Development and validation of multiplex assays for mouse and human IgG and IgA to Neisseria gonorrhoeae antigens.

There is an urgent need for vaccines against Neisseria gonorrhoeae (Ng), the causative agent of gonorrhea. Vaccination with an outer-membrane vesicle (OMV)-based Neisseria meningitidis (Nm) vaccine provides some protection from Ng; however, the mechanisms underlying this cross-protection are unknown. To address this need, we developed multiplexed bead-based assays for the relative quantification of human and mouse IgG and IgA against Ng antigens. The assays were evaluated for analyte independence, dilutional linearity, specificity, sensitivity, intra- and inter-assay variability, and robustness to sample storage conditions. The assay was then used to test samples from mice and humans immunized with an Nm-OMV vaccine.

Evaluating vaccine-elicited antibody activities against Neisseria gonorrhoeae: cross-protective responses elicited by the 4CMenB meningococcal vaccine.

The bacterial pathogen Neisseria gonorrhoeae is an urgent global health problem due to increasing numbers of infections, coupled with rampant antibiotic resistance. Vaccines against gonorrhea are being prioritized to combat drug-resistant N. gonorrhoeae. Meningococcal serogroup B vaccines such as four-component meningococcal B vaccine (4CMenB) are predicted by epidemiology studies to cross-protect individuals from natural infection with N. gonorrhoeae and elicit antibodies that cross-react with N. gonorrhoeae. Evaluation of vaccine candidates for gonorrhea requires a suite of assays for predicting efficacy in vitro and in animal models of infection, including the role of antibodies elicited by immunization. Here, we present the development and optimization of assays to evaluate antibody functionality after immunization of mice: antibody binding to intact N. gonorrhoeae, serum bactericidal activity, and opsonophagocytic killing activity using primary human neutrophils [polymorphonuclear leukocytes (PMNs)]. These assays were developed with purified antibodies against N. gonorrhoeae and used to evaluate serum from mice that were vaccinated with 4CMenB or given alum as a negative control. Results from these assays will help prioritize gonorrhea vaccine candidates for advanced preclinical to early clinical studies and will contribute to identifying correlates and mechanisms of immune protection against N. gonorrhoeae.

Evaluating vaccine-elicited antibody activities against Neisseria gonorrhoeae: cross-protective responses elicited by the 4CMenB meningococcal vaccine.

The bacterial pathogen Neisseria gonorrhoeae is an urgent global health problem due to increasing numbers of infections, coupled with rampant antibiotic resistance. Vaccines against gonorrhea are being prioritized to combat drug-resistant N. gonorrhoeae. Meningococcal serogroup B vaccines such as 4CMenB are predicted by epidemiology studies to cross-protect individuals from natural infection with N. gonorrhoeae and elicit antibodies that cross-react with N. gonorrhoeae. Evaluation of vaccine candidates for gonorrhea requires a suite of assays for predicting efficacy in vitro and in animal models of infection, including the role of antibodies elicited by immunization. Here we present assays to evaluate antibody functionality after immunization: antibody binding to intact N. gonorrhoeae, serum bactericidal activity, and opsonophagocytic killing activity using primary human neutrophils (polymorphonuclear leukocytes). These assays were developed with purified antibodies against N. gonorrhoeae and used to evaluate serum from mice that were vaccinated with 4CMenB or given alum as a negative control. Results from these assays will help prioritize gonorrhea vaccine candidates for advanced preclinical to early clinical study and will contribute to identifying correlates and mechanisms of immune protection against N. gonorrhoeae .

A Novel Oral GyrB/ParE Dual Binding Inhibitor Effective against Multidrug-Resistant Neisseria gonorrhoeae and Other High-Threat Pathogens.

