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

mBio ◽  
2015 ◽  
Vol 6 (3) ◽  
Author(s):  
Glen P. Carter ◽  
Anjana Chakravorty ◽  
Tu Anh Pham Nguyen ◽  
Steven Mileto ◽  
Fernanda Schreiber ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Animal Models ◽  
Virulence Factors ◽  
Host Response ◽  
Organ Damage ◽  
Intestinal Damage ◽  
Content Type ◽  
Animal Infection ◽  
Antibiotic Associated Diarrhea ◽  
First Time

ABSTRACTClostridium difficileis 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 ofC. difficileinfection (CDI). Finally, we show that TcdB is the primary factor responsible for inducing thein vivohost 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 ofC. difficileand provide new insights into the host response toC. difficileduring infection. The results also highlight the critical nature of using appropriate and, when possible, multiple animal infection models when studying bacterial virulence mechanisms.IMPORTANCEClostridium difficileis 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 ofC. difficileinfections (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 toC. difficileduring infection and should guide the rational development of urgently required nonantibiotic therapeutics for the treatment of CDI.

scholarly journals Observations on the Role of TcdE Isoforms in Clostridium difficile Toxin Secretion

2015 ◽  
Vol 197 (15) ◽  
pp. 2600-2609 ◽  
Author(s):  
Revathi Govind ◽  
Leah Fitzwater ◽  
Rebekah Nichols
Keyword(s):  
Amino Acids ◽  
Clostridium Difficile ◽  
Cell Viability ◽  
Virulence Factors ◽  
Start Codon ◽  
Nosocomial Pathogen ◽  
Content Type ◽  
Antibiotic Associated Diarrhea ◽  
Toxin Secretion

ABSTRACTClostridium difficileis a major nosocomial pathogen and the principal causative agent of antibiotic-associated diarrhea. The toxigenicC. difficilestrains that cause disease secrete virulence factors, toxin A and toxin B, that cause colonic injury and inflammation.C. difficiletoxins have no export signature and are secreted by an unusual mechanism that involves TcdE, a holin-like protein. We isolated a TcdE mutant of the epidemic R20291 strain with impaired toxin secretion, which was restored by complementation with functional TcdE. In the TcdE open reading frame (ORF), we identified three possible translation start sites; each translated isoform may play a specific role in TcdE-controlled toxin release. We created plasmid constructs that express only one of the three TcdE isoforms and complemented the TcdE mutant with these isoforms. Western blot analysis of the complemented strains demonstrated that TcdE is translated efficiently from the start codon at the 25th and 27th positions in the predicted ORF, producing proteins with 142 amino acids (TcdE142) and 140 amino acids (TcdE140), respectively. TcdE166was not detected when expressed from its own ribosomal binding site (RBS). The effects of all three TcdE isoforms onC. difficilecell viability and toxin release were determined. Among the three isoforms, overexpression of TcdE166and TcdE142had a profound effect on cell viability compared to the TcdE140isoform. Similarly, TcdE166and TcdE142facilitated toxin release more efficiently than did TcdE140. The importance of these variations among TcdE isoforms and their role in toxin release are discussed.IMPORTANCEC. difficileis a nosocomial pathogen that has become the most prevalent cause of antibiotic-associated diarrhea in North America and in several countries in Europe. Most strains ofC. difficileproduce two high-molecular-weight toxins that are regarded as the primary virulence factors. The mechanism by which these large toxins are secreted from bacterial cells is not yet clear but involves TcdE, a holin-like protein. In this work, we show that TcdE could be translated from three different start codons, resulting in the production of three TcdE isoforms. Furthermore, we investigated the role of these isoforms in toxin release and cell lysis inC. difficile. An understanding of TcdE-dependent toxin secretion may be helpful for the development of strategies for preventing and treatingC. difficileinfections.

scholarly journals Finding of Agr Phase Variants in Staphylococcus aureus

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Vishal Gor ◽  
Aya J. Takemura ◽  
Masami Nishitani ◽  
Masato Higashide ◽  
Veronica Medrano Romero ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Virulence Factors ◽  
Phase Variation ◽  
Accessory Gene ◽  
Content Type ◽  
Accessory Gene Regulator ◽  
Immune Mediated ◽  
A Minor ◽  
First Time ◽  
Phase Variants

