C. Difficile Infection Mouse Model Research Articles

Host metabolic reprogramming ameliorates disease severity and colitis during Clostridioides difficileinfection

Abstract Clostridioides difficile is an anaerobic, opportunistic bacterium that can cause severe infections in the gastrointestinal tract upon disruption of gut microbiome. C. difficile infection (CDI) affects 450,000 people yearly only in the U.S. The high recurrence rates associated to antibiotic regimen, the current standard of care, highlight the need to investigate novel antimicrobial free therapeutic approaches for the treatment of CDI. Here, we have conducted a global transcriptomics analysis and metabolic computational modeling, using our developed M 2pipeline, of an in vivo murine 10-day time course study of CDI to identify novel host metabolic mechanisms with therapeutic potential. Our analysis has reported a biphasic modulation of colonic metabolism, characterized by increased glycolysis during the induction of effector responses, while mitochondrial metabolism is favored in the recovery phase of the infection, when regulatory and healing processes are predominant. Inhibition of glycolysis through administration of 2-Deoxy-D-glucose (2-DG) ameliorates diseases severity, protects from mortality, and reduces colitis in a mouse model of CDI. In vitro treatment with 2-DG, an inhibitor of glycolysis, in T-84 cells exposed to C. difficile toxins A and B protects epithelial barrier integrity and decreases secretion of inflammatory mediators. In conclusion, host immunometabolic reprogramming represents a promising novel antimicrobial-free therapeutic approach for CDI. Supported by a grant from DTRA (HDTRA1-20-1-0021)

Butyrate Differentiates Permissiveness to Clostridioides difficile Infection and Influences Growth of Diverse C. difficile Isolates.

A disrupted "dysbiotic" gut microbiome engenders susceptibility to the diarrheal pathogen Clostridioides difficile by impacting the metabolic milieu of the gut. Diet, in particular the microbiota-accessible carbohydrates (MACs) found in dietary fiber, is one of the most powerful ways to affect the composition and metabolic output of the gut microbiome. As such, diet is a powerful tool for understanding the biology of C. difficile and for developing alternative approaches for coping with this pathogen. One prominent class of metabolites produced by the gut microbiome is short-chain fatty acids (SCFAs), the major metabolic end products of MAC metabolism. SCFAs are known to decrease the fitness of C. difficile in vitro, and high intestinal SCFA concentrations are associated with reduced fitness of C. difficile in animal models of C. difficile infection (CDI). Here, we use controlled dietary conditions (8 diets that differ only by MAC composition) to show that C. difficile fitness is most consistently impacted by butyrate, rather than the other two prominent SCFAs (acetate and propionate), during murine model CDI. We similarly show that butyrate concentrations are lower in fecal samples from humans with CDI than in those from healthy controls. Finally, we demonstrate that butyrate impacts growth in diverse C. difficile isolates. These findings provide a foundation for future work which will dissect how butyrate directly impacts C. difficile fitness and will lead to the development of diverse approaches distinct from antibiotics or fecal transplant, such as dietary interventions, for mitigating CDI in at-risk human populations. IMPORTANCE Clostridioides difficile is a leading cause of infectious diarrhea in humans, and it imposes a tremendous burden on the health care system. Current treatments for C. difficile infection (CDI) include antibiotics and fecal microbiota transplant, which contribute to recurrent CDIs and face major regulatory hurdles, respectively. Therefore, there is an ongoing need to develop new ways to cope with CDI. Notably, a disrupted "dysbiotic" gut microbiota is the primary risk factor for CDI, but we incompletely understand how a healthy microbiota resists CDI. Here, we show that a specific molecule produced by the gut microbiota, butyrate, is negatively associated with C. difficile burdens in humans and in a mouse model of CDI and that butyrate impedes the growth of diverse C. difficile strains in pure culture. These findings help to build a foundation for designing alternative, possibly diet-based, strategies for mitigating CDI in humans.

Peroxisome proliferator-activated receptor-γ as the gatekeeper of tight junction in Clostridioides difficile infection.

