Anaerobic Pathogen Clostridium Perfringens Research Articles

Temperature-regulated heterogeneous extracellular matrix gene expression defines biofilm morphology in Clostridium perfringens

Cells in biofilms dynamically adapt to surrounding environmental conditions, which alters biofilm architecture. The obligate anaerobic pathogen Clostridium perfringens shows different biofilm structures in different temperatures. Here we find that the temperature-regulated production of extracellular polymeric substance (EPS) is necessary for morphological changes in biofilms. We identify BsaA proteins as an EPS matrix necessary for pellicle biofilm formation at lower temperature and find that extracellularly secreted BsaA protein forms filamentous polymers. We show that sipW-bsaA operon expression is bimodal, and the EPS-producing population size is increased at a lower temperature. This heterogeneous expression of the EPS gene requires a two-component system. We find that EPS-producing cells cover EPS-nonproducing cells attaching to the bottom surface. In the deletion mutant of pilA2, encoding a type IV pilin, the EPS gene expression is ON in the whole population. This heterogeneity is further regulated by the cleavage of the pilA2 mRNA by RNase Y, causing temperature-responsive EPS expression in biofilms. As temperature is an environmental cue, C. perfringens may modulate EPS expression to induce morphological changes in biofilm structure as a strategy for adapting to interhost and external environments.

Immunization of mice against alpha, beta, and epsilon toxins of Clostridium perfringens using recombinant rCpa-b-x expressed by Bacillus subtilis

The anaerobic pathogen Clostridium perfringens is the most potent cause of intestinal diseases, such as enterotoxemia, hemorrhagic enteritis, and lamb dysentery, in sheep. Three toxinotypes (B, C, and D) are usually the cause of these diseases and are mainly mediated via three important exotoxins: alpha toxin (CPA), beta toxin (CPB), and epsilon toxin (ETX). We have designed a chimeric protein, rCpa-b-x, that contains the C-terminal binding region of CPA, partial sequence of CPB, and ETX (Cpa247-370, Cpb108-305, and EtxH118P, respectively) according to the principle of structural vaccinology. The rCpa-b-x protein was then expressed by pHT43 plasmid in vivo using Bacillus subtilis as a delivery vector (Bs-pHT43-Cpa-b-x). The immunological activity of the rCpa-b-x protein was verified by western blot and its immunological efficacy was evaluated in a murine model. Oral administration with a recombinant agent caused local mucosal and systemic immune responses, and serum lgG and intestinal mucosal secretory IgA (sIgA) antibody titers were significantly increased. Levels of IL-2, IL-4, and IFN-γ were significantly higher in lymphocytes isolated from the Bs-pHT43-Cpa-b-x group compared with levels from the control groups. The percentages of CD4+ and CD8+ T lymphocytes in the Bs-pHT43-Cpa-b-x and inactivated vaccine (IV) groups were in the normal range. Mice of vaccine groups and control groups were challenged with 1x LD100 unit filtrate containing alpha, beta, and epsilon toxins. Mice in the Bs-pHT43-Cpa-b-x group were found to have lower rates of morbidity. The active immunization of mice with Bs-pHT43-Cpa-b-x still maintained 85% to 90% survival at the end of the 10-day observation period, whereas mice of control groups died within two to five days. The results of this study demonstrate the effectiveness of Bs-pHT43-Cpa-b-x in preventing C. perfringens infection in mice, and that Bs-pHT43-Cpa-b-x could be considered a potential vaccine against C. perfringens.

The anaerobic pathogen Clostridium perfringens colonizes and expresses its toxins in the gut of the nematode Caenorhabditis elegans.

The anaerobic pathogen Clostridium perfringens colonizes and expresses its toxins in the gut of the nematode Caenorhabditis elegans.

Role of RNase Y in Clostridium perfringens mRNA Decay and Processing.

RNases have important roles in RNA degradation and turnover in all organisms. C. perfringens is a Gram-positive anaerobic spore-forming bacterial pathogen that produces numerous extracellular enzymes and toxins, and it is linked to digestive disorders and disease. A highly conserved endoribonuclease, RNase Y, affects the expression of hundreds of genes, including toxin genes, and studying these effects is useful for understanding C. perfringens specifically and RNases generally. Moreover, RNase Y is involved in processing specific transcripts, and we observed that this processing in C. perfringens results in the stabilization of mRNAs encoding a toxin and bacterial extracellular apparatus pili. Our study shows that RNase activity is associated with gene expression, helping to determine the growth, proliferation, and virulence of C. perfringens.

