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.

Targeting Iron - Respiratory Reciprocity Promotes Bacterial Death.

Discovering new bacterial signaling pathways offers unique antibiotic strategies. Here, through an unbiased resistance screen of 3,884 gene knockout strains, we uncovered a previously unknown non-lytic bactericidal mechanism that sequentially couples three transporters and downstream transcription to lethally suppress respiration of the highly virulent P. aeruginosa strain PA14 - one of three species on the WHO's 'Priority 1: Critical' list. By targeting outer membrane YaiW, cationic lacritin peptide 'N-104' translocates into the periplasm where it ligates outer loops 4 and 2 of the inner membrane transporters FeoB and PotH, respectively, to suppress both ferrous iron and polyamine uptake. This broadly shuts down transcription of many biofilm-associated genes, including ferrous iron-dependent TauD and ExbB1. The mechanism is innate to the surface of the eye and is enhanced by synergistic coupling with thrombin peptide GKY20. This is the first example of an inhibitor of multiple bacterial transporters.

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.

A qnr-plasmid allows aminoglycosides to induce SOS in Escherichia coli.

The plasmid-mediated quinolone resistance (PMQR) genes have been shown to promote high-level bacterial resistance to fluoroquinolone antibiotics, potentially leading to clinical treatment failures. In Escherichia coli, sub-minimum inhibitory concentrations (sub-MICs) of the widely used fluoroquinolones are known to induce the SOS response. Interestingly, the expression of several PMQR qnr genes is controlled by the SOS master regulator, LexA. During the characterization of a small qnrD-plasmid carried in E. coli, we observed that the aminoglycosides become able to induce the SOS response in this species, thus leading to the elevated transcription of qnrD. Our findings show that the induction of the SOS response is due to nitric oxide (NO) accumulation in the presence of sub-MIC of aminoglycosides. We demonstrated that the NO accumulation is driven by two plasmid genes, ORF3 and ORF4, whose products act at two levels. ORF3 encodes a putative flavin adenine dinucleotide (FAD)-binding oxidoreductase which helps NO synthesis, while ORF4 codes for a putative fumarate and nitrate reductase (FNR)-type transcription factor, related to an O2-responsive regulator of hmp expression, able to repress the Hmp-mediated NO detoxification pathway of E. coli. Thus, this discovery, that other major classes of antibiotics may induce the SOS response could have worthwhile implications for antibiotic stewardship efforts in preventing the emergence of resistance.

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.

Immunoglobulin GM and KM allotypes are associated with antibody responses to Pseudomonas aeruginosa antigens in chronically infected cystic fibrosis patients.

BACKGROUND: Chronic infection with Pseudomonas aeruginosa (P. aeruginosa) is a leading cause of death in patients with cystic fibrosis (CF). Immunobiology of P. aeruginosa infection is complex and not well understood. Chronically infected CF patients generate high levels of antibodies to P. aeruginosa, but this response does not lead to clinical improvement. Therefore, additional studies aimed at identification and understanding of the host factors that influence naturally occurring immune responses to P. aeruginosa are needed. In this investigation, we evaluated the contribution of immunoglobulin GM (γ marker) and KM (κ marker) allotypes to the antibody responses to P. aeruginosa lipopolysaccharide (LPS) O1, O6, O11, and alginate antigens and the broadly-conserved surface polysaccharide expressed by many microbial pathogens, poly-N-acetyl-D-glucosamine (PNAG), in 58 chronically infected CF patients. METHODS: IgG1 markers GM 3 and 17 and IgG2 markers GM 23- and 23+ were determined by a pre-designed TaqMan® genotyping assay. The κ chain determinants KM 1 and 3 were characterized by PCR-RFLP. Antibodies to the LPS O antigens, alginate, and PNAG were measured by an ELISA. RESULTS: Several significant associations were noted with KM alleles. Particular KM 1/3 genotypes were individually and epistatically (with GM 3/17) associated with the level of IgG antibodies to O1, O11, alginate, and PNAG antigens. CONCLUSIONS: Immunoglobulin GM and KM genotypes influence the magnitude of humoral immunity to LPS O, alginate, and PNAG antigens. These results, if confirmed in a larger study population, will be helpful in devising novel immunotherapeutic approaches against P. aeruginosa.

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.

COVID-19 is a systemic vascular hemopathy: insight for mechanistic and clinical aspects.

Coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is presenting as a systemic disease associated with vascular inflammation and endothelial injury. Severe forms of SARS-CoV-2 infection induce acute respiratory distress syndrome (ARDS) and there is still an ongoing debate on whether COVID-19 ARDS and its perfusion defect differs from ARDS induced by other causes. Beside pro-inflammatory cytokines (such as interleukin-1 ß [IL-1ß] or IL-6), several main pathological phenomena have been seen because of endothelial cell (EC) dysfunction: hypercoagulation reflected by fibrin degradation products called D-dimers, micro- and macrothrombosis and pathological angiogenesis. Direct endothelial infection by SARS-CoV-2 is not likely to occur and ACE-2 expression by EC is a matter of debate. Indeed, endothelial damage reported in severely ill patients with COVID-19 could be more likely secondary to infection of neighboring cells and/or a consequence of inflammation. Endotheliopathy could give rise to hypercoagulation by alteration in the levels of different factors such as von Willebrand factor. Other than thrombotic events, pathological angiogenesis is among the recent findings. Overexpression of different proangiogenic factors such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF-2) or placental growth factors (PlGF) have been found in plasma or lung biopsies of COVID-19 patients. Finally, SARS-CoV-2 infection induces an emergency myelopoiesis associated to deregulated immunity and mobilization of endothelial progenitor cells, leading to features of acquired hematological malignancies or cardiovascular disease, which are discussed in this review. Altogether, this review will try to elucidate the pathophysiology of thrombotic complications, pathological angiogenesis and EC dysfunction, allowing better insight in new targets and antithrombotic protocols to better address vascular system dysfunction. Since treating SARS-CoV-2 infection and its potential long-term effects involves targeting the vascular compartment and/or mobilization of immature immune cells, we propose to define COVID-19 and its complications as a systemic vascular acquired hemopathy.

