scholarly journals Inflammasome Activation Contributes to Interleukin-23 Production in Response to Clostridium difficile

mBio ◽  
2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Carrie A. Cowardin ◽  
Sarah A. Kuehne ◽  
Erica L. Buonomo ◽  
Chelsea S. Marie ◽  
Nigel P. Minton ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Disease Severity ◽  
Tissue Damage ◽  
The United States ◽  
Host Cells ◽  
Inflammasome Activation ◽  
Content Type ◽  
Danger Signals ◽  
Interleukin 23 ◽  
Molecular Patterns

ABSTRACT  Clostridium difficileis the most common hospital-acquired pathogen, causing antibiotic-associated diarrhea in over 250,000 patients annually in the United States. Disease is primarily mediated by toxins A and B, which induce potent proinflammatory signaling in host cells and can activate an ASC-containing inflammasome. Recent findings suggest that the intensity of the host response to infection correlates with disease severity. Our lab has identified the proinflammatory cytokine interleukin-23 (IL-23) as a pathogenic mediator during C. difficile infection (CDI). The mechanisms by which C. difficile induces IL-23, however, are not well understood, and the role of toxins A and B in this process is unclear. Here, we show that toxins A and B alone are not sufficient for IL-23 production but synergistically increase the amount of IL-23 produced in response to MyD88-dependent danger signals, including pathogen-associated molecular patterns (PAMPs) and host-derived damage associated molecular patterns (DAMPs). Danger signals also enhanced the secretion of IL-1β in response to toxins A and B, and subsequent IL-1 receptor signaling accounted for the majority of the increase in IL-23 that occurred in the presence of the toxins. Inhibition of inflammasome activation in the presence of extracellular K+likewise decreased IL-23 production. Finally, we found that IL-1β was increased in the serum of patients with CDI, suggesting that this systemic response could influence downstream production of pathogenic IL-23. Identification of the synergy of danger signals with toxins A and B via inflammasome signaling represents a novel finding in the mechanistic understanding of C. difficile-induced inflammation.IMPORTANCEClostridium difficileis among the leading causes of death due to health care-associated infection, and factors determining disease severity are not well understood. C. difficile secretes toxins A and B, which cause inflammation and tissue damage, and recent findings suggest that some of this tissue damage may be due to an inappropriate host immune response. We have found that toxins A and B, in combination with both bacterium- and host-derived danger signals, can induce expression of the proinflammatory cytokines IL-1β and IL-23. Our results demonstrate that IL-1β signaling enhances IL-23 production and could lead to increased pathogenic inflammation during CDI.

scholarly journals Expression, Purification, and Biological Characterization of Babesia microti Apical Membrane Antigen 1

2015 ◽  
Vol 83 (10) ◽  
pp. 3890-3901 ◽  
Author(s):  
Prasun Moitra ◽  
Hong Zheng ◽  
Vivek Anantharaman ◽  
Rajdeep Banerjee ◽  
Kazuyo Takeda ◽  
...  
Keyword(s):  
Apical Membrane ◽  
The United States ◽  
Host Cells ◽  
Health Concern ◽  
Babesia Microti ◽  
Type I ◽  
Content Type ◽  
Apical Membrane Antigen 1 ◽  
Apical Membrane Antigen ◽  
Extracellular Region

