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.

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 β-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.

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 β-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 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.

How to build a virulence factor: the role of three novel enzymes in the biosynthesis of the Group A Carbohydrate

The Group A carbohydrate (GAC), a bacterial surface polysaccharide, is an essential virulence factor of Streptococcus pyogenes required for growth and infection of humans.In terms of its chemical composition, this peptidoglycan-anchored polymer is mainly formed by a string of rhamnose sugars, with alternated modifications of N-acetylglucosamine and glycerolphosphate. The rhamnose polysaccharide (RhaPS) that forms the backbone chain is synthesised intracellularly by the sequential action of three rhamnosyltransferases named GacB, GacC and GacG. Importantly, deletion of any of these rhamnosyltransferases causes bacterialdeath. In this work, we used an interdisciplinary approach to demonstrate that: 1) GacB is a novel enzyme that initiates the RhaPS biosynthesis; 2) GacC catalyses the formation of a unique stem; 3) GacG elongates the RhaPS string by adding a yet unknown number of rhamnoses. Here, we also show that homologs from different streptococcal species can substitute GacB and GacC in the RhaPS production. In particular, we demonstrate that several human pathogens from the Streptococcus genus encompassed in the Lancefield serotyping scheme, and the dental pathogen Streptococcus mutans can replace S. pyogenes’ enzymes. In contrast, the homologs from S. pneumoniae sp. D39 did not, suggesting a different structural arrangement for its surface carbohydrate. Our results highlight the importance of the group carbohydrate biosynthesis pathways in the Streptococcus genus and open the door for the future development of multi-target compounds that could inhibit these enzymes in Streptococcus pyogenes and other pathogenic streptococci of clinical and veterinary importance.

Synthetic Heparan Sulfate Mimetic Pixatimod (PG545) Potently Inhibits SARS-CoV-2 By Disrupting The Spike-ACE2 interaction

SummaryHeparan sulfate (HS) is a cell surface polysaccharide recently identified as a co-receptor with the ACE2 protein for recognition of the S1 spike protein on SARS-CoV2 virus, revealing an attractive new target for therapeutic intervention. Clinically-used heparins demonstrate relevant inhibitory activity, but world supplies are limited, necessitating a synthetic solution. The HS mimetic pixatimod is synthetic drug candidate for cancer with immunomodulatory and heparanase-inhibiting properties. Here we show that pixatimod binds directly to the SARS-CoV-2 spike protein receptor binding domain (S1-RBD), altering its conformation and destabilizing its structure. Molecular modelling identified a binding site overlapping with the ACE2 receptor site. Consistent with this, pixatimod inhibits binding of S1-RBD to ACE2-expressing cells and displays a direct mechanism of action by inhibiting binding of S1-RBD to human ACE2. Assays with four different clinical isolates of live SARS-CoV-2 virus show that pixatimod potently inhibits infection of Vero cells at doses well within its safe therapeutic dose range. This demonstration of potent anti-SARS-CoV-2 activity establishes that synthetic HS mimetics can target the HS-Spike protein-ACE2 axis. Together with other known activities of pixatimod our data provides a strong rationale for its further investigation as a potential multimodal therapeutic to address the COVID-19 pandemic.

Complete Structure of the Enterococcal Polysaccharide Antigen (EPA) of Vancomycin-Resistant Enterococcus faecalis V583 Reveals that EPA Decorations Are Teichoic Acids Covalently Linked to a Rhamnopolysaccharide Backbone

ABSTRACT All enterococci produce a complex polysaccharide called the enterococcal polysaccharide antigen (EPA). This polymer is required for normal cell growth and division and for resistance to cephalosporins and plays a critical role in host-pathogen interaction. The EPA contributes to host colonization and is essential for virulence, conferring resistance to phagocytosis during the infection. Recent studies revealed that the “decorations” of the EPA polymer, encoded by genetic loci that are variable between isolates, underpin the biological activity of this surface polysaccharide. In this work, we investigated the structure of the EPA polymer produced by the high-risk enterococcal clonal complex Enterococcus faecalis V583. We analyzed purified EPA from the wild-type strain and a mutant lacking decorations and elucidated the structure of the EPA backbone and decorations. We showed that the rhamnan backbone of EPA is composed of a hexasaccharide repeat unit of C2- and C3-linked rhamnan chains, partially substituted in the C3 position by α-glucose (α-Glc) and in the C2 position by β-N-acetylglucosamine (β-GlcNAc). The so-called “EPA decorations” consist of phosphopolysaccharide chains corresponding to teichoic acids covalently bound to the rhamnan backbone. The elucidation of the complete EPA structure allowed us to propose a biosynthetic pathway, a first essential step toward the design of antimicrobials targeting the synthesis of this virulence factor. IMPORTANCE Enterococci are opportunistic pathogens responsible for hospital- and community-acquired infections. All enterococci produce a surface polysaccharide called EPA (enterococcal polysaccharide antigen) required for biofilm formation, antibiotic resistance, and pathogenesis. Despite the critical role of EPA in cell growth and division and as a major virulence factor, no information is available on its structure. Here, we report the complete structure of the EPA polymer produced by the model strain E. faecalis V583. We describe the structure of the EPA backbone, made of a rhamnan hexasaccharide substituted by Glc and GlcNAc residues, and show that teichoic acids are covalently bound to this rhamnan chain, forming the so-called “EPA decorations” essential for host colonization and pathogenesis. This report represents a key step in efforts to identify the structural properties of EPA that are essential for its biological activity and to identify novel targets to develop preventive and therapeutic approaches against enterococci.

Global control of bacterial nitrogen and carbon metabolism by a PTSNtr-regulated switch

The nitrogen-related phosphotransferase system (PTSNtr) of Rhizobium leguminosarum bv. viciae 3841 transfers phosphate from PEP via PtsP and NPr to two output regulators, ManX and PtsN. ManX controls central carbon metabolism via the tricarboxylic acid (TCA) cycle, while PtsN controls nitrogen uptake, exopolysaccharide production, and potassium homeostasis, each of which is critical for cellular adaptation and survival. Cellular nitrogen status modulates phosphorylation when glutamine, an abundant amino acid when nitrogen is available, binds to the GAF sensory domain of PtsP, preventing PtsP phosphorylation and subsequent modification of ManX and PtsN. Under nitrogen-rich, carbon-limiting conditions, unphosphorylated ManX stimulates the TCA cycle and carbon oxidation, while unphosphorylated PtsN stimulates potassium uptake. The effects are reversed with the phosphorylation of ManX and PtsN, occurring under nitrogen-limiting, carbon-rich conditions; phosphorylated PtsN triggers uptake and nitrogen metabolism, the TCA cycle and carbon oxidation are decreased, while carbon-storage polymers such as surface polysaccharide are increased. Deleting the GAF domain from PtsP makes cells “blind” to the cellular nitrogen status. PTSNtr constitutes a switch through which carbon and nitrogen metabolism are rapidly, and reversibly, regulated by protein:protein interactions. PTSNtr is widely conserved in proteobacteria, highlighting its global importance.