poly-N-acetylglucosamine Research Articles

N-acyl homoserine lactones lactonase est816 suppresses biofilm formation and periodontitis in rats mediated by Aggregatibacter actinomycetemcomitans

ABSTRACT Aims The current study aimed to explore the adjuvant therapeutic effect of N-acyl homoserine lactones (AHLs)-lactonase est816 on Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) biological behaviors and periodontitis progression. Methods The inhibitory properties of est816 were detected by live/dead bacterial staining, scanning electron microscope (SEM), crystal-violet staining and reverse-transcription quantitative PCR (RT-qPCR). Biocompatibility of est816 on human gingival fibroblasts (HGFs) and human gingival epithelial cells (HGEs) was evaluated by CCK8 and ELISA. The ligature-induced periodontitis model was established in rats. Micro computed tomography and immunohistochemical and histological staining served to evaluate the effect of est816 on the prevention of periodontitis in vivo. Results est816 significantly attenuated biofilm formation, reduced the mRNA expression of cytolethal distending toxin, leukotoxin and poly-N-acetyl glucosamine (PNAG) and downregulated expressions of interleukin-6 and tumor necrosis factor-α with low cell toxicity. In vivo investigations revealed est816 decreased alveolar bone resorption, suppressed matrix metalloproteinase-9 expression and increased osteoprotegerin expression. Conclusion est816 inhibited A. actinomycetemcomitans biofilm formation and virulence release, resulting in anti-inflammation and soothing of periodontitis in rats, indicating that est816 could be investigated in further research on periodontal diseases.

New Molecular Mechanism of Superbiofilm Elaboration in a Staphylococcus aureus Clinical Strain.

Previously, we reported a novel regulator of biofilm (rob) with a nonsense mutation in the superbiofilm-elaborating strain JP080. Intriguingly, the complementation of JP080 with wild-type rob did not completely abolish its superbiofilm-elaborating phenotype. Therefore, we searched for other possible mutation(s) using complete genome sequence data and found a missense mutation in the gene icaR, which altered its 35th amino acid (Ala35Thr). To further study the mechanism of superbiofilm elaboration in JP080, we reconstructed the same mutations of rob and icaR in the strain FK300 and analyzed the phenotypes. The mutation of rob (A331T) increased biofilm elaboration, as previously demonstrated; similarly, an icaR mutation increased poly-N-acetylglucosamine and biofilm production in strain FK300. Furthermore, our analyses indicated that the double mutant of rob and icaR produced significantly more biofilms than the single mutants. Additionally, gel shift analysis revealed that the icaR from JP080 lost its ability to bind to the ica promoter region. These findings suggest that the icaR mutation in JP080 may result in a nonfunctional protein. We compared ica operon expression in an icaR single mutant, rob single mutant, and rob and icaR double mutant to the wild type. The rob and icaR mutants showed increased ica operon transcription by approximately 19- and 79-fold, respectively. However, the rob and icaR double mutant showed an approximately 350-fold increase, indicating the synergistic effects of icaR and rob on JP080 biofilm elaboration. Consequently, we concluded that the double mutations rob and icaR synergistically increased ica operon transcription, resulting in a superbiofilm phenotype in Staphylococcus aureus. IMPORTANCE Poly-N-acetylglucosamine (PNAG) is a major component of S. aureus biofilm. PNAG production is mediated by the products of four genes, icaADBC encoded in the ica operon, and the major negative regulator of this operon is IcaR encoded just upstream of icaADBC. Previously, we reported another negative regulator, Rob, through gene expression analysis of clinically isolated superbiofilm-elaborating strain JP080. The rob gene is encoded at different loci distant from the ica operon. Here, we report that JP080 also carried a mutation in icaR and demonstrated that IcaR and Rob synergistically regulate PNAG production. We successfully reconstructed these mutations in a wild type, and the double mutant resulted in superbiofilm-elaborating phenotype. We clearly show that loss of function of both IcaR and Rob is the very reason that JP080 is showing the superbiofilm-elaborating phenotype. This study clearly demonstrated there are at least two independent regulators synergistically fine-tuning PNAG production and suggested the complex regulatory mechanism of biofilm production.

Non-deacetylated poly-N-acetylglucosamine-hyperproducing Staphylococcus aureus undergoes immediate autoaggregation upon vortexing.

