Poly-ß-(1→6)-N-acetyl-D-glucosamine mediates surface attachment, biofilm formation, and biocide resistance in Cutibacterium acnes.

Background: The commensal skin bacterium Cutibacterium acnes plays a role in the pathogenesis of acne vulgaris and also causes opportunistic infections of implanted medical devices due to its ability to form biofilms on biomaterial surfaces. Poly-ß-(1→6)-N-acetyl-D-glucosamine (PNAG) is an extracellular polysaccharide that mediates biofilm formation and biocide resistance in a wide range of bacterial pathogens. The objective of this study was to determine whether C. acnes produces PNAG, and whether PNAG contributes to C. acnes biofilm formation and biocide resistance in vitro. Methods: PNAG was detected on the surface of C. acnes cells by fluorescence confocal microscopy using the antigen-specific human IgG1 monoclonal antibody F598. PNAG was detected in C. acnes biofilms by measuring the ability of the PNAG-specific glycosidase dispersin B to inhibit biofilm formation and sensitize biofilms to biocide killing. Results: Monoclonal antibody F598 bound to the surface of C. acnes cells. Dispersin B inhibited attachment of C. acnes cells to polystyrene rods, inhibited biofilm formation by C. acnes in glass and polypropylene tubes, and sensitized C. acnes biofilms to killing by benzoyl peroxide and tetracycline. Conclusion: C. acnes produces PNAG, and PNAG contributes to C. acnes biofilm formation and biocide resistance in vitro. PNAG may play a role in C. acnes skin colonization, biocide resistance, and virulence in vivo.

A comprehensive synthetic library of poly-N-acetyl glucosamines enabled vaccine against lethal challenges of Staphylococcus aureus.

Poly-ß-(1-6)-N-acetylglucosamine (PNAG) is an important vaccine target, expressed on many pathogens. A critical hurdle in developing PNAG based vaccine is that the impacts of the number and the position of free amine vs N-acetylation on its antigenicity are not well understood. In this work, a divergent strategy is developed to synthesize a comprehensive library of 32 PNAG pentasaccharides. This library enables the identification of PNAG sequences with specific patterns of free amines as epitopes for vaccines against Staphylococcus aureus (S. aureus), an important human pathogen. Active vaccination with the conjugate of discovered PNAG epitope with mutant bacteriophage Qß as a vaccine carrier as well as passive vaccination with diluted rabbit antisera provides mice with near complete protection against infections by S. aureus including methicillin-resistant S. aureus (MRSA). Thus, the comprehensive PNAG pentasaccharide library is an exciting tool to empower the design of next generation vaccines.

A high-throughput sequencing approach identifies immunotherapeutic targets for bacterial meningitis in neonates.

BACKGROUND: Worldwide, Escherichia coli is the leading cause of neonatal Gram-negative bacterial meningitis, but full understanding of the pathogenesis of this disease is not yet achieved. Moreover, to date, no vaccine is available against bacterial neonatal meningitis. METHODS: Here, we used Transposon Sequencing of saturated banks of mutants (TnSeq) to evaluate E. coli K1 genetic fitness in murine neonatal meningitis. We identified E. coli K1 genes encoding for factors important for systemic dissemination and brain infection, and focused on products with a likely outer-membrane or extra-cellular localization, as these are potential vaccine candidates. We used in vitro and in vivo models to study the efficacy of active and passive immunization. RESULTS: We selected for further study the conserved surface polysaccharide Poly-ß-(1-6)-N-Acetyl Glucosamine (PNAG), as a strong candidate for vaccine development. We found that PNAG was a virulence factor in our animal model. We showed that both passive and active immunization successfully prevented and/or treated meningitis caused by E. coli K1 in neonatal mice. We found an excellent opsonophagocytic killing activity of the antibodies to PNAG and in vitro these antibodies were also able to decrease binding, invasion and crossing of E. coli K1 through two blood brain barrier cell lines. Finally, to reinforce the potential of PNAG as a vaccine candidate in bacterial neonatal meningitis, we demonstrated that Group B Streptococcus, the main cause of neonatal meningitis in developed countries, also produced PNAG and that antibodies to PNAG could protect in vitro and in vivo against this major neonatal pathogen. INTERPRETATION: Altogether, these results indicate the utility of a high-throughput DNA sequencing method to identify potential immunotherapy targets for a pathogen, including in this study a potential broad-spectrum target for prevention of neonatal bacterial infections. FUNDINGS: ANR Seq-N-Vaq, Charles Hood Foundation, Hearst Foundation, and Groupe Pasteur Mutualité.

