ORCID Profile
0000-0002-9275-3979
Current Organisation
Monash University
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Microbial Genetics | Structural Biology (incl. Macromolecular Modelling) | Microbiology | Bacteriology
Publisher: Springer Science and Business Media LLC
Date: 12-08-2019
DOI: 10.1038/S41564-019-0519-1
Abstract: Spore-forming bacteria encompass a erse range of genera and species, including important human and animal pathogens, and food contaminants. Clostridioides difficile is one such bacterium and is a global health threat because it is the leading cause of antibiotic-associated diarrhoea in hospitals. A crucial mediator of C. difficile disease initiation, dissemination and re-infection is the formation of spores that are resistant to current therapeutics, which do not target sporulation. Here, we show that cephamycin antibiotics inhibit C. difficile sporulation by targeting spore-specific penicillin-binding proteins. Using a mouse disease model, we show that combined treatment with the current standard-of-care antibiotic, vancomycin, and a cephamycin prevents disease recurrence. Cephamycins were found to have broad applicability as an anti-sporulation strategy, as they inhibited sporulation in other spore-forming pathogens, including the food contaminant Bacillus cereus. This study could directly and immediately affect treatment of C. difficile infection and advance drug development to control other important spore-forming bacteria that are problematic in the food industry (B. cereus), are potential bioterrorism agents (Bacillus anthracis) and cause other animal and human infections.
Publisher: BMJ
Date: 23-08-2018
DOI: 10.1136/INJURYPREV-2016-042151
Abstract: To curb high rates of alcohol-related violence and injury in Indigenous communities, alcohol management plans (AMPs) were implemented in 2002-2003 and tightened in 2008. This project compares injury presentations and alcohol involvement from two Indigenous Cape York communities, one that entered full prohibition and one that did not. Aclinical file audit was performed for the period 2006-2011, capturing changes in alcohol availability. Medical files were searched for injury presentation documenting type of injury, cause of injury (including alcohol), date of injury and outcomes of all presenting injuries for the time period 1 January 2006 to 31 December 2011, capturing the major changes of the 2008 AMP restrictions. Findings indicated injury presentation rates were higher in both communities before prohibition than afterwards andreduction was more pronounced in community 2 (prohibition). Ongoing research is imperative, as this area is characterised by a near-absence of evidence.
Publisher: MDPI AG
Date: 10-02-2015
Publisher: Elsevier BV
Date: 12-2014
DOI: 10.1016/J.ANAEROBE.2014.09.007
Abstract: We compared the identification of Clostridium species using mass spectrometry by two different Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) platforms (Bruker MS and Vitek MS) against 16S rRNA sequencing as the reference standard. We then examined the impact of different s le preparations and (on one of those platforms) age of bacterial colonial growth on the performance of the MALDI-TOF MS systems. We identified 10 different species amongst the 52 isolates by 16S rRNA sequencing, with Clostridium perfringens the most prevalent (n=30). Spectrometric analysis using Vitek MS correctly speciated 47/52 (90.4%) isolates and was not affected by the s le preparation used. Performance of the Bruker MS was dependent on s le preparation with correct speciation obtained for 36 of 52 (69.2%) isolates tested using the Direct Transfer [DT] protocol, but all 52 (100%) isolates were correctly speciated using either an Extended Direct Transfer [EDT] or a Full Formic Extraction [EX] protocol. We then examined the effect of bacterial colonial growth age on the performance of Bruker MS and found substantial agreement in speciation using DT (Kappa=0.62, 95% CI: 0.46-0.75), almost perfect agreement for EDT (Kappa=0.94, 95% CI: 0.86-1.00) and exact agreement for EX (Kappa=1.00) between different days.