The intraerythrocytic apicomplexanBabesia microti, the primary causative agent of human babesiosis, is a major public health concern in the United States and elsewhere. Apicomplexans utilize a multiprotein complex that includes a type I membrane protein called apical membrane antigen 1 (AMA1) to invade host cells. We have isolated the full-lengthB. microtiAMA1 (BmAMA1) gene and determined its nucleotide sequence, as well as the amino acid sequence of the AMA1 protein. This protein contains an N-terminal signal sequence, an extracellular region, a transmembrane region, and a short conserved cytoplasmic tail. It shows the same domain organization as the AMA1 orthologs from piroplasm, coccidian, and haemosporidian apicomplexans but differs from all other currently known piroplasmida, including otherBabesiaandTheileriaspecies, in lacking two conserved cysteines in highly variable domain III of the extracellular region. Minimal polymorphism was detected in BmAMA1 gene sequences of parasite isolates from six babesiosis patients from Nantucket. Immunofluorescence microscopy studies showed that BmAMA1 is localized on the cell surface and cytoplasm near the apical end of the parasite. Native BmAMA1 from parasite lysate and refolded recombinant BmAMA1 (rBmAMA1) expressed inEscherichia colireacted with a mouse anti-BmAMA1 antibody using Western blotting.In vitrobinding studies showed that both native BmAMA1 and rBmAMA1 bind to human red blood cells (RBCs). This binding is trypsin and chymotrypsin treatment sensitive but neuraminidase independent. Incubation ofB. microtiparasites in human RBCs with a mouse anti-BmAMA1 antibody inhibited parasite growth by 80% in a 24-h assay. Based on its antigenically conserved nature and potential role in RBC invasion, BmAMA1 should be evaluated as a vaccine candidate.

scholarly journals The Pathogenic Potential of Proteus mirabilis Is Enhanced by Other Uropathogens during Polymicrobial Urinary Tract Infection

2016 ◽  
Vol 85 (2) ◽  
Author(s):  
Chelsie E. Armbruster ◽  
Sara N. Smith ◽  
Alexandra O. Johnson ◽  
Valerie DeOrnellas ◽  
Kathryn A. Eaton ◽  
...  
Keyword(s):  
Urinary Tract Infection ◽  
Urinary Tract ◽  
Tract Infection ◽  
Disease Severity ◽  
Proteus Mirabilis ◽  
Tissue Damage ◽  
Urease Activity ◽  
Pathogenic Potential ◽  
Content Type

ABSTRACT Urinary catheter use is prevalent in health care settings, and polymicrobial colonization by urease-positive organisms, such as Proteus mirabilis and Providencia stuartii, commonly occurs with long-term catheterization. We previously demonstrated that coinfection with P. mirabilis and P. stuartii increased overall urease activity in vitro and disease severity in a model of urinary tract infection (UTI). In this study, we expanded these findings to a murine model of catheter-associated UTI (CAUTI), delineated the contribution of enhanced urease activity to coinfection pathogenesis, and screened for enhanced urease activity with other common CAUTI pathogens. In the UTI model, mice coinfected with the two species exhibited higher urine pH values, urolithiasis, bacteremia, and more pronounced tissue damage and inflammation compared to the findings for mice infected with a single species, despite having a similar bacterial burden within the urinary tract. The presence of P. stuartii, regardless of urease production by this organism, was sufficient to enhance P. mirabilis urease activity and increase disease severity, and enhanced urease activity was the predominant factor driving tissue damage and the dissemination of both organisms to the bloodstream during coinfection. These findings were largely recapitulated in the CAUTI model. Other uropathogens also enhanced P. mirabilis urease activity in vitro, including recent clinical isolates of Escherichia coli, Enterococcus faecalis, Klebsiella pneumoniae, and Pseudomonas aeruginosa. We therefore conclude that the underlying mechanism of enhanced urease activity may represent a widespread target for limiting the detrimental consequences of polymicrobial catheter colonization, particularly by P. mirabilis and other urease-positive bacteria.

scholarly journals Evaluation of Portability and Cost of a Fluorescent PCR Ribotyping Protocol for Clostridium difficile Epidemiology

2015 ◽  
Vol 53 (4) ◽  
pp. 1192-1197 ◽  
Author(s):  
Jonathan N. V. Martinson ◽  
Susan Broadaway ◽  
Egan Lohman ◽  
Christina Johnson ◽  
M. Jahangir Alam ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Low Cost ◽  
Cost Effective ◽  
Pcr Primers ◽  
The United States ◽  
Content Type ◽  
Fluorescent Pcr ◽  
Address Data ◽  
Pcr Ribotyping ◽  
Data Portability

