Patent Application
20230390373
A sequence listing electronically submitted as an ASCII text file named P2021TC1576_ST25.txt, created on May 9, 2023 and having a size of 16000 bytes, is incorporated herein by reference in its entirety.
The invention relates to the field of biomedicine. In particular, the invention relates to a live strain of Staphylococcus aureus and uses thereof. More particularly, the invention relates to a live strain of Staphylococcus aureus which lacks adenosine synthase A (AdsA) activity, to a vaccine against Staphylococcus aureus infection comprising said live strain, and a method for preventing and/or treating Staphylococcus aureus infection in a subject by administering said live strain.
S. aureus is one of the most common causes of community-acquired (CA) and healthcare-associated (HA) bacterial infections (1). S. aureus infection leads to a variety of clinical manifestations ranging from skin and soft-tissue infections to invasive disease including bloodstream infection, endocarditis or sepsis (2). Moreover, the emergence of Methicillin-Resistant S. aureus (MRSA) has further made it a major global health problem (3). Of note is that prior exposure to S. aureus does not confer protection against subsequent S. aureus infection (4). The lack of understanding about how S. aureus constrains protective immunity has impeded the development of efficient treatments against S. aureus infection.
Several host factors to date have been implicated in the protection against S. aureus in different infection models. These include complement system, neutrophils (5), macrophages (6), IL-17A producing γδ+ T cells (7), humoral responses (8), Th1 and Th17 immune responses (6, 9). Nevertheless, humoral responses have long been recognized as a critical indicator of anti-S. aureus immunity, individuals with robust S. aureus specific antibody responses are not exempt from the next infection. In contrast, accumulating evidence has shed light on the role of cellular immunity in preventing the course of S. aureus infection. Patients with disease causing defect in Th17 differentiation often displayed increased susceptibility toward S. aureus infection (10). Th17 immunity can potentiate bacterial killing by enhancing phagocytosis of neutrophils via secreting IL-17 family cytokines (IL-17A and IL-17F) (11). Meanwhile memory Th1 immunity is reported to accelerate the clearance of S. aureus in blood stream infection (BSI) (6). Thus, the large number of recurrent infections in clinical setting implies failure in the establishment of protective T cell responses during S. aureus infection. Thus far, only O-acetyltransferase (OatA) has been proven to suppress the development of protective Th17 immunity by interfering with Th development cytokines milieu (12). Consequently, it is of significant importance to investigate the mechanisms whereby S. aureus counteracts host cellular immunity, contributing to reinfection.
Adenosine synthase A (AdsA) is an important virulence factor by which S. aureus modulates host pro-inflammatory responses, resulting in persistent infection (22). Previous studies have shown that AdsA can inhibit phagocytic clearance (23), secretion of antibacterial peptide sPLA2-IIA (24), and induce apoptosis of macrophages (25) via adenosine signaling or deoxyadenosine signaling. However, the mechanistic interaction of AdsA with host adaptive immunity remains unclear.
In one aspect, the invention provides a vaccine against Staphylococcus aureus infection comprising a live strain of S. aureus, and optionally an adjuvant, wherein the strain lacks adenosine synthase A (AdsA) activity.
In another aspect, the invention provides a live strain of S. aureus for use in preventing and/or treating Staphylococcus aureus infection, wherein the strain lacks adenosine synthase A (AdsA) activity.
In another aspect, the invention provides a method for preventing and/or treating Staphylococcus aureus infection in a subject, which comprises administering an effective amount of a live strain of S. aureus to the subject, wherein the strain lacks adenosine synthase A (AdsA) activity.
In another aspect, the invention provides use of a live strain of S. aureus in preparation of a medicament for preventing and/or treating Staphylococcus aureus infection, wherein the strain lacks adenosine synthase A (AdsA) activity.
In another aspect, the invention provides a kit for immunization against S. aureus infection, comprising a container containing the vaccine of the invention or the live strain of S. aureus of the invention.
Before the aspects of the present invention are described, it must be noted that as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural reference unless the context clearly dictates otherwise. The term “and/or” is intended to encompass any combinations of the items connected by this term, equivalent to listing all the combinations individually. For example, “A, B and/or C” encompasses “A”, “B”, “C”, “A and B”, “A and C”, “B and C”, and “A and B and C”. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs.
