aliquot spread onto YPD plates, and colonies counted after two days. The effect of heparin treatment was determined by the formula % change=[(cfu+heparin)−(cfu−heparin)]/(cfu+heparin).
Heparin binding to trypsin-sensitive lysine and arginine residues in histone H1 unfolds chromatin and increases its accessibility (58). To test whether heparin binding could influence gene expression in C. albicans, qRT-PCR was performed on a selective set of 13 genes involved in adhesion, cell-cell interaction, and biofilm formation after incubating C. albicans with and without 100 units/ml heparin at 37° C. for 75 minutes. Gene expression studies were performed as follows. Overnight cultures (3 ml YPD at 30° C., 225 rpm) were diluted in 25 ml YPD to a nominal concentration of 8×105/ml and grown to OD600 approximately 1.0 (30° C., 225 rpm), collected by centrifugation, and washed in PBS. Cells (5×107) were suspended in 5 ml RPMI1640 with 25 mM MOPS pH7.4, with or without 100 units/ml Sigma heparin in 50 ml polypropylene tubes, and incubated at 37° C. with shaking for 75 minutes. One ml aliquots (approximately 107 cells) were collected by centrifugation, washed once with room temperature PBS, and frozen at −80°. Pellets were thawed on ice and suspended in 1 ml Tri-Reagent (MRC Research) in a 2 ml screw capped tube containing about 0.2 g acid washed glass beads (Sigma) and vortexed three times for 1 minute with a 1 minute rest on ice between each vortex. The lysates were rested for five min at room temperature, centrifuged for five min at 12,000 rpm, and RNA isolated from the supernatant using the DirectZol kit (Zymo), including DNAse digestion, per manufacturer's instructions. cDNA was produced from equivalent quantities of RNA (between 300 and 900 ng) for each treatment using the Maxima Reverse Transcriptase Kit for qRT-PCR (Fermentas). Two μl of 1:5 dilution of cDNA was used in each qPCR reaction with 500 nM of each primer and Fast SYBR Green Master Mix (Life Technologies) on a 7500 FAST Instrument (Applied Biosystems) according to manufacturer's instructions. Relative expression was determined using the ΔΔCt method (60) with 18S RNA as the reference. Primers are shown in Table 5.
In order to understand whether heparin binding motifs 1 and 4 influenced C. albicans pathogenesis in vivo, we employed a rat model of biofilm formation in central venous catheters inserted into the jugular vein, as previously described (61). After insertion into the jugular vein of an anesthetized female Sprague-Dawley rat, a silastic catheter was heparinized with 100 units heparin/mil and remained in place for 24 hours. At the 24 hour timepoint, 500 μl of blood was withdrawn and cultures to insure sterility, and then 1×106 CFU of the desired C. albicans strain was instilled into the catheter and allowed to dwell for 6 hours. The animal was then sacrificed, the catheter was removed aseptically and processed for scanning electron microscopy. Biofilm formation on the intra-luminal surface of the catheter was assessed by scanning electron microscopy (SEM) at 100× and 2000×.
The INT1 wild type strain (WT) produced a profuse biofilm with intertwined hyphae and visible exopolysaccharide matrix (
Inhibition of biofilm formation by the antibody raised against the peptide sequence HKQEKQKKHQIHKV (SEQ ID NO:4) was also tested in the rat central venous catheter model. A 1:10 dilution of affinity-purified IgG against the peptide HKQEKQKKHQIHKV (SEQ ID NO: 4) was incubated with wild type C. albicans at 30° C. for one hour. A 1:10 dilution of pre-immune IgG was used as a control. The strains were then instilled into separate jugular venous catheters in individual rats. After 6 hours, catheters were removed and aseptically processed for scanning electron microscopy (100× and 2000×) as described above.
Central venous catheters from animals that received C. albicans pre-incubated with IgG against HKQEKQKKHQIHKV (SEQ ID NO:4) exhibited substantially reduced biofilm formation.
