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Abstract Background Two ancestral nucleoid-associated proteins called Hha and YdgT contribute to the negative regulation of several virulence-associated genes in Salmonella enterica serovar Typhimurium. Our previous work showed that Hha and YdgT proteins are required for negative regulation of Salmonella Pathogenicity Island-2 and that hha ydgT double mutants are attenuated for murine infection. Interestingly, hha ydgT mutant bacteria exhibited a non-motile phenotype suggesting that Hha and YdgT have a role in flagellar regulation. Results In this study we show that the non-motile phenotype of hha ydgT mutants is due to decreased levels of the master transcriptional regulator FlhD4C2 resulting in down-regulation of class II/III and class III flagellar promoters and lack of surface flagella on these cells. The horizontally acquired pefI-srgD region was found to be partially responsible for this phenotype since deletion of pefI-srgD in a hha ydgT deletion background resulted in transient restoration of class II/III and III transcription, expression of surface flagella, and motility in the quadruple mutant. Conclusion These data extend our current understanding of the mechanisms through which Hha and YdgT regulate flagellar biosynthesis and further describe how S. Typhimurium has integrated horizontal gene acquisitions into ancestral regulatory networks.

Abstract Background We have shown previously that Chlamydophila pneumoniae contains a dual-specific Ser/Thr protein kinase that phosphorylates CdsD, a structural component of the type III secretion apparatus. To further study the role of PknD in growth and development we sought to identify a PknD inhibitor to determine whether PknD activity is required for replication. Results Using an in vitro kinase assay we screened 80 known eukaryotic protein kinase inhibitors for activity against PknD and identified a 3'-pyridyl oxindole compound that inhibited PknD autophosphorylation and phosphorylation of CdsD. The PknD inhibitor significantly retarded the growth rate of C. pneumoniae as evidenced by the presence of very small inclusions with a reduced number of bacteria as seen by electron microscopy. These inclusions contained the normal replicative forms including elementary bodies (EB), intermediate bodies (IB) and reticulate bodies (RB), but lacked persistent bodies (PB), indicating that induction of persistence was not the cause of reduced chlamydial growth. Blind passage of C. pneumoniae grown in the presence of this PknD inhibitor for 72 or 84 hr failed to produce inclusions, suggesting this compound blocks an essential step in the production of infectious chlamydial EB. The compound was not toxic to HeLa cells, did not block activation of the MEK/ERK pathway required for chlamydial invasion and did not block intracellular replication of either Chlamydia trachomatis serovar D or Salmonella enterica sv. Typhimurium suggesting that the inhibitory effect of the compound is specific for C. pneumoniae. Conclusion We have identified a 3'-pyridyl oxindole compound that inhibits the in vitro kinase activity of C. pneumoniae PknD and inhibits the growth and production of infectious C. pneumoniae progeny in HeLa cells. Together, these results suggest that PknD may play a key role in the developmental cycle of C. pneumoniae.

Abstract Background The survival of Salmonella enterica within the intracellular host niche requires highly co-ordinated expression of virulence effectors predominantly regulated by the SsrAB two-component regulatory system. S. enterica serovar Typhimurium mutants lacking the ssrAB genes are avirulent in mice, highlighting the importance of this regulatory system in vivo. Mutants lacking the gene encoding the alternative sigma factor σE (rpoE) are also highly attenuated for intracellular survival, pointing to a potential connection with the SsrAB regulatory system. Results In this study we demonstrate that RpoE is involved in fine-tuning the expression of a subset of SsrB-regulated genes found in the Salmonella pathogenicity island-2 (SPI-2) genetic locus that encodes a horizontally acquired type III secretion system, and unlinked genes integrated into this regulon that are required for virulence in host animals. Conclusion These data point to a potential connection between the virulence phenotype of strains lacking ssrB and rpoE, and highlight new transcriptional regulation that might be essential for appropriate temporal and spatial control of the virulence-associated type III secretion system during host infection.