Drug-resistant Neisseria gonorrhoeae is a serious global health concern. New drugs are needed that can overcome existing drug resistance and limit the development of new resistances. Here, we describe the small molecule tricyclic pyrimidoindole JSF-2414 [8-(6-fluoro-8-(methylamino)-2-((2-methylpyrimidin-5-yl)oxy)-9H-pyrimido[4,5-b]indol-4-yl)-2-oxa-8-azaspiro[4.5]decan-3-yl)methanol], which was developed to target both ATP-binding regions of DNA gyrase (GyrB) and topoisomerase (ParE). JSF-2414 displays potent activity against N. gonorrhoeae, including drug-resistant strains. A phosphate pro-drug, JSF-2659, was developed to facilitate oral dosing. In two different animal models of Neisseria gonorrhoeae vaginal infection, JSF-2659 was highly efficacious in reducing microbial burdens to the limit of detection. The parent molecule also showed potent in vitro activity against high-threat Gram-positive organisms, and JSF-2659 was shown in a deep tissue model of vancomycin-resistant Staphylococcus aureus (VRSA) and a model of Clostridioides difficile-induced colitis to be highly efficacious and protective. JSF-2659 is a novel preclinical drug candidate against high-threat multidrug resistant organisms with low potential to develop new resistance.

Meningococcal Detoxified Outer Membrane Vesicle Vaccines Enhance Gonococcal Clearance in a Murine Infection Model.

BACKGROUND: Despite decades of research efforts, development of a gonorrhea vaccine has remained elusive. Epidemiological studies suggest that detoxified outer membrane vesicle (dOMV) vaccines from Neisseria meningitidis (Nm) may protect against infection with Neisseria gonorrhoeae (Ng). We recently reported that Nm dOMVs lacking the major outer membrane proteins (OMPs) PorA, PorB, and RmpM induced greater antibody cross-reactivity against heterologous Nm strains than wild-type (WT) dOMVs and may represent an improved vaccine against gonorrhea. METHODS: We prepared dOMV vaccines from meningococcal strains that were sufficient or deleted for PorA, PorB, and RmpM. Vaccines were tested in a murine genital tract infection model and antisera were used to identify vaccine targets. RESULTS: Immunization with Nm dOMVs significantly and reproducibly enhanced gonococcal clearance for mice immunized with OMP-deficient dOMVs; significant clearance for WT dOMV-immunized mice was observed in one of two experiments. Clearance was associated with serum and vaginal anti-Nm dOMV immunoglobulin G (IgG) antibodies that cross-reacted with Ng. Serum IgG was used to identify putative Ng vaccine targets, including PilQ, MtrE, NlpD, and GuaB. CONCLUSIONS: Meningococcal dOMVs elicited a protective effect against experimental gonococcal infection. Recognition and identification of Ng vaccine targets by Nm dOMV-induced antibodies supports the development of a cross-protective Neisseria vaccine.

Preclinical Testing of Vaccines and Therapeutics for Gonorrhea in Female Mouse Models of Lower and Upper Reproductive Tract Infection.

Murine models of Neisseria gonorrhoeae lower reproductive tract infection are valuable systems for studying N. gonorrhoeae adaptation to the female host and immune responses to infection. These models have also accelerated preclinical testing of candidate therapeutic and prophylactic products against gonorrhea. However, because N. gonorrhoeae infection is restricted to the murine cervicovaginal region, there is a need for an in vivo system for translational work on N. gonorrhoeae pelvic inflammatory disease (PID). Here we discuss the need for well-characterized preclinical upper reproductive tract infection models for developing candidate products against N. gonorrhoeae PID, and report a refinement of the gonorrhea mouse model that supports sustained upper reproductive tract infection. To establish this new model for vaccine testing, we also tested the licensed meningococcal 4CMenB vaccine, which cross-protects against murine N. gonorrhoeae lower reproductive tract infection, for efficacy against N. gonorrhoeae in the endometrium and oviducts following transcervical or vaginal challenge.

trans-Translation inhibitors bind to a novel site on the ribosome and clear Neisseria gonorrhoeae in vivo.