Biofilms are microbial communities of cells embedded in a matrix of extracellular polymeric substances generated and adhering to each other or to a surface. Cell aggregates formed in the absence of a surface and floating pellicles that form biofilms at the air-liquid interface are also considered to be a type of biofilm. Staphylococcus aureus is a well-known cause of biofilm infections and high-molecular-weight polysaccharides, poly-N-acetylglucosamine (PNAG) is a main constituent of the biofilm. An icaADBC operon comprises major machinery to synthesize and extracellularly secrete PNAG. Extracellular PNAG is partially deacetylated by IcaB deacetylase, and the positively charged PNAG hence interacts with negatively charged cell surface to form the major component of biofilm. We previously reported a new regulator of biofilm (Rob) and demonstrated that Rob binds to a unique 5-bp motif, TATTT, present in intergenic region between icaADBC operon and its repressor gene icaR in Yu et al. The deletion of the 5-bp motif induces excessive adherent biofilm formation. The real function of the 5-bp motif is still unknown. In an attempt to isolate the 5-bp motif deletion mutant, we isolated several non-adherent mutants. They grew normally in turbid broth shaking culture but immediately auto-aggregated upon weak vortexing and sedimented as a lump resulting in a clear supernatant. Whole genome sequencing of the mutants identified they all carried mutations in icaB in addition to deletion of the 5-bp motif. Purification and molecular characterization of auto-aggregating factor in the culture supernatant of the mutant identified that the factor was a massively produced non-deacetylated PNAG. Therefore, we created a double deficient strain of biofilm inhibitory factors (5-bp motif, icaR, rob) and icaB to confirm the aggregation phenomenon. This peculiar phenomenon was only observed in Δ5bpΔicaB double mutant but not in ΔicaR ΔicaB or ΔrobΔicaB mutant. This study explains large amount of extracellularly produced non-deacetylated PNAG by Δ5bpΔicaB double mutation induced rapid auto-aggregation of S. aureus cells by vortexing. This phenomenon indicated that Staphylococcus aureus may form biofilms that do not adhere to solid surfaces and we propose this as a new mechanism of non-adherent biofilm formation of S. aureus.

Exopolysaccharide from Lactobacillus casei NA-2 attenuates Escherichia coli O157:H7 surface adhesion via modulation of membrane surface properties and adhesion-related gene expression

The natural compound, exopolysaccharide from Lactobacillus casei NA-2 (EPS-cn2), has been shown to inhibit biofilm formation by Escherichia coli O157:H7. Although bacterial adhesion to substrate surfaces is a primary, indispensable step in this process, the mechanisms by which EPS-cn2 can block E. coli O157:H7 adhesion to biotic or abiotic surfaces remain unclear. In this study, investigation of E. coli O157:H7 response to EPS-cn2 revealed that 1 mg/mL EPS-cn2 can decrease adherence to polystyrene and confluent Caco-2 cell surfaces to 49.0% (P＜0.0001) and 57.0% (P＜0.01) of that in untreated E. coli O157:H7, respectively. Moreover, EPS-cn2 significantly reduced outer membrane hydrophobicity by 49.0% and decreased the electronegativity of the membrane surface charge by as much as 1.57 mV (P＜0.05) compared to untreated cells. High throughput RNA sequencing indicated that genes responsible for adhesion through extracellular matrix secretion, such as poly-N-acetyl-glucosamine (PNAG) biosynthesis, locus of enterocyte effacement (LEE) proteins and outer membrane protein (OmpT) were all down-regulated in response to EPS-cn2, while chemotaxis and motility-related flagellar assembly genes were differentially up-regulated, suggesting that the EPS-cn2 may serve as an extracellular signal to attenuate adhesion-related gene expression and alter bacterial surface properties in E. coli O157:H7. These findings support the further development of EPS-cn2 for pathogenic biofilm management in clinical and industrial settings, and suggests the further targeting of adhesion-related genes to limit the persistence of this highly pathogenic strain in sensitive environments.

Quorum Sensing in ESKAPE Bugs: A Target for Combating Antimicrobial Resistance and Bacterial Virulence.