Clostridium difficileis the most commonly identified pathogen among health care-associated infections in the United States. There is a need for accurate and low-cost typing tools that produce comparable data across studies (i.e., portable data) to help characterize isolates during epidemiologic investigations ofC. difficileoutbreaks and sporadic cases of disease. The most popularC. difficile-typing technique is PCR ribotyping, and we previously developed methods using fluorescent PCR primers and amplicon sizing on a Sanger-style sequencer to generate fluorescent PCR ribotyping data. This technique has been used to characterize tens of thousands ofC. difficileisolates from cases of disease. Here, we present validation of a protocol for the cost-effective generation of fluorescent PCR ribotyping data. A key component of this protocol is the ability to accurately identify PCR ribotypes against an online database (http://walklab.rcg.montana.edu) at no cost. We present results from a blinded multicenter study to address data portability across four different laboratories and three different sequencing centers. Our standardized protocol and centralized database for typing ofC. difficilepathogens will increase comparability between studies so that important epidemiologic linkages between cases of disease and patterns of emergence can be rapidly identified.

scholarly journals Comparison of Multilocus Sequence Typing and the Xpert C. difficile/Epi Assay for Identification of Clostridium difficile 027/NAP1/BI

2015 ◽  
Vol 54 (3) ◽  
pp. 775-778 ◽  
Author(s):  
Tracy McMillen ◽  
Mini Kamboj ◽  
N. Esther Babady
Keyword(s):  
United States ◽  
Clostridium Difficile ◽  
Confidence Interval ◽  
Multilocus Sequence Typing ◽  
The United States ◽  
Content Type ◽  
Presumptive Identification ◽  
Good Agreement

Clostridium difficile027/NAP1/BI is the most commonC. difficilestrain in the United States. The XpertC. difficile/Epi assay allows rapid, presumptive identification ofC. difficileNAP1. We compared XpertC. difficile/Epi to multilocus sequence typing for identification ofC. difficileNAP1 and found “very good” agreement at 97.9% (κ = 0.86; 95% confidence interval, 0.80 to 0.91).

scholarly journals Intrarectal Instillation of Clostridium difficile Toxin A Triggers Colonic Inflammation and Tissue Damage: Development of a Novel and Efficient Mouse Model of Clostridium difficile Toxin Exposure

2012 ◽  
Vol 80 (12) ◽  
pp. 4474-4484 ◽  
Author(s):  
Simon A. Hirota ◽  
Vadim Iablokov ◽  
Sarah E. Tulk ◽  
L. Patrick Schenck ◽  
Helen Becker ◽  
...  
Keyword(s):  
Epithelial Cell ◽  
Clostridium Difficile ◽  
Inflammatory Mediators ◽  
Tissue Damage ◽  
Intestinal Epithelial Cell ◽  
Intestinal Epithelial ◽  
Toxin A ◽  
Colonic Inflammation ◽  
Content Type ◽  
Clostridium Difficile Toxin

ABSTRACTClostridium difficile, a major cause of hospital-acquired diarrhea, triggers disease through the release of two toxins, toxin A (TcdA) and toxin B (TcdB). These toxins disrupt the cytoskeleton of the intestinal epithelial cell, increasing intestinal permeability and triggering the release of inflammatory mediators resulting in intestinal injury and inflammation. The most prevalent animal model to study TcdA/TcdB-induced intestinal injury involves injecting toxin into the lumen of a surgically generated “ileal loop.” This model is time-consuming and exhibits variability depending on the expertise of the surgeon. Furthermore, the target organ ofC. difficileinfection (CDI) in humans is the colon, not the ileum. In the current study, we describe a new model of CDI that involves intrarectal instillation of TcdA/TcdB into the mouse colon. The administration of TcdA/TcdB triggered colonic inflammation and neutrophil and macrophage infiltration as well as increased epithelial barrier permeability and intestinal epithelial cell death. The damage and inflammation triggered by TcdA/TcdB isolates from the VPI and 630 strains correlated with the concentration of TcdA and TcdB produced. TcdA/TcdB exposure increased the expression of a number of inflammatory mediators associated with human CDI, including interleukin-6 (IL-6), gamma interferon (IFN-γ), and IL-1β. Finally, we were able to demonstrate that TcdA was much more potent at inducing colonic injury than was TcdB but TcdB could act synergistically with TcdA to exacerbate injury. Taken together, our data indicate that the intrarectal murine model provides a robust and efficient system to examine the effects of TcdA/TcdB on the induction of inflammation and colonic tissue damage in the context of human CDI.