Experimental Urethral Infection with Neisseria gonorrhoeae.

Gonorrhea rates and antibiotic resistance are both increasing. Neisseria gonorrhoeae (Ng) is an exclusively human pathogen and is exquisitely adapted to its natural host. Ng can subvert immune responses and undergoes frequent antigenic variation, resulting in limited immunity and protection from reinfection. Previous gonococcal vaccine efforts have been largely unsuccessful, and the last vaccine to be tested in humans was more than 35 years ago. Advancing technologies and the threat of untreatable gonorrhea have fueled renewed pursuit of a vaccine as a long-term sustainable solution for gonorrhea control. Despite the development of a female mouse model of genital gonococcal infection two decades ago, correlates of immunity or protection remain largely unknown, making the gonococcus a challenging vaccine target. The controlled human urethral infection model of gonorrhea (Ng CHIM) has been used to study gonococcal pathogenesis and the basis of anti-gonococcal immunity. Over 200 participants have been inoculated without serious adverse events. The Ng CHIM replicates the early natural course of urethral infection. We are now at an inflexion point to pivot the use of the model for vaccine testing to address the urgency of improved gonorrhea control. Herein we discuss the need for gonorrhea vaccines, and the advantages and limitations of the Ng CHIM in accelerating the development of gonorrhea vaccines.

Immunization against a Conserved Surface Polysaccharide Stimulates Bovine Antibodies with Opsonic Killing Activity but Does Not Protect against Babesia bovis Challenge.

Arthropod-borne apicomplexan pathogens remain a great concern and challenge for disease control in animals and humans. In order to prevent Babesia infection, the discovery of antigens that elicit protective immunity is essential to establish approaches to stop disease dissemination. In this study, we determined that poly-N-acetylglucosamine (PNAG) is conserved among tick-borne pathogens including B. bovis, B. bigemina, B. divergens, B. microti, and Babesia WA1. Calves immunized with synthetic ß-(1→6)-linked glucosamine oligosaccharides conjugated to tetanus toxoid (5GlcNH2-TT) developed antibodies with in vitro opsonophagocytic activity against Staphylococcus aureus. Sera from immunized calves reacted to B. bovis. These results suggest strong immune responses against PNAG. However, 5GlcNH2-TT-immunized bovines challenged with B. bovis developed acute babesiosis with the cytoadhesion of infected erythrocytes to brain capillary vessels. While this antigen elicited antibodies that did not prevent disease, we are continuing to explore other antigens that may mitigate these vector-borne diseases for the cattle industry.

Randomized, controlled trial comparing Rhodococcus equi and poly-N-acetyl glucosamine hyperimmune plasma to prevent R equi pneumonia in foals.

BACKGROUND: Hyperimmune plasma raised against ß-1→6-poly-N-acetyl glucosamine (PNAG HIP) mediates more opsonophagocytic killing of Rhodococcus equi (R equi) than does R equi hyperimmune plasma (RE HIP) in vitro. The relative efficacy of PNAG HIP and RE HIP to protect foals against R equi pneumonia, however, has not been evaluated. HYPOTHESIS: Transfusion with PNAG HIP will be superior to RE HIP in foals for protection against R equi pneumonia in a randomized, controlled, blinded clinical trial. ANIMALS: Four hundred sixty Quarter Horse and Thoroughbred foals at 5 large breeding farms in the United States. METHODS: A randomized, controlled, blinded clinical trial was conducted in which foals were transfused within 24 hours after birth with 2 L of either RE HIP or PNAG HIP. Study foals were monitored through weaning for clinical signs of pneumonia by farm veterinarians. The primary outcome was the proportion of foals that developed pneumonia after receiving each type of plasma. RESULTS: The proportion of foals that developed pneumonia was the same between foals transfused with RE HIP (14%; 32/228) and PNAG HIP (14%; 30/215). CONCLUSIONS AND CLINICAL IMPORTANCE: Results indicate that PNAG HIP was not superior to a commercially available, United States Department of Agriculture-licensed RE HIP product for protecting foals against R equi pneumonia under field conditions.