Simple SummaryQuorum sensing in ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) bacteria denotes a global threat to public health. The acquisition of antimicrobial resistance genes, virulence production, and biofilm formation by ESKAPE pathogens has reduced the treatment options for serious infections. QS has been well recognized as being involved in the pathogenesis and antibiotic resistance. More understanding of QS mechanistic would also aid in the prediction of underlying or even unknown mechanisms of antimicrobial resistance and bacterial pathogenesis. In this review, we describe the known antibiotic resistance and pathogenesis caused by QS as well as the strategies to control QS in these pathogens.A clique of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. (ESKAPE) bugs is the utmost causative agent responsible for multidrug resistance in hospital settings. These microorganisms employ a type of cell–cell communication termed ‘quorum sensing (QS) system’ to mediate population density and synchronously control the genes that modulate drug resistance and pathogenic behaviors. In this article, we focused on the present understanding of the prevailing QS system in ESKAPE pathogens. Basically, the QS component consisted of an autoinducer synthase, a ligand (e.g., acyl homoserine lactones/peptide hormones), and a transcriptional regulator. QS mediated expression of the bacterial capsule, iron acquisition, adherence factors, synthesis of lipopolysaccharide, poly-N-acetylglucosamine (PNAG) biosynthesis, motility, as well as biofilm development allow bacteria to promote an antimicrobial-resistant population that can escape the action of traditional drugs and endorse a divergent virulence production. The increasing prevalence of these harmful threats to infection control, as well as the urgent need for effective antimicrobial strategies to combat them, serve to highlight the important anti-QS strategies developed to address the difficulty of treating microorganisms.

The endoglycosidase activity of Dispersin B is mediated through electrostatic interactions with cationic poly-β-(1→6)-N-acetylglucosamine.

Bacterial biofilms consist of bacterial cells embedded within a self-produced extracellular polymeric substance (EPS) composed of exopolysaccharides, extra cellular DNA, proteins and lipids. The enzyme Dispersin B (DspB) is a CAZy type 20 β-hexosaminidase enzyme that catalyses the hydrolysis of poly-N-acetylglucosamine (PNAG), a major biofilm polysaccharide produced by a wide variety of biofilm-forming bacteria. Native PNAG is partially de-N-acetylated, and the degree of deacetylation varies between species and dependent on the environment. We have previously shown that DspB is able to perform both endo- and exo-glycosidic bond cleavage of PNAG depending on the de-N-acetylation patterns present in the PNAG substrate. Here, we used a combination of synthetic PNAG substrate analogues, site-directed mutagenesis and in vitro biofilm dispersal assay to investigate the molecular basis for the endo-glycosidic cleavage activity of DspB and the importance of this activity for dispersal of PNAG-dependent Staphylococcus epidermidis biofilms. We found that D242 contributes to the endoglycosidase activity of DspB through electrostatic interactions with cationic substrates in the -2 binding site. A DspBD242N mutant was highly deficient in endoglycosidase activity while maintaining exoglycosidase activity. When used to disperse S. epidermidis biofilms, this DspBD242N mutant resulted in an increase in residual biofilm biomass after treatment when compared to wild-type DspB. These results suggest that the de-N-acetylation of PNAG in S. epidermidis biofilms is not uniformly distributed and that the endoglycosidase activity of DspB is required for efficient biofilm dispersal.

3-Phenyllactic acid generated in medicinal plant extracts fermented with plant-derived lactic acid bacteria inhibits the biofilm synthesis of Aggregatibacter actinomycetemcomitans.