Staphylococcus aureus is a common human pathogen, capable of causing diverse illnesses with possibility of recurrent infections, and adenosine synthase A (AdsA) is a potent S. aureus virulence factor. The present inventors surprisingly found that a live strain of S. aureus lacking AdsA activity can protect mice against wildtype S. aureus infection (see such as, Example 5,
Accordingly, in one aspect, the invention provides a vaccine against Staphylococcus aureus infection comprising a live strain of S. aureus, wherein the strain lacks adenosine synthase A (AdsA) activity.
Adenosine synthase A (AdsA) is an important virulence factor of S. aureus. An exemplary AdsA of S. aureus comprises an amino acid sequence of SEQ ID NO:46. But it is well known to a person skilled in the art that the AdsA of S. aureus may have minor differences from SEQ ID NO:46 due to polymorphyism between strains, while retain the same or similar functions.
In some embodiments, the live strain of S. aureus comprises a deletion of an AdsA gene encoding AdsA. The AdsA gene may be completely deleted from the S. aureus strain so that no AdsA protein is present in the strain. The AdsA gene may also be partially deleted so that merely a truncated AdsA protein without activity is present in the strain, for example, at least a portion of AdsA responsible for adenosine production is deleted.
In some embodiments, the live strain of S. aureus comprises a mutation in an AdsA gene encoding AdsA. Such a mutation can be addition, substitution, or deletion of one or more nucleotides. In some embodiments, said mutation is a frame-shift mutation, which results in mistranslation of the AdsA protein.
In some embodiments, the mutation in the AdsA gene results in a deletion of a portion of AdsA responsible for adenosine production.
In some embodiments, the AdsA activity is responsible for attenuation of NLRP-3 mediated IL-1β production in an inflammatory cell via the adenosine/A2AR pathway during Staphylococcus aureus infection.
Preferably, the deletion of the AdsA gene is carried out by means of a strategy that avoids the reversal of the mutated strain to the wild phenotype.
In some embodiments, the strategy chosen to prevent the reversal of the mutated strain to the wild phenotype is the double homologous recombination.
In some embodiments, the mutation/deletion of the AdsA gene is carried out by targeted mutation, such as via CRISPR, TALEN or ZFN technologies.
In some embodiments, the vaccine may further comprise an adjuvant. As used herein, “adjuvant” refers to additional components in a vaccine to enhance the immune response, or ancillary molecules added to the vaccine or generated by the body after the respective induction by such additional components, like but not restricted to interferons, interleukins or growth factors. “Adjuvants” as used herein, can include aluminum hydroxide and aluminum phosphate, saponins, water-in-oil emulsion, oil-in-water emulsion, water-in-oil-in-water emulsion.
In some embodiments, the vaccine further comprises a pharmaceutically acceptable carrier. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, coatings, antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. Non-limiting examples of pharmaceutically acceptable carriers include water, NaCl, physiological saline, lactated Ringer's, normal sucrose, normal glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavoring agents, salt solutions (such as Ringer's solution), alcohol, oil, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethyl cellulose, polyvinylpyrrolidone and coloring agents.
The live strain of S. aureus can be derived from any S. aureus strains, such as those well known in the art. For example, the live strain of the invention may be derived from Staphylococcus aureus USA300, Newman, ATCC29213, and the like.
In some embodiments, the Staphylococcus aureus infection is a skin infection, soft-tissue infection, or invasive disease. In some embodiments, the invasive disease is bloodstream infection, endocarditis or sepsis.
In some embodiments, the Staphylococcus aureus infection is methicillin-resistant S. aureus (MRSA) infection or methicillin-sensitive S. aureus (MSSA) infection. In some preferred embodiments, the infection is a recurring S. aureus infection.
In some embodiments, the vaccine is formulated in a form for intramuscular administration, intraperitoneal administration, subcutaneous administration, oral administration or intranasal administration. In one embodiment, the vaccine is not for intravenous administration.