Publisher: Future Medicine Ltd
Date: 02-2011
DOI: 10.2217/FMB.10.169
Publisher: MDPI AG
Date: 17-06-2016
Publisher: Mary Ann Liebert Inc
Date: 12-2019
Abstract: The antifibrinolytic agent, tranexamic acid (TXA), an inhibitor of plasmin formation, currently is evaluated to reduce bleeding in various conditions, including traumatic brain injury (TBI). Because plasmin is implicated in inflammation and immunity, we investigated the effects of plasmin inhibition on the immune response after TBI in the presence or absence of induced pneumonia. Wild-type mice treated with vehicle or TXA or mice deficient in plasminogen (plg
Publisher: Elsevier BV
Date: 10-2002
DOI: 10.1016/S0022-2836(02)00850-1
Abstract: The VirSR two-component signal transduction pathway regulates virulence and toxin production in Clostridium perfringens, the causative agent of gas gangrene. The response regulator, VirR, binds to repeat sequences located upstream of the promoter and is directly responsible for the transcriptional activation of pfoA, the structural gene for the cholesterol-dependent cytolysin, perfringolysin O. Comparative sequence analysis of the 236 amino acid residue VirR protein revealed a two-domain structure: a typical N-terminal response regulator domain and an uncharacterised C-terminal domain. Database searching revealed that over 40 other proteins, many of which appeared to be response regulators or transcriptional activators, had homology with the VirR C-terminal domain (VirRc). Multiple sequence alignment of this VirRc family revealed a highly conserved region that was designated the FxRxHrS motif. By deletion analysis this motif was shown to be essential for the functional integrity of the VirR protein. Alanine scanning mutagenesis and subsequent phenotypic analysis indicated that conserved residues located within the motif were required for activity. These residues extended from L179 to N194. More detailed site-directed mutagenesis showed that amino acid residues R186, H188 and S190 were essential for activity since even conservative substitutions in these positions resulted in non-functional proteins. Three of the mutant proteins, R186K, S190A and S190C, were purified and shown by in vitro gel shift analysis to be unable to bind to the specific target DNA with the same efficiency as the wild-type protein. These data reveal for the first time that VirRc functions as a DNA binding domain in which the highly conserved FxRxHrS motif has a functional role. These studies have important implications for this new family of transcriptional factors since they imply that the conserved FxRxHrS motif may be involved in DNA binding in all of these proteins, irrespective of their biological role.
Publisher: Elsevier BV
Date: 12-1996
Publisher: Springer Science and Business Media LLC
Date: 04-03-2019
Publisher: Public Library of Science (PLoS)
Date: 29-07-2011
Publisher: Oxford University Press (OUP)
Date: 18-02-2014
Abstract: Gas gangrene is a potentially fatal disease that is primarily caused by the ubiquitous, anaerobic bacteria Clostridium perfringens and Clostridium septicum. Treatment is limited to antibiotic therapy, debridement of the infected tissue, and, in severe cases, utation. The need for new treatment approaches is compelling. Opioid-based analgesics such as buprenorphine and morphine also have immunomodulatory properties, usually leading to faster disease progression. However, here we show that mice pretreated with buprenorphine and morphine do not die from clostridial myonecrosis. Treatment with buprenorphine after the onset of infection also arrested disease development. Protection against myonecrotic disease was specific to C. perfringens-mediated myonecrosis buprenorphine did not protect against disease caused by C. septicum infection even though infections due to both species are very similar. These data provide the first evidence of a protective role for opioids during infection and suggest that new therapeutic strategies may be possible for the treatment of C. perfringens-mediated myonecrosis.
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.C.6532560
Abstract: AbstractPurpose: Fluorescence-guided surgery using tumor-targeted contrast agents has been developed to improve the completeness of oncologic resections. Quenched activity–based probes that fluoresce after covalently binding to tumor-specific enzymes have been proposed to improve specificity, but none have been tested in humans. Here, we report the successful clinical translation of a cathepsin activity–based probe (VGT-309) for fluorescence-guided surgery. Experimental Design: We optimized the specificity, dosing, and timing of VGT-309 in preclinical models of lung cancer. To evaluate clinical feasibility, we conducted a canine study of VGT-309 during pulmonary tumor resection. We then conducted a randomized, double-blind, dose-escalation study in healthy human volunteers receiving VGT-309 to evaluate safety. Finally, we tested VGT-309 in humans undergoing lung cancer surgery. Results: In preclinical models, we found highly specific tumor cell labeling that was blocked by a broad spectrum cathepsin inhibitor. When evaluating VGT-309 for guidance during resection of canine tumors, we found that the probe selectively labeled tumors and demonstrated high tumor-to-background ratio (TBR range: 2.15–3.71). In the Phase I human study, we found that VGT-309 was safe at all doses studied. In the ongoing Phase II trial, we report two cases in which VGT-309 localized visually occult, non-palpable tumors (TBRs = 2.83 and 7.18) in real time to illustrate its successful clinical translation and potential to improve surgical management. Conclusions: This first-in-human study demonstrates the safety and feasibility of VGT-309 to label human pulmonary tumors during resection. These results may be generalizable to other cancers due to cathepsin overexpression in many solid tumors. /
Publisher: Elsevier BV
Date: 02-2000
Publisher: American Society for Microbiology
Date: 11-2006
DOI: 10.1128/IAI.00806-06
Abstract: The Clostridium perfringens alpha-toxin has previously been implicated as the major virulence factor in necrotic enteritis in chickens, although definitive proof has not been reported. In this study an alpha-toxin mutant was constructed in a virulent chicken isolate and shown to retain full virulence in a chicken disease model. These results demonstrated that alpha-toxin is not an essential virulence factor in the pathogenesis of necrotic enteritis in chickens.
