SummaryClostridium perfringens type C isolates, which cause enteritis necroticans in humans and enteritis and enterotoxaemias of domestic animals, typically produce (at minimum) beta toxin (CPB), alpha toxin (CPA) and perfringolysin O (PFO) during log-phase growth. To assist development of improved vaccines and therapeutics, we evaluated the contribution of these three toxins to the intestinal virulence of type C disease isolate CN3685. Similar to natural type C infection, log-phase vegetative cultures of wild-type CN3685 caused haemorrhagic necrotizing enteritis in rabbit ileal loops. When isogenic toxin null mutants were prepared using TargeTron ® technology, even a double cpa/pfoA null mutant of CN3685 remained virulent in ileal loops. However, two independent cpb null mutants were completely attenuated for virulence in this animal model. Complementation of a cpb mutant restored its CPB production and intestinal virulence. Additionally, pre-incubation of wild-type CN3685 with a CPB-neutralizing monoclonal antibody rendered the strain avirulent for causing intestinal pathology. Finally, highly purified CPB reproduced the intestinal damage of wild-type CN3685 and that damage was prevented by pre-incubating purified CPB with a CPB monoclonal antibody. These results indicate that CPB is both required and sufficient for CN3685-induced enteric pathology, supporting a key role for this toxin in type C intestinal pathogenesis.

Streptococcus pneumoniae is the leading cause of death in children worldwide and forms highly organized biofilms in the nasopharynx, lungs, and middle ear mucosa. The luxS-controlled quorum-sensing (QS) system has recently been implicated in virulence and persistence in the nasopharynx, but its role in biofilms has not been studied. Here we show that this QS system plays a major role in the control of S. pneumoniae biofilm formation. Our results demonstrate that the luxS gene is contained by invasive isolates and normal-flora strains in a region that contains genes involved in division and cell wall biosynthesis. The luxS gene was maximally transcribed, as a monocistronic message, in the early mid-log phase of growth, and this coincides with the appearance of early biofilms. Demonstrating the role of the LuxS system in regulating S. pneumoniae biofilms, at 24 h postinoculation, two different D39⌬luxS mutants produced ϳ80% less biofilm biomass than wild-type (WT) strain D39 did. Complementation of these strains with luxS, either in a plasmid or integrated as a single copy in the genome, restored their biofilm level to that of the WT. Moreover, a soluble factor secreted by WT strain D39 or purified AI-2 restored the biofilm phenotype of D39⌬luxS. Our results also demonstrate that during the early mid-log phase of growth, LuxS regulates the transcript levels of lytA, which encodes an autolysin previously implicated in biofilms, and also the transcript levels of ply, which encodes the pneumococcal pneumolysin. In conclusion, the luxS-controlled QS system is a key regulator of early biofilm formation by S. pneumoniae strain D39.

BackgroundAlthough useful for probing bacterial pathogenesis and physiology, current random mutagenesis systems suffer limitations for studying the toxin-producing bacterium Clostridium perfringens.Methodology/Principal FindingsAn EZ-Tn5-based random mutagenesis approach was developed for use in C. perfringens. This mutagenesis system identified a new regulatory locus controlling toxin production by strain 13, a C. perfringens type A strain. The novel locus, encoding proteins with homology to the AgrB and AgrD components of the Agr quorum sensing system of Staphylococcus aureus and two hypothetical proteins, was found to regulate early production of both alpha toxin and perfringolysin O (PFO) by strain 13. PFO production by the strain 13 ΔagrB mutant could be restored by genetic complementation or by physical complementation, i.e. by co-culture of the strain 13 ΔagrB mutant with a pfoA mutant of either strain 13 or C. perfringens type C CN3685. A similar AgrB- and AgrD-encoding locus is identifiable in all sequenced C. perfringens strains, including type B, C, D, and E isolates, suggesting this regulatory locus contributes to toxin regulation by most C. perfringens strains. In strain 13, the agrB and agrD genes were found to be co-transcribed in an operon with two upstream genes encoding hypothetical proteins.Conclusions/SignificanceThe new Tn5-based random mutagenesis system developed in this study is more efficient and random than previously reported C. perfringens random mutagenesis approaches. It allowed identification of a novel C. perfringens toxin regulatory locus with homology to the Agr system of S. aureus and which functions as expected of an Agr-like quorum sensing system. Since previous studies have shown that alpha toxin and perfringolysin O are responsible for strain 13-induced clostridial myonecrosis in the mouse model, the new agr regulatory locus may have importance for strain 13 virulence.