ABSTRACT Staphylococcus aureus is an important human pathogen whose success is largely attributed to its vast arsenal of virulence factors that facilitate its invasion into, and survival within, the human host. The expression of these virulence factors is controlled by the quorum sensing accessory gene regulator (Agr) system. However, a large proportion of clinical S. aureus isolates are consistently found to have a mutationally inactivated Agr system. These mutants have a survival advantage in the host but are considered irreversible mutants. Here we show, for the first time, that a fraction of Agr-negative mutants can revert their Agr activity. By serially passaging Agr-negative strains and screening for phenotypic reversion of hemolysis and subsequent sequencing, we identified two mutational events responsible for reversion: a genetic duplication plus inversion event and a poly(A) tract alteration. Additionally, we demonstrate that one clinical Agr-negative methicillin-resistant S. aureus (MRSA) isolate could reproducibly generate Agr-revertant colonies with a poly(A) tract genetic mechanism. We also show that these revertants activate their Agr system upon phagocytosis. We propose a model in which a minor fraction of Agr-negative S. aureus strains are phase variants that can revert their Agr activity and may act as a cryptic insurance strategy against host-mediated stress. IMPORTANCE Staphylococcus aureus is responsible for a broad range of infections. This pathogen has a vast arsenal of virulence factors at its disposal, but avirulent strains are frequently isolated as the cause of clinical infections. These isolates have a mutated agr locus and have been believed to have no evolutionary future. Here we show that a fraction of Agr-negative strains can repair their mutated agr locus with mechanisms resembling phase variation. The agr revertants sustain an Agr OFF state as long as they exist as a minority but can activate their Agr system upon phagocytosis. These revertant cells might function as a cryptic insurance strategy to survive immune-mediated host stress that arises during infection.

scholarly journals Evidence ofIn VivoProphage Induction during Clostridium difficile Infection

2012 ◽  
Vol 78 (21) ◽  
pp. 7662-7670 ◽  
Author(s):  
Mathieu Meessen-Pinard ◽  
Ognjen Sekulovic ◽  
Louis-Charles Fortier
Keyword(s):  
Clostridium Difficile ◽  
Bioinformatics Analyses ◽  
Content Type ◽  
Unique Character ◽  
Viral Particles ◽  
Potential Impact ◽  
First Time ◽  
Culture Supernatants

ABSTRACTProphages contribute to the evolution and virulence of most bacterial pathogens, but their role inClostridium difficileis unclear. Here we describe the isolation of fourMyoviridaephages, ϕMMP01, ϕMMP02, ϕMMP03, and ϕMMP04, that were recovered as free viral particles in the filter-sterilized stool supernatants of patients suffering fromC. difficileinfection (CDI). Furthermore, identical prophages were found in the chromosomes ofC. difficileisolated from the corresponding fecal samples. We therefore provide, for the first time, evidence ofin vivoprophage induction during CDI. We completely sequenced the genomes of ϕMMP02 and ϕMMP04, and bioinformatics analyses did not reveal the presence of virulence factors but underlined the unique character of ϕMMP04. We also studied the mobility of ϕMMP02 and ϕMMP04 prophagesin vitro. Both prophages were spontaneously induced, with 4 to 5 log PFU/ml detected in the culture supernatants of the corresponding lysogens. When lysogens were grown in the presence of subinhibitory concentrations of ciprofloxacin, moxifloxacin, levofloxacin, or mitomycin C, the phage titers further increased, reaching 8 to 9 log PFU/ml in the case of ϕMMP04. In summary, our study highlights the extensive genetic diversity and mobility ofC. difficileprophages. Moreover, antibiotics known to represent risk factors for CDI, such as quinolones, can stimulate prophage mobilityin vitroand probablyin vivoas well, which underscores their potential impact on phage-mediated horizontal gene transfer events and the evolution ofC. difficile.

scholarly journals Clostridium difficileLipoprotein GerS Is Required for Cortex Modification and Thus Spore Germination

mSphere ◽  
2018 ◽  
Vol 3 (3) ◽  
Author(s):  
Oscar R. Diaz ◽  
Cameron V. Sayer ◽  
David L. Popham ◽  
Aimee Shen
Keyword(s):  
Clostridium Difficile ◽  
Spore Germination ◽  
Protective Layer ◽  
Thick Layer ◽  
Lytic Enzyme ◽  
Gram Positive ◽  
Content Type ◽  
Clostridioides Difficile ◽  
Antibiotic Associated Diarrhea ◽  
Biochemical Analyses