Publisher: Wiley
Date: 1995
DOI: 10.1111/J.1365-2958.1995.TB02234.X
Abstract: The pathogenesis of clostridial myonecrosis, or gas gangrene, involves the growth of the anaerobic bacterium Clostridium perfringens in the infected tissues and the elaboration of numerous extracellular toxins and enzymes. The precise role of each of these toxins in tissue invasion and necrosis has not been determined. To enable genetic approaches to be used to study C. perfringens pathogenesis we developed an allelic exchange method which involved the transformation of C. perfringens cells with a suicide plasmid carrying a gene insertionally inactivated with an erythromycin-resistance determinant. The frequency with which double reciprocal crossover events were observed was increased to a workable level by increasing the amount of homologous DNA located on either side of the inactivated gene. Allelic exchange was used to isolate mutations in the chromosomal pfoA gene, which encodes an oxygen-labile haemolysin known as theta-toxin or perfringolysin O, and in the chromosomal plc gene, which encodes the alpha-toxin or phospholipase C. The resultant mutants failed to produce detectable theta-toxin or alpha-toxin activity, respectively, and could be complemented by recombinant plasmids that carried the respective wild-type genes. The resultant strains were virulence tested in a mouse myonecrosis model. The results showed that the plc mutants had demonstrably reduced virulence and therefore provided definitive genetic evidence for the essential role of alpha-toxin in gas gangrene or clostridial myonecrosis.
Publisher: Public Library of Science (PLoS)
Date: 09-06-2009
Publisher: Future Medicine Ltd
Date: 03-2014
DOI: 10.2217/FMB.13.168
Abstract: ABSTRACT: Clostridium perfringens uses its arsenal of toxins to cause histotoxic and intestinal infections in humans and animals. It has been unclear why this bacterium produces so many different toxins, especially since many target the plasma membrane of host cells. However, it is now established that C. perfringens uses chromosomally encoded alpha toxin (a phospholipase C) and perfringolysin O (a pore-forming toxin) during histotoxic infections. In contrast, this bacterium causes intestinal disease by employing toxins encoded by mobile genetic elements, including C. perfringens enterotoxin, necrotic enteritis toxin B-like, epsilon toxin and beta toxin. Like perfringolysin O, the toxins with established roles in intestinal disease form membrane pores. However, the intestinal disease-associated toxins vary in their target specificity, when they are produced (sporulation vs vegetative growth), and in their sensitivity to intestinal proteases. Producing many toxins with erse characteristics likely imparts virulence flexibility to C. perfringens so it can cause an array of diseases.
Publisher: Springer Science and Business Media LLC
Date: 16-05-2015
Publisher: American Society for Microbiology
Date: 10-2009
DOI: 10.1128/IAI.00548-09
Abstract: The essential toxin in Clostridium perfringens -mediated gas gangrene or clostridial myonecrosis is alpha-toxin, although other toxins and extracellular enzymes may also be involved. In many bacterial pathogens extracellular sialidases are important virulence factors, and it has been suggested that sialidases may play a role in gas gangrene. C. perfringens strains have combinations of three different sialidase genes, two of which, nanI and nanJ , encode secreted sialidases. The nanI and nanJ genes were insertionally inactivated by homologous recombination in derivatives of sequenced strain 13 and were shown to encode two functional secreted sialidases, NanI and NanJ. Analysis of these derivatives showed that NanI was the major sialidase in this organism. Mutation of nanI resulted in loss of most of the secreted sialidase activity, and the residual activity was eliminated by subsequent mutation of the nanJ gene. Only a slight reduction in the total sialidase activity was observed in a nanJ mutant. Cytotoxicity assays using the B16 melanoma cell line showed that supernatants containing NanI or overexpressing NanJ enhanced alpha-toxin-mediated cytotoxicity. Finally, the ability of nanI , nanJ , and nanIJ mutants to cause disease was assessed in a mouse myonecrosis model. No attenuation of virulence was observed for any of these strains, providing evidence that neither the NanI sialidase nor the NanJ sialidase is essential for virulence.
Publisher: Wiley
Date: 22-07-2005
DOI: 10.1111/J.1365-2958.2005.04774.X
Abstract: Clostridium septicum is the causative agent of spontaneous gas gangrene or atraumatic myonecrosis, a sudden and frequently fatal infection that is increasingly associated with malignancy of the colon. Little is known about the disease process although the focus of virulence studies has been the alpha-toxin, a pore-forming cytolysin that is encoded by the csa gene and secreted as an inactive protoxin. Until now a lack of techniques for the genetic manipulation of C. septicum has hindered the use of molecular approaches to understand pathogenesis. By introducing plasmids by conjugation from Escherichia coli, we have developed methods for the genetic manipulation of C. septicum and constructed a chromosomal csa mutant by allelic exchange. Virulence testing of an isogenic series of strains consisting of the wild type, the csa mutant, and a csa mutant complemented with the wild-type csa gene revealed that the development of fulminant myonecrosis in mice was dependent on the ability to produce a functional haemolytic alpha-toxin. Furthermore, the inhibition of leukocyte influx into the lesion, which is very typical of clostridial myonecrosis, was also dependent on the ability to produce alpha-toxin. This study represents the first definitive identification of a virulence factor in this organism and opens the way for further studies that will delineate the role of other putative virulence factors in this significant pathogen.