In some embodiments, the vaccine is in a lyophilized form, which can be reconstituted before use.
In another aspect, the invention provides a live strain of S. aureus for use in preventing and/or treating Staphylococcus aureus infection, wherein the strain lacks adenosine synthase A (AdsA) activity.
In some embodiments, the strain of S. aureus comprises a deletion of an AdsA gene encoding AdsA.
In some embodiments, the strain of S. aureus comprises a mutation in an AdsA gene encoding AdsA.
In some embodiments, the mutation in the AdsA gene results in a deletion of a portion of AdsA responsible for adenosine production.
In some embodiments, the AdsA activity is responsible for attenuation of NLRP-3 mediated IL-1β production in an inflammatory cell via the adenosine/A2AR pathway during Staphylococcus aureus infection.
In some embodiments, the strain is derived from Staphylococcus aureus USA300, Newman, or ATCC29213.
In some embodiments, the Staphylococcus aureus infection is a skin infection, soft-tissue infection, or invasive disease.
In some embodiments, the invasive disease is bloodstream infection, endocarditis or sepsis.
In some embodiments, the Staphylococcus aureus infection is methicillin-resistant S. aureus (MRSA) infection or methicillin-sensitive S. aureus (MSSA) infection. In some preferred embodiments, the infection is a recurring S. aureus infection.
In some embodiments, the strain is administered intramuscularly, intraperitoneally, subcutaneously, orally or intranasally. In one embodiment, the strain is not for intravenous administration.
In some embodiments, the live strain is in a lyophilized form, which can be reconstituted before use.
In another aspect, the invention provides a method for preventing and/or treating Staphylococcus aureus infection in a subject, which comprises administering an effective amount of a live strain of S. aureus to the subject, wherein the strain lacks adenosine synthase A (AdsA) activity.
As used herein, “effective amount” refers to an amount of a substance, compound, material, or composition containing a compound (such as the live strain of the invention of the vaccine of the invention) which is at least sufficient to produce a therapeutic effect after administration to a subject. Therefore, it is an amount necessary to prevent, cure, improve, retard or partially retard the symptoms of a disease or disorder, such as S. aureus infection.
The actual dosage of the live strain or vaccine of the present invention to be administered to a subject can be determined according to the following physical and physiological factors: weight, sex, severity of symptoms, type of diseases to be treated, previous or current therapeutic intervention, unknown etiological disease of the patient, administration time, administration route and the like. In any case, the amount of the live strains in the vaccine and the appropriate dose for an individual subject will be determined by the medical personnel responsible for administration.
In some embodiments, the strain of S. aureus comprises a deletion of an AdsA gene encoding AdsA.
In some embodiments, the strain of S. aureus comprises a mutation in an AdsA gene encoding AdsA.
In some embodiments, the mutation in the AdsA gene results in a deletion of a portion of AdsA responsible for adenosine production.
In some embodiments, the AdsA activity is responsible for attenuation of NLRP-3 mediated IL-1β production in an inflammatory cell via the adenosine/A2AR pathway during Staphylococcus aureus infection.
In some embodiments, the strain is derived from Staphylococcus aureus USA300, Newman, or ATCC29213.
In some embodiments, the Staphylococcus aureus infection is a skin infection, soft-tissue infection, or invasive disease.
In some embodiments, the invasive disease is bloodstream infection, endocarditis or sepsis.
In some embodiments, the Staphylococcus aureus infection is methicillin-resistant S. aureus (MRSA) infection or methicillin-sensitive S. aureus (MSSA) infection. In some preferred embodiments, the infection is a recurring S. aureus infection.
In some embodiments, the strain is administered intramuscularly, intraperitoneally, subcutaneously, orally or intranasally. In one embodiment, the strain is not administered intravenously.
In some embodiments, the strain is in a lyophilized form, which can be reconstituted before use.
In another aspect, the invention provides use of a live strain of S. aureus in preparation of a medicament for preventing and/or treating Staphylococcus aureus infection, wherein the strain lacks adenosine synthase A (AdsA) activity.
In some embodiments, the live strain of S. aureus comprises a deletion of an AdsA gene encoding AdsA.