Bacterial ribosome rescue pathways that remove ribosomes stalled on mRNAs during translation have been proposed as novel antibiotic targets because they are essential in bacteria and are not conserved in humans. We previously reported the discovery of a family of acylaminooxadiazoles that selectively inhibit trans-translation, the main ribosome rescue pathway in bacteria. Here, we report optimization of the pharmacokinetic and antibiotic properties of the acylaminooxadiazoles, producing MBX-4132, which clears multiple-drug resistant Neisseria gonorrhoeae infection in mice after a single oral dose. Single particle cryogenic-EM studies of non-stop ribosomes show that acylaminooxadiazoles bind to a unique site near the peptidyl-transfer center and significantly alter the conformation of ribosomal protein bL27, suggesting a novel mechanism for specific inhibition of trans-translation by these molecules. These results show that trans-translation is a viable therapeutic target and reveal a new conformation within the bacterial ribosome that may be critical for ribosome rescue pathways.

Molecular Features of Cephalosporins Important for Activity against Antimicrobial-Resistant Neisseria gonorrhoeae.

The increasing prevalence of Neisseria gonorrhoeae strains exhibiting decreased susceptibility to extended-spectrum cephalosporins (ESCs) presents a challenge for the successful treatment of gonorrhea infections. To address this challenge, we evaluated a panel of 23 cephalosporins against penicillin-binding protein 2 (PBP2) from the ESC-resistant (ESCR) N. gonorrhoeae strain H041 to determine which molecular features are important for antimicrobial activity. Structure-activity relationships (SARs) developed from acylation rate constants against PBP2 and antimicrobial susceptibilities against the H041 strain of N. gonorrhoeae, and interpreted against docking models, reveal that cephalosporins possessing large, lipophilic R1 side chains and electronegative R2 side chains with planar groups are associated with higher acylation rates against PBP2, but also that these same amphipathic features can lower antimicrobial activity. Based on these studies, we tested cefoperazone, one of the most effective ESCs for targeting PBP2, in the female mouse model infected with H041 and showed that it was equally or more effective than ceftriaxone or gentamicin for clearing infections. Taken together, our results reveal that two U.S. Food and Drug Administration (FDA)-approved agents (cefoperazone, ceftaroline) and one FDA-qualified infectious disease product (ceftobiprole) have potential as first-line treatments for gonorrhea and provide a framework for the future design of cephalosporins with improved activity against ESC-resistant N. gonorrhoeae.

The serogroup B meningococcal outer membrane vesicle-based vaccine 4CMenB induces cross-species protection against Neisseria gonorrhoeae.

There is a pressing need for a gonorrhea vaccine due to the high disease burden associated with gonococcal infections globally and the rapid evolution of antibiotic resistance in Neisseria gonorrhoeae (Ng). Current gonorrhea vaccine research is in the stages of antigen discovery and the identification of protective immune responses, and no vaccine has been tested in clinical trials in over 30 years. Recently, however, it was reported in a retrospective case-control study that vaccination of humans with a serogroup B Neisseria meningitidis (Nm) outer membrane vesicle (OMV) vaccine (MeNZB) was associated with reduced rates of gonorrhea. Here we directly tested the hypothesis that Nm OMVs induce cross-protection against gonorrhea in a well-characterized female mouse model of Ng genital tract infection. We found that immunization with the licensed Nm OMV-based vaccine 4CMenB (Bexsero) significantly accelerated clearance and reduced the Ng bacterial burden compared to administration of alum or PBS. Serum IgG and vaginal IgA and IgG that cross-reacted with Ng OMVs were induced by 4CMenB vaccination by either the subcutaneous or intraperitoneal routes. Antibodies from vaccinated mice recognized several Ng surface proteins, including PilQ, BamA, MtrE, NHBA (known to be recognized by humans), PorB, and Opa. Immune sera from both mice and humans recognized Ng PilQ and several proteins of similar apparent molecular weight, but MtrE was only recognized by mouse serum. Pooled sera from 4CMenB-immunized mice showed a 4-fold increase in serum bactericidal50 titers against the challenge strain; in contrast, no significant difference in bactericidal activity was detected when sera from 4CMenB-immunized and unimmunized subjects were compared. Our findings directly support epidemiological evidence that Nm OMVs confer cross-species protection against gonorrhea, and implicate several Ng surface antigens as potentially protective targets. Additionally, this study further defines the usefulness of murine infection model as a relevant experimental system for gonorrhea vaccine development.

