The food-borne pathogen Campylobacter jejuni is dependent on a functional flagellum for motility and the export of virulence proteins that promote maximal host cell invasion. Both the flagellar and non-flagellar proteins exported via the flagellar type III secretion system contain a sequence within the amino-terminus that directs their export from the bacterial cell. Accordingly, we developed a genetic screen to identify C. jejuni genes that encode a type III secretion amino-terminal sequence that utilizes the flagellar type III secretion system of Yersinia enterocolitica and a phospholipase reporter (yplA). We screened a library of 321 C. jejuni genes and identified proteins with putative type III secretion amino-terminal sequences. One gene identified by the screen was Cj1242. We generated a mutation in Cj1242, and performed growth rate, motility, secretion and INT 407 cell adherence and internalization assays. The C. jejuni Cj1242 mutant was not altered in growth rate or motility when compared with the wild-type strain, but displayed an altered secretion profile and a reduction in host cell internalization. Based on the phenotype of the C. jejuni Cj1242 mutant, we designated the protein Campylobacter invasion antigen C (CiaC). Collectively, our findings indicate that CiaC is a potentially important virulence factor.

Background:The role of the MmpL11 transporter in mycobacteria is not understood. Results: Mycobacterium smegmatis mmpL11 mutants accumulate mycolic acid precursors and fail to transport monomeromycolyl diacylglycerol and mycolate ester wax to the bacterial surface. Conclusion: MmpL11 contributes to mycobacterial cell wall biosynthesis. Significance: MmpL11 plays a conserved role in mycobacterial cell wall biogenesis that is important for M. tuberculosis virulence.

The modification of metabolic pathways to allow for a dormant lifestyle appears to be an important feature for the survival of pathogenic bacteria within their host. One regulatory mechanism for persistent Mycobacterium tuberculosis infections is the stringent response. In this study, we analyze the stringent response of a nonpathogenic, saprophytic mycobacterial species, Mycobacterium smegmatis. The use of M. smegmatis as a tool for studying the mycobacterial stringent response was demonstrated by measuring the expression of two M. tuberculosis genes, hspX and eis, in M. smegmatis in the presence and absence of rel Msm . The stringent response plays a role in M. smegmatis cellular and colony formation that is suggestive of changes in the bacterial cell wall structure.The ability of Mycobacterium tuberculosis to persist in the human host is a major challenge for both vaccine-and drugbased strategies for controlling the spread of tuberculosis (TB) (17). Persistent M. tuberculosis cells are capable of initiating active growth in the host, a condition known as reactivation TB. Although it is somewhat controversial, it is generally believed that the site of viable M. tuberculosis in the host is inside caseous, necrotic, granulomatous lesions in the lungs. It has been reported that M. tuberculosis cells persisting inside granulomas lack the acid-fast staining characteristic of bacteria recovered from sputa and lesions of patents with active disease, indicating that changes occur in the bacterial cell wall during metabolic adaptation to a state of dormancy in the host (18,22). Various models have been developed to create in vitro growth conditions that mimic the presumed state of M. tuberculosis inside granulomas, including oxygen limitation (32, 33) and nutrient starvation (1,20). For example, when M. tuberculosis cultures are suspended in distilled water, they appear to lose their acid-fast staining ability, but the cells can remain viable for over 2 years in this extreme nutrient-deprived environment (22). This implies that in vitro observations of mycobacterial physiology may provide important insights into understanding how pathogenic mycobacteria survive in hosts. In addition, several mutants of M. tuberculosis that are impaired in de novo biosynthesis of various amino acids and vitamins (14) are attenuated in mouse models of tuberculosis infection, suggesting that the organism resides in a nutrient-poor environment. This supports the significance of in vitro nutrient starvation models for understanding in vivo persistence.Recently, the stringent response of M. tuberculosis has been shown to play an important role in the in vitro and in vivo survival of this bacterium (9, 29). Escherichia coli has two homologous genes that are responsible for initiating the stringent response, namely, relA and spoT. Collectively, RelA and SpoT can sense nutrient deprivation and respond by synthesizing guanosine tetraphosphate (ppGpp) and guanosine pentaphosphate (pppGpp), which can alter the promoter specificity of RNA polymeras...

The mycobacterial cell wall is crucial to the host-pathogen interface, because it provides a barrier against antibiotics and the host immune response. In addition, cell wall lipids are mycobacterial virulence factors. The mycobacterial membrane protein large (MmpL) proteins are cell wall lipid transporters that are important for basic mycobacterial physiology and Mycobacterium tuberculosis pathogenesis. MmpL3 and MmpL11 are conserved across pathogenic and nonpathogenic mycobacteria, a feature consistent with an important role in the basic physiology of the bacterium. MmpL3 is essential and transports trehalose monomycolate to the mycobacterial surface. In this report, we characterize the role of MmpL11 in M. tuberculosis. M. tuberculosis mmpL11 mutants have altered biofilms associated with lower levels of mycolic acid wax ester and long-chain triacylglycerols than those for wild-type bacteria. While the growth rate of the mmpL11 mutant is similar to that of wild-type M. tuberculosis in macrophages, the mutant exhibits impaired survival in an in vitro granuloma model. Finally, we show that the survival or recovery of the mmpL11 mutant is impaired when it is incubated under conditions of nutrient and oxygen starvation. Our results suggest that MmpL11 and its cell wall lipid substrates are important for survival in the context of adaptive immune pressure and for nonreplicating persistence, both of which are critically important aspects of M. tuberculosis pathogenicity.

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