- Volume 156, Issue 9, 2010
Volume 156, Issue 9, 2010
- Microbial Pathogenicity
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The 33 carboxyl-terminal residues of Spa40 orchestrate the multi-step assembly process of the type III secretion needle complex in Shigella flexneri
The type III secretion apparatus (T3SA) is a central virulence factor of many Gram-negative bacteria. Its overall morphology consists of a cytoplasmic region, inner- and outer-membrane sections and an extracellular needle. In Shigella, the length of the needle is regulated by Spa32. To understand better the role of Spa32 we searched for its interacting partners using a two-hybrid screen in yeast. We found that Spa32 interacts with the 33 C-terminal residues (CC*) of Spa40, a member of the conserved FlhB/YscU family. Using a GST pull-down assay we confirmed this interaction and identified additional interactions between Spa40 and the type III secretion components Spa33, Spa47, MxiK, MxiN and MxiA. Inactivation of spa40 abolished protein secretion and led to needleless structures. Genetic and functional analyses were used to investigate the roles of residues L310 and V320, located within the CC* domain of Spa40, in the assembly of the T3SA. Spa40 cleavage, at the conserved NPTH motif, is required for assembly of the T3SA and for its interaction with Spa32, Spa33 and Spa47. In contrast, unprocessed forms of Spa40 interacted only with MxiA, MxiK and MxiN. Our data suggest that the conformation of the cytoplasmic domain of Spa40 defines the multi-step assembly process of the T3SA.
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ApuA, a multifunctional α-glucan-degrading enzyme of Streptococcus suis, mediates adhesion to porcine epithelium and mucus
More LessWe have identified apuA in Streptococcus suis, which encodes a bifunctional amylopullulanase with conserved α-amylase and pullulanase substrate-binding domains and catalytic motifs. ApuA exhibited properties typical of a Gram-positive surface protein, with a putative signal sequence and LPKTGE cell-wall-anchoring motif. A recombinant protein containing the predicted N-terminal α-amylase domain of ApuA was shown to have α-(1,4) glycosidic activity. Additionally, an apuA mutant of S. suis lacked the pullulanase α-(1,6) glycosidic activity detected in a cell-surface protein extract of wild-type S. suis. ApuA was required for normal growth in complex medium containing pullulan as the major carbon source, suggesting that this enzyme plays a role in nutrient acquisition in vivo via the degradation of glycogen and food-derived starch in the nasopharyngeal and oral cavities. ApuA was shown to promote adhesion to porcine epithelium and mucus in vitro, highlighting a link between carbohydrate utilization and the ability of S. suis to colonize and infect the host.
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Mycobacterium tuberculosis protein kinase K confers survival advantage during early infection in mice and regulates growth in culture and during persistent infection: implications for immune modulation
More LessMycobacterium tuberculosis serine/threonine protein kinases (STPKs) are key regulators of growth and metabolism; however, evidence for their roles in virulence is limited. In a preliminary screen based on comparative expression between strains H37Rv and H37Ra, six STPK genes, pknD, pknG, pknH, pknJ, pknK and pknL, showed higher expression in H37Rv. In the second screen, STPK expression was analysed in H37Rv-infected human macrophages. Interestingly, significant expression of pknK was detected only at 18 h post-infection, suggesting its involvement in early infection events. We have investigated the roles of PknK in vitro and in vivo. PknK levels were induced under stationary phase and deletion of pknK resulted in increased resistance of the mutant to acidic pH, hypoxia, oxidative and stationary-phase stresses in vitro. These results, together with the increased survival of the ΔpknK strain during persistent infection in mice, reveal a role for PknK in adaptive mechanisms that slow the growth of mycobacteria. A novel finding of this study was the inhibition of growth of ΔpknK strain during acute infection in mice that correlated with the significant upregulation of tumour necrosis factor as well as the simultaneous downregulation of interleukin-12p40, interferon-γ and induced nitric oxide synthase transcripts. Finally, we provide evidence for the localization of PknK during infection and discuss its implications in pathogenesis.