ABSTRACTClostridium difficile, also known asClostridioides difficile, is a Gram-positive, spore-forming bacterium that is a leading cause of antibiotic-associated diarrhea.C. difficileinfections begin when its metabolically dormant spores germinate to form toxin-producing vegetative cells. Successful spore germination depends on the degradation of the cortex, a thick layer of modified peptidoglycan that maintains dormancy. Cortex degradation is mediated by the SleC cortex lytic enzyme, which is thought to recognize the cortex-specific modification muramic-δ-lactam.C. difficilecortex degradation also depends on thePeptostreptococcaceae-specific lipoprotein GerS for unknown reasons. In this study, we tested whether GerS regulates production of muramic-δ-lactam and thus controls the ability of SleC to recognize its cortex substrate. By comparing the muropeptide profiles of ΔgerSspores to those of spores lacking either CwlD or PdaA, both of which mediate cortex modification inBacillus subtilis, we determined thatC. difficileGerS, CwlD, and PdaA are all required to generate muramic-δ-lactam. Both GerS and CwlD were needed to cleave the peptide side chains from N-acetylmuramic acid, suggesting that these two factors act in concert. Consistent with this hypothesis, biochemical analyses revealed that GerS and CwlD directly interact and that CwlD modulates GerS incorporation into mature spores. Since ΔgerS, ΔcwlD, and ΔpdaAspores exhibited equivalent germination defects, our results indicate thatC. difficilespore germination depends on cortex-specific modifications, reveal GerS as a novel regulator of these processes, and highlight additional differences in the regulation of spore germination inC. difficilerelative toB. subtilisand other spore-forming organisms.IMPORTANCEThe Gram-positive, spore-forming bacteriumClostridium difficileis a leading cause of antibiotic-associated diarrhea. BecauseC. difficileis an obligate anaerobe, its aerotolerant spores are essential for transmitting disease, and their germination into toxin-producing cells is necessary for causing disease. Spore germination requires the removal of the cortex, a thick layer of modified peptidoglycan that maintains spore dormancy. Cortex degradation is mediated by the SleC hydrolase, which is thought to recognize cortex-specific modifications. Cortex degradation also requires the GerS lipoprotein for unknown reasons. In our study, we tested whether GerS is required to generate cortex-specific modifications by comparing the cortex composition of ΔgerSspores to the cortex composition of spores lacking two putative cortex-modifying enzymes, CwlD and PdaA. These analyses revealed that GerS, CwlD, and PdaA are all required to generate cortex-specific modifications. Since loss of these modifications in ΔgerS, ΔcwlD, and ΔpdaAmutants resulted in spore germination and heat resistance defects, the SleC cortex lytic enzyme depends on cortex-specific modifications to efficiently degrade this protective layer. Our results further indicate that GerS and CwlD are mutually required for removing peptide chains from spore peptidoglycan and revealed a novel interaction between these proteins. Thus, our findings provide new mechanistic insight intoC. difficilespore germination.

scholarly journals Clostridium difficile Toxins TcdA and TcdB Cause Colonic Tissue Damage by Distinct Mechanisms

2016 ◽  
Vol 84 (10) ◽  
pp. 2871-2877 ◽  
Author(s):  
Nicole M. Chumbler ◽  
Melissa A. Farrow ◽  
Lynne A. Lapierre ◽  
Jeffrey L. Franklin ◽  
D. Borden Lacy
Keyword(s):  
Clostridium Difficile ◽  
Apoptotic Cell ◽  
Large Body ◽  
Health Care Facilities ◽  
Dependent Manner ◽  
Content Type ◽  
Culture Model ◽  
Care Facilities ◽  
Antibiotic Associated Diarrhea ◽  
A Cell

As the major cause of antibiotic-associated diarrhea,Clostridium difficileis a serious problem in health care facilities worldwide.C. difficileproduces two large toxins, TcdA and TcdB, which are the primary virulence factors in disease. The respective functions of these toxins have been difficult to discern, in part because the cytotoxicity profiles for these toxins differ with concentration and cell type. The goal of this study was to develop a cell culture model that would allow a side-by-side mechanistic comparison of the toxins. Conditionally immortalized, young adult mouse colonic (YAMC) epithelial cells demonstrate an exquisite sensitivity to both toxins with phenotypes that agree with observations in tissue explants. TcdA intoxication results in an apoptotic cell death that is dependent on the glucosyltransferase activity of the toxin. In contrast, TcdB has a bimodal mechanism; it induces apoptosis in a glucosyltransferase-dependent manner at lower concentrations and glucosyltransferase-independent necrotic death at higher concentrations. The direct comparison of the responses to TcdA and TcdB in cells and colonic explants provides the opportunity to unify a large body of observations made by many independent investigators.

scholarly journals The HtrA-Like Protease CD3284 Modulates Virulence of Clostridium difficile

