Glen P. Carter

Toxin B is essential for virulence of Clostridium difficile

Clostridium difficile is the leading cause of infectious diarrhoea in hospitals worldwide, because of its virulence, spore-forming ability and persistence. C. difficile-associated diseases are induced by antibiotic treatment or disruption of the normal gastrointestinal flora. Recently, morbidity and mortality resulting from C. difficile-associated diseases have increased significantly due to changes in the virulence of the causative strains and antibiotic usage patterns. Since 2002, epidemic toxinotype III NAP1/027 strains, which produce high levels of the major virulence factors, toxin A and toxin B, have emerged. These toxins have 63% amino acid sequence similarity and are members of the large clostridial glucosylating toxin family, which are monoglucosyltransferases that are pro-inflammatory, cytotoxic and enterotoxic in the human colon. Inside host cells, both toxins catalyse the transfer of glucose onto the Rho family of GTPases, leading to cell death. However, the role of these toxins in the context of a C. difficile infection is unknown. Here we describe the construction of isogenic tcdA and tcdB (encoding toxin A and B, respectively) mutants of a virulent C. difficile strain and their use in the hamster disease model to show that toxin B is a key virulence determinant. Previous studies showed that purified toxin A alone can induce most of the pathology observed after infection of hamsters with C. difficile and that toxin B is not toxic in animals unless it is co-administered with toxin A, suggesting that the toxins act synergistically. Our work provides evidence that toxin B, not toxin A, is essential for virulence. Furthermore, it is clear that the importance of these toxins in the context of infection cannot be predicted exclusively from studies using purified toxins, reinforcing the importance of using the natural infection process to dissect the role of toxins in disease.

The Anti-Sigma Factor TcdC Modulates Hypervirulence in an Epidemic BI/NAP1/027 Clinical Isolate of Clostridium difficile

Nosocomial infections are increasingly being recognised as a major patient safety issue. The modern hospital environment and associated health care practices have provided a niche for the rapid evolution of microbial pathogens that are well adapted to surviving and proliferating in this setting, after which they can infect susceptible patients. This is clearly the case for bacterial pathogens such as Methicillin Resistant Staphylococcus aureus (MRSA) and Vancomycin Resistant Enterococcus (VRE) species, both of which have acquired resistance to antimicrobial agents as well as enhanced survival and virulence properties that present serious therapeutic dilemmas for treating physicians. It has recently become apparent that the spore-forming bacterium Clostridium difficile also falls within this category. Since 2000, there has been a striking increase in C. difficile nosocomial infections worldwide, predominantly due to the emergence of epidemic or hypervirulent isolates that appear to possess extended antibiotic resistance and virulence properties. Various hypotheses have been proposed for the emergence of these strains, and for their persistence and increased virulence, but supportive experimental data are lacking. Here we describe a genetic approach using isogenic strains to identify a factor linked to the development of hypervirulence in C. difficile. This study provides evidence that a naturally occurring mutation in a negative regulator of toxin production, the anti-sigma factor TcdC, is an important factor in the development of hypervirulence in epidemic C. difficile isolates, presumably because the mutation leads to significantly increased toxin production, a contentious hypothesis until now. These results have important implications for C. difficile pathogenesis and virulence since they suggest that strains carrying a similar mutation have the inherent potential to develop a hypervirulent phenotype.

The role of toxin A and toxin B in the virulence of Clostridium difficile

During the past decade, there has been a striking increase in Clostridium difficile nosocomial infections worldwide predominantly due to the emergence of epidemic or hypervirulent isolates, leading to an increased research focus on this bacterium. Particular interest has surrounded the two large clostridial toxins encoded by most virulent isolates, known as toxin A and toxin B. Toxin A was thought to be the major virulence factor for many years; however, it is becoming increasingly evident that toxin B plays a much more important role than anticipated. It is clear that further experiments are required to accurately determine the relative roles of each toxin in disease, especially in more clinically relevant current epidemic isolates.

Defining the Roles of TcdA and TcdB in Localized Gastrointestinal Disease, Systemic Organ Damage, and the Host Response during Clostridium difficile Infections