Publisher: American Society for Microbiology
Date: 03-2011
DOI: 10.1128/IAI.00968-10
Abstract: Clostridium sordellii is an important pathogen of humans and animals, causing a range of diseases, including myonecrosis, sepsis, and shock. Although relatively rare in humans, the incidence of disease is increasing, and it is associated with high mortality rates, approaching 70%. Currently, very little is known about the pathogenesis of C. sordellii infections or disease. Previous work suggested that the lethal large clostridial glucosylating toxin TcsL is the major virulence factor, but a lack of genetic tools has hindered our ability to conclusively assign a role for TcsL or, indeed, any of the other putative virulence factors produced by this organism. In this study, we have developed methods for the introduction of plasmids into C. sordellii using RP4-mediated conjugation from Escherichia coli and have successfully used these techniques to insertionally inactivate the tcsL gene in the reference strain ATCC 9714, using targetron technology. Virulence testing revealed that the production of TcsL is essential for the development of lethal infections by C. sordellii ATCC 9714 and also contributes significantly to edema seen during uterine infection. This study represents the first definitive identification of a virulence factor in C. sordellii and opens the way for in-depth studies of this important human pathogen at the molecular level.
Publisher: American Society for Microbiology
Date: 12-2001
DOI: 10.1128/IAI.69.12.7904-7910.2001
Abstract: To examine the synergistic effects of alpha-toxin and perfringolysin O in clostridial myonecrosis, homologous recombination was used to construct an alpha-toxin deficient derivative of a perfringolysin O mutant of Clostridium perfringens . The subsequent strain was complemented with separate plasmids that carried the alpha-toxin structural gene ( plc) , the perfringolysin O gene ( pfoA ), or both toxin genes, and the resultant isogenic strains were examined in a mouse myonecrosis model. Synergistic effects were clearly observed in these experiments. Infection with the control strain, which did not produce either toxin, resulted in very minimal gross pathological changes, whereas the isogenic strain that was reconstituted for both toxins produced a pathology that was clearly more severe than when alpha-toxin alone was reconstituted. These changes were most apparent in the rapid spread of the disease, the gross pathology of the footpad and in the rate at which the mice had to be euthanatized for ethical reasons. Elimination of both alpha-toxin and perfringolysin O production removed most of the histopathological features typical of clostridial myonecrosis. These effects were restored when the mutant was complemented with the alpha-toxin structural gene, but reconstituting only perfringolysin O activity produced vastly different results, with regions of coagulative necrosis, apparently enhanced by vascular disruption, being observed. Reconstitution of both alpha-toxin and perfringolysin O activity produced histopathology most similar to that observed with the alpha-toxin reconstituted strain. The spreading of myonecrosis was very rapid in these tissues, and coagulative necrosis appeared to be restricted to the lumen of the blood vessels. The results of these virulence experiments clearly support the hypothesis that alpha-toxin and perfringolysin O have a synergistic effect in the pathology of gas gangrene.
Publisher: American Society for Microbiology
Date: 15-06-2014
DOI: 10.1128/AEM.04285-13
Abstract: TnpX is a site-specific recombinase responsible for the excision and insertion of the transposons Tn 4451 and Tn 4453 in Clostridium perfringens and Clostridium difficile , respectively. Here, we exploit phenotypic features of TnpX to facilitate genetic mutagenesis and complementation studies. Genetic manipulation of bacteria often relies on the use of antibiotic resistance genes however, a limited number are available for use in the clostridia. The ability of TnpX to recognize and excise specific DNA fragments was exploited here as the basis of an antibiotic resistance marker recycling system, specifically to remove antibiotic resistance genes from plasmids in Escherichia coli and from marked chromosomal C. perfringens mutants. This methodology enabled the construction of a C. perfringens plc virR double mutant by allowing the removal and subsequent reuse of the same resistance gene to construct a second mutation. Genetic complementation can be challenging when the gene of interest encodes a product toxic to E. coli . We show that TnpX represses expression from its own promoter, P attCI , which can be exploited to facilitate the cloning of recalcitrant genes in E. coli for subsequent expression in the heterologous host C. perfringens . Importantly, this technology expands the repertoire of tools available for the genetic manipulation of the clostridia.