In some embodiments, the live strain of S. aureus comprises a mutation in an AdsA gene encoding AdsA.
In some embodiments, the mutation in the AdsA gene results in a deletion of a portion of AdsA responsible for adenosine production.
In some embodiments, the AdsA activity is responsible for attenuation of NLRP-3 mediated IL-1β production in an inflammatory cell via the adenosine/A2AR pathway during Staphylococcus aureus infection.
In some embodiments, the strain is derived from Staphylococcus aureus USA300, Newman, or ATCC29213.
In some embodiments, the Staphylococcus aureus infection is a skin infection, soft-tissue infection, or invasive disease.
In some embodiments, the invasive disease is bloodstream infection, endocarditis or sepsis.
In some embodiments, the Staphylococcus aureus infection is methicillin-resistant S. aureus (MRSA) infection or methicillin-sensitive S. aureus (MSSA) infection. In some preferred embodiments, the infection is a recurring S. aureus infection.
In some embodiments, the live strain of S. aureus is in the form for intramuscular administration, intraperitoneal administration, subcutaneous administration, oral administration, or intranasal administration.
In some embodiments, the live strain is in a lyophilized form, which can be reconstituted before use.
In another aspect, the invention provides a kit for immunization against S. aureus infection, comprising a container containing the vaccine of the invention or the live strain of S. aureus of the invention.
In another aspect, the invention provides a method of enhancing IL-1β production and/or Th1/Th17 responses by inhibiting A2a receptor.
In another aspect, the invention provides a method to downregulate S. aureus-specific Th1/Th17 responses by inhibiting NLRP3 and/or caspase-1.
A further understanding of the present invention may be obtained by reference to the specific examples set forth herein, which are only intended to illustrate the invention, and are not intended to limit the scope of the invention. It is apparent that various modifications and variations may be made to the present invention without departing from the spirit of the invention, and such modifications and variations are therefore also within the scope of the present invention.
The Reagents and Primers as used are described in Table 1 below.
THP1 were purchased from the American Type Culture Collection (ATCC) and cultured in RPMI-1640 supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS), 100 U/ml penicillin and 0.1 mg/ml streptomycin. Before infection experiment, THP1 were differentiated into macrophages with 50 nM Phorbol 12-myristate 13-acetate (PMA) for 24 hours. After stimulation, cells were washed with 1640-RPMI medium and cultured with medium without PMA for 24 hours.
Human peripheral blood mononuclear cells (PBMC) were isolated from human buffy coat (provided by Department of Microbiology, The University of Hong Kong, Li Ka Shing Faculty of Medicine) by Ficoll-Paque gradient protocol. In brief, 30 mL of 1:1 PBS diluted buffy coat from healthy donors were layered on Ficoll-Paque Plus (GE Healthcare, Life Sciences) and centrifuged at 450×g for 30 min at room temperature. Separated layers of PBMC were collected and then washed 2 times with RPMI-1640 medium. After washing, the cells were resuspended in 4 mL red blood cell lysing buffer (Biolegend, RBC Lysis Buffer) and incubated for 5 min at room temperature. Following two subsequent washes, the cell pellet was resuspended in RPMI-1640 media supplemented with 10% FBS, 100 U/ml penicillin and 0.1 mg/ml streptomycin for further infection experiments. To differentiate human monocytes-derived macrophages (HMDM), isolated PBMC were seeded on poly-L-lysine coated coverslips in 24 well plate and cultured in RPMI-1640 media supplemented with L-glutamine, 10% FBS, 1×penicillin/streptomycin, 10 mM HEPES, 50 ng/mL hGM-CSF (PeproTech) for up to 7 days differentiation.
Bone marrow cells extracted from femur of 8-12 weeks old female BALB/c mice were culture in RPMI-1640 medium supplemented with L-glutamine, 10% heat inactivated-FBS, 1×penicillin/streptomycin, 10 mM HEPES, 50 μM-β mercaptoethanol, 20 ng/ml mGM-CSF (PeproTech) for up to 7 days differentiation.
