scholarly journals The Clostridium difficile Dlt Pathway Is Controlled by the Extracytoplasmic Function Sigma Factor σVin Response to Lysozyme

2016 ◽  
Vol 84 (6) ◽  
pp. 1902-1916 ◽  
Author(s):  
Emily C. Woods ◽  
Kathryn L. Nawrocki ◽  
Jose M. Suárez ◽  
Shonna M. McBride
Keyword(s):  
Cell Wall ◽  
Clostridium Difficile ◽  
Sigma Factor ◽  
Intestinal Tract ◽  
Dependent Manner ◽  
Hamster Model ◽  
Content Type ◽  
Rapid Amplification ◽  
Extracytoplasmic Function Sigma Factor

Clostridium difficile(also known asPeptoclostridium difficile) is a major nosocomial pathogen and a leading cause of antibiotic-associated diarrhea throughout the world. Colonization of the intestinal tract is necessary forC. difficileto cause disease. Host-produced antimicrobial proteins (AMPs), such as lysozyme, are present in the intestinal tract and can deter colonization by many bacterial pathogens, and yetC. difficileis able to survive in the colon in the presence of these AMPs. Our prior studies established that the Dlt pathway, which increases the surface charge of the bacterium by addition ofd-alanine to teichoic acids, is important forC. difficileresistance to a variety of AMPs. We sought to determine what genetic mechanisms regulate expression of the Dlt pathway. In this study, we show that adltnull mutant is severely attenuated for growth in lysozyme and that expression of thedltDABCoperon is induced in response to lysozyme. Moreover, we found that a mutant lacking the extracytoplasmic function (ECF) sigma factor σVdoes not inducedltexpression in response to lysozyme, indicating that σVis required for regulation of lysozyme-dependentd-alanylation of the cell wall. Using reporter gene fusions and 5′ RACE (rapid amplification of cDNA ends) analysis, we identified promoter elements necessary for lysozyme-dependent and lysozyme-independentdltexpression. In addition, we observed that both asigVmutant and adltmutant are more virulent in a hamster model of infection. These findings demonstrate that cell walld-alanylation inC. difficileis induced by lysozyme in a σV-dependent manner and that this pathway impacts virulencein vivo.

scholarly journals MBX-500, a Hybrid Antibiotic withIn VitroandIn VivoEfficacy against Toxigenic Clostridium difficile

2012 ◽  
Vol 56 (9) ◽  
pp. 4786-4792 ◽  
Author(s):  
Michelle M. Butler ◽  
Dean L. Shinabarger ◽  
Diane M. Citron ◽  
Ciarán P. Kelly ◽  
Sofya Dvoskin ◽  
...  
Keyword(s):  
Weight Gain ◽  
Clostridium Difficile ◽  
Severe Disease ◽  
Normal Weight ◽  
New Drugs ◽  
Hamster Model ◽  
Resistance Development ◽  
Content Type

ABSTRACTClostridium difficileinfection (CDI) causes moderate to severe disease, resulting in diarrhea and pseudomembranous colitis. CDI is difficult to treat due to production of inflammation-inducing toxins, resistance development, and high probability of recurrence. Only two antibiotics are approved for the treatment of CDI, and the pipeline for therapeutic agents contains few new drugs. MBX-500 is a hybrid antibacterial, composed of an anilinouracil DNA polymerase inhibitor linked to a fluoroquinolone DNA gyrase/topoisomerase inhibitor, with potential as a new therapeutic for CDI treatment. Since MBX-500 inhibits three bacterial targets, it has been previously shown to be minimally susceptible to resistance development. In the present study, thein vitroandin vivoefficacies of MBX-500 were explored against the Gram-positive anaerobe,C. difficile. MBX-500 displayed potency across nearly 50 isolates, including those of the fluoroquinolone-resistant, toxin-overproducing NAP1/027 ribotype, performing as well as comparator antibiotics vancomycin and metronidazole. Furthermore, MBX-500 was a narrow-spectrum agent, displaying poor activity against many other gut anaerobes. MBX-500 was active in acute and recurrent infections in a toxigenic hamster model of CDI, exhibiting full protection against acute infections and prevention of recurrence in 70% of the animals. Hamsters treated with MBX-500 displayed significantly greater weight gain than did those treated with vancomycin. Finally, MBX-500 was efficacious in a murine model of CDI, again demonstrating a fully protective effect and permitting near-normal weight gain in the treated animals. These selective anti-CDI features support the further development of MBX 500 for the treatment of CDI.