Antibody activities in hyperimmune plasma against the Rhodococcus equi virulence -associated protein A or poly-N-acetyl glucosamine are associated with protection of foals against rhodococcal pneumonia.

The efficacy of transfusion with hyperimmune plasma (HIP) for preventing pneumonia caused by Rhodococcus equi remains ill-defined. Quarter Horse foals at 2 large breeding farms were randomly assigned to be transfused with 2 L of HIP from adult donors hyperimmunized either with R. equi (RE HIP) or a conjugate vaccine eliciting antibody to the surface polysaccharide ß-1→6-poly-N-acetyl glucosamine (PNAG HIP) within 24 hours of birth. Antibody activities against PNAG and the rhodococcal virulence-associated protein A (VapA), and to deposition of complement component 1q (C՛1q) onto PNAG were determined by ELISA, and then associated with either clinical pneumonia at Farm A (n = 119) or subclinical pneumonia at Farm B (n = 114). Data were analyzed using multivariable logistic regression. Among RE HIP-transfused foals, the odds of pneumonia were approximately 6-fold higher (P = 0.0005) among foals with VapA antibody activity ≤ the population median. Among PNAG HIP-transfused foals, the odds of pneumonia were approximately 3-fold (P = 0.0347) and 11-fold (P = 0.0034) higher for foals with antibody activities ≤ the population median for PNAG or C՛1q deposition, respectively. Results indicated that levels of activity of antibodies against R. equi antigens are correlates of protection against both subclinical and clinical R. equi pneumonia in field settings. Among PNAG HIP-transfused foals, activity of antibodies with C՛1q deposition (an indicator of functional antibodies) were a stronger predictor of protection than was PNAG antibody activity alone. Collectively, these findings suggest that the amount and activity of antibodies in HIP (i.e., plasma volume and/or antibody activity) is positively associated with protection against R. equi pneumonia in foals.

Serum Antibody Activity against Poly-N-Acetyl Glucosamine (PNAG), but Not PNAG Vaccination Status, Is Associated with Protecting Newborn Foals against Intrabronchial Infection with Rhodococcus equi.

Rhodococcus equi is a prevalent cause of pneumonia in foals worldwide. Our laboratory has demonstrated that vaccination against the surface polysaccharide ß-1→6-poly-N-acetylglucosamine (PNAG) protects foals against intrabronchial infection with R. equi when challenged at age 28 days. However, it is important that the efficacy of this vaccine be evaluated in foals when they are infected at an earlier age, because foals are naturally exposed to virulent R. equi in their environment from birth and because susceptibility is inversely related to age in foals. Using a randomized, blind experimental design, we evaluated whether maternal vaccination against PNAG protected foals against intrabronchial infection with R. equi 6 days after birth. Vaccination of mares per se did not significantly reduce the incidence of pneumonia in foals; however, activities of antibody against PNAG or for deposition of complement component 1q onto PNAG was significantly (P < 0.05) higher among foals that did not develop pneumonia than among foals that developed pneumonia. Results differed between years, with evidence of protection during 2018 but not 2020. In the absence of a licensed vaccine, further evaluation of the PNAG vaccine is warranted, including efforts to optimize the formulation and dose of this vaccine. IMPORTANCE Pneumonia caused by R. equi is an important cause of disease and death in foals worldwide for which a licensed vaccine is lacking. Foals are exposed to R. equi in their environment from birth, and they appear to be infected soon after parturition at an age when innate and adaptive immune responses are diminished. Results of this study indicate that higher activity of antibodies recognizing PNAG was associated with protection against R. equi pneumonia, indicating the need for further optimization of maternal vaccination against PNAG to protect foals against R. equi pneumonia.