S. aureus strains USA300 and its isogenic adsA variant were grown in Brain Heart Infusion (BHI) at 37° C. Unmarked, non-polar deletion of adsA was constructed using plasmid pKOR1 as described previously (26). Briefly, 5′- and 3′-flanking regions of adsA was PCR amplified from chromosomal DNA of S. aureus strain USA300 with primers adsA-UF (5′ CGGAATTCTGCGGCTCATGCAATGAC 3′), adsA-UR (5′ GGCACTGACATGTTCGAGACTTGCCATAATC 3′), adsA-DF (5′ AGTCTCGAACATGTCAGTGCCTAAAGGTAG 3′), adsA-DR (5′ GGGGTACCTCCCTACAGCTAAAATGG 3′) and the individual PCR products were mixed to generate an in-frame deletion pattern of adsA. The overlapping amplicon containing the in-frame deletion pattern was sub-cloned into pKOR1, to generate pKOR1-ΔadsA. The recombinant plasmid pKOR1-ΔadsA was firstly introduced into DH5a, followed by electro-transformed into S. aureus RN4220 and subsequently into USA300. The selection of allelic replacement was performed as described previously, and the deletion of adsA was further confirmed by PCR using primers adsA-UF/adsA-DR and inner primers adsA-IF (5′ TATCCATGGCCGACTAGC 3′)/adsA-IR (5′ ACCTGTTTGTGCCAATGC 3′) specific for the deleted sequence and DNA sequencing.
All animals care and experiments were performed in accordance with the Association for Assessment and Accreditation of Laboratory Animal Care guidelines (www.aaalac.org) and with approval from our institutional animal care and use committee. BALB/c mice were provided from the Laboratory Animal Unit of the University of Hong Kong. Mice were housed in specific-pathogen free facilities and 8 to 12-week old female mice were utilized for all in vitro and in vivo experiments.
One day before bacterial infection experiments, S. aureus strains were inoculated and cultured with BHI broth for overnight. Next day, overnight culture of bacteria strains were sub-cultured in fresh BHI broth at a dilution of 1:100 and grown at 37° C. Following 3 hours of culturing, S. aureus were harvested and washed for two to three times in cold PBS by centrifugation. Finally, S. aureus strains were diluted with desired volume of PBS, yielding an OD600 of 0.5 (1×108 CFU/ml), and further centrifuged and resuspended at desired bacterial concentration. The number of bacteria was determined by serial dilution and colony formation on BHI agar plates. Mammalian cells were plated in 24-well plates at a number of 4×105 per well and infected with S. aureus strains in antibiotic free medium at the indicated MOI.
Protocol for harvest and calculation of wild type and variant S. aureus strains was the same as described above. To induce systemic blood infection model of S. aureus in
Animals were sacrificed at indicated time points in re-infection model. Spleens were harvested and grinded for cells suspension. After centrifugation, splenocytes were experienced red blood cell lysing, washes and filtering, and single cells suspension was cultured in RPMI-1640 media supplemented with 10% FBS, 100 U/ml penicillin and mg/ml streptomycin. For re-stimulation, splenocytes were seeded in 24 well plates at 4×105 cells/well and stimulated with heat-killed S. aureus at a MOI of 5 for 4 days. Culture supernatants were collected for measurement of cytokines by ELISA.
After differentiation, BMDC were plated in 24-well plates at a number of 4×105 cells in each well and infected with S. aureus strains at the indicated MOI. For surface marker analysis, cells were detached with PBS containing 5 mM EDTA and were incubated in FACS buffer (PBS containing 3% FBS and 0.1% sodium azide). After incubation with purified neutralizing monoclonal antibodies against CD16:CD32 (Fc Block; Biolegend) for 15 minutes at 4° C., cells were staining with specific antibodies for 30 minutes at 4° C. in the dark. The following antibody were used for flow cytometry analysis: Anti-Mouse I-A/I-E FITC (cat. 553623; BD Biosciences), Anti-Mouse CD86 PE (cat. 553692; BD Biosciences), Anti-Mouse CD40 PE (cat. 553791; BD Biosciences). The stained cells were then analyzed using a flow cytometer (ACEA NovoCyte Quanteon) and FlowJo 10.4.0 software (TreeStar, Co).