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 Protection of Hamsters from Mortality by Reducing Fecal Moxifloxacin Concentration with DAV131A in a Model of Moxifloxacin-Induced Clostridium difficile Colitis

2017 ◽  
Vol 61 (10) ◽  
Author(s):  
Charles Burdet ◽  
Sakina Sayah-Jeanne ◽  
Thu Thuy Nguyen ◽  
Christine Miossec ◽  
Nathalie Saint-Lu ◽  
...  
Keyword(s):  
Mathematical Modeling ◽  
Body Weight ◽  
Clostridium Difficile ◽  
Mortality Rates ◽  
Dependent Manner ◽  
Hamster Model ◽  
Content Type ◽  
Clostridium Difficile Colitis ◽  
Dose Dependent ◽  
G 14

ABSTRACT Lowering the gut exposure to antibiotics during treatments can prevent microbiota disruption. We evaluated the effects of an activated charcoal-based adsorbent, DAV131A, on the fecal free moxifloxacin concentration and mortality in a hamster model of moxifloxacin-induced Clostridium difficile infection. A total of 215 hamsters receiving moxifloxacin subcutaneously (day 1 [D1] to D5) were orally infected at D3 with C. difficile spores. They received various doses (0 to 1,800 mg/kg of body weight/day) and schedules (twice a day [BID] or three times a day [TID]) of DAV131A (D1 to D8). Moxifloxacin concentrations and C. difficile counts were determined at D3, and mortality was determined at D12. We compared mortality rates, moxifloxacin concentrations, and C. difficile counts according to DAV131A regimen and modeled the links between DAV131A regimen, moxifloxacin concentration, and mortality. All hamsters that received no DAV131A died, but none of those that received 1,800 mg/kg/day died. Significant dose-dependent relationships between DAV131A dose and (i) mortality, (ii) moxifloxacin concentration, and (iii) C. difficile count were evidenced. Mathematical modeling suggested that (i) lowering the moxifloxacin concentration at D3, which was 58 μg/g (95% confidence interval [CI] = 50 to 66 μg/g) without DAV131A, to 17 μg/g (14 to 21 μg/g) would reduce mortality by 90%; and (ii) this would be achieved with a daily DAV131A dose of 703 mg/kg (596 to 809 mg/kg). In this model of C. difficile infection, DAV131A reduced mortality in a dose-dependent manner by decreasing the fecal free moxifloxacin concentration.

scholarly journals Pharmacological, Toxicological, and Dose Range Assessment of OG716, a Novel Lantibiotic for the Treatment ofClostridium difficile-Associated Infection

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
Mark E. Pulse ◽  
William J. Weiss ◽  
Johan A. Kers ◽  
Anthony W. DeFusco ◽  
Jae H. Park ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Dose Range ◽  
Hamster Model ◽  
Ofclostridium Difficile ◽  
Golden Syrian Hamster ◽  
Content Type ◽  
Lead Compounds ◽  
Novel Antibiotics

ABSTRACTLantibiotics present an attractive scaffold for the development of novel antibiotics. We report here a novel lantibiotic for the treatment ofClostridium difficileinfection. The lead compounds were selected from a library of over 700 single- and multiple-substitution variants of the lantibiotic mutacin 1140 (MU1140). The best performersin vitroandin vivowere further used to challenge Golden Syrian hamsters orally in a Golden Syrian hamster model ofClostridium difficile-associated disease (CDAD) in a dose-response format, resulting in the selection of OG716 as the lead compound. This lantibiotic was characterized by a 50% effective dose of 23.85 mg/kg of body weight/day (10.97 μmol/kg/day) in this model. Upon oral administration of the maximum feasible dose (≥1,918 mg/kg/day), no observable toxicities or side effects were noted, and no effect on intestinal motility was observed. Compartmentalization to the gastrointestinal tract was confirmed. MU1140-derived variants offer a large pipeline for the development of novel antibiotics for the treatment of several indications and are particularly attractive considering their novel mechanism of action. Based on the currently available data, OG716 has an acceptable profile for further development for the treatment of CDAD.

