SummaryAll known virulence genes of Listeria monocytogenes are under positive regulation by the transcription factor PrfA. Previous work employing the L. monocytogenes strain NCTC7973 suggested that the disaccharide cellobiose might serve as a specific 'signature molecule' which functions to prevent activation of the PrfAcontrolled regulon in a soil environment. We have examined three other L. monocytogenes strains, 10403S, LO28 and EGD, all commonly regarded as wild-type isolates, and find that NCTC7973 is anomalous with respect to the effect of carbohydrates on the expression of PrfA-controlled gene expression. In the case of 10403S, LO28 and EGD, several other readily metabolized mono-and disaccharides are as effective as cellobiose in repressing expression of the PrfA-controlled gene hly, indicating that the cellobiose effect is not specific, and suggesting that NCTC7973 may be a partially deregulated variant. Moreover, concentrations of cellobiose and other sugars required for repression of hly expression (> 1 mM) were found to significantly enhance growth of L. monocytogenes cultures, suggesting that the repression phenomenon probably results from a metabolic effect of sugar utilization rather than a signal-sensing response. Thus the previously reported cellobiose effect may reflect an aspect of a more global mechanism of catabolite repression in L. monocytogenes. Although cellobiose represses expression of hly and plcA at the level of transcript accumulation, quantitative Western blot analysis indicates that cellobiose has no effect on PrfA levels. These results are consistent with a model in which PrfA activity is controlled by interaction with a hypothetical cofactor, the synthesis or depletion of which is responsive to the presence of readily metabolized carbohydrates.

The induction of cytokine synthesis by flagellin is mediated by a Toll-like receptor 5 (TLR5) signaling pathway. Although flagellin activation of the IL-1R-associated kinase and induction of TNF-α synthesis are dependent on TLR5 and not TLR4, we have found that flagellin stimulates NO in macrophages via a pathway that requires TLR5 and TLR4. Flagellin induced NO synthesis in HeNC2 cells, a murine macrophage cell line that expresses wild-type TLR4, but not in TLR4-mutant or -deficient GG2EE and 10ScNCr/23 cells. Flagellin stimulated an increase in inducible NO synthase (iNOS) mRNA and activation of the iNOS promoter. TLR5 forms heteromeric complexes with TLR4 as well as homomeric complexes. IFN-γ permitted GG2EE and 10ScNCr/23 cells to produce NO in response to flagellin. Flagellin stimulated IFN-β synthesis and Stat1 activation. The effect of flagellin on iNOS gene expression was inhibited by a Stat1 mutant protein. Taken together, these results support the conclusions that flagellin induces distinct patterns of inflammatory mediators depending on the nature of the TLR5 signaling complex and that the induction of NO by flagellin involves signaling via TLR5/TLR4 complexes.

Listeria monocytogenes is a Gram-positive intracytosolic pathogen that causes severe disease in pregnant and immunocompromised individuals. We found that L. monocytogenes lacking the lipoate protein ligase LplA1 was defective for growth specifically in the host cytosol and was less virulent in animals by a factor of 300. A major target for LplA1, the E2 subunit of pyruvate dehydrogenase (PDH), lacked a critical lipoyl modification when the DeltalplA1 strain was grown intracellularly, which suggests that abortive growth was due to loss of PDH function. Thus, the use of host-derived lipoic acid may be a critical process for in vivo replication of bacterial pathogens.

Listeria monocytogenes requires listeriolysin O (LLO) and ActA, the products of hly and actA, respectively, to establish a productive intracellular infection. LLO is essential for vacuolar lysis and entry into the cytosol, while ActA is required for bacterial spread to adjacent cells. We have used a transcriptional reporter gene system to compare the expression ofactA and hly during intracellular growth to that during growth in broth cultures. The hly andactA genes were transcriptionally fused toEscherichia coli lacZ and Bacillus pumilus cat-86 (cat), and the fusions were integrated in single copies into the L. monocytogenes chromosome. A chloramphenicol resistance assay indicated that the hlyfusion but not the actA fusion was significantly activated in Luria-Bertani (LB) broth, and this finding correlated with LLO and ActA levels detectable in broth cultures. Quantitation of promoter activity on the basis of β-galactosidase activity revealed up to 10-fold-higher level of expression of the hly fusion relative to the actA fusion in LB broth. In contrast, both fusions were active in the cytosol of J774 cells, and the activity of the actA fusion was approximately 3-fold higher than that of the hly fusion under these conditions. However, quantitative immunoprecipitation of ActA and LLO from infected J774 cells demonstrated approximately 70-fold more cytosolic ActA than cytosolic LLO. Finally, in comparison to induction in broth cultures,actA was highly induced (226-fold) and hly was moderately induced (20-fold) in J774 cells. Collectively, these results indicate that actA and hly are differentially regulated in response to the growth environment and that both genes are preferentially expressed during intracellular growth. Further, while the lower level of production of ActA than of LLO in broth can be accounted for by transcriptional regulation, the relative abundance of intracellular ActA compared to that of intracellular LLO is a function of additional, possibly host-mediated, factors.

No abstract