Culture supernatants of relevant cells were collected and centrifuged at 13000 rpm for 4 min to get rid of cell debris and bacteria. Levels of LDH in culture supernatants were measured by CytoTox 96 Non-Radioactive Cytotoxicity Assay kit (Promega). Cell viability was measured by the CellTiter Glo Luminescent Cell Viability Assay (Promega). ELISA assay was conducted according manufactures' instructions.
All THP1 knock-out cell lines in this study were generated by Cas9-encoding lentiCRISPRv2 vector from Zhang Feng lab (Addgene plasmid #52961). Single guide RNAs (sgRNAs) targeting human AIM2, NLRP3, PYCARD and caspase-1 were designed utilizing online sgRNA Designer from Broad Institute. All sgRNAs were annealed and cloned into plasmid lentiCRISPRv2 according to Zhang Feng's protocol.
The Lentiviral particles were produced from HEK293T cells transfected with lentiCRISPRv2 vector, and two packaging plasmids pMD2.G and psPAX2 (Addgene plasmids #12259 and #12260) using PEI-MAX (Polysciences) and were further concentrated by ultracentrifugation. THP-1 cells were transduced by spinoculation in the presence of 8 μg/mL polybrene. A polyclonal population was selected using 1 mg/ml puromycin for at least one week. Genetic ablation was verified by Western blot analysis.
All siRNAs were designed according to previous published studies and synthesized by by GenePharma (Shanghai, China). The control siRNA (negative control) was provided by GenePharma. Sequence of siRNAs were listed in Table 1. Lipofectamine® RNAiMAX Reagent (Invitrogen) were used for transient transfection of siRNAs into BMDC. 48-72 hours after transfection, BMDC were prepared for bacterial infection experiment.
For detection of cleaved form of caspase-1, cell culture supernatants were precipitated by methanol-chloroform method. Briefly, supernatant was mixed with an equal volume of methanol and 0.25 volumes of chloroform, vortexed and centrifuge for 15 min at 20000 g. The upper phase was discarded and the interphase was mixed with methanol. After centrifugation for 5 min at 20000 g, the pellet was resuspended in 2×SDS-PAGE sample buffer and boiled for 5 min at 100° C. Protein samples were separated by 15% SDS-PAGE gels and were transferred onto PVDF membranes. Total cell lysates lysed by RIPA buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate and 0.5 mM EDTA) supplemented with 1× protease inhibitor cocktail (Roche) were boiled for 5 min at 100° C. Lysate aliquots were separated by SDS-PAGE gels and transferred onto PVDF membranes. Blots were probed with primary antibody: rabbit anti-caspase-1 (1:1000 dilution, 179515 from Abcam, USA), mouse anti-β actin (1:5000 dilution, A5316 from Sigma). Anti-rabbit or mouse antibodies conjugated to HRP were used as secondary reagents.
Total RNA was extracted from cells using TRIzol Reagent (Invitrogen) and 1 μg total RNA was used for reverse transcription (Takara) according to manufactures' instructions. The cDNA was then used for quantitative RT-PCR to analyze relevant mRNA expression using Applied Biosystems StepOnePlus™ Real-Time PCR System and SYBR Premix Ex Taq kit (Takara) according to the manufacturer's instruction. Primers for interest of genes are listed in Table 1. The data were normalized to GAPDH and fold change in gene expression was calculated by comparative CT method (2−ΔΔCT).
Data are presented as means±SD. Data from in vitro experiments were assumed to follow a normal distribution. Therefore, to compare means from two groups Unpaired Student's test was used. One-way analysis of variance (ANOVA) with Bonferroni correction was utilized to compare means among multiple groups. Data from in vitro experiments normally do not follow normal distribution. Accordingly, non-parametric Mann-Whitney U-test was used. For survival analysis, Log-rank test was used. All statistical tests were performed by GraphPad Prism 8.0 and Microsoft Excel. P value less than 0.05 was considered to be statistically significant.