scholarly journals A Combination of Three Fully Human Toxin A- and Toxin B-Specific Monoclonal Antibodies Protects against Challenge with Highly Virulent Epidemic Strains of Clostridium difficile in the Hamster Model

2015 ◽  
Vol 22 (7) ◽  
pp. 711-725 ◽  
Author(s):  
Natalie G. Anosova ◽  
Leah E. Cole ◽  
Lu Li ◽  
Jinrong Zhang ◽  
Anna M. Brown ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Clostridium Difficile ◽  
Toxin A ◽  
Healthy Human ◽  
Hamster Model ◽  
Toxin B ◽  
Content Type ◽  
Highly Virulent

ABSTRACTClostridium difficileinfection (CDI) is the principal cause of nosocomial diarrhea and pseudomembranous colitis associated with antibiotic therapy. Recent increases in the number of outbreaks attributed to highly virulent antibiotic-resistant strains underscore the importance of identifying efficacious alternatives to antibiotics to control this infection. CDI is mediated by two large exotoxins, toxins A and B. Strong humoral toxin-specific immune responses are associated with recovery and a lack of disease recurrence, whereas insufficient humoral responses are associated with recurrent CDI. Multiple approaches targeting these toxins, including intravenous immunoglobulin, neutralizing polymers, active vaccines, and, most recently, monoclonal antibodies (MAbs), have been explored, with various degrees of success. In this study, we describe the characterization of the first MAbs isolated from healthy human donors using a high-throughput B-cell cloning strategy. The MAbs were selected based on their ability to inhibit the actions of toxins A and Bin vitroand because of theirin vivoefficacy in a hamster challenge model. A potent 2-MAb cocktail was identified and then further potentiated by the addition of a second anti-toxin B MAb. This 3-MAb combination protected animals against mortality and also reduced the severity and duration of diarrhea associated with challenge with highly virulent strains ofC. difficiletoxinotypes 0 and III. This highly efficacious cocktail consists of one MAb specific to the receptor binding domain of toxin A and two MAbs specific to nonoverlapping regions of the glucosyltransferase domain of toxin B. This MAb combination offers great potential as a nonantibiotic treatment for the prevention of recurrent CDI.

scholarly journals The Yeast-Phase Virulence Requirement for α-Glucan Synthase Differs among Histoplasma capsulatum Chemotypes

2010 ◽  
Vol 10 (1) ◽  
pp. 87-97 ◽  
Author(s):  
Jessica A. Edwards ◽  
Elizabeth A. Alore ◽  
Chad A. Rappleye
Keyword(s):  
Cell Wall ◽  
Histoplasma Capsulatum ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Glucan Synthase ◽  
Content Type ◽  
I Factor ◽  
Yeast Phase

ABSTRACTHistoplasma capsulatumstrains can be classified into two chemotypes based on cell wall composition. The cell wall of chemotype II yeast contains a layer of α-(1,3)-glucan that masks immunostimulatory β-(1,3)-glucans from detection by the Dectin-1 receptor on host phagocytes. This α-(1,3)-glucan cell wall component is essential for chemotype IIHistoplasmavirulence. In contrast, chemotype I yeast cells lack α-(1,3)-glucanin vitro, yet they remain fully virulentin vivo. Analysis of the chemotype I α-glucan synthase (AGS1) locus revealed a 2.7-kb insertion in the promoter region that diminishesAGS1expression. Nonetheless,AGS1mRNA can be detected during respiratory infection with chemotype I yeast, suggesting that α-(1,3)-glucan could be produced duringin vivogrowth despite its absencein vitro. To directly test whetherAGS1contributes to chemotype I strain virulence, we preventedAGS1function by RNA interference and by insertional mutation. Loss ofAGS1function in chemotype I does not impair the cytotoxicity ofags1(−) mutant yeast to cultured macrophages, nor does it affect the intracellular growth of yeast. In a murine model of histoplasmosis, theags1(−) chemotype I mutant strains show no defect in lung infection or in extrapulmonary dissemination. Together, these studies demonstrate thatAGS1expression is dispensable for chemotype I yeast virulence, in contrast to the case for chemotype II yeast. Despite the absence of cell wall α-(1,3)-glucan, chemotype I yeast can avoid detection by Dectin-1 in a growth stage-dependent manner. This suggests the production of a uniqueHistoplasmachemotype I factor that, at least partially, circumvents the α-(1,3)-glucan requirement for yeast virulence.