Deletion of major porins from meningococcal outer membrane vesicle vaccines enhances reactivity against heterologous serogroup B Neisseria meningitidis strains.

Detergent-extracted detoxified outer membrane vesicle (dOMV) vaccines are effective at preventing invasive serogroup B meningococcal (MenB) disease caused by the homologous Neisseria meningitidis strain from which they are produced, but offer limited protection from heterologous strains. Differences in vaccine efficacy are partially due to strain-specific variations in the antigenic sequence types and expression levels of outer membrane proteins (OMPs), including the immunodominant OMP PorA. In this study, dOMV vaccines deficient in major OMPs, including PorA, PorB, and RmpM were isolated and used to immunize rabbits and mice. Serum samples were obtained from each animal and tested for antibody responses against five MenB strains. Immunization with wild type dOMVs elicited antibodies to major antigens including PorA, PorB, RmpM, and lipooligosaccharide (LOS), and demonstrated limited bactericidal activity against heterologous strains. In contrast, OMP-deficient dOMV vaccines elicited broadly cross-reactive bactericidal antibodies, with PorA/PorB-dual deficient dOMVs inducing antibodies exhibiting the greatest cross-reactivity. Enhanced killing of heterologous strains correlated with binding to unique protein bands in immunoblots, suggestive of improved immunogenicity of antigens under-represented in the wild type vaccine.

Could Dampening Expression of the Neisseria gonorrhoeae mtrCDE-Encoded Efflux Pump Be a Strategy To Preserve Currently or Resurrect Formerly Used Antibiotics To Treat Gonorrhea?

Neisseria gonorrhoeae has developed resistance to every antibiotic introduced for treatment of gonorrhea since 1938, and concern now exists that gonorrheal infections may become refractory to all available antibiotics approved for therapy. The current recommended dual antibiotic treatment regimen of ceftriaxone (CRO) and azithromycin (AZM) is threatened with the emergence of gonococcal strains displaying resistance to one or both of these antibiotics. Non-beta-lactamase resistance to penicillin and third-generation cephalosporins, as well as low-level AZM resistance expressed by gonococci, requires overexpression of the mtrCDE-encoded efflux pump, which in wild-type (WT) strains is subject to transcriptional repression by MtrR. Since earlier studies showed that loss of MtrCDE renders gonococci hypersusceptible to beta-lactams and macrolides, we hypothesized that transcriptional dampening of mtrCDE would render an otherwise resistant strain susceptible to these antibiotics as assessed by antibiotic susceptibility testing and during experimental infection. In order to test this hypothesis, we ectopically expressed a WT copy of the mtrR gene, which encodes the repressor of the mtrCDE efflux pump operon, in N. gonorrhoeae strain H041, the first reported gonococcal strain to cause a third-generation-cephalosporin-resistant infection. We now report that MtrR production can repress the expression of mtrCDE, increase antimicrobial susceptibility in vitro, and enhance beta-lactam efficacy in eliminating gonococci as assessed in a female mouse model of lower genital tract infection. We propose that strategies that target the MtrCDE efflux pump should be considered to counteract the increasing problem of antibiotic-resistant gonococci.IMPORTANCE The emergence of gonococcal strains resistant to past or currently used antibiotics is a global public health concern, given the estimated 78 million infections that occur annually. The dearth of new antibiotics to treat gonorrhea demands that alternative curative strategies be considered to counteract antibiotic resistance expressed by gonococci. Herein, we show that decreased expression of a drug efflux pump that participates in gonococcal resistance to antibiotics can increase gonococcal susceptibility to beta-lactams and macrolides under laboratory conditions, as well as improve antibiotic-mediated clearance of gonococci from the genital tract of experimentally infected female mice.

Pharmacokinetic Data Are Predictive of In Vivo Efficacy for Cefixime and Ceftriaxone against Susceptible and Resistant Neisseria gonorrhoeae Strains in the Gonorrhea Mouse Model.