Vaccination of yearling horses against poly-N-acetyl glucosamine fails to protect against infection with Streptococcus equi subspecies equi.

Strangles is a common disease of horses with worldwide distribution caused by the bacterium Streptococcus equi subspecies equi (SEE). Although vaccines against strangles are available commercially, these products have limitations in safety and efficacy. The microbial surface antigen ß 1→6 poly-N-acetylglucosamine (PNAG) is expressed by SEE. Here we show that intramuscular (IM) injection alone or a combination of IM plus intranasal (IN) immunization generated antibodies to PNAG that functioned to deposit complement and mediate opsonophagocytic killing of SEE ex vivo. However, immunization strategies targeting PNAG either by either IM only injection or a combination of IM and IN immunizations failed to protect yearling horses against infection following contact with infected horses in an experimental setting. We speculate that a protective vaccine against strangles will require additional components, such as those targeting SEE enzymes that degrade or inactivate equine IgG.

A Conserved Streptococcal Virulence Regulator Controls the Expression of a Distinct Class of M-Like Proteins.

Streptococcus equi subspecies zooepidemicus (SEZ) are group C streptococci that are important pathogens of economically valuable animals such as horses and pigs. Here, we found that many SEZ isolates bind to a monoclonal antibody that recognizes poly-N-acetylglucosamine (PNAG), a polymer that is found as a surface capsule-like structure on diverse microbes. A fluorescence-activated cell sorting-based transposon insertion sequencing (Tn-seq) screen, coupled with whole-genome sequencing, was used to search for genes for PNAG biosynthesis. Surprisingly, mutations in a gene encoding an M-like protein, szM, and the adjacent transcription factor, designated sezV, rendered strains PNAG negative. SezV was required for szM expression and transcriptome analysis showed that SezV has a small regulon. SEZ strains with inactivating mutations in either sezV or szM were highly attenuated in a mouse model of infection. Comparative genomic analyses revealed that linked sezV and szM homologues are present in all SEZ, S. equi subspecies equi (SEE), and M18 group A streptococcal (GAS) genomes in the database, but not in other streptococci. The antibody to PNAG bound to a wide range of SEZ, SEE, and M18 GAS strains. Immunochemical studies suggest that the SzM protein may be decorated with a PNAG-like oligosaccharide although an intact oligosaccharide substituent could not be isolated. Collectively, our findings suggest that the szM and sezV loci define a subtype of virulent streptococci and that an antibody to PNAG may have therapeutic applications in animal and human diseases caused by streptococci bearing SzM-like proteins.IMPORTANCE M proteins are surface-anchored virulence factors in group A streptococci, human pathogens. Here, we identified an M-like protein, SzM, and its positive regulator, SezV, in Streptococcus equi subspecies zooepidemicus (SEZ), an important group of pathogens for domesticated animals, including horses and pigs. SzM and SezV homologues were found in the genomes of all SEZ and S. equi subspecies equi and M18 group A streptococcal strains analyzed but not in other streptococci. Mutant SEZ strains lacking either sezV or szM were highly attenuated in a mouse model of infection. Collectively, our findings suggest that SezV-related regulators and the linked SzM family of M-like proteins define a new subset of virulent streptococci.

Immunization against poly-N-acetylglucosamine reduces neutrophil activation and GVHD while sparing microbial diversity.