Pseudomonas aeruginosa-induced nociceptor activation increases susceptibility to infection.

We report a rapid reduction in blink reflexes during in vivo ocular Pseudomonas aeruginosa infection, which is commonly attributed and indicative of functional neuronal damage. Sensory neurons derived in vitro from trigeminal ganglia (TG) were able to directly respond to P. aeruginosa but reacted significantly less to strains of P. aeruginosa that lacked virulence factors such as pili, flagella, or a type III secretion system. These observations led us to explore the impact of neurons on the host's susceptibility to P. aeruginosa keratitis. Mice were treated with Resiniferatoxin (RTX), a potent activator of Transient Receptor Potential Vanilloid 1 (TRPV1) channels, which significantly ablated corneal sensory neurons, exhibited delayed disease progression that was exemplified with decreased bacterial corneal burdens and altered neutrophil trafficking. Sensitization to disease was due to the increased frequencies of CGRP-induced ICAM-1+ neutrophils in the infected corneas and reduced neutrophil bactericidal activities. These data showed that sensory neurons regulate corneal neutrophil responses in a tissue-specific matter affecting disease progression during P. aeruginosa keratitis. Hence, therapeutic modalities that control nociception could beneficially impact anti-infective therapy.

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.

Glycomics Microarrays Reveal Differential In Situ Presentation of the Biofilm Polysaccharide Poly-N-acetylglucosamine on Acinetobacter baumannii and Staphylococcus aureus Cell Surfaces.

The biofilm component poly-N-acetylglucosamine (PNAG) is an important virulence determinant in medical-device-related infections caused by ESKAPE group pathogens including Gram-positive Staphylococcus aureus and Gram-negative Acinetobacter baumannii. PNAG presentation on bacterial cell surfaces and its accessibility for host interactions are not fully understood. We employed a lectin microarray to examine PNAG surface presentation and interactions on methicillin-sensitive (MSSA) and methicillin-resistant S. aureus (MRSA) and a clinical A. baumannii isolate. Purified PNAG bound to wheatgerm agglutinin (WGA) and succinylated WGA (sWGA) lectins only. PNAG was the main accessible surface component on MSSA but was relatively inaccessible on the A. baumannii surface, where it modulated the presentation of other surface molecules. Carbohydrate microarrays demonstrated similar specificities of S. aureus and A. baumannii for their most intensely binding carbohydrates, including 3' and 6'sialyllactose, but differences in moderately binding ligands, including blood groups A and B. An N-acetylglucosamine-binding lectin function which binds to PNAG identified on the A. baumannii cell surface may contribute to biofilm structure and PNAG surface presentation on A. baumannii. Overall, these data indicated differences in PNAG presentation and accessibility for interactions on Gram-positive and Gram-negative cell surfaces which may play an important role in biofilm-mediated pathogenesis.

Broadly protective semi-synthetic glycoconjugate vaccine against pathogens capable of producing poly-ß-(1→6)-N-acetyl-d-glucosamine exopolysaccharide.

Poly-ß-(1→6)-N-acetylglucosamine (PNAG) was first discovered as a major component of biofilms formed by Staphylococcus aureus and some other staphylococci but later this exopolysaccharide was also found to be produced by pathogens of various nature. This common antigen is considered as a promising target for construction of a broadly protective vaccine. Extensive studies of PNAG, its de-N-acetylated derivative (dPNAG, containing around 15% of residual N-acetates) and their conjugates with Tetanus Toxoid (TT) revealed the crucial role of de-N-acetylated glucosamine units for the induction of protective immunity. Conjugates of synthetic penta- (5GlcNH2) and nona-ß-(1→6)-d-glucosamines (9GlcNH2) were tested in vitro and in different animal models and proved to be effective in passive and active protection against different microbial pathogens. Presently conjugate 5GlcNH2-TT is being produced under GMP conditions and undergoes safety and effectiveness evaluation in humans and economically important animals. Current review summarizes all stages of this long-termed study.

Antibody recognition of bacterial surfaces and extracellular polysaccharides.

Because of the ongoing increase in antibiotic-resistant microbes, new strategies such as therapeutic antibodies and effective vaccines are required. Bacterial carbohydrates are known to be particularly antigenic, and several monoclonal antibodies that target bacterial polysaccharides have been generated, with more in current development. This review examines the known 3D crystal structures of anti-bacterial antibodies and the structural basis for carbohydrate recognition and explores the potential mechanisms for antibody-dependent bacterial cell death. Understanding the key interactions between an antibody and its polysaccharide target on the surface of bacteria or in biofilms can provide essential information for the development of more specific and effective antibody therapeutics as well as carbohydrate-based vaccines.

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.