NanR, a Transcriptional Regulator That Binds to the Promoters of Genes Involved in Sialic Acid Metabolism in the Anaerobic Pathogen Clostridium perfringens.

Among many other virulence factors, Clostridium perfringens produces three sialidases NanH, NanI and NanJ. NanH lacks a secretion signal peptide and is predicted to be an intracellular enzyme, while NanI and NanJ are secreted. Previously, we had identified part of an operon encoding NanE (epimerase) and NanA (sialic acid lyase) enzymes. Further analysis of the entire operon suggests that it encodes a complete pathway for the transport and metabolism of sialic acid along with a putative transcriptional regulator, NanR. The addition of 30 mM N-acetyl neuraminic acid (Neu5Ac) to a semi-defined medium significantly enhanced the growth yield of strain 13, suggesting that Neu5Ac can be used as a nutrient. C. perfringens strain 13 lacks a nanH gene, but has NanI- and NanJ-encoding genes. Analysis of nanI, nanJ, and nanInanJ mutants constructed by homologous recombination revealed that the expression of the major sialidase, NanI, was induced by the addition of Neu5Ac to the medium, and that in separate experiments, the same was true of a nanI-gusA transcriptional fusion. For the nanI and nanJ genes, primer extension identified three and two putative transcription start sites, respectively. Gel mobility shift assays using purified NanR and DNA from the promoter regions of the nanI and nanE genes showed high affinity, specific binding by NanR. We propose that NanR is a global regulator of sialic acid-associated genes and that it responds, in a positive feedback loop, to the concentration of sialic acid in the cell.

Two novel membrane proteins, TcpD and TcpE, are essential for conjugative transfer of pCW3 in Clostridium perfringens.

The anaerobic pathogen Clostridium perfringens encodes either toxin genes or antibiotic resistance determinants on a unique family of conjugative plasmids that have a novel conjugation region, the tcp locus. Studies of the paradigm conjugative plasmid from C. perfringens, the 47-kb tetracycline resistance plasmid pCW3, have identified several tcp-encoded proteins that are involved in conjugative transfer and form part of the transfer apparatus. In this study, the role of the conserved hypothetical proteins TcpD, TcpE, and TcpJ was examined. Mutation and complementation analyses showed that TcpD and TcpE were essential for the conjugative transfer of pCW3, whereas TcpJ was not required. To analyze the TcpD and TcpE proteins in C. perfringens, functional hemagglutinin (HA)-tagged derivatives were constructed. Western blots showed that TcpD and TcpE localized to the cell envelope fraction independently of the presence of other pCW3-encoded proteins. Finally, examination of the subcellular localization of TcpD and TcpE by immunofluorescence showed that these proteins were concentrated at both poles of C. perfringens donor cells, where they are postulated to form essential components of the multiprotein complex that comprises the transfer apparatus.

The Putative Coupling Protein TcpA Interacts with Other pCW3-Encoded Proteins To Form an Essential Part of the Conjugation Complex

Conjugative plasmids encode antibiotic resistance determinants or toxin genes in the anaerobic pathogen Clostridium perfringens. The paradigm conjugative plasmid in this bacterium is pCW3, a 47-kb tetracycline resistance plasmid that encodes the unique tcp transfer locus. The tcp locus consists of 11 genes, intP and tcpA-tcpJ, at least three of which, tcpA, tcpF, and tcpH, are essential for the conjugative transfer of pCW3. In this study we examined protein-protein interactions involving TcpA, the putative coupling protein. Use of a bacterial two-hybrid system identified interactions between TcpA and TcpC, TcpG, and TcpH. This analysis also demonstrated TcpA, TcpC, and TcpG self-interactions, which were confirmed by chemical cross-linking studies. Examination of a series of deletion and site-directed derivatives of TcpA identified the domains and motifs required for these interactions. Based on these results, we have constructed a model for this unique conjugative transfer apparatus.

Antibacterial activity of two Phlomis essential oils against food pathogens

Phlomis species from the Lamiaceae family are widely distributed in Turkey. In this study, the essential oils of Phlomis russeliana (Sims.) Bentham and Phlomis grandiflora H.S. Thompson var. grandiflora collected from North and Southern parts of Turkey, were obtained by hydrodistillation of the aerial parts. The essential oils were subsequently analysed by gas chromatography (GC) and gas chromatography–mass spectrometry (GC/MS). The major constituents of P. russeliana essential oil were identified as sesquiterpenes β-caryophyllene (23%), germacrene-D (15%), and caryophyllene oxide (8%). Analysis of P. grandiflora var. grandiflora oil also showed oxygenated sesquiterpenes such as β-eudesmol (42%) and α-eudesmol (16%) as major constituents. Furthermore, essential oils were tested in vitro against common food borne bacteria such as Aeromonas hydrophila, Bacillus cereus, Escherichia coli O157:H7, Listeria monocytogenes, Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhimurium, Yersinia enterocolitica, and the anaerobic pathogen Clostridium perfringens using the micro-broth dilution assay. When compared with antimicrobial standards weak to moderate (125 to >1000 μg/ml) minimum inhibitory concentrations (MIC) were observed. The results show that Phlomis essential oils might be an alternative to conventional antimicrobials in various foods.

