The Streptococcos suis sortases SrtB and SrtF are essential for disease in pigs.

The porcine pathogen Streptococcus suis colonizes the upper respiratory tracts of pigs, potentially causing septicaemia, meningitis and death, thus placing a severe burden on the agricultural industry worldwide. It is also a zoonotic pathogen that is known to cause systemic infections and meningitis in humans. Understanding how S. suis colonizes and interacts with its hosts is relevant for future strategies of drug and vaccine development. As with other Gram-positive bacteria, S. suis utilizes enzymes known as sortases to attach specific proteins bearing cell wall sorting signals to its surface, where they can play a role in host-pathogen interactions. The surface proteins of bacteria are often important in adhesion to and invasion of host cells. In this study, markerless in-frame deletion mutants of the housekeeping sortase srtA and the two pilus-associated sortases, srtB and srtF, were generated and their importance in S. suis infections was investigated. We found that all three of these sortases are essential to disease in pigs, concluding that their cognate-sorted proteins may also be useful in protecting pigs against infection

Clostridioides difficile para-Cresol Production Is Induced by the Precursor para-Hydroxyphenylacetate.

Clostridioides difficile is an etiological agent for antibiotic-associated diarrheal disease. C. difficile produces a phenolic compound, para-cresol, which selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. C. difficile decarboxylates para-hydroxyphenylacetate (p-HPA) to produce p-cresol by the action of the HpdBCA decarboxylase encoded by the hpdBCA operon. Here, we investigate regulation of the hpdBCA operon and directly compare three independent reporter systems; SNAP-tag, glucuronidase gusA, and alkaline phosphatase phoZ reporters to detect basal and inducible expression. We show that expression of hpdBCA is upregulated in response to elevated p-HPA. In silico analysis identified three putative promoters upstream of hpdBCA operon-P1, P2, and P?54; only the P1 promoter was responsible for both basal and p-HPA-inducible expression of hpdBCA We demonstrated that turnover of tyrosine, a precursor for p-HPA, is insufficient to induce expression of the hpdBCA operon above basal levels because it is inefficiently converted to p-HPA in minimal media. We show that induction of the hpdBCA operon in response to p-HPA occurs in a dose-dependent manner. We also identified an inverted palindromic repeat (AAAAAG-N13-CTTTTT) upstream of the hpdBCA start codon (ATG) that is essential for inducing transcription of the hpdBCA operon in response to p-HPA, which drives the production of p-cresol. This provides insights into the regulatory control of p-cresol production, which affords a competitive advantage for C. difficile over other intestinal bacteria, promoting dysbiosis.IMPORTANCE Clostridioides difficile infection results from antibiotic-associated dysbiosis. para-Cresol, a phenolic compound produced by C. difficile, selectively targets gammaproteobacteria in the gut, facilitating dysbiosis. Here, we demonstrate that expression of the hpdBCA operon, encoding the HpdBCA decarboxylase which converts p-HPA to p-cresol, is upregulated in response to elevated exogenous p-HPA, with induction occurring between >0.1 and ?0.25?mg/ml. We determined a single promoter and an inverted palindromic repeat responsible for basal and p-HPA-inducible hpdBCA expression. We identified turnover of tyrosine, a p-HPA precursor, does not induce hpdBCA expression above basal level, indicating that exogenous p-HPA was required for p-cresol production. Identifying regulatory controls of p-cresol production will provide novel therapeutic targets to prevent p-cresol production, reducing C. difficile's competitive advantage

The regulation of flagellar filament assembly in caulobacter crescentus

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

The post-translational modification of the Clostridium difficile flagellin affects motility, cell surface properties and virulence.