Microbial invasion into the intestinal mucosa after allogeneic hematopoietic cell transplantation (allo-HCT) triggers neutrophil activation and requires antibiotic interventions to prevent sepsis. However, antibiotics lead to a loss of microbiota diversity, which is connected to a higher incidence of acute graft-versus-host disease (aGVHD). Antimicrobial therapies that eliminate invading bacteria and reduce neutrophil-mediated damage without reducing the diversity of the microbiota are therefore highly desirable. A potential solution would be the use of antimicrobial antibodies that target invading pathogens, ultimately leading to their elimination by innate immune cells. In a mouse model of aGVHD, we investigated the potency of active and passive immunization against the conserved microbial surface polysaccharide poly-N-acetylglucosamine (PNAG) that is expressed on numerous pathogens. Treatment with monoclonal or polyclonal antibodies to PNAG (anti-PNAG) or vaccination against PNAG reduced aGVHD-related mortality. Anti-PNAG treatment did not change the intestinal microbial diversity as determined by 16S ribosomal DNA sequencing. Anti-PNAG treatment reduced myeloperoxidase activation and proliferation of neutrophil granulocytes (neutrophils) in the ileum of mice developing GVHD. In vitro, anti-PNAG treatment showed high antimicrobial activity. The functional role of neutrophils was confirmed by using neutrophil-deficient LysMcreMcl1fl/fl mice that had no survival advantage under anti-PNAG treatment. In summary, the control of invading bacteria by anti-PNAG treatment could be a novel approach to reduce the uncontrolled neutrophil activation that promotes early GVHD and opens a new avenue to interfere with aGVHD without affecting commensal intestinal microbial diversity.

In vitro evaluation of complement deposition and opsonophagocytic killing of Rhodococcus equi mediated by poly-N-acetyl glucosamine hyperimmune plasma compared to commercial plasma products.

BACKGROUND: The bacterium Rhodococcus equi can cause severe pneumonia in foals. The absence of a licensed vaccine and limited effectiveness of commercial R. equi hyperimmune plasma (RE-HIP) create a great need for improved prevention of this disease. HYPOTHESIS: Plasma hyperimmune to the capsular polysaccharide poly-N-acetyl glucosamine (PNAG) would be significantly more effective than RE-HIP at mediating complement deposition and opsonophagocytic killing (OPK) of R. equi. ANIMALS: Venipuncture was performed on 9 Quarter Horses. METHODS: The ability of the following plasma sources to mediate complement component 1 (C1) deposition onto either PNAG or R. equi was determined by ELISA: (1) PNAG hyperimmune plasma (PNAG-HIP), (2) RE-HIP, and (3) standard non-hyperimmune commercial plasma (SP). For OPK, each plasma type was combined with R. equi, equine complement, and neutrophils isolated from horses (n = 9); after 4 hours, the number of R. equi in each well was determined by quantitative culture. Data were analyzed using linear mixed-effects regression with significance set at P < .05. RESULTS: The PNAG-HIP and RE-HIP were able to deposit significantly (P < .05) more complement onto their respective targets than the other plasmas. The mean proportional survival of R. equi opsonized with PNAG-HIP was significantly (P < .05) less (14.7%) than that for SP (51.1%) or RE-HIP (42.2%). CONCLUSIONS AND CLINICAL IMPORTANCE: Plasma hyperimmune to PNAG is superior to RE-HIP for opsonizing and killing R. equi in vitro. Comparison of these 2 plasmas in field trials is warranted because of the reported incomplete effectiveness of RE-HIP.

PolyGlcNAc-containing exopolymers enable surface penetration by non-motile Enterococcus faecalis.

Bacterial pathogens have evolved strategies that enable them to invade tissues and spread within the host. Enterococcus faecalis is a leading cause of local and disseminated multidrug-resistant hospital infections, but the molecular mechanisms used by this non-motile bacterium to penetrate surfaces and translocate through tissues remain largely unexplored. Here we present experimental evidence indicating that E. faecalis generates exopolysaccharides containing ß-1,6-linked poly-N-acetylglucosamine (polyGlcNAc) as a mechanism to successfully penetrate semisolid surfaces and translocate through human epithelial cell monolayers. Genetic screening and molecular analyses of mutant strains identified glnA, rpiA and epaX as genes critically required for optimal E. faecalis penetration and translocation. Mechanistically, GlnA and RpiA cooperated to generate uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) that was utilized by EpaX to synthesize polyGlcNAc-containing polymers. Notably, exogenous supplementation with polymeric N-acetylglucosamine (PNAG) restored surface penetration by E. faecalis mutants devoid of EpaX. Our study uncovers an unexpected mechanism whereby the RpiA-GlnA-EpaX metabolic axis enables production of polyGlcNAc-containing polysaccharides that endow E. faecalis with the ability to penetrate surfaces. Hence, targeting carbohydrate metabolism or inhibiting biosynthesis of polyGlcNAc-containing exopolymers may represent a new strategy to more effectively confront enterococcal infections in the clinic.