ABSTRACT Clostridium difficile is a leading cause of antibiotic-associated diarrhea, a significant animal pathogen, and a worldwide public health burden. Most disease-causing strains secrete two exotoxins, TcdA and TcdB, which are considered to be the primary virulence factors. Understanding the role that these toxins play in disease is essential for the rational design of urgently needed new therapeutics. However, their relative contributions to disease remain contentious. Using three different animal models, we show that TcdA+ TcdB− mutants are attenuated in virulence in comparison to the wild-type (TcdA+ TcdB+) strain, whereas TcdA− TcdB+ mutants are fully virulent. We also show for the first time that TcdB alone is associated with both severe localized intestinal damage and systemic organ damage, suggesting that this toxin might be responsible for the onset of multiple organ dysfunction syndrome (MODS), a poorly characterized but often fatal complication of C. difficile infection (CDI). Finally, we show that TcdB is the primary factor responsible for inducing the in vivo host innate immune and inflammatory responses. Surprisingly, the animal infection model used was found to profoundly influence disease outcomes, a finding which has important ramifications for the validation of new therapeutics and future disease pathogenesis studies. Overall, our results show unequivocally that TcdB is the major virulence factor of C. difficile and provide new insights into the host response to C. difficile during infection. The results also highlight the critical nature of using appropriate and, when possible, multiple animal infection models when studying bacterial virulence mechanisms. IMPORTANCE Clostridium difficile is a leading cause of antibiotic-associated diarrhea and an important hospital pathogen. TcdA and TcdB are thought to be the primary virulence factors responsible for disease symptoms of C. difficile infections (CDI). However, the individual contributions of these toxins to disease remain contentious. Using three different animal models of infection, we show for the first time that TcdB alone causes severe damage to the gut, as well as systemic organ damage, suggesting that this toxin might be responsible for MODS, a serious but poorly understood complication of CDI. These findings provide important new insights into the host response to C. difficile during infection and should guide the rational development of urgently required nonantibiotic therapeutics for the treatment of CDI. Clostridium difficile is a leading cause of antibiotic-associated diarrhea and an important hospital pathogen. TcdA and TcdB are thought to be the primary virulence factors responsible for disease symptoms of C. difficile infections (CDI). However, the individual contributions of these toxins to disease remain contentious. Using three different animal models of infection, we show for the first time that TcdB alone causes severe damage to the gut, as well as systemic organ damage, suggesting that this toxin might be responsible for MODS, a serious but poorly understood complication of CDI. These findings provide important new insights into the host response to C. difficile during infection and should guide the rational development of urgently required nonantibiotic therapeutics for the treatment of CDI.

Emergence of a Ribotype 244 Strain of Clostridium difficile Associated With Severe Disease and Related to the Epidemic Ribotype 027 Strain

BACKGROUND We identified 12 patients with Clostridium difficile infection between July 2011 and March 2012 from whom an unusual C. difficile strain was isolated. This strain had a single-nucleotide deletion of the tcdC gene at position 117 and binary toxin genes, which are characteristic of the hypervirulent ribotype (RT) 027 strain. METHODS A retrospective cohort study of 12 patients infected with C. difficile RT244 and 24 patients infected with non-RT244/non-RT027 strains matched for place of diagnosis and time of collection of specimen was performed. We performed whole-genome sequencing to understand the relationship of the RT244 strain to other C. difficile strains and further understand its virulence potential. RESULTS Clostridium difficile RT244 was associated with more severe disease and a higher mortality rate. Phylogenomic analysis using core genome single-nucleotide polymorphisms showed that RT244 is in the same genetic clade (clade 2) as RT027 but is distinct from all RT027 strains. The pathogenicity locus of the RT244 strain encodes a variant toxin B, and this was confirmed by demonstration of Clostridium sordellii-like cytopathic effect on Vero cells. Toxin B production in culture supernatants was lower than that seen with a RT027 strain. CONCLUSIONS Our findings demonstrate the pathogenic potential of this RT244 C. difficile strain and emphasize the importance of ongoing surveillance for emergent strains.

The VirSR Two-Component Signal Transduction System Regulates NetB Toxin Production in Clostridium perfringens

ABSTRACT Clostridium perfringens causes several diseases in domestic livestock, including necrotic enteritis in chickens, which is of concern to the poultry industry due to its health implications and associated economic cost. The novel pore-forming toxin NetB is a critical virulence factor in the pathogenesis of this disease. In this study, we have examined the regulation of NetB toxin production. In C. perfringens, the quorum sensing-dependent VirSR two-component signal transduction system regulates genes encoding several toxins and extracellular enzymes. Analysis of the sequence upstream of the netB gene revealed the presence of potential DNA binding sites, or VirR boxes, that are recognized by the VirR response regulator. In vitro binding experiments showed that purified VirR was able to recognize and bind to these netB-associated VirR boxes. Furthermore, using a reporter gene assay, the netB VirR boxes were shown to be functional. Mutation of the virR gene in two avian C. perfringens strains was shown to significantly reduce the production of the NetB toxin; culture supernatants derived from these strains were no longer cytotoxic to Leghorn male hepatoma cells. Complementation with the virRS operon restored the toxin phenotypes to wild type. The results also showed that the VirSR two-component system regulates the expression of netB at the level of transcription. We postulate that in the gastrointestinal tract of infected birds, NetB production is upregulated when the population of C. perfringens cells reaches a threshold level that leads to activation of the VirSR system.