Clostridium difficile is a prominent nosocomial pathogen, proliferating and causing enteric disease in individuals with a compromised gut microflora. We characterized the post-translational modification of flagellin in C. difficile 630. The structure of the modification was solved by nuclear magnetic resonance and shown to contain an N-acetylglucosamine substituted with a phosphorylated N-methyl-l-threonine. A reverse genetics approach investigated the function of the putative four-gene modification locus. All mutants were found to have truncated glycan structures by LC-MS/MS, taking into account bioinformatic analysis, we propose that the open reading frame CD0241 encodes a kinase involved in the transfer of the phosphate to the threonine, the CD0242 protein catalyses the addition of the phosphothreonine to the N-acetylglucosamine moiety and CD0243 transfers the methyl group to the threonine. Some mutations affected motility and caused cells to aggregate to each other and abiotic surfaces. Altering the structure of the flagellin modification impacted on colonization and disease recurrence in a murine model of infection, showing that alterations in the surface architecture of C. difficile vegetative cells can play a significant role in disease. We show that motility is not a requirement for colonization, but that colonization was compromised when the glycan structure was incomplete

The quantitation of biofilm formation by crystal violet in three day old and six day old biofilms.

<p>A regression analysis was performed to determine whether there were any strain specific differences in the levels of biofilm formation at three days compared to the R20291reference. This was repeated for the six day old biofilms. <i>p</i><0.05 indicates a significant difference from the reference R20291. The COV indicates whether the difference in biofilm formation is higher (positive number) or lower (negative number) compared to the R20291reference. A regression analysis was also used to determine whether there were significant changes in the biofilm formation from three days to six days, <i>p</i><0.01.</p

The effect of oxygen stress on viability of cells within a biofilm.

<p>Regression analyses were performed in three categories; a) were there strain dependent differences in survival in both the intact, disrupted and control biofilms. b) were there any significant differences in survival of vegetative cells or spores in intact and disrupted biofilms compared to the untreated control and c) were there any differences in vegetative cell or spore survival in intact biofilms compared to disrupted biofilms in response to oxygen stress. <i>p</i><0.05 is significant (in bold).</p

Maturation of the <i>C. difficile</i> biofilm from 24 hours to six days.

<p>The biofilms observed in the tissue culture flasks were produced after incubation for either; 24 hours, three days or six days, before they were mechanically detached from the plastic by agitation as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050527#pone-0050527-g001" target="_blank">figure 1</a>. The top panel corresponds to the biofilm mass observed in strain R20291 and the bottom corresponds to 630Δerm.</p

Enumeration of vegetative cells and spores encased within a biofilm.

<p>Biofilms were matured for three and six days post seeding in TC flasks for strains R20291, R20291_<i>spo0A</i>::CT, R20291_<i>spo0A</i>::CT::p<i>spo0A</i> and 630Δerm. The biofilms were detached from the plastic and dispursed by vortexing. These suspensions were used to determine the proportion of vegetative cells and spores per ml of biofilm (10 ml total volume). A partial F-test revealed strain specific differences at three days and six days, indicated with * (<i>p</i><0.001). Regression analysis was used to determine whether there were significant differences in the levels of spores and vegetative cells at each time point, compared to the R20291 reference, as indicated by ∧ for spores (<i>p</i><0.05) and ∼ for vegetative cells (<i>p</i><0.05). Differences between the cell types in six day old biofilms compared to three day old biofilms are listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0050527#pone-0050527-t002" target="_blank">table 2</a>.</p

Strains and plasmids used in this study.

<p>Strains and plasmids used in this study.</p

Crystal violet quantification of biofilm formation.

<p>a) Visualisation of three day old biofilms attached to the bottom of the individual wells in a 24 well plate, compared to a media blank control. b) Graphical representation of the optical density readings OD595 nm derived from methanol elution of the crystal violet stain. A partial F-test was performed to determine if there were strain specific differences in the level of biofilm formation, indicated with * (<i>p</i><0.05). A regression analysis was performed to determine whether there were any specific strain differences compared to the reference indicated with ∧ (<i>p</i><0.05). The reference was 630Δerm for the first graph, and R20291 for the <i>spo0A</i> mutant and wild-type comparison. c) Visualisation of six day old biofilms attached to the bottom of a 24 well plate as outlined in (a). d) Graphical representation of the optical density readings OD595 nm derived from methanol elution of the crystal violet stain, as outlined in (b). Statistical analysis was performed as outlined above.</p