There is a pressing need for drug development for gonorrhea. Here we describe a pharmacokinetic (PK)/pharmacodynamic (PD) analysis of extended-spectrum cephalosporins (ESC) against drug-susceptible and drug-resistant gonococcal strains in a murine genital tract infection model. The PK determined in uninfected mice displayed a clear dose-response in plasma levels following single doses of ceftriaxone (CRO) (intraperitoneal) or cefixime (CFM) (oral). The observed doses required for efficacy against ESC-susceptible (ESCs) strain FA1090 were 5 mg/kg of body weight (CRO) and 12 mg/kg (CFM); these doses had estimated therapeutic times (the time that the free drug concentration remains above the MIC [fTMIC]) of 24 h and 37 h, respectively. No single dose of CRO or CFM was effective against ESC-resistant (ESCr) strain H041. However, fractionation (three times a day every 8 h [TIDq8h]) of a 120-mg/kg dose of CRO resulted in estimated therapeutic times in the range of 23 h and cleared H041 infection in a majority (90%) of mice, comparable to the findings for gentamicin. In contrast, multiple CFM doses of 120 or 300 mg/kg administered TIDq8h cleared infection in ≤50% of mice, with the therapeutic times estimated from single-dose PK data being 13 and 27 h, respectively. This study reveals a clear relationship between plasma ESC levels and bacterial clearance rates in the gonorrhea mouse model. The PK/PD relationships observed in mice reflected those observed in humans, with in vivo efficacy against an ESCs strain requiring doses that yielded an fTMIC in excess of 20 to 24 h. PK data also accurately predicted the failure of single doses of ESCs against an ESCr strain and were useful in designing effective dosing regimens.

Aminomethyl Spectinomycins as Therapeutics for Drug-Resistant Gonorrhea and Chlamydia Coinfections.

Bacterial sexually transmitted infections are widespread and common, with Neisseria gonorrhoeae (gonorrhea) and Chlamydia trachomatis (chlamydia) being the two most frequent causes. If left untreated, both infections can cause pelvic inflammatory disease, infertility, ectopic pregnancy, and other sequelae. The recommended treatment for gonorrhea is ceftriaxone plus azithromycin (to empirically treat chlamydial coinfections). Antibiotic resistance to all existing therapies has developed in gonorrheal infections. The need for new antibiotics is great, but the pipeline for new drugs is alarmingly small. The aminomethyl spectinomycins, a new class of semisynthetic analogs of the antibiotic spectinomycin, were developed on the basis of a computational analysis of the spectinomycin binding site of the bacterial 30S ribosome and structure-guided synthesis. The compounds display particular potency against common respiratory tract pathogens as well as the sexually transmitted pathogens that cause gonorrhea and chlamydia. Here, we demonstrate the in vitro potencies of several compounds of this class against both bacterial species; the compounds displayed increased potencies against N. gonorrhoeae compared to that of spectinomycin and, significantly, demonstrated activity against C. trachomatis that is not observed with spectinomycin. Efficacies of the compounds were compared to those of spectinomycin and gentamicin in a murine model of infection caused by ceftriaxone/azithromycin-resistant N. gonorrhoeae; the aminomethyl spectinomycins significantly reduced the colonization load and were as potent as the comparator compounds. In summary, data produced by this study support aminomethyl spectinomycins as a promising replacement for spectinomycin and antibiotics such as ceftriaxone for treating drug-resistant gonorrhea, with the added benefit of treating chlamydial coinfections.

In Vitro Susceptibility of Neisseria gonorrhoeae Strains to Mupirocin, an Antibiotic Reformulated for Parenteral Administration in Nanoliposomes.

Neisseria gonorrhoeae is an urgent antibiotic-resistant threat. This study determined the MICs of mupirocin to be 0.0039 to 0.0625 µg/ml for 94 N. gonorrhoeae strains. Cross-resistance with other antibiotics was not detected. Mupirocin, which is currently limited to topical administration, demonstrated activity by injection when delivered in nanoliposomes. The nanoliposomal formulation of mupirocin is a potential treatment for drug-resistant N. gonorrhoeae.

Antibacterial activity of resazurin-based compounds against Neisseria gonorrhoeae in vitro and in vivo.