Clostridioides difficile is a major causative pathogen of nosocomial antibiotic-associated diarrhea and severe colitis. Despite the use of vancomycin and fidaxomicin as standard drugs for the treatment of C. difficile infection (CDI), clinical relapse rates remain high. Therefore, new alternative therapeutics to treat CDI are urgently required. The nuclear receptor, peroxisome proliferator-activated receptor-γ (PPAR-γ), is mainly expressed in the adipose tissue and modulates lipid metabolism and insulin sensitization. Previous studies have shown that PPAR-γ is highly expressed in colonic tissues and regulates tight junction function in epithelial cells. However, the role of PPAR-γ in CDI pathogenesis remains unclear. In this study, we used a mouse model of CDI and found that both expression levels of PPAR-γ and the tight junction protein, occludin, were decreased in colonic tissues. Furthermore, to investigate the role of PPAR-γ in CDI, we used PPAR-γ defective mice and found that intestinal permeability and bacterial dissemination in these mice were significantly higher than those in wild-type mice during CDI. Administration of the PPAR-γ agonist, pioglitazone, to activate PPAR-γ activity improved the phenotypes of CDI, including bodyweight loss, inflammation, and intestinal integrity. Taken together, these results demonstrate that PPAR-γ is a potential therapeutic target in CDI, as it modulates colonic inflammation and integrity.

The Role of Toll-Like Receptor-2 in Clostridioides difficile Infection: Evidence From a Mouse Model and Clinical Patients.

Background Clostridioides difficile is the leading cause of nosocomial infectious diarrhea. Toll-like receptors (TLRs) are the major components of innate immunity that sense pathogens. The relationship between TLRs and C. difficile infection (CDI) was analyzed in clinical patients and a mouse model.Materials and MethodsA prospective investigation was conducted in medical wards of Tainan Hospital, Ministry of Health and Welfare, Tainan, Taiwan, from January 2011 to January 2013. Adult patients were followed up for the development of CDI. Single nucleotide polymorphisms (SNPs) of TLR2 and TLR4 were analyzed to assess the relationship between genetic polymorphisms and the development of CDI. A mouse model of CDI was used to investigate the pathogenic role of TLRs in CDI, TLR2 and TLR4 knockout (Tlr2-/- and Tlr4-/-) mice.ResultsIn the prospective study, 556 patients were enrolled, and 6.5% (36) of patients, accounting for 3.59 episodes per 1000 patient-days, developed CDI. Of 539 patients with available blood samples, the TLR2 rs3804099 polymorphism was more often noted in those with CDI than in those without CDI (64.5% vs. 46.1%; P = 0.046) but was not significant in multivariate analysis. Because the TLR2 rs3804099 polymorphism was moderately associated with CDI, the role of TLR2 and TLR4 was further evaluated in a mouse model. Both Tlr2-/- and Tlr4-/- mice showed more severe CDI disease than wild-type mice in terms of body weight change and fecal content five days after oral challenge with C. difficile. Furthermore, Tlr2-/- mice suffered from more severe disease than Tlr4-/- mice, as evidenced by stool consistency, cecum weight, and survival rate.ConclusionThe TLR2 rs3804099 polymorphism is marginally associated with the development of CDI, and the pathogenic role of TLR2 is further supported by a mouse model.

Age-associated defects in the adaptive immune response to Clostridium difficile infection impair the development of a protective immune response in the elderly

Abstract Clostridium difficile is the number one cause of healthcare-associated infection in the United States and the most lethal acute enteric pathogen. The elderly (≥65) have the greatest risk of C. difficile infection (CDI) and severe morbidity and mortality due to CDI. Additionally, most recurrent CDIs occur within the elderly. To better understand why the elderly are at greater risk of severe and recurrent CDI, our lab has conducted a longitudinal study of the aging immune response to primary and recurrent CDI in patients at Hahnemann University Hospital. Both elderly and young CDI patients were enrolled in the study, and stool and blood samples were collected at enrollment and 12 days after diagnosis of CDI. Analysis of the samples looking at the humoral response in elderly versus young CDI patients shows that the early humoral response in elderly patients is impaired when compared to young CDI patients. In addition, recurrent CDI patients have similarly defective humoral responses. Finally, preliminary work in an aging mouse model of CDI further supports that age-related defects within the adaptive immune response impair the development of a protective immune response. Since the adaptive immune response to CDI plays an important role in neutralizing toxins produced by C. difficile, an impaired humoral response in both aged and recurrent CDI patients may contribute to the increased risk of infection, morbidity, and mortality observed in elderly patients. Future studies will seek to further characterize the aging immune response using patient samples as well as aging mouse models of CDI.

Fecal host biomarkers predicting severity of Clostridioides difficile infection.

