The Clostridium difficile cell wall protein CwpV is antigenically variable between strains, but exhibits conserved aggregation-promoting function.

Clostridium difficile is the main cause of antibiotic-associated diarrhea, leading to significant morbidity and mortality and putting considerable economic pressure on healthcare systems. Current knowledge of the molecular basis of pathogenesis is limited primarily to the activities and regulation of two major toxins. In contrast, little is known of mechanisms used in colonization of the enteric system. C. difficile expresses a proteinaceous array on its cell surface known as the S-layer, consisting primarily of the major S-layer protein SlpA and a family of SlpA homologues, the cell wall protein (CWP) family. CwpV is the largest member of this family and is expressed in a phase variable manner. Here we show CwpV promotes C. difficile aggregation, mediated by the C-terminal repetitive domain. This domain varies markedly between strains; five distinct repeat types were identified and were shown to be antigenically distinct. Other aspects of CwpV are, however, conserved. All CwpV types are expressed in a phase variable manner. Using targeted gene knock-out, we show that a single site-specific recombinase RecV is required for CwpV phase variation. CwpV is post-translationally cleaved at a conserved site leading to formation of a complex of cleavage products. The highly conserved N-terminus anchors the CwpV complex to the cell surface. Therefore CwpV function, regulation and processing are highly conserved across C. difficile strains, whilst the functional domain exists in at least five antigenically distinct forms. This hints at a complex evolutionary history for CwpV.

Distinctive profiles of infection and pathology in hamsters infected with Clostridium difficile strains 630 and B1.

Currently, the Golden Syrian hamster is widely considered an important model of Clostridium difficile disease, as oral infection of this animal pretreated with antibiotics reproduces many of the symptoms observed in humans. Two C. difficile strains, B1 and 630, showed significant differences in the progression and severity of disease in this model. B1-infected hamsters exhibited more severe pathology and a shorter time to death than hamsters infected with 630. Histological changes in the gut did not correlate with absolute numbers of C. difficile bacteria, but there were clear differences in the distribution of bacteria within gut tissues. Light, scanning, and transmission electron microscopy revealed high numbers of B1 bacteria at the mucosal surface of the tissue, whereas 630 bacteria were more frequently associated with the crypt regions. Both B1 and 630 bacteria were frequently observed within polymorphonuclear leukocytes, although, interestingly, a space frequently separated B1 bacteria from the phagosome wall, a phenomenon not observed with 630. However, pilus-like structures were detected on 630 located in the crypts of the gut tissue. Furthermore, B1 bacteria, but not 630 bacteria, were found within nonphagocytic cells, including enterocytes and muscle cells.

A novel genetic switch controls phase variable expression of CwpV, a Clostridium difficile cell wall protein.

Clostridium difficile is a nosocomial pathogen that can cause severe gastrointestinal infections. C. difficile encodes a family of cell wall proteins, some of which are implicated in pathogenesis. Here we have characterized CwpV, the largest member of this family. CwpV is surface expressed and post-translationally processed in a manner analogous to the major S-layer protein SlpA. Expression of cwpV is phase variable, with approximately 5% of cells in a population expressing the protein under standard laboratory growth conditions. Upstream of cwpV, inverted repeats flank a 195 bp sequence which undergoes DNA inversion. Use of a gusA transcriptional reporter demonstrated that phase variation is mediated by DNA inversion; in one orientation cwpV is expressed while in the opposite orientation the gene is silent. The inversion region contains neither the promoter nor any of the open reading frame, therefore this system differs from previously described phase variation mechanisms. The cwpV promoter is located upstream of the inversion region and we propose a model of phase variation based on intrinsic terminator formation in the OFF transcript. A C. difficile site-specific recombinase able to catalyse the inversion has been identified.

KOPS-guided DNA translocation by FtsK safeguards Escherichia coli chromosome segregation.

The septum-located DNA translocase, FtsK, acts to co-ordinate the late steps of Escherichia coli chromosome segregation with cell division. The FtsK gamma regulatory subdomain interacts with 8 bp KOPS DNA sequences, which are oriented from the replication origin to the terminus region (ter) in each arm of the chromosome. This interaction directs FtsK translocation towards ter where the final chromosome unlinking by decatenation and chromosome dimer resolution occurs. Chromosome dimer resolution requires FtsK translocation along DNA and its interaction with the XerCD recombinase bound to the recombination site, dif, located within ter. The frequency of chromosome dimer formation is approximately 15% per generation in wild-type cells. Here we characterize FtsK alleles that no longer recognize KOPS, yet are proficient for translocation and chromosome dimer resolution. Non-directed FtsK translocation leads to a small reduction in fitness in otherwise normal cell populations, as a consequence of approximately 70% of chromosome dimers being resolved to monomers. More serious consequences arise when chromosome dimer formation is increased, or their resolution efficiency is impaired because of defects in chromosome organization and processing. For example, when Cre-loxP recombination replaces XerCD-dif recombination in dimer resolution, when functional MukBEF is absent, or when replication terminates away from ter.

Microarray analysis of the transcriptional responses of Clostridium difficile to environmental and antibiotic stress.