scholarly journals Conserved Oligopeptide Permeases Modulate Sporulation Initiation in Clostridium difficile

2014 ◽  
Vol 82 (10) ◽  
pp. 4276-4291 ◽  
Author(s):  
Adrianne N. Edwards ◽  
Kathryn L. Nawrocki ◽  
Shonna M. McBride
Keyword(s):  
Clostridium Difficile ◽  
Hamster Model ◽  
Content Type ◽  
Gastrointestinal Pathogen ◽  
Dormant Spore ◽  
Sporulation Initiation ◽  
Spore Forming Bacteria

ABSTRACTThe anaerobic gastrointestinal pathogenClostridium difficilemust form a metabolically dormant spore to survive in oxygenic environments and be transmitted from host to host. The regulatory factors by whichC. difficileinitiates and controls the early stages of sporulation inC. difficileare not highly conserved in otherClostridiumorBacillusspecies. Here, we investigated the role of two conserved oligopeptide permeases, Opp and App, in the regulation of sporulation inC. difficile. These permeases are known to positively affect sporulation inBacillusspecies through the import of sporulation-specific quorum-sensing peptides. In contrast to other spore-forming bacteria, we discovered that inactivating these permeases inC. difficileresulted in the earlier expression of early sporulation genes and increased sporulationin vitro. Furthermore, disruption ofoppandappresulted in greater virulence and increased the amounts of spores recovered from feces in the hamster model ofC. difficileinfection. Our data suggest that Opp and App indirectly inhibit sporulation, likely through the activities of the transcriptional regulator SinR and its inhibitor, SinI. Taken together, these results indicate that the Opp and App transporters serve a different function in controlling sporulation and virulence inC. difficilethan inBacillus subtilisand suggest that nutrient availability plays a significant role in pathogenesis and sporulationin vivo. This study suggests a link between the nutritional status of the environment and sporulation initiation inC. difficile.

scholarly journals Macrophage Reporter Cell Assay for Screening Immunopharmacological Activity of Cell Wall-Active Antifungals

2014 ◽  
Vol 58 (3) ◽  
pp. 1738-1743 ◽  
Author(s):  
Russell E. Lewis ◽  
Guangling Liao ◽  
Katherine Young ◽  
Cameron Douglas ◽  
Dimitrios P. Kontoyiannis
Keyword(s):  
Cell Wall ◽  
Transcriptional Activation ◽  
Yeast Cells ◽  
Dependent Manner ◽  
Activator Protein 1 ◽  
Chitin Synthesis ◽  
Synthesis Inhibitor ◽  
Content Type

ABSTRACTAntifungal exposure can elicit immunological effects that contribute to activityin vivo, but this activity is rarely screenedin vitroin a fashion analogous to MIC testing. We used RAW 264.7 murine macrophages that express a secreted embryonic alkaline phosphatase (SEAP) gene induced by transcriptional activation of NF-κB and activator protein 1 (AP-1) to develop a screen for immunopharmacological activity of cell wall-active antifungal agents. Isolates ofCandida albicansandAspergillus fumigatusthat conditionally express genes involved in cell wall synthesis were also tested with the reporter macrophages. We found that growth of fungi in subinhibitory concentrations of glucan synthesis inhibitors (caspofungin and enfumafungin A) or repression of the β-glucan catalytic subunit of glucan synthase,FKS1, increased macrophage NF-κB/AP-1 activation in a dectin-1-dependent manner. This pattern of activation was also transiently observed with repression of chitin synthesis inC. albicansor when yeast cells were incubated in low concentrations of the chitin synthesis inhibitor nikkomycin Z.

scholarly journals Efficacy of LFF571 in a Hamster Model of Clostridium difficile Infection

2012 ◽  
Vol 56 (8) ◽  
pp. 4459-4462 ◽  
Author(s):  
Anna Trzasko ◽  
Jennifer A. Leeds ◽  
Jens Praestgaard ◽  
Matthew J. LaMarche ◽  
David McKenney
Keyword(s):  
Clostridium Difficile ◽  
Survival Data ◽  
Cox Regression ◽  
Toxigenic Strain ◽  
Hamster Model ◽  
Content Type ◽  
Pooled Data ◽  
Risk Of Death ◽  
End Of Treatment