scholarly journals Multicenter, Double-Blind, Randomized, Phase 2 Study Evaluating the Novel Antibiotic Cadazolid in Patients with Clostridium difficile Infection

2015 ◽  
Vol 59 (10) ◽  
pp. 6266-6273 ◽  
Author(s):  
Thomas Louie ◽  
Carl Erik Nord ◽  
George H. Talbot ◽  
Mark Wilcox ◽  
Dale N. Gerding ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Clinical Response ◽  
Recurrence Rate ◽  
Primary Endpoint ◽  
Clinical Cure ◽  
The United States ◽  
Phase 2 ◽  
Double Blind ◽  
Content Type ◽  
Phase 2 Study

ABSTRACTCadazolid, a novel fluoroquinolone-oxazolidinone antibiotic, exhibits potentin vitroactivity againstClostridium difficile, including the epidemic BI/NAP1/027 strain. This multicenter, randomized, double-blind, active reference group, phase 2 study evaluated the efficacy and safety of oral cadazolid in treatment of adult patients withC. difficileinfection (CDI). Eligible patients with first occurrence/first recurrence of CDI were randomized 1:1:1:1 to 250, 500, or 1,000 mg cadazolid twice daily (BID) or oral 125 mg vancomycin four times daily (QID) for 10 days. The primary endpoint was clinical cure at test of cure (48 ± 24 h after the end of treatment; modified intent-to-treat population), defined as resolution of diarrhea with no further CDI treatment required. Secondary endpoints included recurrence rate, sustained clinical response (clinical cure without recurrence), and time to diarrhea resolution. Of 84 patients enrolled, 20, 22, 20, and 22 received 250, 500, or 1,000 mg cadazolid BID or 125 mg vancomycin QID, respectively. The primary endpoint was achieved in 76.5% (80% confidence interval [CI], 58.4, 89.3), 80.0% (63.9, 91.0), 68.4% (51.1, 82.5), and 68.2% (52.3, 81.3) of patients, respectively. There was no evidence of a cadazolid dosage-dependent response. Each dosage of cadazolid resulted in a lower recurrence rate than with vancomycin (18.2 to 25.0% versus 50%). Consequently, higher sustained clinical response rates were observed with cadazolid (46.7 to 60.0%) than with vancomycin (33.3%). The times to diarrhea resolution were similar for cadazolid and vancomycin. Cadazolid was well tolerated, with no safety signal observed. The results of this phase 2 study support further clinical development of cadazolid. (This study has been registered in the United States at ClinicalTrials.gov under registration no. NCT01222702 and in Europe with the European Medicines Agency under registration no. EUDRA-CT 2010-020941-29.)

scholarly journals Interaction of Mycoplasma hominis PG21 with Human Dendritic Cells: Interleukin-23-Inducing Mycoplasmal Lipoproteins and Inflammasome Activation of the Cell

2017 ◽  
Vol 199 (15) ◽  
Author(s):  
J. Goret ◽  
L. Béven ◽  
B. Faustin ◽  
C. Contin-Bordes ◽  
C. Le Roy ◽  
...  
Keyword(s):  
Immune Response ◽  
Dendritic Cells ◽  
Molecular Mass ◽  
Host Cell ◽  
Mycoplasma Hominis ◽  
Inflammasome Activation ◽  
Content Type ◽  
Interleukin 23 ◽  
Extracellular Side ◽  
Human Dendritic Cells