In vitro study results showed that C. albicans binds heparin through HBM in Int1 (
The in vivo studies of biofilm formation in heparinized central venous catheters in rats showed an obvious reduction in biofilm formation after mutation of lysine residues 805/806 in Motif 1 (
Putative linear HBM are also present in Staphylococcus epidermidis and Staphylococcus aureus (Long and Hostetter unpublished data), two organisms that are even more common causes of catheter-associated infection (43), as shown in Tables I-IV. For example, putative linear HBM were identified in the following cell wall or putative cell wall proteins, where the motif and the beginning position of the motif is indicated in parenthesis, from methicillin resistant Staphylococcus aureus, strain 252 (MRSA252): sasC (LKKNKY; 4; and IRKYKV; 11), isdB (YKKAKT; 289), sasF (SRRNKL; 618), glcB (IRKFKL; 415), sasA (MHHTHS; 1263), SAR0879 (LKKIKG; 573), SAR0986 (YRHLKP; 754), SAR1559 (IRKAHQ; 206), SAR2393 (PKRKVVKI; 149), and sasG (VRKARS; 140); from methicillin sensitive Staphylococcus aureus, strain 476 (MSSA476): SAS2383 (VRKARS; 140; and VKKSKI; 1319), SAS1682 (LKKNKY; 4; and IRKYKV; 11), SAS1063 (YKKAKT; 282), SAS1657 (YHKAKT; 484), SAS2532 (SRRNKL; 626), SAS2540 (MHHTHS; 2187), SAS0082 (LKKIKG; 573), SAS1011 (IRKAHQ; 206), SAS2035 (PKRKVVKI; 149), and SAS2424 (IRKFKL; 415); and from Staphylococcus epidermidis, strain RP62A: SERP1316 (FRKQKF; 4; VHRLKV; 352; and IHKIKP; 3234), SERP0660 (LKKWKV; 4; and IRRAHQ; 212), SERP0719 (TRKNHY; 13), SERP1482 (VKRFKN; 1730), SERP1654 (MKKSKV; 1), SERP2264 (MKRIKT; 393), SERP0207 (NRKNKN; 887), and SERP1691 (PKKIKN; 72).
In one embodiment, an antibody is generated against a linear heparin binding motif which is conserved among MSSA, MRSA, and S. epidermidis. In one embodiment, the conserved heparin binding motif is selected from the group consisting of LKKNKY, LKKNKY, LKKWKV, LKKIKG, LKKIKG, VRKARS, VRKARS, YKKAKT, YKKAKT, YHKAKT, PKRKVVKI, PKRKVVKI, IRKAHQ, IRKAHQ, and IRRAHQ.
Putative linear HBM were identified in the following cell wall or putative cell wall proteins from various yeast species, as shown in Table V where a check mark indicates that the motif is identical to the motif found in C. albicans, including C. dubliniensis (Int1 (YKKRFFKL), Eno1 (AKKGKF), and Tdh3 (GHKIKV) proteins), C. parapsilosis (Tdh3 protein (GHKIKV)), C. tropicalis (Int1 (FKRRFFKL), and Tdh3 (GHKIKV) proteins), C. glabrata (Int1 (FKKRFFTL) protein), Lodderomyces elongisporus (Int1 (FKKFIFKL) and Tdh3 (GHKIKV) proteins), and A. nidulans (Int1 (FKKRFFKL) protein). In embodiments, an antibody directed to a region of these proteins containing the putative HBM or the region of these proteins containing the putative HBM may be used in the described methods.
Staphylococcus epidermidis, strain RP62A
Lodderomyces
C. albicans XXXXXXX
C. albicans
C. dubliniensis
C. parapsilosis
C. tropicalis
C. glabrata
elongisporus
A. nidulans
The interaction of C. albicans with heparin or heparin-like compounds may play a key role in clinical settings where heparin is used (e.g. central venous catheters, dialysis catheters) or possibly in tissues where heparan sulfates are preferentially expressed.
Because antibodies raised against a linear heparin binding motif inhibited binding of C. albicans to heparin, the heparin binding peptide HKQEKQKKHQIHKV (SEQ ID NO: 4) could be used as an immunizing antigen in immunocompetent patients to elicit antibodies that protect against heparin binding.