ABSTRACTLFF571 is a novel semisynthetic thiopeptide antibiotic with potent activity against a variety of Gram-positive pathogens, includingClostridium difficile.In vivoefficacy of LFF571 was compared to vancomycin in a hamster model ofC. difficileinfection (CDI). Infection was induced in Golden Syrian hamsters using a toxigenic strain ofC. difficile. Treatment started 24 h postinfection and consisted of saline, vancomycin, or LFF571. Cox regression was used to analyze survival data from a cohort of animals evaluated across seven serial experimental groups treated with vancomycin at 20 mg/kg, LFF571 at 5 mg/kg, or vehicle alone. Survival was right censored; animals were not observed beyond day 21. At death or end of study, cecal contents were tested forC. difficiletoxins A and B. In summary, the data showed that 5 mg/kg LFF571 decreased the risk of death by 79% (P< 0.0001) and 69% (P= 0.0022) compared with saline and 20 mg/kg vancomycin, respectively. Further analysis of the pooled data indicated that the survival benefit of LFF571 treatment at 5 mg/kg compared to vancomycin at 20 mg/kg was due primarily to a decrease in the risk of recurrence after end of treatment. Animals successfully treated with LFF571 or vancomycin had no detectableC. difficiletoxin. Overall, LFF571 was more efficacious at the end of the study, at a lower dose, and with fewer recurrences, than vancomycin in the hamster model of CDI. LFF571 is being assessed in humans for safety and efficacy in the treatment ofC. difficileinfections.

scholarly journals In VivoStudies Suggest that Induction of VanS-Dependent Vancomycin Resistance Requires Binding of the Drug to d-Ala-d-Ala Termini in the Peptidoglycan Cell Wall

2013 ◽  
Vol 57 (9) ◽  
pp. 4470-4480 ◽  
Author(s):  
Min Jung Kwun ◽  
Gabriela Novotna ◽  
Andrew R. Hesketh ◽  
Lionel Hill ◽  
Hee-Jeon Hong
Keyword(s):  
Cell Wall ◽  
Transcriptional Activation ◽  
Streptomyces Coelicolor ◽  
Constitutive Expression ◽  
Transcriptional Response ◽  
Vancomycin Resistance ◽  
Content Type ◽  
Expression Of Genes ◽  
Peptidoglycan Cell Wall

ABSTRACTVanRS two-component regulatory systems are key elements required for the transcriptional activation of inducible vancomycin resistance genes in bacteria, but the precise nature of the ligand signal that activates these systems has remained undefined. Using the resistance system inStreptomyces coelicoloras a model, we have undertaken a series ofin vivostudies which indicate that the VanS sensor kinase in VanB-type resistance systems is activated by vancomycin in complex with thed-alanyl-d-alanine (d-Ala-d-Ala) termini of cell wall peptidoglycan (PG) precursors. Complementation of an essentiald-Ala-d-Ala ligase activity by constitutive expression ofvanAencoding a bifunctionald-Ala-d-Ala andd-alanyl-d-lactate (d-Ala-d-Lac) ligase activity allowed construction of strains that synthesized variable amounts of PG precursors containingd-Ala-d-Ala. Assays quantifying the expression of genes under VanRS control showed that the response to vancomycin in these strains correlated with the abundance ofd-Ala-d-Ala-containing PG precursors; strains producing a lower proportion of PG precursors terminating ind-Ala-d-Ala consistently exhibited a lower response to vancomycin. Pretreatment of wild-type cells with vancomycin or teicoplanin to saturate and mask thed-Ala-d-Ala binding sites in nascent PG also blocked the transcriptional response to subsequent vancomycin exposure, and desleucyl vancomycin, a vancomycin analogue incapable of interacting withd-Ala-d-Ala residues, failed to inducevangene expression. Activation of resistance by a vancomycin–d-Ala-d-Ala PG complex predicts a limit to the proportion of PG that can be derived from precursors terminating ind-Ala-d-Lac, a restriction also enforced by the bifunctional activity of the VanA ligase.

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