In the present study, the effect of PLA on a periodontic pathogen, Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), the biofilm, and virulence-related genes was investigated. We confirmed that two lactic acid bacteria (LAB) strains isolated from plant sources, Lactiplantibacillus plantarum MSC-C2 and Pediococcus pentosaceus K40, secrete PLA into the de Man, Rogosa & Sharpe (MRS) broth when supplemented with phenyl pyruvic acid (PPA) as a precursor to PLA. Moreover, PLA was generated in the fermentation broths of two medicinal plant extracts, Paeonia lactiflora Pall (PR) and Carthamus tinctorius (CT), when used by each LAB strain and each extract supplemented with PPA. We determined that the minimum inhibitory concentration (MIC) of PLA against A. actinomycetemcomitans was 20 mM. PLA significantly decreased biofilm formation and suppressed the transcription of pgA, ltxA, and cdtB genes, which encode the poly-N-acetylglucosamine (PGA) polysaccharide of biofilm matrix and exotoxins leukotoxin and cytolethal distending toxin (CDT), respectively. The PLA produced by the MSC-C2 and K40 strains was increased several times by the addition of PPA to the MRS broth. The anti-biofilm effect of the extracts from the fermentation broth was proportional to the increasing PLA concentration, while a cumulatively higher effect than that of PLA alone suggested a combinational effect of PLA and the other metabolites, such as lactic acid (LA). Among the two medicinal plants, PLA, produced after the addition of PPA, was higher in PR extract in case of both the LAB strains. PLA production by the MSC-C2 strain in the PR extract reached 4.8 ± 0.23 mM, which was obviously higher than that in the MRS broth (3.88 ± 0.12 mM) supplemented with 1 mg/ml PPA. The activity to inhibit biofilm formation in the fermented PR extract was clearly high. PLA formed in the fermented PR extract downregulated the dispersin B encoding the dspB gene together with pgA, ltxA, and cdtB. In conclusion, this study shows a promising activity of PLA against the A. actinomycetemcomitans biofilm and virulence genes. In addition, the combinational effect of PLA and the medicinal plant extract can be achieved by fermentation with a specific plant-derived LAB strain.

Deacetylated-poly-N-acetylglucosamine-folic Acid as a Nanocarrier for Delivering miR-196a Inhibitor to Anticancer Activity

Background:MiR-196a is particularly noticeable in the development of liver cancer. However, the rapid degradation by ribonuclease (RNase) imposes a limit on the miRNA gene therapy applications.Aims:To design a novel gene-targeting nano system for liver cancer treatment.Study Design:Cell culture study and animal experimentation.Methods:Deacetylated (DEAC)-poly-N-acetylglucosamine (PNAG)-folic acid (FA) was prepared via ethyl (dimethylaminopropyl) carbodiimide/N-hydroxysuccinimide reaction, and miR-196a inhibitor (miR-196a I)/DEAC-PNAG-FA was prepared through self-assembly. The characterization and nucleic acid protection of the self-assembly system were also determined. The biological function and related mechanism of the prepared system were studied at cellular and molecular levels. Mice were established as a xenotransplantation model to evaluate the anticancer capacity of miR-196a I/DEAC-PNAG-FA in vivo.Results:The morphology of miR-196a I/DEAC-PNAG-FA was uniform, and its particle size was approximately 70–100 nm. A nanocarrier with an N/P ratio of 200:1 can maximize the nucleic acid carrying capacity of the self-assembly system. The nanosystem can protect miRNA from RNase degradation and could be internalized rapidly within 4 h. The self-assembly system significantly enhanced the apoptosis-inducing effect of miR-196a I on HepG2 cells (P = 0.003). Molecular biological analyses confirmed that the apoptosis-inducing effect of the nanosystem was due to the inhibition of miR-196a gene expression in HepG2 cells, which upregulate the expression of pro-apoptotic proteins FOXO1 (P < 0.001), Bax (P < 0.001), Ki67 (P < 0.001), and proliferating cell nuclear antigen (P < 0.001), and inhibit the expression of apoptosis inhibitory protein Bcl-2 (P < 0.001). Moreover, compared with free miR-196a inhibitor or miR-196a I/DEAC-PNAG, miR-196a I/DEAC-PNAG-FA can more effectively inhibit tumor growth in vivo (P = 0.026).Conclusion:The newly prepared self-assembly targeting system can effectively induce apoptosis and abrogate tumor growth, which may open a new approach for liver cancer treatment.

Deinococcus radiodurans Exopolysaccharide Inhibits Staphylococcus aureus Biofilm Formation