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Mutation of the gene encoding a major outer-membrane protein in Xanthomonas campestris pv. campestris causes pleiotropic effects, including loss of pathogenicity
More LessXanthomonas campestris pv. campestris (Xcc) is the phytopathogen that causes black rot in crucifers. The xanthan polysaccharide and extracellular enzymes produced by this organism are virulence factors, the expression of which is upregulated by Clp (CRP-like protein) and DSF (diffusible signal factor), which is synthesized by RpfF. It is also known that biofilm formation/dispersal, regulated by the effect of controlled synthesis of DSF on cell–cell signalling, is required for virulence. Furthermore, a deficiency in DSF causes cell aggregation with concomitant production of a gum-like substance that can be dispersed by addition of DSF or digested by exogenous endo-β-1,4-mannanase expressed by Xcc. In this study, Western blotting of proteins from a mopB mutant (XcMopB) showed Xcc MopB to be the major outer-membrane protein (OMP); Xcc MopB shared over 97 % identity with homologues from other members of Xanthomonas. Similarly to the rpfF mutant, XcMopB formed aggregates with simultaneous production of a gummy substance, but these aggregates could not be dispersed by DSF or endo-β-1,4-mannanase, indicating that different mechanisms were involved in aggregation. In addition, XcMopB showed surface deformation, altered OMP composition, impaired xanthan production, increased sensitivity to stressful conditions including SDS, elevated temperature and changes in pH, reduced adhesion and motility and defects in pathogenesis. The finding that the major OMP is required for pathogenicity is unprecedented in phytopathogenic bacteria.
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Interaction of Rickettsia felis with histone H2B facilitates the infection of a tick cell line
Haematophagous arthropods are the primary vectors in the transmission of Rickettsia, yet the molecular mechanisms mediating the rickettsial infection of arthropods remain elusive. This study utilized a biotinylated protein pull-down assay together with LC-MS/MS to identify interaction between Ixodes scapularis histone H2B and Rickettsia felis. Co-immunoprecipitation of histone with rickettsial cell lysate demonstrated the association of H2B with R. felis proteins, including outer-membrane protein B (OmpB), a major rickettsial adhesin molecule. The rickettsial infection of tick ISE6 cells was reduced by approximately 25 % via RNA-mediated H2B-depletion or enzymic treatment of histones. The interaction of H2B with the rickettsial adhesin OmpB suggests a role for H2B in mediating R. felis internalization into ISE6 cells.
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The type III secretion system of Vibrio alginolyticus induces rapid apoptosis, cell rounding and osmotic lysis of fish cells
Vibrio alginolyticus is a Gram-negative bacterium and has been recognized as an opportunistic pathogen in humans as well as marine animals. However, the virulence mechanisms for this species of Vibrio have not been elucidated. This study characterized multiple mechanisms that induce cell death in fish cells upon infection with a V. alginolyticus strain, ZJO. The bacterium required its type III secretion system (T3SS) to cause rapid death of infected fish cells. Dying cells exhibited some features of apoptotic cells, such as membrane blebbing, nuclear condensation and DNA fragmentation. Further studies showed that caspase-3 was activated by the T3SS of the ZJO strain, confirming that infection with V. alginolyticus rapidly induces T3SS-dependent apoptosis in fish cells. Infection with the ZJO strain also led to membrane pore formation and release of cellular contents from infected fish cells, as evidenced by lactate dehydrogenase release and the uptake of a membrane-impermeable dye. Importantly, inhibition of apoptosis did not prevent ZJO-infected cells from releasing cellular contents and did not block cell rounding. Taken together, these data demonstrate that infection with V. alginolyticus may promote at least three different T3SS-dependent events, which lead to the death of fish cells. This study provides an important insight into the mechanism used by Vibrio species to cause host-cell death.
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- Physiology And Biochemistry
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Escherichia coli K-12 YfgF is an anaerobic cyclic di-GMP phosphodiesterase with roles in cell surface remodelling and the oxidative stress response
More LessThe Escherichia coli K-12 yfgF gene encodes a protein with domains associated with cyclic di-GMP signalling: GGDEF (associated with diguanylate cyclase activity) and EAL (associated with cyclic di-GMP phosphodiesterase activity). Here, it is shown that yfgF is expressed under anaerobic conditions from a class II FNR (regulator of fumarate and nitrate reduction)-dependent promoter. Anaerobic expression of yfgF is greatest in stationary phase, and in cultures grown at 28 °C, suggesting that low growth rates promote yfgF expression. Mutation of yfgF resulted in altered cell surface properties and enhanced sensitivity when anaerobic cultures were exposed to peroxides. The purified YfgF GGDEF-EAL (YfgFGE) and EAL (YfgFE) domains possessed cyclic di-GMP-specific phosphodiesterase activity, but lacked diguanylate cyclase activity. However, the catalytically inactive GGDEF domain was required for YfgFGE dimerization and enhanced cyclic di-GMP phosphodiesterase activity in the presence of physiological concentrations of Mg2+. The cyclic di-GMP phosphodiesterase activity of YfgFGE and YfgFE was inhibited by the product of the reaction, 5′-phosphoguanylyl-(3′–5′)-guanosine (pGpG). Thus, it is shown that the yfgF gene encodes an anaerobic cyclic di-GMP phosphodiesterase that is involved in remodelling the cell surface of E. coli K-12 and in the response to peroxide shock, with implications for integrating three global regulatory networks, i.e. oxygen regulation, cyclic di-GMP signalling and the oxidative stress response.
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Volumes and issues
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