Clostridium difficile is a spore-forming anaerobic bacterium that is an emerging nosocomial threat; incidence of infection in hospitals is increasing, both in frequency and severity, resulting in considerable morbidity and mortality. In order to adapt to the intestinal environment, C. difficile must react to the many stresses involved with colonization, including exposure to antibiotics, which represents the most frequent precipitating agent of C. difficile infection. The responses of C. difficile to environmental shocks (heat, pH and oxidative shock) and to growth in the presence of subinhibitory concentrations of antibiotics (amoxicillin, clindamycin and metronidazole) were investigated using the C. difficile 630 microarray developed by the Bacterial Microarray Group at St George's, University of London, UK (BmuG@S). Significantly regulated genes and operons were identified that are unique to or common between different stresses. The transcriptional profiles of C. difficile 630 are similar after growth in the presence of amoxicillin and clindamycin: both increased transcription of ribosomal protein genes and altered transcription of genes encoding surface-associated proteins. In contrast, metronidazole treatment resulted in minor changes in transcription patterns. The general stress response is observed after heat shock and acid shock. Heat shock also affected transcription of several biochemical pathways. Exposure to atmospheric oxygen induced a large number of electron transporters. This study provides a starting point for detailed analyses of numerous genes whose expression is affected by stress and may therefore be involved in adaptation to the host environment.

Synergistic up-regulation of epithelial cell matrix metalloproteinase-9 secretion in tuberculosis.

Mycobacterium tuberculosis (MTb) kills approximately 2 million people each year. MTb must drive host tissue destruction to disseminate and also to cause pulmonary cavitation. Matrix metalloproteinase-9 (MMP-9, gelatinase B) is implicated in this Tb-related immunopathology. We demonstrate that conditioned media from MTb-infected monocytes (CoMTb), but not direct infection with MTb, up-regulates MMP-9 gene expression and secretion from primary human bronchial epithelial cells (NHBE). MMP-9 secretion was increased 8.7-fold by CoMTb (P < 0.05) as assayed by gelatin zymography. A549 and 16HBE14o epithelial cell MMP secretion was significantly less than primary NHBE secretion. MMP-9 secretion was decreased 53.2% by inhibition of the p38 mitogen-activated protein kinase (MAPK) by SB203580 (P < 0.01) and 48.3% by inhibition of extracellular signal-regulated kinase with PD98059 (P < 0.05). MMP-9 secretion was prostaglandin independent. TNF-alpha was necessary but not sufficient for MMP-9 up-regulation by the monocyte-epithelial cell network. Soluble factors derived from Tb culture synergized with TNF-alpha to increase MMP-9 secretion by NHBE 6-fold (P < 0.01 compared with either stimulus alone). Together, these data reveal a new mechanism by which host- and pathogen-derived factors act together in MTb infection to drive MAPK-dependent MMP-9 secretion from respiratory epithelial cells.

Kinetoplastid PPEF phosphatases: dual acylated proteins expressed in the endomembrane system of Leishmania.

Bioinformatic analyses have been used to identify potential downstream targets of the essential enzyme N-myristoyl transferase in the TriTryp species, Leishmania major, Trypanosoma brucei and Trypanosoma cruzi. These database searches predict approximately 60 putative N-myristoylated proteins with high confidence, including both previously characterised and novel molecules. One of the latter is an N-myristoylated protein phosphatase which has high sequence similarity to the Protein Phosphatase with EF-Hand (PPEF) proteins identified in sensory cells of higher eukaryotes. In L. major and T. brucei, the PPEF-like phosphatases are encoded by single-copy genes and are constitutively expressed in all parasite life cycle stages. The N-terminus of LmPPEF is a substrate for N-myristoyl transferase and is also palmitoylated in vivo. The wild type protein has been localised to the endocytic system by immunofluorescence. The catalytic and fused C-terminal domains of the kinetoplastid and other eukaryotic PPEFs share high sequence similarity, but unlike their higher eukaryotic relatives, the C-terminal parasite EF-hand domains are degenerate and do not bind calcium.

Mycobacterium tuberculosis up-regulates matrix metalloproteinase-1 secretion from human airway epithelial cells via a p38 MAPK switch.

Pulmonary cavitation is vital to the persistence and spread of Mycobacterium tuberculosis (MTb), but mechanisms underlying this lung destruction are poorly understood. Fibrillar type I collagen provides the lung's tensile strength, and only matrix metalloproteinases (MMPs) can degrade it at neutral pH. We investigated MTb-infected lung tissue and found that airway epithelial cells adjacent to tuberculosis (Tb) granulomas expressed a high level of MMP-1 (interstitial collagenase). Conditioned media from MTb-infected monocytes (CoMTb) up-regulated epithelial cell MMP-1 promoter activity, gene expression, and secretion, whereas direct MTb infection did not. CoMTb concurrently suppressed tissue inhibitor of metalloprotease-1 (TIMP-1) secretion, further promoting matrix degradation, and in Tb patients very low TIMP-1 expression was detected. MMP-1 up-regulation required synergy between TNF-alpha and G protein-coupled receptor signaling pathways. CoMTb stimulated p38 MAPK phosphorylation, and this is the point of TNF-alpha synergy with G protein-coupled receptor activation. Furthermore, p38 phosphorylation was the switch up-regulating MMP-1 activity and decreasing TIMP-1 secretion. Activated p38 localized to MMP-1-secreting airway epithelial cells in Tb patients. These data reveal a monocyte-epithelial cell network whereby MTb may drive tissue destruction, and they demonstrate that p38 phosphorylation is a key regulatory point in the generation of a matrix-degrading phenotype.