PNAG-specific equine IgG1 mediates significantly greater opsonization and killing of Prescottella equi (formerly Rhodococcus equi) than does IgG4/7.

Prescottella equi (formerly Rhodococcus equi) is a facultative intracellular bacterial pathogen that causes severe pneumonia in foals 1-6 months of age, whereas adult horses are highly resistant to infection. We have shown that vaccinating pregnant mares against the conserved surface polysaccharide capsule, ß-1 → 6-linked poly-N-acetyl glucosamine (PNAG), elicits opsonic killing antibody that transfers via colostrum to foals and protects them against experimental infection with virulent. R. equi. We hypothesized that equine IgG1 might be more important than IgG4/7 for mediating protection against R. equi infection in foals. To test this hypothesis, we compared complement component 1 (C1) deposition and polymorphonuclear cell-mediated opsonophagocytic killing (OPK) mediated by IgG1 or IgG4/7 enriched from either PNAG hyperimmune plasma (HIP) or standard plasma. Subclasses IgG1 and IgG4/7 from PNAG HIP and standard plasma were precipitated onto a diethylaminoethyl ion exchange column, then further enriched using a protein G Sepharose column. We determined C1 deposition by enzyme-linked immunosorbent assay (ELISA) and estimated OPK by quantitative microbiologic culture. Anti-PNAG IgG1 deposited significantly (P < 0.05) more C1 onto PNAG than did IgG4/7 from PNAG HIP or subclasses IgG1 and IgG4/7 from standard plasma. In addition, IgG1 from PNAG HIP mediated significantly (P < 0.05) greater OPK than IgG4/7 from PNAG HIP or IgG1 and IgG4/7 from standard plasma. Our findings indicate that anti-PNAG IgG1 is a correlate of protection against R. equi in foals, which has important implications for understanding the immunopathogenesis of R. equi pneumonia, and as a tool for assessing vaccine efficacy and effectiveness when challenge is not feasible.

Antibody to Poly-N-acetyl glucosamine provides protection against intracellular pathogens: Mechanism of action and validation in horse foals challenged with Rhodococcus equi.

Immune correlates of protection against intracellular bacterial pathogens are largely thought to be cell-mediated, although a reasonable amount of data supports a role for antibody-mediated protection. To define a role for antibody-mediated immunity against an intracellular pathogen, Rhodococcus equi, that causes granulomatous pneumonia in horse foals, we devised and tested an experimental system relying solely on antibody-mediated protection against this host-specific etiologic agent. Immunity was induced by vaccinating pregnant mares 6 and 3 weeks prior to predicted parturition with a conjugate vaccine targeting the highly conserved microbial surface polysaccharide, poly-N-acetyl glucosamine (PNAG). We ascertained antibody was transferred to foals via colostrum, the only means for foals to acquire maternal antibody. Horses lack transplacental antibody transfer. Next, a randomized, controlled, blinded challenge was conducted by inoculating at ~4 weeks of age ~10(6) cfu of R. equi via intrabronchial challenge. Eleven of 12 (91%) foals born to immune mares did not develop clinical R. equi pneumonia, whereas 6 of 7 (86%) foals born to unvaccinated controls developed pneumonia (P = 0.0017). In a confirmatory passive immunization study, infusion of PNAG-hyperimmune plasma protected 100% of 5 foals against R. equi pneumonia whereas all 4 recipients of normal horse plasma developed clinical disease (P = 0.0079). Antibodies to PNAG mediated killing of extracellular and intracellular R. equi and other intracellular pathogens. Killing of intracellular organisms depended on antibody recognition of surface expression of PNAG on infected cells, along with complement deposition and PMN-assisted lysis of infected macrophages. Peripheral blood mononuclear cells from immune and protected foals released higher levels of interferon-γ in response to PNAG compared to controls, indicating vaccination also induced an antibody-dependent cellular release of this critical immune cytokine. Overall, antibody-mediated opsonic killing and interferon-γ release in response to PNAG may protect against diseases caused by intracellular bacterial pathogens.