Clostridioides difficile is a major cause of health care–associated diarrhea. Severity ranges from mild to life-threatening, but this variability remains poorly understood. Microbiologic diagnosis of C. difficile infection (CDI) is straightforward but offers little insight into the patient’s prognosis or into pathophysiologic determinants of clinical trajectory. The aim of this study was to discover host-derived, CDI-specific fecal biomarkers involved in disease severity. Subjects without and with CDI diarrhea were recruited. CDI severity was based on Infectious Diseases Society of America/Society for Healthcare Epidemiology of America criteria. We developed a liquid chromatography tandem mass spectrometry approach to identify host-derived protein biomarkers from stool and applied it to diagnostic samples for cohort-wise comparison (CDI-negative vs. nonsevere CDI vs. severe CDI). Selected biomarkers were orthogonally confirmed and subsequently verified in a CDI mouse model. We identified a protein signature from stool, consisting of alpha-2-macroglobulin (A2MG), matrix metalloproteinase-7 (MMP-7), and alpha-1-antitrypsin (A1AT), that not only discriminates CDI-positive samples from non-CDI ones but also is potentially associated with disease severity. In the mouse model, this signature with the murine homologs of the corresponding proteins was also identified. A2MG, MMP-7, and A1AT serve as biomarkers in patients with CDI and define novel components of the host response that may determine disease severity.

Auranofin, at clinically achievable dose, protects mice and prevents recurrence from Clostridioides difficile infection

Clostridioides difficile is the leading cause of nosocomial infections and a worldwide urgent public health threat. Without doubt, there is an urgent need for new effective anticlostridial agents due to the increasing incidence and severity of C. difficile infection (CDI). The aim of the present study is to investigate the in vivo efficacy of auranofin (rheumatoid arthritis FDA-approved drug) in a CDI mouse model and establish an adequate dosage for treatment. The effects of increased C. difficile inoculum, and pre-exposure to simulated gastric intestinal fluid (SGF) and simulated intestinal fluid (SIF), on the antibacterial activity of auranofin were investigated. Auranofin’s in vitro antibacterial activity was stable in the presence of high bacterial inoculum size compared to vancomycin and fidaxomicin. Moreover, it maintained its anti-C. difficile activity after being exposed to SGF and SIF. Upon testing in a CDI mouse model, auranofin at low clinically achievable doses (0.125 mg/kg and 0.25 mg/kg) significantly protected mice against CDI with 100% and 80% survival, respectively. Most importantly, auranofin (0.125 mg/kg and 0.25 mg/kg) significantly prevented CDI recurrence when compared with vancomycin. Collectively, these results indicate that auranofin could potentially provide an effective, safe and quick supplement to the current approaches for treating CDI.

In Vivo Targeting of Clostridioides difficile Using Phage-Delivered CRISPR-Cas3 Antimicrobials.

Clostridioides difficile is an important nosocomial pathogen that causes approximately 500,000 cases of C. difficile infection (CDI) and 29,000 deaths annually in the United States. Antibiotic use is a major risk factor for CDI because broad-spectrum antimicrobials disrupt the indigenous gut microbiota, decreasing colonization resistance against C. difficile Vancomycin is the standard of care for the treatment of CDI, likely contributing to the high recurrence rates due to the continued disruption of the gut microbiota. Thus, there is an urgent need for the development of novel therapeutics that can prevent and treat CDI and precisely target the pathogen without disrupting the gut microbiota. Here, we show that the endogenous type I-B CRISPR-Cas system in C. difficile can be repurposed as an antimicrobial agent by the expression of a self-targeting CRISPR that redirects endogenous CRISPR-Cas3 activity against the bacterial chromosome. We demonstrate that a recombinant bacteriophage expressing bacterial genome-targeting CRISPR RNAs is significantly more effective than its wild-type parent bacteriophage at killing C. difficile both in vitro and in a mouse model of CDI. We also report that conversion of the phage from temperate to obligately lytic is feasible and contributes to the therapeutic suitability of intrinsic C. difficile phages, despite the specific challenges encountered in the disease phenotypes of phage-treated animals. Our findings suggest that phage-delivered programmable CRISPR therapeutics have the potential to leverage the specificity and apparent safety of phage therapies and improve their potency and reliability for eradicating specific bacterial species within complex communities, offering a novel mechanism to treat pathogenic and/or multidrug-resistant organisms.IMPORTANCEClostridioides difficile is a bacterial pathogen responsible for significant morbidity and mortality across the globe. Current therapies based on broad-spectrum antibiotics have some clinical success, but approximately 30% of patients have relapses, presumably due to the continued perturbation to the gut microbiota. Here, we show that phages can be engineered with type I CRISPR-Cas systems and modified to reduce lysogeny and to enable the specific and efficient targeting and killing of C. difficilein vitro and in vivo. Additional genetic engineering to disrupt phage modulation of toxin expression by lysogeny or other mechanisms would be required to advance a CRISPR-enhanced phage antimicrobial for C. difficile toward clinical application. These findings provide evidence into how phage can be combined with CRISPR-based targeting to develop novel therapies and modulate microbiomes associated with health and disease.