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.C.6532560.V1
Abstract: AbstractPurpose: Fluorescence-guided surgery using tumor-targeted contrast agents has been developed to improve the completeness of oncologic resections. Quenched activity–based probes that fluoresce after covalently binding to tumor-specific enzymes have been proposed to improve specificity, but none have been tested in humans. Here, we report the successful clinical translation of a cathepsin activity–based probe (VGT-309) for fluorescence-guided surgery. Experimental Design: We optimized the specificity, dosing, and timing of VGT-309 in preclinical models of lung cancer. To evaluate clinical feasibility, we conducted a canine study of VGT-309 during pulmonary tumor resection. We then conducted a randomized, double-blind, dose-escalation study in healthy human volunteers receiving VGT-309 to evaluate safety. Finally, we tested VGT-309 in humans undergoing lung cancer surgery. Results: In preclinical models, we found highly specific tumor cell labeling that was blocked by a broad spectrum cathepsin inhibitor. When evaluating VGT-309 for guidance during resection of canine tumors, we found that the probe selectively labeled tumors and demonstrated high tumor-to-background ratio (TBR range: 2.15–3.71). In the Phase I human study, we found that VGT-309 was safe at all doses studied. In the ongoing Phase II trial, we report two cases in which VGT-309 localized visually occult, non-palpable tumors (TBRs = 2.83 and 7.18) in real time to illustrate its successful clinical translation and potential to improve surgical management. Conclusions: This first-in-human study demonstrates the safety and feasibility of VGT-309 to label human pulmonary tumors during resection. These results may be generalizable to other cancers due to cathepsin overexpression in many solid tumors. /
Publisher: Public Library of Science (PLoS)
Date: 18-04-2018
Publisher: EMBO
Date: 19-04-2023
Abstract: Inflammasome signaling is a central pillar of innate immunity triggering inflammation and cell death in response to microbes and danger signals. Here, we show that two virulence factors from the human bacterial pathogen Clostridium perfringens are nonredundant activators of the NLRP3 inflammasome in mice and humans. C. perfringens lecithinase (also known as phospolipase C) and C. perfringens perfringolysin O induce distinct mechanisms of activation. Lecithinase enters LAMP1 + vesicular structures and induces lysosomal membrane destabilization. Furthermore, lecithinase induces the release of the inflammasome‐dependent cytokines IL‐1β and IL‐18, and the induction of cell death independently of the pore‐forming proteins gasdermin D, MLKL and the cell death effector protein ninjurin‐1 or NINJ1. We also show that lecithinase triggers inflammation via the NLRP3 inflammasome in vivo and that pharmacological blockade of NLRP3 using MCC950 partially prevents lecithinase‐induced lethality. Together, these findings reveal that lecithinase activates an alternative pathway to induce inflammation during C. perfringens infection and that this mode of action can be similarly exploited for sensing by a single inflammasome.
Publisher: American Society for Microbiology
Date: 07-03-2018
Abstract: A major virulence factor in Clostridium sordellii -mediated infection is the toxin TcsL, which is encoded within a region of the genome called the pathogenicity locus (PaLoc). C. sordellii isolates carry the PaLoc on the pCS1 family of plasmids, of which there are four characterized members. Here, we determined the potential mobility of pCS1 plasmids and characterized a fifth unique pCS1 member. Using a derivative of the pCS1-1 plasmid from strain ATCC 9714 which had been marked with the ermB erythromycin resistance gene, conjugative transfer into a recipient C. sordellii isolate, R28058, was demonstrated. Bioinformatic analysis of pCS1-1 identified a novel conjugation gene cluster defined as the C. sordellii transfer ( cst ) locus. Interruption of genes within the cst locus resulted in loss of pCS1-1 transfer, which was restored upon complementation in trans . These studies provided clear evidence that genes within the cst locus are essential for the conjugative transfer of pCS1-1. The cst locus is present on all pCS1 subtypes, and homologous loci were identified on toxin-encoding plasmids from Clostridium perfringens and Clostridium botulinum and also carried within genomes of Clostridium difficile isolates, indicating that it is a widespread clostridial conjugation locus. The results of this study have broad implications for the dissemination of toxin genes and, potentially, antibiotic resistance genes among members of a erse range of clostridial pathogens, providing these microorganisms with a survival advantage within the infected host. IMPORTANCE C. sordellii is a bacterial pathogen that causes severe infections in humans and animals, with high mortality rates. While the pathogenesis of C. sordellii infections is not well understood, it is known that the toxin TcsL is an important virulence factor. Here, we have shown the ability of a plasmid carrying the tcsL gene to undergo conjugative transfer between distantly related strains of C. sordellii , which has far-reaching implications for the ability of C. sordellii to acquire the capacity to cause disease. Plasmids that carry tcsL encode a previously uncharacterized conjugation locus, and in idual genes within this locus were shown to be required for conjugative transfer. Furthermore, homologues on toxin plasmids from other clostridial species were identified, indicating that this region represents a novel clostridial conjugation locus. The results of this study have broad implications for the dissemination of virulence genes among members of a erse range of clostridial pathogens.