Deinococcus radiodurans is an extremely resistant bacterium against extracellular stress owing to on its unique physiological functions and the structure of its cellular constituents. Interestingly, it has been reported that the pattern of alteration in Deinococcus proportion on the skin is negatively correlated with skin inflammatory diseases, whereas the proportion of Staphylococcus aureus was increased in patients with chronic skin inflammatory diseases. However, the biological mechanisms of deinococcal interactions with other skin commensal bacteria have not been studied. In this study, we hypothesized that deinococcal cellular constituents play a pivotal role in preventing S. aureus colonization by inhibiting biofilm formation. To prove this, we first isolated cellular constituents, such as exopolysaccharide (DeinoPol), cell wall (DeinoWall), and cell membrane (DeinoMem), from D. radiodurans and investigated their inhibitory effects on S. aureus colonization and biofilm formation in vitro and in vivo. Among them, only DeinoPol exhibited an anti-biofilm effect without affecting bacterial growth and inhibiting staphylococcal colonization and inflammation in a mouse skin infection model. Moreover, the inhibitory effect was impaired in the Δdra0033 strain, a mutant that cannot produce DeinoPol. Remarkably, DeinoPol not only interfered with S. aureus biofilm formation at early and late stages but also disrupted a preexisting biofilm by inhibiting the production of poly-N-acetylglucosamine (PNAG), a key molecule required for S. aureus biofilm formation. Taken together, the present study suggests that DeinoPol is a key molecule in the negative regulation of S. aureus biofilm formation by D. radiodurans. Therefore, DeinoPol could be applied to prevent and/or treat infections or inflammatory diseases associated with S. aureus biofilms.

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.

Vaccination against the broadly expressed microbial antigen PNAG prevents cognitive decline in the APP-PS1 mouse model of Alzheimer's disease.

While there is ample evidence for a role of the microbiota in cognitive decline in Alzheimer's Disease (AD), there is little insight into how the microbiota mediates neuronal dysfunction and cognitive decline. A conserved surface polysaccharide, poly-N-acetyl glucosamine (PNAG) is produced by a broad range of microbial cells, but not mammals. We have detected the presence of PNAG-positive, likely microbiota-derived cells or fragments closely associated with amyloid-ß deposits in the brains of deceased AD individuals and cognitively impaired APP-PS1 mice, but not non-AD controls. These fragments potentially provide an inflammatory stimulus critical for neural tissue destruction and cognitive impairment. We tested whether vaccination against PNAG prevented microbial access to brains of APP-PS1 mice and impacted cognitive decline. Microbial factors were detected with a human IgG monoclonal antibody (MAb) to PNAG in brain tissues from human AD and APP/PS1 mice. Two cohorts of APP/PS1 mice and wild type (WT) littermate controls were vaccinated with either a PNAG vaccine (5GlcNH2 -TT) or TT (tetanus toxin) only as a control, five times starting at either 5 weeks or 5 months of age. Only PNAG-immunized mice made specific antibodies over the experimental period. Water T maze testing of mice was performed at 12-15 months of age. Brains were harvested to detect both PNAG and amyloid-ß immunohistochemically. PNAG-positive microbial factors were elevated in areas with amyloid-ß deposits in brain tissue from human AD cases and APP/PS1 mice. Non-AD age- and sex-matched controls had little detectable brain PNAG, even around amyloid-ß deposits. Active PNAG vaccination at both 5 weeks and 5 months of age prevented cognitive decline in APP-PS1 mice. Only 10% or 30% of non-immune APP/PS1 mice reached criteria by day 5 of reversal, respectively, compared to 90% and 100% of PNAG vaccinated APP/PS1 mice. Immunohistochemistry indicated decreased presence of PNAG-positive material in PNAG vaccinated mice and a modest reduction in amyloid-ß deposits. Antibodies to PNAG induced at 5 weeks or 5 months of age prevented cognitive decline and brain pathology in APP/PS1 mice, suggesting that microbial factors that enter the brain are critical for cognitive decline that may be prevented by vaccination.

Regulation of Biofilm Exopolysaccharide Production by Cyclic Di-Guanosine Monophosphate.

Many bacterial species in nature possess the ability to transition into a sessile lifestyle and aggregate into cohesive colonies, known as biofilms. Within a biofilm, bacterial cells are encapsulated within an extracellular polymeric substance (EPS) comprised of polysaccharides, proteins, nucleic acids, lipids, and other small molecules. The transition from planktonic growth to the biofilm lifecycle provides numerous benefits to bacteria, such as facilitating adherence to abiotic surfaces, evasion of a host immune system, and resistance to common antibiotics. As a result, biofilm-forming bacteria contribute to 65% of infections in humans, and substantially increase the energy and time required for treatment and recovery. Several biofilm specific exopolysaccharides, including cellulose, alginate, Pel polysaccharide, and poly-N-acetylglucosamine (PNAG), have been shown to play an important role in bacterial biofilm formation and their production is strongly correlated with pathogenicity and virulence. In many bacteria the biosynthetic machineries required for assembly of these exopolysaccharides are regulated by common signaling molecules, with the second messenger cyclic di-guanosine monophosphate (c-di-GMP) playing an especially important role in the post-translational activation of exopolysaccharide biosynthesis. Research on treatments of antibiotic-resistant and biofilm-forming bacteria through direct targeting of c-di-GMP signaling has shown promise, including peptide-based treatments that sequester intracellular c-di-GMP. In this review, we will examine the direct role c-di-GMP plays in the biosynthesis and export of biofilm exopolysaccharides with a focus on the mechanism of post-translational activation of these pathways, as well as describe novel approaches to inhibit biofilm formation through direct targeting of c-di-GMP.