Bacteroides fragilis Prevents Clostridium difficile Infection in a Mouse Model by Restoring Gut Barrier and Microbiome Regulation.

Clostridium difficile is currently the leading cause of nosocomial infection. Antibiotics remain the first-line therapy for C. difficile-associated diseases (CDAD), despite the risks of resistance promotion and further gut microbiota perturbation. Notably, the abundance of Bacteroides fragilis was reported to be significantly decreased in CDAD patients. This study aimed to clarify the prophylactic effects of B. fragilis strain ZY-312 in a mouse model of C. difficile infection (CDI). The CDI mouse model was successfully created using C. difficile strain VPI 10463 spores, as confirmed by lethal diarrhea (12.5% survival rate), serious gut barrier disruption, and microbiota disruption. CDI model mice prophylactically treated with B. fragilis exhibited significantly higher survival rates (100% in low dosage group, 87.5% in high dosage group) and improved clinical manifestations. Histopathological analysis of colon and cecum tissue samples revealed an intact gut barrier with strong ZO-1 and Muc-2 expression. The bacterial diversity and relative abundance of gut microbiota were significantly improved. Interestingly, the relative abundance of Akkermansia muciniphila was positively correlated with B. fragilis treatment. In vitro experiments showed that B. fragilis inhibited C. difficile adherence, and attenuated the decrease in CDI-induced transepithelial electrical resistance, ZO-1 and MUC-2 loss, and apoptosis, suggesting that B. fragilis protected against CDI possibly by resisting pathogen colonization and improving gut barrier integrity and functions. In summary, B. fragilis exerted protective effects on a CDI mouse model by modulating gut microbiota and alleviating barrier destruction, thereby relieving epithelial stress and pathogenic colitis triggered by C. difficile. This study provides an alternative preventative measure for CDI and lays the foundations for further investigations of the relationships among opportunistic pathogens, commensal microbiota, and the gut barrier.

Characterization of the virulence of a non-RT027, non-RT078 and binary toxin-positive Clostridium difficile strain associated with severe diarrhea

The expression of the Clostridium difficile binary toxin CDT is generally observed in the RT027 (ST1) and RT078 (ST11) C. difficile isolates, which are associated with severe C. difficile infection (CDI). However, we recently reported that the non-RT027 and non-RT078 C. difficile strain LC693 (TcdA+TcdB+ CDT+, ST201) caused severe diarrhea in a patient in Xiangya Hospital in China. C.difficile LC693 is a member of Clade 3, and in this study, we identified LC693 as RT871 and compared its virulence and pathogenicity to those of C.difficile R20291 (TcdA+TcdB+CDT+, ST1/RT027), UK6 (TcdA+TcdB+CDT+, ST35/RT027), CD630 (TcdA+TcdB+CDT−, ST54, RT012), and 1379 (TcdA+TcdB+CDT−, ST54/RT012), with strain 1379 being an epidemic C.difficile isolate from the same hospital. LC693 displayed a higher sporulation rate than R20291, CD630 or strain 1379. LC693 was comparable to R20291 with respect to spore germination, motility, and biofilm formation, but showed a faster germination rate, higher motility and a higher biofilm formation capability compared to CD630 and strain 1379. The adherence of spores to human gut epithelial cells was similar for all strains.The total toxin release of LC693 was lower than that of R20291, but higher than that of CD630 and strain 1379. Finally, in a mouse model of CDI, LC693 was capable of causing moderate to severe disease. Our findings demonstrate the pathogenicity of non-RT027 and non-RT078 binary toxin-positive C. difficile strains. Furthermore, our data indicate that LC693 may be more virulent than strain 1379, an epidemic strain from the same hospital, and provide the first phenotypic characterization of a non-RT027 and non-RT078 binary toxin-positive ST201 isolate.