2014 ◽  
Vol 82 (10) ◽  
pp. 4222-4232 ◽  
Author(s):  
Dennis Bakker ◽  
Anthony M. Buckley ◽  
Anne de Jong ◽  
Vincent J. C. van Winden ◽  
Joost P. A. Verhoeks ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Virulence Factors ◽  
Pathogenic Bacteria ◽  
Microarray Data Analysis ◽  
Toxin A ◽  
Hamster Model ◽  
Golden Syrian Hamster ◽  
Content Type

ABSTRACTIn the past decade,Clostridium difficilehas emerged as an important gut pathogen. Symptoms ofC. difficileinfection range from mild diarrhea to pseudomembranous colitis. Besides the two main virulence factors toxin A and toxin B, other virulence factors are likely to play a role in the pathogenesis of the disease. In other Gram-positive and Gram-negative pathogenic bacteria, conserved high-temperature requirement A (HtrA)-like proteases have been shown to have a role in protein homeostasis and quality control. This affects the functionality of virulence factors and the resistance of bacteria to (host-induced) environmental stresses. We found that theC. difficile630 genome encodes a single HtrA-like protease (CD3284; HtrA) and have analyzed its rolein vivoandin vitrothrough the creation of an isogenic ClosTron-basedhtrAmutant ofC. difficilestrain 630Δerm(wild type). In contrast to the attenuated phenotype seen withhtrAdeletion in other pathogens, this mutant showed enhanced virulence in the Golden Syrian hamster model of acuteC. difficileinfection. Microarray data analysis showed a pleiotropic effect ofhtrAon the transcriptome ofC. difficile, including upregulation of the toxin A gene. In addition,the htrAmutant showed reduced spore formation and adherence to colonic cells. Together, our data show thathtrAcan modulate virulence inC. difficile.

scholarly journals Secreted Compounds of the Probiotic Bacillus clausii Strain O/C Inhibit the Cytotoxic Effects Induced by Clostridium difficile and Bacillus cereus Toxins

2016 ◽  
Vol 60 (6) ◽  
pp. 3445-3454 ◽  
Author(s):  
Gabrielle Ripert ◽  
Silvia M. Racedo ◽  
Anne-Marie Elie ◽  
Claudine Jacquot ◽  
Philippe Bressollier ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Bacillus Cereus ◽  
Serine Protease ◽  
Rho Gtpase ◽  
Cytotoxic Effects ◽  
Content Type ◽  
Beneficial Effects ◽  
Bacillus Clausii ◽  
Antibiotic Associated Diarrhea ◽  
Intestinal Pathogens

Although the use of probiotics based onBacillusstrains to fight off intestinal pathogens and antibiotic-associated diarrhea is widespread, the mechanisms involved in producing their beneficial effects remain unclear. Here, we studied the ability of compounds secreted by the probioticBacillus clausiistrain O/C to counteract the cytotoxic effects induced by toxins of two pathogens,Clostridium difficileandBacillus cereus, by evaluating eukaryotic cell viability and expression of selected genes. Coincubation ofC. difficileandB. cereustoxic culture supernatants with theB. clausiisupernatant completely prevented the damage induced by toxins in Vero and Caco-2 cells. The hemolytic effect ofB. cereuswas also avoided by the probiotic supernatant. Moreover, in these cells, the expression ofrhoB, encoding a Rho GTPase target forC. difficiletoxins, was normalized whenC. difficilesupernatant was pretreated using theB. clausiisupernatant. All of the beneficial effects observed with the probiotic were abolished by the serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF). Suspecting the involvement of a secreted protease in this protective effect, a protease was purified from theB. clausiisupernatant and identified as a serine protease (M-protease; GenBank accession numberQ99405). Experiments on Vero cells demonstrated the antitoxic activity of the purified protease against pathogen supernatants. This is the first report showing the capacity of a protease secreted by probiotic bacteria to inhibit the cytotoxic effects of toxinogenicC. difficileandB. cereusstrains. This extracellular compound could be responsible, at least in part, for the protective effects observed for this human probiotic in antibiotic-associated diarrhea.

scholarly journals Signature-Tagged Mutagenesis of Pasteurella multocida Identifies Mutants Displaying Differential Virulence Characteristics in Mice and Chickens

2003 ◽  
Vol 71 (9) ◽  
pp. 5440-5446 ◽  
Author(s):  
Marina Harper ◽  
John D. Boyce ◽  
Ian W. Wilkie ◽  
Ben Adler
Keyword(s):  
Animal Models ◽  
Virulence Factors ◽  
Causative Agent ◽  
Pasteurella Multocida ◽  
Disease Model ◽  
Competitive Growth ◽  
Fowl Cholera ◽  
First Time ◽  
Chicken Model ◽  
Specific Virulence