Immunization with outer membrane vesicles displaying conserved surface polysaccharide antigen elicits broadly antimicrobial antibodies.

Many microbial pathogens produce a ß-(1→6)-linked poly-N-acetyl-d-glucosamine (PNAG) surface capsule, including bacterial, fungal, and protozoan cells. Broadly protective immune responses to this single conserved polysaccharide antigen in animals are possible but only when a deacetylated poly-N-acetyl-d-glucosamine (dPNAG; <30% acetate) glycoform is administered as a conjugate to a carrier protein. Unfortunately, conventional methods for natural extraction or chemical synthesis of dPNAG and its subsequent conjugation to protein carriers can be technically demanding and expensive. Here, we describe an alternative strategy for creating broadly protective vaccine candidates that involved coordinating recombinant poly-N-acetyl-d-glucosamine (rPNAG) biosynthesis with outer membrane vesicle (OMV) formation in laboratory strains of Escherichia coli The glycosylated outer membrane vesicles (glycOMVs) released by these engineered bacteria were decorated with the PNAG glycopolymer and induced high titers of PNAG-specific IgG antibodies after immunization in mice. When a Staphylococcus aureus enzyme responsible for PNAG deacetylation was additionally expressed in these cells, glycOMVs were generated that elicited antibodies to both highly acetylated PNAG (∼95-100% acetate) and a chemically deacetylated dPNAG derivative (∼15% acetate). These antibodies mediated efficient in vitro killing of two distinct PNAG-positive bacterial species, namely S. aureus and Francisella tularensis subsp. holarctica, and mice immunized with PNAG-containing glycOMVs developed protective immunity against these unrelated pathogens. Collectively, our results reveal the potential of glycOMVs for targeting this conserved polysaccharide antigen and engendering protective immunity against the broad range of pathogens that produce surface PNAG.

Structural basis for antibody targeting of the broadly expressed microbial polysaccharide poly-N-acetylglucosamine.

In response to the widespread emergence of antibiotic-resistant microbes, new therapeutic agents are required for many human pathogens. A non-mammalian polysaccharide, poly-N-acetyl-d-glucosamine (PNAG), is produced by bacteria, fungi, and protozoan parasites. Antibodies that bind to PNAG and its deacetylated form (dPNAG) exhibit promising in vitro and in vivo activities against many microbes. A human IgG1 mAb (F598) that binds both PNAG and dPNAG has opsonic and protective activities against multiple microbial pathogens and is undergoing preclinical and clinical assessments as a broad-spectrum antimicrobial therapy. Here, to understand how F598 targets PNAG, we determined crystal structures of the unliganded F598 antigen-binding fragment (Fab) and its complexes with N-acetyl-d-glucosamine (GlcNAc) and a PNAG oligosaccharide. We found that F598 recognizes PNAG through a large groove-shaped binding site that traverses the entire light- and heavy-chain interface and accommodates at least five GlcNAc residues. The Fab-GlcNAc complex revealed a deep binding pocket in which the monosaccharide and a core GlcNAc of the oligosaccharide were almost identically positioned, suggesting an anchored binding mechanism of PNAG by F598. The Fab used in our structural analyses retained binding to PNAG on the surface of an antibiotic-resistant, biofilm-forming strain of Staphylococcus aureus Additionally, a model of intact F598 binding to two pentasaccharide epitopes indicates that the Fab arms can span at least 40 GlcNAc residues on an extended PNAG chain. Our findings unravel the structural basis for F598 binding to PNAG on microbial surfaces and biofilms.

The exceptionally broad-based potential of active and passive vaccination targeting the conserved microbial surface polysaccharide PNAG.