Publisher: Elsevier BV
Date: 05-1997
Abstract: Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene and mediates infection and disease by producing numerous extracellular toxins, including alpha-toxin, theta-toxin and kappa-toxin. Tn916-mutagenesis was used to isolate mutants defective in their ability to produce either alpha-toxin or theta-toxin. Nine independently derived mutants were isolated. In four of these mutants Tn916 had inserted at sites located 193 bp or 198 bp upstream of the theta-toxin structural gene, pfoA. Four mutants contained large deletions, three in regions which encompassed the theta-toxin structural and regulatory genes pfoA and pfoR, respectively, and the kappa-toxin structural gene, colA, and one in a region encompassing the alpha-toxin structural gene, plc. These mutants should prove to be invaluable for further genetic studies aimed at determining the role of these toxins in virulence.
Publisher: Elsevier BV
Date: 03-2019
DOI: 10.1016/J.PLASMID.2019.02.002
Abstract: Conjugative transfer is a major contributor to the dissemination of antibiotic resistance and virulence genes in the human and animal pathogen, Clostridium perfringens. The C. perfringens plasmid pCW3 is the archetype of an extensive family of highly related conjugative toxin and antibiotic resistance plasmids found in this bacterium. These plasmids were thought to constitute the only conjugative plasmid family in C. perfringens. Recently, another series of C. perfringens plasmids, the pCP13-like family, have been shown to harbour important toxin genes, including genes that encode the novel binary clostridial enterotoxin, BEC. Based on early bioinformatics analysis this plasmid family was thought to be non-conjugative. Here we demonstrate that pCP13 is in fact conjugative, transfers at high frequency and that the newly defined Pcp conjugation locus encodes putative homologues of a type 4 secretion system (T4SS), one of which, PcpB4, was shown to be essential for transfer. The T4SS of pCP13 also appears to be evolutionarily related to conjugative toxin plasmids from other clostridia-like species, including Paeniclostridium (formerly Clostridium) sordellii, Clostridioides (formerly Clostridium) difficile and Clostridium botulinum. Therefore, it is clear that there are two distinct families of conjugative plasmids in C. perfringens: the pCW3 family and the pCP13 family. This study has significant implications for our understanding of the movement of toxin genes both within C. perfringens, but also potentially to other pathogenic clostridia.
Publisher: American Association of Avian Pathologists (AAAP)
Date: 09-2015
Publisher: Informa UK Limited
Date: 03-09-2014
Publisher: Public Library of Science (PLoS)
Date: 16-09-2016
Publisher: Wiley
Date: 06-1994
DOI: 10.1111/J.1365-2958.1994.TB01063.X
Abstract: The anaerobic bacterium Clostridium perfringens mediates clostridial myonecrosis, or gas gangrene, by producing a number of extracellular toxins and enzymes. Transposon mutagenesis with Tn916 was used to isolate a pleiotropic mutant of C. perfringens that produced reduced levels of phospholipase C, protease and sialidase, and did not produce any detectable perfringolysin O activity. Southern hybridization revealed that a single copy of Tn916 had inserted into a 2.7 kb HindIII fragment in the C. perfringens chromosome. A 4.3kb PstI fragment, which spanned the Tn916 insertion site, was cloned from the wild-type strain. When subcloned into a shuttle vector and introduced into C. perfringens this fragment was able to complement the Tn916-derived mutation. Transformation of the mutant with plasmids containing the 2.7 kb HindIII fragment, or the 4.3 kb PstI fragment, resulted in toxin and enzyme levels greater than or equal to those of the wild-type strain. The PstI fragment was sequenced and found to potentially encode seven open reading frames, two of which appeared to be arranged in an operon and shared sequence similarity with members of two-component signal transduction systems. The putative virR gene encoded a protein with a deduced molecular weight of 30,140, and with sequence similarity to activators in the response regulator family of proteins. The next gene, virS, into which Tn916 had inserted, was predicted to encode a membrane-spanning protein with a deduced molecular weight of 51,274. The putative VirS protein had sequence similarity to sensor proteins and also contained a histidine residue highly conserved in the histidine protein kinase family of sensor proteins. Virulence studies carried out using a mouse model implicated the virS gene in the pathogenesis of histotoxic C. perfringens infections. It was concluded that a two-component sensor regulator system that activated the expression of a number of extracellular toxins and enzymes involved in virulence had been cloned and sequenced. A model that described the regulation of extracellular toxin production in C. perfringens was constructed.
Publisher: American Society for Microbiology
Date: 25-10-2017
Abstract: Clostridium sordellii is a significant pathogen with mortality rates approaching 100%. It is the bacterial spore that is critical in initiating infection and disease. An understanding of spore structures as well as spore morphology across a range of strains may lead to a better understanding of C. sordellii infection and disease. However, the structural characteristics of the C. sordellii spores are limited. In this work, we have addressed this lack of detail and characterized the C. sordellii spore morphology. The use of traditional and advanced microscopy techniques has provided detailed new observations of C. sordellii spore structural features, which serve as a reference point for structural studies of spores from other bacterial species.