ABSTRACT Mycoplasma hominis lacks a cell wall, and lipoproteins anchored to the extracellular side of the plasma membrane are in direct contact with the host components. A Triton X-114 extract of M. hominis enriched with lipoproteins was shown to stimulate the production of interleukin-23 (IL-23) by human dendritic cells (hDCs). The inflammasome activation of the host cell has never been reported upon M. hominis infection. We studied here the interaction between M. hominis PG21 and hDCs by analyzing both the inflammation-inducing mycoplasmal lipoproteins and the inflammasome activation of the host cell. IL-23-inducing lipoproteins were determined using a sequential extraction strategy with two nondenaturing detergents, Sarkosyl and Triton X-114, followed by SDS-PAGE separation and mass spectrometry identification. The activation of the hDC inflammasome was assessed using PCR array and enzyme-linked immunosorbent assay (ELISA). We defined a list of 24 lipoproteins that could induce the secretion of IL-23 by hDCs, 5 with a molecular mass between 20 and 35 kDa and 19 with a molecular mass between 40 and 100 kDa. Among them, lipoprotein MHO_4720 was identified as potentially bioactive, and a synthetic lipopeptide corresponding to the N-terminal part of the lipoprotein was subsequently shown to induce IL-23 release by hDCs. Regarding the hDC innate immune response, inflammasome activation with caspase-dependent production of IL-1β was observed. After 24 h of coincubation of hDCs with M. hominis, downregulation of the NLRP3-encoding gene and of the adaptor PYCARD-encoding gene was noticed. Overall, this study provides insight into both protagonists of the interaction of M. hominis and hDCs. IMPORTANCE Mycoplasma hominis is a human urogenital pathogen involved in gynecologic and opportunistic infections. M. hominis lacks a cell wall, and its membrane contains many lipoproteins that are anchored to the extracellular side of the plasma membrane. In the present study, we focused on the interaction between M. hominis and human dendritic cells and examined both sides of the interaction, the mycoplasmal lipoproteins involved in the activation of the host cell and the immune response of the cell. On the mycoplasmal side, we showed for the first time that M. hominis lipoproteins with high molecular mass were potentially bioactive. On the cell side, we reported an activation of the inflammasome, which is involved in the innate immune response.

scholarly journals Clostridium difficile Toxin A Attenuates Wnt/β-Catenin Signaling in Intestinal Epithelial Cells

2014 ◽  
Vol 82 (7) ◽  
pp. 2680-2687 ◽  
Author(s):  
Bruno Bezerra Lima ◽  
Bárbara Faria Fonseca ◽  
Nathália da Graça Amado ◽  
Débora Moreira Lima ◽  
Ronaldo Albuquerque Ribeiro ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Epithelial Cells ◽  
Wnt Signaling ◽  
Rho Gtpases ◽  
Small Gtpases ◽  
Host Cells ◽  
Reporter Assay ◽  
Content Type ◽  
Protein Levels ◽  
Dependent Inhibition

ABSTRACTClostridium difficiletoxins A and B (TcdA and TcdB) are homologous glycosyltransferases that inhibit a group of small GTPases within host cells, but several mechanisms underlying their pathogenic activity remain unclear. In this study, we evaluated the effects of TcdA on the Wnt/β-catenin pathway, the major driving force behind the proliferation of epithelial cells in colonic crypts. IEC-6 and RKO cells stimulated with Wnt3a-conditioned medium were incubated with 10, 50, and 100 ng/ml of TcdA for 24 h, resulting in a dose-dependent inhibition of the Wnt signaling, as demonstrated by a T-cell factor (TCF) reporter assay. This was further confirmed by immunofluorescence staining for nuclear localization of β-catenin and Western blotting for β-catenin and c-Myc (encoded by a Wnt target gene). Moreover, our Western blot analysis showed a decrease in the β-catenin protein levels, which was reversed by z-VAD-fmk, a pan-caspase inhibitor. Nonetheless, TcdA was still able to inhibit the Wnt/β-catenin pathway even in the presence of z-VAD-fmk, lithium chloride (a GSK3β inhibitor), or constitutively active β-catenin, as determined by a TCF reporter assay. Furthermore, preincubation of RKO cells with TcdA for 12 h also attenuated Wnt3a-mediated activation of Wnt signaling, suggesting that inactivation of Rho GTPases plays a significant role in that inhibition. Taken together, these findings suggest that attenuation of the Wnt signaling by TcdA is important for TcdA antiproliferative effects.

scholarly journals The Gut Microbiota Is Associated with Clearance ofClostridium difficileInfection Independent of Adaptive Immunity

mSphere ◽  
2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Jhansi L. Leslie ◽  
Kimberly C. Vendrov ◽  
Matthew L. Jenior ◽  
Vincent B. Young
Keyword(s):  
United States ◽  
Immune Response ◽  
Clostridium Difficile ◽  
Gut Microbiota ◽  
Adaptive Immunity ◽  
Adaptive Immune Response ◽  
The United States ◽  
Content Type ◽  
Adaptive Immune