The role of toxin A and toxin B in Clostridium difficile-associated disease

Recently, we constructed and characterised isogenic tcdA and tcdB mutants of a virulent Clostridium difficile strain and used a hamster model of disease to demonstrate that toxin B, not toxin A, is essential for virulence of this emerging pathogen. Earlier studies had shown that purified toxin A alone was able to induce C. difficile disease pathology and that purified toxin B was not effective unless it was co-administered with toxin A, suggesting that the toxins act synergistically. In this addendum we discuss this paradigm-shifting conclusion in the context of current strain epidemiology, particularly with respect to naturally occurring toxin A-negative, toxin B-positive isolates and the NAP1/027 epidemic isolates. The role of toxin receptors and how variant toxins might exert their effects is also discussed in relation to the published data. We conclude that it is critical to use the natural infection process to dissect the role of toxins in disease, and that future studies are contingent on such work. The impact and importance of animal models of C. difficile virulence are therefore considered within this frame of reference.

Spo0A differentially regulates toxin production in evolutionarily diverse strains of Clostridium difficile.

Clostridium difficile is an important pathogen of humans and animals, representing a significant global healthcare problem. The last decade has seen the emergence of epidemic BI/NAP1/027 and ribotype 078 isolates, associated with the onset of more severe disease and higher rates of morbidity and mortality. However, little is known about these isolates at the molecular level, partly due to difficulties in the genetic manipulation of these strains. Here we report the development of an optimised Tn916-mediated plasmid transfer system, and the use of this system to construct and complement spo0A mutants in a number of different C. difficile strain backgrounds. Spo0A is a global regulator known to control sporulation, but may also be involved in the regulation of potential virulence factors and other phenotypes. Recent studies have failed to elucidate the role of Spo0A in toxin A and toxin B production by C. difficile, with conflicting data published to date. In this study, we aimed to clarify the role of Spo0A in production of the major toxins by C. difficile. Through the construction and complementation of spo0A mutants in two ribotype 027 isolates, we demonstrate that Spo0A acts as a negative regulator of toxin A and toxin B production in this strain background. In addition, spo0A was disrupted and subsequently complemented in strain 630Δerm and, for the first time, in a ribotype 078 isolate, JGS6133. In contrast to the ribotype 027 strains, Spo0A does not appear to regulate toxin production in strain 630Δerm. In strain JGS6133, Spo0A appears to negatively regulate toxin production during early stationary phase, but has little effect on toxin expression during late stationary phase. These data suggest that Spo0A may differentially regulate toxin production in phylogenetically distinct C. difficile strain types. In addition, Spo0A may be involved in regulating some aspects of C. difficile motility.

Clostridium difficile virulence factors: Insights into an anaerobic spore-forming pathogen

The worldwide emergence of epidemic strains of Clostridium difficile linked to increased disease severity and mortality has resulted in greater research efforts toward determining the virulence factors and pathogenesis mechanisms used by this organism to cause disease. C. difficile is an opportunist pathogen that employs many factors to infect and damage the host, often with devastating consequences. This review will focus on the role of the 2 major virulence factors, toxin A (TcdA) and toxin B (TcdB), as well as the role of other putative virulence factors, such as binary toxin, in C. difficile-mediated infection. Consideration is given to the importance of spores in both the initiation of disease and disease recurrence and also to the role that surface proteins play in host interactions.

TcsL Is an Essential Virulence Factor in Clostridium sordellii ATCC 9714

ABSTRACT Clostridium sordellii is an important pathogen of humans and animals, causing a range of diseases, including myonecrosis, sepsis, and shock. Although relatively rare in humans, the incidence of disease is increasing, and it is associated with high mortality rates, approaching 70%. Currently, very little is known about the pathogenesis of C. sordellii infections or disease. Previous work suggested that the lethal large clostridial glucosylating toxin TcsL is the major virulence factor, but a lack of genetic tools has hindered our ability to conclusively assign a role for TcsL or, indeed, any of the other putative virulence factors produced by this organism. In this study, we have developed methods for the introduction of plasmids into C. sordellii using RP4-mediated conjugation from Escherichia coli and have successfully used these techniques to insertionally inactivate the tcsL gene in the reference strain ATCC 9714, using targetron technology. Virulence testing revealed that the production of TcsL is essential for the development of lethal infections by C. sordellii ATCC 9714 and also contributes significantly to edema seen during uterine infection. This study represents the first definitive identification of a virulence factor in C. sordellii and opens the way for in-depth studies of this important human pathogen at the molecular level.