Publisher: American Society for Microbiology
Date: 03-2019
DOI: 10.1128/IAI.00676-18
Abstract: Clostridium difficile is a major cause of hospital-acquired antibiotic-associated diarrhea. C. difficile produces two cytotoxins, TcdA and TcdB both toxins are multidomain proteins that lead to cytotoxicity through the modification and inactivation of small GTPases of the Rho/Rac family.
Publisher: American Society for Microbiology
Date: 04-2002
DOI: 10.1128/JB.184.7.2034-2038.2002
Abstract: The pathogenesis of Clostridium perfringens -mediated gas gangrene or clostridial myonecrosis involves the extracellular toxins alpha-toxin and perfringolysin O. Previous studies (T. Shimizu, A. Okabe, J. Minami, and H. Hayashi, Infect. Immun. 59:137-142, 1991) carried out with Escherichia coli suggested that the perfringolysin O structural gene, pfoA , was positively regulated by the product of the upstream pfoR gene. In an attempt to confirm this hypothesis in C . perfringens , a pfoR-pfoA deletion mutant was complemented with isogenic pfoA + shuttle plasmids that varied only in their ability to encode an intact pfoR gene. No difference in the ability to produce perfringolysin O was observed for C . perfringens strains carrying these plasmids. In addition, chromosomal pfoR mutants were constructed by homologous recombination in C . perfringens . Again no difference in perfringolysin O activity was observed. Since it was not possible to alter perfringolysin O expression by mutation of pfoR , it was concluded that the pfoR gene product is unlikely to have a role in the regulation of pfoA expression in C . perfringens .
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.22487646
Abstract: Supplementary Data from A Cathepsin-Targeted Quenched Activity–Based Probe Facilitates Enhanced Detection of Human Tumors during Resection
Publisher: Elsevier BV
Date: 2014
Publisher: American Association for Cancer Research (AACR)
Date: 31-03-2023
DOI: 10.1158/1078-0432.22487646.V1
Abstract: Supplementary Data from A Cathepsin-Targeted Quenched Activity–Based Probe Facilitates Enhanced Detection of Human Tumors during Resection
Publisher: Elsevier BV
Date: 10-2016
DOI: 10.1016/J.ANAEROBE.2016.05.005
Abstract: Bacterial pathogens have adopted numerous mechanisms for acquiring iron from host proteins during an infection, including the direct acquisition of ferric iron from heme-associated proteins or from iron-scavenging siderophores. Ferric iron then is transported into the cytosol, where it can be utilized by the bacterial pathogen. Under anaerobic conditions bacteria can also transport ferrous iron using the transmembrane complex FeoAB, but little is known about iron transport systems in anaerobic bacteria such as the pathogenic clostridia. In this study we sought to characterize the iron acquisition process in Clostridium perfringens. Bioinformatic analysis of the Clostridium perfringens strain 13 genome sequence revealed that it has seven potential iron acquisition systems: three siderophore-mediated systems, one ferric citrate uptake system, two heme-associated acquisition systems and one ferrous iron uptake system (FeoAB). The relative level of expression of these systems was determined using quantitative real-time RT-PCR assays that were specific for one gene from each system. Each of these genes was expressed, with the feoAB genes generating the most abundant iron-uptake related transcripts. To further examine the role of this system in the growth of C. perfringens, insertional inactivation was used to isolate a chromosomal feoB mutant. Growth of this mutant in the presence and absence of iron revealed that it had altered growth properties and a markedly reduced total iron and manganese content compared to the wild type effects that were reversed upon complementation with the wild-type feoB gene. These studies suggest that under anaerobic conditions FeoB is the major protein required for the uptake of iron into the cell and that it may play an important role in the pathogenesis of C. perfringens infections.
Publisher: Cold Spring Harbor Laboratory
Date: 06-07-2006
DOI: 10.1101/GR.5238106
Abstract: Clostridium perfringens is a Gram-positive, anaerobic spore-forming bacterium commonly found in soil, sediments, and the human gastrointestinal tract. C. perfringens is responsible for a wide spectrum of disease, including food poisoning, gas gangrene (clostridial myonecrosis), enteritis necroticans, and non-foodborne gastrointestinal infections. The complete genome sequences of Clostridium perfringens strain ATCC 13124, a gas gangrene isolate and the species type strain, and the enterotoxin-producing food poisoning strain SM101, were determined and compared with the published C. perfringens strain 13 genome. Comparison of the three genomes revealed considerable genomic ersity with unique “genomic islands” identified, with the majority of these islands unusually clustered on one replichore. PCR-based analysis indicated that the large genomic islands are widely variable across a large collection of C. perfringens strains. These islands encode genes that correlate to differences in virulence and phenotypic characteristics of these strains. Significant differences between the strains include numerous novel mobile elements and genes encoding metabolic capabilities, strain-specific extracellular polysaccharide capsule, sporulation factors, toxins, and other secreted enzymes, providing substantial insight into this medically important bacterial pathogen.