Streptococcus pneumoniae (the pneumococcus) is a common commensal inhabitant of the nasopharynx and a frequent etiologic agent in serious diseases such as pneumonia, otitis media, bacteremia, and meningitis. Multiple pneumococcal strains can colonize the nasopharynx, which is also home to many other bacterial species. Intraspecies and interspecies interactions influence pneumococcal carriage in important ways. Co-colonization by two or more pneumococcal strains has implications for vaccine serotype replacement, carriage detection, and pneumonia diagnostics. Interactions between the pneumococcus and other bacterial species alter carriage prevalence, modulate virulence, and affect biofilm formation. By examining these interactions, this review highlights how the bacterial ecosystem of the nasopharynx changes the nature and course of pneumococcal carriage.

Clostridium perfringens type A strains producing enterotoxin (CPE) cause one of the most common bacterial food-borne illnesses, as well as many cases of non-food-borne human gastrointestinal disease. Recent studies have shown that an Agr-like quorum-sensing system controls production of chromosomally encoded alphatoxin and perfringolysin O by C. perfringens, as well as sporulation by Clostridium botulinum and Clostridium sporogenes. The current study explored whether the Agr-like quorum-sensing system also regulates sporulation and production of two plasmid-encoded toxins (CPE and beta2 toxin) that may contribute to the pathogenesis of non-food-borne human gastrointestinal disease strain F5603. An isogenic agrB null mutant was inhibited for production of beta2 toxin during vegetative growth and in sporulating culture, providing the first evidence that, in C. perfringens, this system can control production of plasmid-encoded toxins as well as chromosomally encoded toxins. This mutant also showed reduced production of alpha-toxin and perfringolysin O during vegetative growth. Importantly, when cultured in sporulation medium, the mutant failed to efficiently form spores and was blocked for CPE production. Complementation partially or fully reversed all phenotypic changes in the mutant, confirming that they were specifically due to inactivation of the agr locus. Western blots suggest that this loss of sporulation and sporulation-specific CPE production for the agrB null mutant involves, at least in part, Agr-mediated regulation of production of Spo0A and alternative sigma factors, which are essential for C. perfringens sporulation.The Gram-positive, anaerobic, spore-forming bacterium Clostridium perfringens is an important human and medical pathogen, causing both histotoxic infections (e.g., traumatic gas gangrene) and infections originating in the intestines (e.g., human food poisoning). Isolates of this bacterium are commonly assigned to one of five types (A to E) based upon their production of four typing toxins (alpha-, beta-, epsilon-, and iota-toxin). Although not part of the toxinotyping classification system, C. perfringens enterotoxin (CPE) is one of the most important toxins produced by this bacterium. CPE-producing type A strains are responsible for C. perfringens type A food poisoning, which is the second most commonly identified bacterial food-borne disease in the United States, where nearly a million cases occur annually (24). Besides causing food poisoning, CPE-producing type A strains are also responsible for 5 to 15% of all cases of human non-food-borne gastrointestinal (GI) diseases (2). Molecular Koch's postulate studies (22) have shown that CPE is very important for the GI pathogenesis of both C. perfringens type A food poisoning and CPE-associated non-food-borne human GI diseases. However, it has been proposed that beta2 toxin (CPB2) may also contribute to the pathogenesis of some cases of non-food-borne human GI disease (6).Sporulation offers two critical contributions to the GI diseases caused...