Neisseria gonorrhoeae is the cause of the second most common sexually transmitted bacterial infection, with ca. 80 million new cases of gonorrhoea reported annually. The recent emergence of clinical isolates resistant to the last monotherapy against this bacterium, the cephalosporins, illustrates the need for new antigonococcal agents. Here we have characterised a new group of antimicrobials based on the compound resazurin that exhibits robust activity against N. gonorrhoeae in vitro. Resazurin inhibits the growth of a broad range of N. gonorrhoeae isolates, including those resistant to multiple antibiotics. Furthermore, treatment of human endometrial cells infected with N. gonorrhoeae with resazurin significantly reduces the number of intracellular bacteria. Whilst resazurin exhibited potent in vitro antimicrobial activity, in vivo resazurin did not limit the colonisation of mice with N. gonorrhoeae following vaginal infection. The ineffectiveness of resazurin in vivo is likely due to its interaction with serum albumin, which completely diminishes its antimicrobial activity. However, treatment of mice with a resazurin analogue (resorufin pentyl ether) that maintains its antimicrobial activity in the presence of serum albumin approached a significant decrease in the percentage of mice vaginally colonised. This treatment also decreased vaginal colonisation by N. gonorrhoeae over time. Together, these data suggest that resazurin derivatives have potential for the treatment of gonorrhoea.

Detection of group A Streptococcus in tonsils from pediatric patients reveals high rate of asymptomatic streptococcal carriage.

BACKGROUND: Group A Streptococcus (GAS) causes acute tonsillopharyngitis in children, and approximately 20% of this population are chronic carriers of GAS. Antibacterial therapy has previously been shown to be insufficient at clearing GAS carriage. Bacterial biofilms are a surface-attached bacterial community that is encased in a matrix of extracellular polymeric substances. Biofilms have been shown to provide a protective niche against the immune response and antibiotic treatments, and are often associated with recurrent or chronic bacterial infections. The objective of this study was to test the hypothesis that GAS is present within tonsil tissue at the time of tonsillectomy. METHODS: Blinded immunofluorescent and histological methods were employed to evaluate palatine tonsils from children undergoing routine tonsillectomy for adenotonsillar hypertrophy or recurrent GAS tonsillopharyngitis. RESULTS: Immunofluorescence analysis using anti-GAS antibody was positive in 11/30 (37%) children who had tonsillectomy for adenotonsillar hypertrophy and in 10/30 (33%) children who had tonsillectomy for recurrent GAS pharyngitis. Fluorescent microscopy with anti-GAS and anti-cytokeratin 8 & 18 antibodies revealed GAS was localized to the tonsillar reticulated crypts. Scanning electron microscopy identified 3-dimensional communities of cocci similar in size and morphology to GAS. The characteristics of these communities are similar to GAS biofilms from in vivo animal models. CONCLUSION: Our study revealed the presence of GAS within the tonsillar reticulated crypts of approximately one-third of children who underwent tonsillectomy for either adenotonsillar hypertrophy or recurrent GAS tonsillopharyngitis at the Wake Forest School of Medicine. TRIAL REGISTRATION: The tissue collected was normally discarded tissue and no patient identifiers were collected. Thus, no subjects were formally enrolled.

Srv mediated dispersal of streptococcal biofilms through SpeB is observed in CovRS+ strains.

Group A Streptococcus (GAS) is a human specific pathogen capable of causing both mild infections and severe invasive disease. We and others have shown that GAS is able to form biofilms during infection. That is to say, they form a three-dimensional, surface attached structure consisting of bacteria and a multi-component extracellular matrix. The mechanisms involved in regulation and dispersal of these GAS structures are still unclear. Recently we have reported that in the absence of the transcriptional regulator Srv in the MGAS5005 background, the cysteine protease SpeB is constitutively produced, leading to increased tissue damage and decreased biofilm formation during a subcutaneous infection in a mouse model. This was interesting because MGAS5005 has a naturally occurring mutation that inactivates the sensor kinase domain of the two component regulatory system CovRS. Others have previously shown that strains lacking covS are associated with decreased SpeB production due to CovR repression of speB expression. Thus, our results suggest the inactivation of srv can bypass CovR repression and lead to constitutive SpeB production. We hypothesized that Srv control of SpeB production may be a mechanism to regulate biofilm dispersal and provide a mechanism by which mild infection can transition to severe disease through biofilm dispersal. The question remained however, is this mechanism conserved among GAS strains or restricted to the unique genetic makeup of MGAS5005. Here we show that Srv mediated control of SpeB and biofilm dispersal is conserved in the invasive clinical isolates RGAS053 (serotype M1) and MGAS315 (serotype M3), both of which have covS intact. This work provides additional evidence that Srv regulated control of SpeB may mediate biofilm formation and dispersal in diverse strain backgrounds.