Publisher: Public Library of Science (PLoS)
Date: 11-04-2008
Publisher: American Association for Cancer Research (AACR)
Date: 06-07-2022
DOI: 10.1158/1078-0432.CCR-22-1215
Abstract: Fluorescence-guided surgery using tumor-targeted contrast agents has been developed to improve the completeness of oncologic resections. Quenched activity–based probes that fluoresce after covalently binding to tumor-specific enzymes have been proposed to improve specificity, but none have been tested in humans. Here, we report the successful clinical translation of a cathepsin activity–based probe (VGT-309) for fluorescence-guided surgery. We optimized the specificity, dosing, and timing of VGT-309 in preclinical models of lung cancer. To evaluate clinical feasibility, we conducted a canine study of VGT-309 during pulmonary tumor resection. We then conducted a randomized, double-blind, dose-escalation study in healthy human volunteers receiving VGT-309 to evaluate safety. Finally, we tested VGT-309 in humans undergoing lung cancer surgery. In preclinical models, we found highly specific tumor cell labeling that was blocked by a broad spectrum cathepsin inhibitor. When evaluating VGT-309 for guidance during resection of canine tumors, we found that the probe selectively labeled tumors and demonstrated high tumor-to-background ratio (TBR range: 2.15–3.71). In the Phase I human study, we found that VGT-309 was safe at all doses studied. In the ongoing Phase II trial, we report two cases in which VGT-309 localized visually occult, non-palpable tumors (TBRs = 2.83 and 7.18) in real time to illustrate its successful clinical translation and potential to improve surgical management. This first-in-human study demonstrates the safety and feasibility of VGT-309 to label human pulmonary tumors during resection. These results may be generalizable to other cancers due to cathepsin overexpression in many solid tumors.
Publisher: Elsevier BV
Date: 05-2015
DOI: 10.1016/J.RESMIC.2014.09.003
Abstract: Clostridium sordellii is gram positive bacterial pathogen of humans and animals. While the incidence of human-related C. sordellii infection is low, the mortality rate associated with infection is high. Of particular concern are C. sordellii infections after child-birth or medical abortion, which have an almost 100% mortality rate. Recent genetic and epidemiological work has increased our understanding of how this pathogen has evolved and how it causes disease. This review will summarise studies involving the genetics of C. sordellii, including an antibiotic resistance profile, the genetic determinants of virulence and mutagenesis of C. sordellii.
Publisher: Elsevier BV
Date: 11-2014
DOI: 10.1016/J.IJMM.2014.08.008
Abstract: The clostridia cause many human and animal diseases, resulting in significant morbidity and mortality. Host damage results from the action of potent exotoxins, an important group of which is the large clostridial toxins (LCTs) produced by Clostridium difficile, Clostridium sordellii, Clostridium perfringens and Clostridium novyi. Knowledge of the structure and function of these toxins has been attained, however, apart from C. difficile, the regulatory pathways that control LCT production remain largely unknown. Here we show that LCT production in C. sordellii and C. perfringens is temporally regulated and repressed by glucose in a similar manner to C. difficile. Furthermore, we show that the TpeL-encoding gene of C. perfringens is located in an uncharacterized Pathogenicity Locus (PaLoc), along with accessory genes predicted to encode a bacteriophage holin-type protein and a TcdR-family alternative sigma factor, TpeR. Inactivation of tpeR demonstrated that TpeR is critical for C. perfringens TpeL production, in a similar manner to C. difficile TcdR and C. sordellii TcsR, but cross-complementation showed that TpeR is not functionally interchangeable with TcdR or TcsR. Although conserved mechanisms are employed by the clostridia to control LCT production there are important functional differences that distinguish members of the TcdR-family of clostridial alternative sigma factors.
Publisher: American Society for Microbiology
Date: 11-2010
DOI: 10.1128/JCM.01724-10
Abstract: Clostridium perfringens has been associated with necrotizing enterocolitis (NEC), which is a serious disease of neonates. Our study describes the novel use of selective tryptose sulfite cycloserine with egg yolk agar (TSC-EYA) during a nursery outbreak. This medium provides a rapid, sensitive, and accurate presumptive identification of C. perfringens .
Publisher: MDPI AG
Date: 17-12-2022
DOI: 10.3390/GENES6041347
Start Date: 2013
End Date: 2015
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2011
End Date: 2014
Funder: National Health and Medical Research Council
View Funded ActivityStart Date: 2021
End Date: 12-2023
Amount: $633,006.00
Funder: Australian Research Council
View Funded Activity