A challenging component of vaccine development is the large serologic diversity of protective antigens. Remarkably, there is a conserved surface/capsular polysaccharide, one of the most effective vaccine targets, expressed by a large number of bacterial, fungal and eukaryotic pathogens: poly-N-acetyl glucosamine (PNAG). Natural antibodies to PNAG are poorly effective at mediating in vitro microbial killing or in vivo protection. Removing most of the acetate substituents to produce a deacetylated glycoform, or using synthetic oligosaccharides of poly-ß-1-6-linked glucosamine conjugated to carrier proteins, results in vaccines that elicit high levels of broad-based immunity. A fully human monoclonal antibody is highly active in laboratory and preclinical studies and has been successfully tested in a phase-I setting. Both the synthetic oligosaccharide conjugate vaccine and MAb will be further tested in humans starting in 2016; but, even if effective against only a fraction of the PNAG-producing pathogens, a major advance in vaccine-preventable diseases will occur.

Extended-spectrum antibodies protective against carbapenemase-producing Enterobacteriaceae.

BACKGROUND: Carbapenem-resistant Enterobacteriaceae (CRE) are responsible for worldwide outbreaks and antibiotic treatments are problematic. The polysaccharide poly-(ß-1,6)-N-acetyl glucosamine (PNAG) is a vaccine target detected on the surface of numerous pathogenic bacteria, including Escherichia coli. Genes encoding PNAG biosynthetic proteins have been identified in two other main pathogenic Enterobacteriaceae, Enterobacter cloacae and Klebsiella pneumoniae. We hypothesized that antibodies to PNAG might be a new therapeutic option for the different pan-resistant pathogenic species of CRE. METHODS: PNAG production was detected by confocal microscopy and its role in the formation of the biofilm (for E. cloacae) and as a virulence factor (for K. pneumoniae) was analysed. The in vitro (opsonophagocytosis killing assay) and in vivo (mouse models of peritonitis) activity of antibodies to PNAG were studied using antibiotic-susceptible and -resistant E. coli, E. cloacae and K. pneumoniae. A PNAG-producing strain of Pseudomonas aeruginosa, an organism that does not naturally produce this antigen, was constructed by adding the pga locus to a strain with inactive alg genes responsible for the production of P. aeruginosa alginate. Antibodies to PNAG were tested in vitro and in vivo as above. RESULTS: PNAG is a major component of the E. cloacae biofilm and a virulence factor for K. pneumoniae. Antibodies to PNAG mediated in vitro killing (>50%) and significantly protected mice against the New Delhi metallo-ß-lactamase-producing E. coli (P = 0.02), E. cloacae (P = 0.0196) and K. pneumoniae (P = 0.006), against K. pneumoniae carbapenemase (KPC)-producing K. pneumoniae (P = 0.02) and against PNAG-producing P. aeruginosa (P = 0.0013). Thus, regardless of the Gram-negative bacterial species, PNAG expression is the sole determinant of the protective efficacy of antibodies to this antigen. CONCLUSIONS: Our findings suggest antibodies to PNAG may provide extended-spectrum antibacterial protective activity.

Emergence of Antimicrobial-Resistant Escherichia coli of Animal Origin Spreading in Humans.

In the context of the great concern about the impact of human activities on the environment, we studied 403 commensal Escherichia coli/Escherichia clade strains isolated from several animal and human populations that have variable contacts to one another. Multilocus sequence typing (MLST) showed a decrease of diversity 1) in strains isolated from animals that had an increasing contact with humans and 2) in all strains that had increased antimicrobial resistance. A specific B1 phylogroup clonal complex (CC87, Institut Pasteur schema nomenclature) of animal origin was identified and characterized as being responsible for the increased antimicrobial resistance prevalence observed in strains from the environments with a high human-mediated antimicrobial pressure. CC87 strains have a high capacity of acquiring and disseminating resistance genes with specific metabolic and genetic determinants as demonstrated by high-throughput sequencing and phenotyping. They are good mouse gut colonizers but are not virulent. Our data confirm the predominant role of human activities in the emergence of antimicrobial resistance in the environmental bacterial strains and unveil a particular E. coli clonal complex of animal origin capable of spreading antimicrobial resistance to other members of microbial communities.