Dispersal of Group A streptococcal biofilms by the cysteine protease SpeB leads to increased disease severity in a murine model.

Group A Streptococcus (GAS) is a Gram-positive human pathogen best known for causing pharyngeal and mild skin infections. However, in the 1980's there was an increase in severe GAS infections including cellulitis and deeper tissue infections like necrotizing fasciitis. Particularly striking about this elevation in the incidence of severe disease was that those most often affected were previously healthy individuals. Several groups have shown that changes in gene content or regulation, as with proteases, may contribute to severe disease; yet strains harboring these proteases continue to cause mild disease as well. We and others have shown that group A streptococci (MGAS5005) reside within biofilms both in vitro and in vivo. That is to say that the organism colonizes a host surface and forms a 3-dimensional community encased in a protective matrix of extracellular protein, DNA and polysaccharide(s). However, the mechanism of assembly or dispersal of these structures is unclear, as is the relationship of these structures to disease outcome. Recently we reported that allelic replacement of the streptococcal regulator srv resulted in constitutive production of the streptococcal cysteine protease SpeB. We further showed that the constitutive production of SpeB significantly decreased MGAS5005Δsrv biofilm formation in vitro. Here we show that mice infected with MGAS5005Δsrv had significantly larger lesion development than wild-type infected animals. Histopathology, Gram-staining and immunofluorescence link the increased lesion development with lack of disease containment, lack of biofilm formation, and readily detectable levels of SpeB in the tissue. Treatment of MGAS5005Δsrv infected lesions with a chemical inhibitor of SpeB significantly reduced lesion formation and disease spread to wild-type levels. Furthermore, inactivation of speB in the MGAS5005Δsrv background reduced lesion formation to wild-type levels. Taken together, these data suggest a mechanism by which GAS disease may transition from mild to severe through the Srv mediated dispersal of GAS biofilms.

Loss of the group A Streptococcus regulator Srv decreases biofilm formation in vivo in an otitis media model of infection.

Group A Streptococcus (GAS) is a common causative agent of pharyngitis, but the role of GAS in otitis media is underappreciated. In this study, we sought to test the hypothesis that GAS colonizes the middle ear and establishes itself in localized, three-dimensional communities representative of biofilms. To test this hypothesis, the middle ears of chinchillas were infected with either a strain of GAS capable of forming biofilms in vitro (MGAS5005) or a strain deficient in biofilm formation due to the lack of the transcriptional regulator Srv (MGAS5005 Δsrv). Infection resulted in the formation of large, macroscopic structures within the middle ears of MGAS5005- and MGAS5005 Δsrv-infected animals. Plate counts, scanning electron microscopy, LIVE/DEAD staining, and Gram staining revealed a difference in the distributions of MGAS5005 versus MGAS5005 Δsrv in the infected samples. High numbers of CFU of MGAS5005 Δsrv were isolated from the middle ear effusion, and MGAS5005 Δsrv was found randomly distributed throughout the excised macroscopic structure. In contrast, MGAS5005 was found in densely packed microcolonies indicative of biofilms within the excised material from the middle ear. CFU levels of MGAS5005 from the effusion were significantly lower than that of MGAS5005 Δsrv early during the course of infection. Allelic replacement of the chromosomally encoded streptococcal cysteine protease (speB) in the MGAS5005 Δsrv background restored biofilm formation in vivo. Interestingly, our results suggest that GAS naturally forms a biofilm during otitis media but that biofilm formation is not required to establish infection following transbullar inoculation of chinchillas.