bBacteria within biofilms are protected from multiple stresses, including immune responses and antimicrobial agents. The biofilm-forming ability of bacterial pathogens has been associated with increased antibiotic resistance and chronic recurrent infections. Although biofilms have been well studied for several gut pathogens, little is known about biofilm formation by anaerobic gut species. The obligate anaerobe Clostridium difficile causes C. difficile infection (CDI), a major health care-associated problem primarily due to the high incidence of recurring infections. C. difficile colonizes the gut when the normal intestinal microflora is disrupted by antimicrobial agents; however, the factors or processes involved in gut colonization during infection remain unclear. We demonstrate that clinical C. difficile strains, i.e., strain 630 and the hypervirulent strain R20291, form structured biofilms in vitro, with R20291 accumulating substantially more biofilm. Microscopic and biochemical analyses show multiple layers of bacteria encased in a biofilm matrix containing proteins, DNA, and polysaccharide. Employing isogenic mutants, we show that virulence-associated proteins, Cwp84, flagella, and a putative quorum-sensing regulator, LuxS, are all required for maximal biofilm formation by C. difficile. Interestingly, a mutant in Spo0A, a transcription factor that controls spore formation, was defective for biofilm formation, indicating a possible link between sporulation and biofilm formation. Furthermore, we demonstrate that bacteria in clostridial biofilms are more resistant to high concentrations of vancomycin, a drug commonly used for treatment of CDI. Our data suggest that biofilm formation by C. difficile is a complex multifactorial process and may be a crucial mechanism for clostridial persistence in the host.
SummaryMacrophage infectivity potentiators (MIPs) are a family of surface-exposed virulence factors of intracellular microorganisms such as Legionella , Chlamydia and Trypanosoma . These proteins display peptidylprolyl cis / trans isomerase (PPIase) activity that is inhibited by immunosuppressants FK506 and rapamycin. Here we describe the identification and characterization in Neisseria gonorrhoeae of Ng-MIP, a surface-exposed lipoprotein with high homology to MIPs. The protein is an homodimer with rapamycininhibited PPIase activity confirming that it is a functional member of the MIP family. A knock-out strain, generated by deletion of the mip gene in N. gonorrhoeae F62 strain, was evaluated for its role in infection of mouse and human macrophages. We show that Ng-MIP promotes the intracellular survival of N. gonorrhoeae in macrophages, highlighting a possible role of this protein in promoting the persistence of gonococcal infection.
Clostridium difficile is emerging worldwide as a major cause of nosocomial infections. The negatively charged PSII polysaccharide has been found in different strains of C. difficile and, thereby, represents an important target molecule for a possible carbohydrate-based vaccine. In order to identify a synthetic fragment that after conjugation to a protein carrier could be able to induce anti-PSII antibodies, we exploited a combination of chemical synthesis with immunochemistry, confocal immunofluorescence microscopy, and solid state NMR. We demonstrate that the phosphate group is crucial in synthetic glycans to mimic the native PSII polysaccharide; both native PSII and a phosphorylated synthetic hexasaccharide repeating unit conjugated to CRM(197) elicit comparable immunogenic responses in mice. This finding can aid design and selection of carbohydrate antigens to be explored as vaccine candidates.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.