For example, a peptide from streptococcal M protein has been used to make a vaccine for rheumatic fever (62). Alternatively, antibodies raised against this peptide in immunized humans can be used for passive immunization. This technology is used with commercially available antibodies such as hepatitis B immune globulin (HBIG) or botulism immune globulin (BabyBIG®) (63). Hepatitis B immune globulin is commercially available; botulism immune globulin is made and distributed by the California Department of Public Health and is FDA-approved. A humanized monoclonal antibody recognizing a desired heparin binding motif could be made using technology for the production of palivizumab (Synagis®), a humanized monoclonal antibody that is given monthly to premature newborns to prevent infection with respiratory syncytial virus (64). Because similar heparin binding motifs are also found in S. epidermidis and S. aureus, using heparin binding motifs as antigens is a first step in developing vaccines against three of the most common causes of central line-associated bloodstream infection.
Antibodies raised against a linear heparin binding motif expressed by a surface protein of C. albicans inhibited adhesion of the yeast to heparin in vitro and abolished biofilm formation in vivo. Similar heparin binding motifs occur in surface proteins from S. epidermidis and S. aureus, two organisms which are also major causes of biofilm-related infections.
In one embodiment, an antibody reactive with a heparin binding motif expressed on the surface of a microorganism is administered to an individual. The microorganism may be, but is not limited to, a Candida species or a Staphylococcus species. In one embodiment, the microorganism is C. albicans, S. epidermidis and/or S. aureus. In one embodiment, the heparin binding motif is identified by a computer-based algorithm analysis of protein sequences expressed by a microorganism. In one embodiment, the heparin binding motif to which the antibody was generated is derived from Int1. In one embodiment, the antibody is directed to the peptide HKQEKQKKHQIHKV (SEQ ID NO: 4), or a fragment of this peptide. In one embodiment, the fragment is at least seven amino acids of the peptide. In one embodiment, the surface expression of the heparin binding motif depends on the life-cycle stage of the microorganism. In one embodiment, the antibody is a polyclonal antibody. In one embodiment, the antibody is a monoclonal antibody.
In one embodiment, a peptide that corresponds to a linear heparin binding motif expressed on the surface of a microorganism, or a portion of the peptide, is provided. In one embodiment, the peptide is used to generate antibodies directed against the peptide, using methods known in the art. Alternatively, a peptide is provided combined with additional carriers or as a component of a complex, e.g., an adjuvant non-toxic to humans. Such an adjuvant could be aluminum hydroxide used in Engerix-B® (commercially available hepatitis B vaccine), and in Comvax® (commercially available vaccine for Haemophilus influenzae type b and hepatitis B). The peptide corresponds to a linear heparin binding motif, or a portion of the motif, of a microorganism. In one embodiment, the microorganism is a Candida species and/or a Staphylococcus species. In one embodiment, the microorganism is C. albicans, S. epidermidis and/or S. aureus. In one embodiment, the heparin binding motif is identified by a computer-based algorithm analysis of protein sequences expressed by a microorganism. In one embodiment, the heparin binding motif is derived from Int1. In one embodiment, the peptide is HKQEKQKKHQIHKV (SEQ ID NO: 4), or a fragment of SEQ ID NO: 4. In one embodiment, the peptide is a fragment of the peptide HKQEKQKKHQIHKV (SEQ ID NO: 4), where the fragment contains at least seven amino acids of the peptide. In one embodiment, the surface expression of the heparin binding motif depends on the life-cycle stage of the microorganism. In one embodiment, the peptide is used to generate an antibody using methods known in the art.
In one embodiment, a method is provided for ameliorating biofilm formation on a surface of an implanted device in a patient. In one embodiment, the method comprises administering an antibody to a patient, and ameliorating biofilm formation on a surface of an implanted device in a patient, wherein the antibody is directed to a heparin binding motif expressed on the surface of a microorganism. In one embodiment, the antibody is administered intravenously at a dose not more than 2 grams/kg given as an infusion over 10-24 hours. Since the half-life of antibody preparations is about four weeks, antibody administration would be repeated at monthly intervals over the life of the catheter. In one embodiment, the antibody has been modified to increase safety and/or efficacy in a human patient. In one embodiment, the antibody has been humanized using methods known in the art. In one embodiment, the described antibody is provided as a pharmaceutical composition with at least one biocompatible excipient, e.g., buffers, preservatives, tonicity adjusting agents, pH adjusting agents, osmolality adjusting agents, etc, as known to one skilled in the art. Without being held to a single theory, the antibody blocks binding between a heparin binding motif-containing protein expressed on the surface of a microorganism and heparin or heparin sulfate, which is expressed by human cells lining the lumen of the implanted device (
The administration of the described antibody is referred to as passive immunization.