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.

ABSTRACTRhodococcus equi is a prevalent cause of pneumonia in foals worldwide. Our laboratory has demonstrated that vaccination against the surface polysaccharide β-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.

A global transcriptomic analysis of Staphylococcus aureus biofilm formation across diverse clonal lineages.

A key characteristic of Staphylococcus aureus infections, and one that also varies phenotypically between clones, is that of biofilm formation, which aids in bacterial persistence through increased adherence and immune evasion. Though there is a general understanding of the process of biofilm formation – adhesion, proliferation, maturation and dispersal – the tightly orchestrated molecular events behind each stage, and what drives variation between S. aureus strains, has yet to be unravelled. Herein we measure biofilm progression and dispersal in real-time across the five major S. aureus CDC-types (USA100-USA500) revealing adherence patterns that differ markedly amongst strains. To gain insight into this, we performed transcriptomic profiling on these isolates at multiple timepoints, compared to planktonically growing counterparts. Our findings support a model in which eDNA release, followed by increased positive surface charge, perhaps drives initial abiotic attachment. This is seemingly followed by cooperative repression of autolysis and activation of poly-N-acetylglucosamine (PNAG) production, which may indicate a developmental shift in structuring the biofilm matrix. As biofilms mature, diminished translational capacity was apparent, with 53 % of all ribosomal proteins downregulated, followed by upregulation of anaerobic respiration enzymes. These findings are noteworthy because reduced cellular activity and an altered metabolic state have been previously shown to contribute to higher antibiotic tolerance and bacterial persistence. In sum, this work is, to our knowledge, the first study to investigate transcriptional regulation during the early, establishing phase of biofilm formation, and to compare global transcriptional regulation both temporally and across multiple clonal lineages.

115-OR: Microbiome Antigens as a Vaccination Strategy in Type 1 Diabetes

Type 1 diabetes (T1D) is an autoimmune disorder that results from T cell-mediated destruction of the insulin-producing β cells of the pancreatic islets. Accumulating evidence from both human and animal studies points to the gut microbiome, the 100 trillion microbes inhabiting our intestinal tract, as a key component driving the inflammatory processes leading to T1D. However, the mechanisms therein are poorly understood. We propose that this inflammatory milieu occurs in the pancreas, where microbes or microbial fragments that translocate from their usual luminal, mucosal sites persist and induce the cytokine and chemokine signals needed for destructive, autoimmune cellular and humoral responses that destroy the insulin producing β cells. By controlling this inflammation via vaccination against a highly conserved microbial surface antigen, poly-N-acetyl glucosamine (PNAG), we can decrease the inflammatory response such that vaccination prevents diabetes. In analyses of human and murine pancreatic islets, we have found that PNAG is readily detectable within diabetic, but not healthy, pancreases and associated with both T cells and polymorphonuclear cells (PMNs). This finding suggests that inducing an effective immune response that can clear PNAG-containing microbes or microbial fragments may be critical in counteracting the microbiota-driven inflammation leading to autoimmunity. We report that NOD female mice vaccinated against PNAG exhibited a drastic reduction in diabetes incidence from in 70% of 21 controls to 17% of 18 PNAG-immune mice. Immunophenotyping on these mice indicates that PNAG vaccination dramatically increases Foxp3+ Tregs as well as CD4+ and CD4- IL-10-producing cells. Taken together, our work provides evidence that the translocation of microbial antigens to the pancreas may drive inflammation leading to T1D, and that vaccination against one particular microbial antigen, PNAG, may lead to protection against this disease. Disclosure A. Kostic: Other Relationship; Self; DeepBiome Therapeutics, Inc., FitBiomics, Inc. Funding American Diabetes Association/Pathway to Stop Diabetes (1-17-INI-13 to A.K.)