Para-cresol production by Clostridium difficile affects microbial diversity and membrane integrity of Gram-negative bacteria.

Clostridium difficile is a Gram-positive spore-forming anaerobe and a major cause of antibiotic-associated diarrhoea. Disruption of the commensal microbiota, such as through treatment with broad-spectrum antibiotics, is a critical precursor for colonisation by C. difficile and subsequent disease. Furthermore, failure of the gut microbiota to recover colonisation resistance can result in recurrence of infection. An unusual characteristic of C. difficile among gut bacteria is its ability to produce the bacteriostatic compound para-cresol (p-cresol) through fermentation of tyrosine. Here, we demonstrate that the ability of C. difficile to produce p-cresol in vitro provides a competitive advantage over gut bacteria including Escherichia coli, Klebsiella oxytoca and Bacteroides thetaiotaomicron. Metabolic profiling of competitive co-cultures revealed that acetate, alanine, butyrate, isobutyrate, p-cresol and p-hydroxyphenylacetate were the main metabolites responsible for differentiating the parent strain C. difficile (630Δerm) from a defined mutant deficient in p-cresol production. Moreover, we show that the p-cresol mutant displays a fitness defect in a mouse relapse model of C. difficile infection (CDI). Analysis of the microbiome from this mouse model of CDI demonstrates that colonisation by the p-cresol mutant results in a distinctly altered intestinal microbiota, and metabolic profile, with a greater representation of Gammaproteobacteria, including the Pseudomonales and Enterobacteriales. We demonstrate that Gammaproteobacteria are susceptible to exogenous p-cresol in vitro and that there is a clear divide between bacterial Phyla and their susceptibility to p-cresol. In general, Gram-negative species were relatively sensitive to p-cresol, whereas Gram-positive species were more tolerant. This study demonstrates that production of p-cresol by C. difficile has an effect on the viability of intestinal bacteria as well as the major metabolites produced in vitro. These observations are upheld in a mouse model of CDI, in which p-cresol production affects the biodiversity of gut microbiota and faecal metabolite profiles, suggesting that p-cresol production contributes to C. difficile survival and pathogenesis.

The role of zinc and nutritional immunity in Clostridium difficile infection

Clostridium difficile in one of the most commonly reported nosocomial pathogens worldwide. Beyond antibiotic use, little is known about the host, microbiota, and environmental factors that contribute to susceptibility to and severity of C. difficile infection (CDI). We recently observed that in a mouse model of CDI, excess dietary zinc (Zn) alters the gut microbiota and decreases resistance to CDI. Moreover, we determined that high levels of Zn exacerbate C. difficile-associated disease and calprotectin-mediated Zn limitation is an essential host response to infection. In this addendum, we discuss how these findings add to our understanding of CDI and consider the potential implications of excess metal intake on the microbiota and infection.

Cefoperazone-treated Mouse Model of Clinically-relevant Clostridium difficile Strain R20291.

Clostridium difficile is an anaerobic, gram-positive, spore-forming enteric pathogen that is associated with increasing morbidity and mortality and consequently poses an urgent threat to public health. Recurrence of a C. difficile infection (CDI) after successful treatment with antibiotics is high, occurring in 20-30% of patients, thus necessitating the discovery of novel therapeutics against this pathogen. Current animal models of CDI result in high mortality rates and thus do not approximate the chronic, insidious disease manifestations seen in humans with CDI. To evaluate therapeutics against C. difficile, a mouse model approximating human disease utilizing a clinically-relevant strain is needed. This protocol outlines the cefoperazone mouse model of CDI using a clinically-relevant and genetically-tractable strain, R20291. Techniques for clinical disease monitoring, C. difficile bacterial enumeration, toxin cytotoxicity, and histopathological changes throughout CDI in a mouse model are detailed in the protocol. Compared to other mouse models of CDI, this model is not uniformly lethal at the dose administered, allowing for the observation of a prolonged clinical course of infection concordant with the human disease. Therefore, this cefoperazone mouse model of CDI proves a valuable experimental platform to assess the effects of novel therapeutics on the amelioration of clinical disease and on the restoration of colonization resistance against C. difficile.