In one embodiment, the method comprises administering a peptide to a patient, where the peptide encompasses a heparin binding motif, or a fragment of the motif, that is expressed on the surface of a microorganism. The peptide is 14 amino acids in length, with amino acids both preceding and following the 8 amino acids of the heparin binding motif. In one embodiment, the peptide is HKQEKQKKHQIHKV (SEQ ID NO: 4), or a fragment thereof. In one embodiment, the described peptide is provided as a pharmaceutical composition comprising at least one biocompatible excipient including but not limited to buffers, stabilizing agents, solubility enhancing agents, tonicity agents, etc. as known to one skilled in the art.
Without being held to a single theory, the peptide serves as an antigen to immunize the patient. It provides immunity by promoting endogenous generation of antibodies directed to the peptide, where the endogenously-produced antibodies block binding between heparin, which is administered through the catheter or is bound to a surface of an implanted medical device, and a heparin binding motif-containing protein expressed on a microorganism surface. Administration of the described peptide is referred to as active immunization. Peptide vaccines are typically administered subcutaneously or intramuscularly. Immunizing dose of the peptide and frequency of immunization are experimentally determined with humans.
The patient being treated may be naturally immunodeficient by having an underdeveloped immune response, e.g., a premature newborn. The patient being treated may have a normal immune response, but may be immunocompromised due to particular circumstances, e.g., as a result of treatment such as chemotherapy, or disease, or burns. In one embodiment, the disclosed antibody is administered to an immunodeficient patient. In one embodiment, the disclosed peptide is administered to an non-immunodeficient patient, e.g., an otherwise well adult or child who will receive long term antibiotics through a central line for the treatment of a serious infection, e.g., endocarditis.
In one embodiment, the implanted medical device, e.g., catheter, central line, hemodialysis catheter, peritoneal catheter, PICC line, plastic catheters such as central venous catheters, urinary tract catheters, central nervous system shunt catheters, peritoneal dialysis catheters, dialysis shunts, etc., is implanted in a vein, artery, or a body cavity. In one embodiment, the implanted medical device is a plastic device. In one embodiment, the implanted device is present in the patient's body for a period of time ranging from minutes to six weeks or longer. In one embodiment, the above described methods may be performed before the device is implanted in the patient. In one embodiment, a first dose of the antibody is administered before the medical device is implanted. In this embodiment, a peripheral intravenous line could administer this first dose of the antibody if the device to be implanted is a catheter.
In one embodiment, the described methods decrease the virulence of a microorganism, i.e., the method decrease the ability of a microorganism to cause infection in a patient.
All references cited are expressly incorporated by reference herein in their entirety.
The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. The ASCII copy, created on Mar. 8, 2013, is named 070248_94_SL.txt and is 30,824 bytes in size.
The embodiments described in the specification are only specific embodiments of the inventors who are skilled in the art and are not limiting. Therefore, various changes, modifications, or alterations to those embodiments may be made without departing from the spirit of the invention or the scope of the following claims.
This application is a Continuation-In-Part of co-pending PCT/US2013/031499 filed Mar. 14, 2013, which claims priority to U.S. application Ser. No. 61/636,243 filed Apr. 20, 2012, each of which is expressly incorporated by reference herein in its entirety.
| Number | Name | Date | Kind |
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| 20030082680 | Hostetter et al. | May 2003 | A1 |
| 20070116706 | Hostetter et al. | May 2007 | A1 |
| 20110189183 | Williamson et al. | Aug 2011 | A1 |
| Number | Date | Country |
|---|---|---|
| WO 2006024631 | Mar 2006 | DK |
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| Number | Date | Country | |
|---|---|---|---|
| 20150050284 A1 | Feb 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61636243 | Apr 2012 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2013/031499 | Mar 2013 | US |
| Child | 14518051 | US |