The anaerobic pathogen Clostridium perfringens can escape the phagosome of macrophages under aerobic conditions

Clostridium perfringens is the most common cause of gas gangrene (clostridial myonecrosis), a disease that begins when ischaemic tissues become contaminated with C. perfringens vegetative cells or spores. An aerotolerant anaerobe, C. perfringens quickly multiplies in ischaemic tissues and spreads to healthy areas, leading to a high level of morbidity and mortality. As a species, the bacterium can synthesize 13 different toxins, and these are thought to be the major virulence factors of the disease. However, we present evidence here that C. perfringens can also persist inside macrophages, under aerobic conditions, by escaping the phagosome into the cytoplasm. C. perfringens was not killed by the cells of a clone (J774-33) of the macrophage-like murine cell line J774A.1 under aerobic or anaerobic conditions, whereas the non-pathogenic bacterium Bacillus subtilis was killed by J774-33 cells under both conditions. Electron microscopy images showed that C. perfringens cells were intact and resided mostly in the cytoplasm of J774-33 cells, whereas B. subtilis was in the phagosome. Immunofluorescence microscopy showed that intracellular C. perfringens bacteria failed to co-localize with the late endosome-lysosomal marker glycoprotein LAMP-1, whereas B. subtilis did co-localize with LAMP-1. C. perfringens also appeared to escape the phagosome of both activated and unactivated mouse peritoneal macrophages, but not as efficiently as was seen with the J774-33 cell line. In addition, cytochalasin D was used to show that phagocytosis of C. perfringens was dependent on actin polymerization and that the bacteria attach to J774-33 cells at distinct areas of the cell membrane. We propose that the ability to escape the phagosome and persist inside macrophages is an important factor in the early stages of a gangrene infection, when bacterial numbers are low and phagocytic cells are present.

Clostridium perfringens urease genes are plasmid borne.

Although many bacteria are ureolytic, and in some cases urease acts as a virulence factor, the urease phenotype has not been analyzed in the anaerobic pathogen Clostridium perfringens. In this study, approximately 2% of C. perfringens strains, representing the principal biotypes, were found to harbor the urease structural genes, ureABC, and these were localized on large plasmids that often encode, in addition, the lethal epsilon or iota toxins or the enterotoxin. This represents the first report of a plasmid-encoded urease in a gram-positive bacterium. The C. perfringens enzyme was highly similar to the ureases of other bacteria and cross-reacted with antibodies raised against the urease purified from Helicobacter pylori. Urease production was inhibited by urea and induced under growth conditions where the availability of nitrogen sources was limiting. To date, this form of regulation has been observed only for chromosomal ureABC genes.

Cloning, mapping, and molecular characterization of the rRNA operons of Clostridium perfringens

All 10 rRNA operons have been situated on the genome map of the anaerobic pathogen Clostridium perfringens. Four of these have been cloned and partially sequenced, and their transcriptional patterns in vivo and in vitro have been examined. Expression of rrnA, rrnB, and rrnE is directed by tandem promoters, P1 and P2, whereas rrnH is the only one to be expressed from a single promoter, which resembles P1. On inspection of the nucleotide sequences of the control regions, several sites which might be involved in the regulation of rrn expression were identified. These include a possible upstream activating region which could be recognized by the C. perfringens equivalent of the Escherichia coli Fis protein and a stringent response target site. Studies of maturation of 16S RNA identified two 5' cleavage sites and sequence analysis showed the dG+dC content of its gene, rrs, to be 52%, which is twice that of the genome.

Genome organization of the anaerobic pathogen Clostridium perfringens.

A physical map of the genome of Clostridium perfringens, an important human pathogen, has been established by pulsed-field gel electrophoresis. Recognition sites for six rare-cutting endonucleases were situated on a single circular chromosome of approximately 3.6 million base pairs thus defining 50 arbitrary genetic intervals of between 10 and 250 kilobase pairs. This considerably facilitated the chromosomal localization of some 24 genes and loci for which probes were available and allowed the construction of the genome map of a clostridial species.