To explore the role of AdsA in modulating inflammatory responses, the inventors first generated an adsA mutant strain based on USA300 background by allelic replacement (26). BALB/c mice were then infected by intravenous (i.v.) injection with 107 CFU of wild-type S. aureus USA300 or its isogenic adsA variant. The survival of the mice was monitored for 14 days. Interestingly, 70% of mice infected with wild-type USA300 survived, whereas mice infected with adsA mutant Staphylococci had all died by day 3 post infection (
There are two major biological roles of inflammasome: (i) the maturation and secretion of a potent inflammatory cytokine, IL-1β and (ii) induction of pyroptosis (16). In mice intravenous infection model, adsA mutant strain evidently improved the production of IL-1β in blood, implying that AdsA might suppress the activity of inflammasome. To examine the effect of AdsA on inflammasome, the inventors measured the viability of HMDM after infection with either S. aureus USA300 or its isogenic adsA variant. The cell viability assay showed that adsA mutant significantly triggered cell death after 8 hours post infection, whereas 70% of HMDM infected by wild type strain remained alive (
Since DC are professional antigen-presenting cell and critical mediator in initiating T lymphocytes lineage differentiation, inflammasome activation in DC could have profound influence on cellular immunity. The inventors therefore sought to delineate the detailed mechanism by which AdsA attenuates inflammasome activation in BMDC with pharmacological inhibitors and siRNA-mediated knockdown studies. Previous report demonstrated that phagocytosis linked PGN degradation is essential to NLRP3 inflammasome activation during S. aureus infection (20). To determine whether NLRP3 inflammasome is affected by AdsA, BMDC infected with wild-type or adsA mutant S. were treated with NLRP3 specific inhibitor MCC950. The results showed that IL-1β release in BMDC during S. aureus infection is primarily induced by NLRP3 inflammasome, as inhibition of NLRP3 can largely dampen IL-1β production to the level similar to caspase-1 inhibition by VX765 (
The immune modulatory characteristics of adenosine are attributed to four trans-membrane receptors: A1, A2A, A2B, and A3 (27). Activation of these receptors can induce pro-inflammatory or anti-inflammatory effect, and the abundance and distribution of four receptors varies in different cell types and tissues. Among them, A2A receptor (A2AR) is known for its anti-inflammatory trait in immune cells. Hence, the inventors try to figure out whether AdsA/adenosine/A2AR signaling is implicated in NLRP3 inflammasome in dendritic cells. The result showed that addition of ZM241385, a pharmacological inhibitor of A2AR improved IL-1β production in BMDC infected with wild type S. aureus to a level comparable to adsA mutant infection (
Increasing evidence showed that inflammasome helps to establish adaptive immunity through promoting production of danger signals or bioactive cytokines. Among them, IL-1β can manipulate extensive immune responses through IL-1R signaling by paracrine or autocrine. Since AdsA can inhibit IL-1β production in BMDC, the inventors next sought to characterize the role of AdsA in modulating function of dendritic cells, especially the cytokines environment for developing protective immunity. The inventors first evaluated the activation and maturation of BMDC under in vitro infection condition. Flow cytometry analysis showed that BMDC infected with adsA variant displayed higher expression of DC maturation markers including CD40, CD86, and major histocompatibility complex (MHC) II in comparison with wild type strain (
To assess the influence of AdsA on adaptive immunity, the inventors adopted a murine reinfection model described elsewhere (6). In this model, BALB/c mice were repeatedly infected by intraperitoneal injection with wild-type S. aureus USA300 or its isogenic adsA variant. Eventually, mice in both groups were re-challenged with a lethal or sublethal dose of wild type S. aureus USA300 (
The role of inflammasome/IL-1β/IL-1R signaling in the development of antigen specific Th17 responses is well defined (17, 28). And our in vitro experiments using BMDC implied that AdsA dampens NLRP3 inflammasome mediated IL-1β release via A2A receptor (
Staphylococcus aureus is characteristic of its capability of evading host immunity, resulting in persistent infection and recurrent infection (4). In particular, subversion from T cell responses was reported to be critical in recurrent S. aureus infection (6, 12). In this study, the inventors demonstrate that AdsA can suppress the production of proinflammatory cytokines which is important for the development of protective T cell responses. Mechanistically, this study also highlights the role of AdsA in the evasion of host protective Th17 immunity by impairing NLRP3 inflammasome mediated IL-1β release via adenosine/A2AR pathway. Our findings potentiate the understanding of host-pathogen interaction during S. aureus infection.