Failure of a novel surface polysaccharide-targeting vaccine to prevent Tritrichomonas infection in beef cattle

Tritrichomonas foetus (T. foetus) is the causative agent of bovine trichomoniasis that has a major impact on production costs for beef cattle producers. Immunization strategies to effectively protect against T. foetus are a high priority. Tritrichomonas foetus expresses a surface polysaccharide, beta 1-6 poly-N-acetyl glucosamine (PNAG), that is synthesized by a broad range of microbial pathogens. Vaccination with a PNAG-specific vaccine has demonstrated protection in pigs and horses. This study attempted to protect pregnant cows from an experimental T. foetus infection by prior vaccination with a PNAG-specific vaccine. Acceptable PNAG-specific antibody titers were achieved after vaccination, but antibodies elicited were non-protective against experimental challenge with T. foetus and in in vitro antibody functional bacterial killing assays.

In Silico Characterization and Phylogenetic Distribution of Extracellular Matrix Components in the Model Rhizobacteria Pseudomonas fluorescens F113 and Other Pseudomonads.

Biofilms are complex structures that are crucial during host–bacteria interaction and colonization. Bacteria within biofilms are surrounded by an extracellular matrix (ECM) typically composed of proteins, polysaccharides, lipids, and DNA. Pseudomonads contain a variety of ECM components, some of which have been extensively characterized. However, neither the ECM composition of plant-associated pseudomonads nor their phylogenetic distribution within the genus has been so thoroughly studied. In this work, we use in silico methods to describe the ECM composition of Pseudomonas fluorescens F113, a plant growth-promoting rhizobacteria and model for rhizosphere colonization. These components include the polysaccharides alginate, poly-N-acetyl-glucosamine (PNAG) and levan; the adhesins LapA, MapA and PsmE; and the functional amyloids in Pseudomonas. Interestingly, we identified novel components: the Pseudomonas acidic polysaccharide (Pap), whose presence is limited within the genus; and a novel type of Flp/Tad pilus, partially different from the one described in P. aeruginosa. Furthermore, we explored the phylogenetic distribution of the most relevant ECM components in nearly 600 complete Pseudomonas genomes. Our analyses show that Pseudomonas populations contain a diverse set of gene/gene clusters potentially involved in the formation of their ECMs, showing certain commensal versus pathogen lifestyle specialization.

Mycoplasma genitalium Biofilms Contain Poly-GlcNAc and Contribute to Antibiotic Resistance.

Mycoplasma genitalium is an important etiologic agent of non-gonococcal urethritis (NGU), known for chronicity and multidrug resistance, in which biofilms may play an integral role. In some bacterial species capable of forming biofilms, extracellular polymeric substances (EPS) composed of poly-N-acetylglucosamine (PNAG) are a crucial component of the matrix. Monosaccharide analysis of M. genitalium strains revealed high abundance of GlcNAc, suggesting a biofilm-specific EPS. Chromatograms also showed high concentrations of galactose and glucose as observed in other mycoplasma species. Fluorescence microscopy of M. genitalium biofilms utilizing fluor-coupled lectins revealed differential staining of biofilm structures. Scanning electron microscopy (SEM) showed increasing maturation over time of bacterial “towers” seen in biofilm development. As seen with Mycoplasma pneumoniae, organisms within fully mature M. genitalium biofilms exhibited loss of cell polarization. Bacteria associated with disrupted biofilms exhibited decreased dose-dependent viability after treatment with antibiotics compared to bacteria with intact biofilms. In addition, growth index analysis demonstrated decreases in metabolism in cultures with disrupted biofilms with antibiotic treatment. Taken together, these data suggest that M. genitalium biofilms are a contributing factor in antibiotic resistance.

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 β 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.

Failure of a novel surface polysaccharide targeting vaccine to prevent Tritrichomonas foetus infection in beef cattle

Tritrichomonas foetus (T. foetus) is the causative agent of bovine trichomoniasis that has a major impact on production costs for beef cattle farmers. Immunization strategies to effectively protect against T. foetus are a high priority. T. foetus expresses a surface polysaccharide, beta 1-6 poly-Nacetyl glucosamine (PNAG). A PNAG-specific vaccine has demonstrated protection in pigs and horses. This study attempted to protect pregnant cows from an experimental T. foetus infection by prior vaccination with a PNAG-specific vaccine.