Effect of age on Clostridium difficile infection in a murine model of infection

Clostridium difficile, a Gram-positive, spore-forming bacillus, is the leading cause of nosocomial antibiotic-associated diarrhea in the U.S. A major risk factor for developing C. difficile infection (CDI) is advanced age (>65 years), which also accounts for 93% of CDI-related deaths. It is unknown why CDI becomes more severe with age. The objective of this study was to develop a CDI mouse model to study the effects of aging on the pathogenesis of CDI. To achieve this goal we compared CDI in aged (12 months) versus young (2 months) C57BL/6J mice. Six days after C. difficile spore challenge, clinical symptoms (e.g. wet tail, hunching, dehydration) were exhibited by a greater proportion of aged mice compared to young mice (p=0.08). Aged mice experienced greater weight loss than younger animals (p=0.013) and shed greater numbers of bacteria into the feces (p=0.023). Not surprisingly, aged mice suffered a higher cumulative mortality (44%) compared to the young (20%). Histological examination of cecum and colon tissue revealed the severity of inflammation and mucosal damage was greater in aged mice than younger animals (p=0.004). Because mortality is typically associated with lower anti-toxin antibody titers, splenic B cells (CD45+CD19+) numbers were measured by flow cytometry. In comparison to young mice, the proportion of splenic B cells was significantly reduced in the aged group (p=0.006). These results demonstrate that the consequences of CDI are more severe in aged mice and associated with B cell changes. Utilizing our model will help to advance our understanding of age-related changes on host immune defenses against CDI, which is crucial for designing new prophylactic and treatment strategies to reduce morbidity and mortality in older adults.

Protection from Clostridium difficile Infection in CD4 T Cell- and Polymeric Immunoglobulin Receptor-Deficient Mice

ABSTRACT Clostridium difficile rivals methicillin-resistant Staphylococcus aureus as the primary hospital-acquired infection. C. difficile infection (CDI) caused by toxins A and/or B can manifest as mild diarrhea to life-threatening pseudomembranous colitis. Although most patients recover fully from CDI, ∼20% undergo recurrent disease. Several studies have demonstrated a correlation between anti-toxin antibody (Ab) and decreased recurrence; however, the contributions of the systemic and mucosal Ab responses remain unclear. Our goal was to use the CDI mouse model to characterize the protective immune response to C. difficile . C57BL/6 mice infected with epidemic C. difficile strain BI17 developed protective immunity against CDI and did not develop CDI upon rechallenge; they generated systemic IgG and IgA as well as mucosal IgA Ab to toxin. To determine if protective immunity to C. difficile could be generated in immunodeficient individuals, we infected CD4 −/− mice and found that they generated both mucosal and serum IgA anti-toxin Abs and were protected from CDI upon rechallenge, with protection dependent on major histocompatibility complex class II (MHCII) expression; no IgG anti-toxin Ab was found. We found that protection was likely due to neutralizing mucosal IgA Ab. In contrast, pIgR −/− mice, which lack the receptor to transcytose polymeric Ab across the epithelium, were also protected from CDI, suggesting that although mucosal anti-toxin Ab may contribute to protection, it is not required. We conclude that protection from CDI can occur by several mechanisms and that the mechanism of protection is determined by the state of immunocompetence of the host.

Adaptive immunity in the mouse model of Clostridium difficile infection (43.4)

Abstract Clostridium difficile rivals MRSA as the most common hospital-acquired infection. C. difficile infection (CDI) can manifest as mild diarrhea to life-threatening psuedomembranous colitis. CDI occurs following a course of antibiotics, and although most patients recover fully from CDI, ~20% undergo recurrent disease caused by toxins A and/or B. Several studies demonstrate a correlation between anti-toxin antibody (Ab) and a decrease in recurrence; however; the contribution of the systemic and mucosal Ab response remains unclear. Our goal was to use the recently described mouse model of CDI to characterize the protective immune response. Mice infected with C. difficile developed protective immunity against CDI and did not develop CDI upon re-challenge. Infected mice generated both systemic and mucosal Ab directed against toxin. We infected CD4-/- mice with C. difficile, expecting they would succumb to CDI. Instead, the mice developed immunity and we found IgA anti-toxin Ab in both serum and the mucosa. pIgR-/- mice also developed serum anti-toxin Ab and were protected from CDI, indicating that mucosal anti-toxin Ab is not required for protection from CDI. Our studies suggest that protective anti-toxin Ab can be induced in T-cell deficient individuals and that systemic anti-toxin immunity may be sufficient for protection from CDI. The results should aid in development of vaccines for CDI, especially for individuals with immunodeficiency.