Being a vital intracellular sensor involved in host-pathogen interaction, inflammasome actively participates in the process of S. aureus pathogenesis (13). Mice deficient in inflammasome had decreased neutrophils recruitment, resulting in impaired bacterial clearance at the site of infection (21). It is well-established that NLRP3 inflammasome is activated in several S. aureus infection murine models. The underlying mechanisms can be divided into two aspects: (1) pore forming toxins (hemolysin, leukocidin and Panton-Valentine leukocidin) produced by S. aureus cause rupture of cellular membrane, leading to potassium efflux which is recognized as a common mechanism for NLRP3 inflammasome activation; (2) phagocytosis and lysosomal degradation of S. aureus peptidoglycan also contributes to NLRP3 inflammasome mediated IL-1β release (20, 29). In the present study, immune cells were stimulated by live S. aureus instead of bacterial culture filtrates containing large amount of PFTs and BMDC treated with MCC950 or cytochalasin D had little IL-1β production, implying that phagocytosis dependent NLRP3 activation predominate in the present in vitro infection assays. The production of IL-1β was also reported to be regulated by RIP1/RIP3/MLKL mediated necroptosis, which constrains excessive inflammasome (30). However, in present work, treatment of necroptosis inhibitor before infection, necrosulfonamide (NSA), did not have apparent effect on S. aureus induced cytotoxicity. The potential explanations may lie in different infection conditions or cell types. The inventors' work also highlights a role of adenosine signaling in AdsA mediated IL-1β inhibition, as verified by adenosine and A2AR antagonist in vitro infection assays. The enzymatic activity of AdsA is well-defined, which can facilitate the degradation of ATP, ADP and AMP to adenosine (31) or conversion of neutrophil extracellular traps (NETs) to deoxyadenosine (25). The results do not exclude the possibility that AdsA may suppress inflammasome in vivo by other mechanisms. In murine model, bacterial infection can increase extracellular ATP levels and NLRP3 inflammasome activation, thereby promoting anti-bacterial immunity (32). Given that AdsA is capable of degrading extracellular ATP, it is possible that AdsA could suppress IL-1β production by decreasing ATP levels in vivo.
Mechanistically, the inventors also provide evidence that AdsA/adenosine/A2AR axis might affect S. aureus induced IL-1β release by interfering with priming signal of NLRP3 inflammasome. It is demonstrated that AdsA or adenosine can act on A2a receptor by inhibiting NF-κB and p38 MAPK activity, both of which were contributing to NLRP3 priming signal (12, 33). In contrast to present findings, other group reported that adenosine and A2a receptor signaling could enhance NLRP3 inflammasome activation by amplifying priming signal (34). In their study, BMDM were treated with adenosine after a long period of LPS priming, which is distinct from the infection conditions in the present study, indicating a complex role of adenosine in the regulation of inflammasome at different stages of bacterial infection. Therefore, the detailed mechanism of adenosine/A2AR axis in the modulation of NLRP3 inflammasome during S. aureus infection merits further investigation.
In summary, as shown in
The foregoing description of the specific embodiments will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the relevant art (s) (including the contents of the documents cited and incorporated by reference herein), readily modify and/or adapt for various applications such specific embodiments, without undue experimentation, without departing from the general concept of the present disclosure. Such adaptations and modifications are therefore intended to be within the meaning and range of equivalents of the disclosed embodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance presented herein, in combination with the knowledge of one skilled in the relevant art (s).
While various embodiments of the present disclosure have been described above, it should be understood that they have been presented by way of examples, and not limitation. It would be apparent to one skilled in the relevant art (s) that various changes in form and detail could be made therein without departing from the spirit and scope of the disclosure. Thus, the present disclosure should not be limited by any of the above-described exemplary embodiments but should be defined only in accordance with the following claims and their equivalents.
All references cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2021/095165 | 5/21/2021 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63028710 | May 2020 | US | |
| 63123635 | Dec 2020 | US |