ABSTRACTClostridium(Clostridioides)difficile, a Gram-positive, anaerobic bacterium, is the leading single cause of nosocomial infections in the United States. A major risk factor forClostridium difficileinfection (CDI) is prior exposure to antibiotics, as they increase susceptibility to CDI by altering the membership of the microbial community enabling colonization. The importance of the gut microbiota in providing protection from CDI is underscored by the reported 80 to 90% success rate of fecal microbial transplants in treating recurrent infections. Adaptive immunity, specifically humoral immunity, is also sufficient to protect from both acute and recurrent CDI. However, the role of the adaptive immune system in mediating clearance ofC. difficilehas yet to be resolved. Using murine models of CDI, we found that adaptive immunity is dispensable for clearance ofC. difficile. However, random forest analysis using only two members of the resident bacterial community correctly identified animals that would go on to clear the infection with 66.7% accuracy. These findings indicate that the indigenous gut microbiota independent of adaptive immunity facilitates clearance ofC. difficilefrom the murine gastrointestinal tract.IMPORTANCEClostridium difficileinfection is a major cause of morbidity and mortality in hospitalized patients in the United States. Currently, the role of the adaptive immune response in modulating levels ofC. difficilecolonization is unresolved. This work suggests that the indigenous gut microbiota is a main factor that promotes clearance ofC. difficilefrom the GI tract. Our results show that clearance ofC. difficilecan occur without contributions from the adaptive immune response. This study also has implications for the design of preclinical studies testing the efficacy of vaccines on clearance of bacterial pathogens, as inherent differences in the baseline community structure of animals may bias findings.

scholarly journals U.S.-Based National Sentinel Surveillance Study for the Epidemiology of Clostridium difficile-Associated Diarrheal Isolates and Their Susceptibility to Fidaxomicin

2015 ◽  
Vol 59 (10) ◽  
pp. 6437-6443 ◽  
Author(s):  
D. R. Snydman ◽  
L. A. McDermott ◽  
N. V. Jacobus ◽  
C. Thorpe ◽  
S. Stone ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Antimicrobial Susceptibility ◽  
The United States ◽  
Surveillance Study ◽  
Binary Toxin ◽  
Toxin Gene ◽  
Gene Profile ◽  
Geographic Variations ◽  
Convenience Sample ◽  
Content Type

ABSTRACTIn 2011 a surveillance study for the susceptibility to fidaxomicin and epidemiology ofClostridium difficileisolates in the United States was undertaken in seven geographically dispersed medical centers. This report encompasses baseline surveillance in 2011 and 2012 on 925 isolates. A convenience sample ofC. difficileisolates or toxin positive stools from patients were referred to a central laboratory. Antimicrobial susceptibility was determined by agar dilution (CLSI M11-A8). Clinical and Laboratory Standards Institute (CLSI), Food and Drug Administration, or European Union of Clinical Antimicrobial Susceptibility Testing (EUCAST) breakpoints were applied where applicable. Toxin gene profiles were characterized by multiplex PCR on each isolate. A random sample of 322 strains, stratified by institution, underwent restriction endonuclease analysis (REA). The fidaxomicin MIC90was 0.5 μg/ml for all isolates regardless of REA type or toxin gene profile, and all isolates were inhibited at ≤1.0 μg/ml. By REA typing, BI strains represented 25.5% of the isolates. The toxin gene profile oftcdA,tcdB, andcdtA/Bpositive with atcdC18-bp deletion correlated with BI REA group. Moxifloxacin and clindamycin resistance was increased among either BI or binary toxin-positive isolates. Metronidazole and vancomycin showed reduced susceptibility (EUCAST criteria) in these isolates. Geographic variations in susceptibility, REA group and binary toxin gene presence were observed. Fidaxomicin activity againstC. difficileisolated in a national surveillance study did not change more than 1 year after licensure. This analysis provides baseline results for future comparisons.

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