ABSTRACT Pasteurella multocida is the causative agent of fowl cholera in birds. Signature-tagged mutagenesis (STM) was used to identify potential virulence factors in a mouse septicemia disease model and a chicken fowl cholera model. A library of P. multocida mutants was constructed with a modified Tn916 and screened for attenuation in both animal models. Mutants identified by the STM screening were confirmed as attenuated by competitive growth assays in both chickens and mice. Of the 15 mutants identified in the chicken model, only 5 were also attenuated in mice, showing for the first time the presence of host-specific virulence factors and indicating the importance of screening for attenuation in the natural host.

scholarly journals Staphylococcal Virulence Factors on the Skin of Atopic Dermatitis Patients

mSphere ◽  
2019 ◽  
Vol 4 (6) ◽  
Author(s):  
Mary C. Moran ◽  
Michael P. Cahill ◽  
Matthew G. Brewer ◽  
Takeshi Yoshida ◽  
Sara Knowlden ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Atopic Dermatitis ◽  
Virulence Factors ◽  
Virulence Factor ◽  
Protein Level ◽  
Content Type ◽  
Factor Production ◽  
First Time ◽  
Virulence Factor Production ◽  
Specific Virulence

ABSTRACT Staphylococcus aureus is the leading cause of skin and soft tissue infections, bacteremia, infective endocarditis, osteoarticular, pleuropulmonary, and device-related infections. Virulence factors secreted by S. aureus, including superantigens and cytotoxins, play significant roles in driving disease. The ability to identify virulence factors present at the site of infection will be an important tool in better identifying and understanding how specific virulence factors contribute to disease. Previously, virulence factor production has been determined by culturing S. aureus isolates and detecting the mRNA of specific virulence factors. We demonstrated for the first time that virulence factors can be directly detected at the protein level from human samples, removing the need to first culture isolated bacteria. Superantigens and cytotoxins were detected and quantified with a Western dot blot assay by using reconstituted skin swabs obtained from patients with atopic dermatitis. This methodology will significantly enhance our ability to investigate the complex host-microbe environment and the effects various therapies have on virulence factor production. Overall, the ability to directly quantify virulence factors present at the site of infection or colonization will enhance our understanding of S. aureus-related diseases and help identify optimal treatments. IMPORTANCE For the first time, we show that secreted staphylococcal virulence factors can be quantified at the protein level directly from skin swabs obtained from the skin of atopic dermatitis patients. This technique eliminates the need to culture Staphylococcus aureus and then test the strain’s potential to produce secreted virulence factors. Our methodology shows that secreted virulence factors are present on the skin of atopic patients and provides a more accurate means of evaluating the physiological impact of S. aureus in inflammatory diseases such as atopic dermatitis.

scholarly journals Role of Enteroaggregative Escherichia coli Virulence Factors in Uropathogenesis

2013 ◽  
Vol 81 (4) ◽  
pp. 1164-1171 ◽  
Author(s):  
Erik J. Boll ◽  
Carsten Struve ◽  
Nadia Boisen ◽  
Bente Olesen ◽  
Steen G. Stahlhut ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Virulence Factors ◽  
Outbreak Strain ◽  
Content Type ◽  
E Coli ◽  
Abiotic Surfaces ◽  
Extraintestinal Disease ◽  
First Time ◽  
Specific Virulence

ABSTRACTA multiresistant clonalEscherichia coliO78:H10 strain qualifying molecularly as enteroaggregativeEscherichia coli(EAEC) was recently shown to be the cause of a community-acquired outbreak of urinary tract infection (UTI) in greater Copenhagen, Denmark, in 1991. This marks the first time EAEC has been associated with an extraintestinal disease outbreak. Importantly, the outbreak isolates were recovered from the urine of patients with symptomatic UTI, strongly implying urovirulence. Here, we sought to determine the uropathogenic properties of the Copenhagen outbreak strain and whether these properties are conferred by the EAEC-specific virulence factors. We demonstrated that through expression of aggregative adherence fimbriae, the principal adhesins of EAEC, the outbreak strain exhibited pronouncedly increased adherence to human bladder epithelial cells compared to prototype uropathogenic strains. Moreover, the strain was able to produce distinct biofilms on abiotic surfaces, including urethral catheters. These findings suggest that EAEC-specific virulence factors increase uropathogenicity and may have played a significant role in the ability of the strain to cause a community-acquired outbreak of UTI. Thus, inclusion of EAEC-specific virulence factors is warranted in future detection and characterization of uropathogenicE. coli.

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