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Salmonella Typhimurium (S. Tm) establishes systemic infection in susceptible hosts by evading the innate immune response and replicating within host phagocytes. Here, we sought to identify inhibitors of intracellular S. Tm replication by conducting parallel chemical screens against S. Tm growing in macrophage-mimicking media and within macrophages. We identify several compounds that inhibit Salmonella growth in the intracellular environment and in acidic, ion-limited media. We report on the antimicrobial activity of the psychoactive drug metergoline, which is specific against intracellular S. Tm. Screening an S. Tm deletion library in the presence of metergoline reveals hypersensitization of outer membrane mutants to metergoline activity. Metergoline disrupts the proton motive force at the bacterial cytoplasmic membrane and extends animal survival during a systemic S. Tm infection. This work highlights the predictive nature of intracellular screens for in vivo efficacy, and identifies metergoline as a novel antimicrobial active against Salmonella.

Technological advances in DNA recovery and sequencing have drastically expanded the scope of genetic analyses of ancient specimens to the extent that full genomic investigations are now feasible and are quickly becoming standard (1). This trend has important implications for infectious disease research because genomic data from ancient microbes may help to elucidate mechanisms of pathogen evolution and adaptation for emerging and re-emerging infections. Here we report a reconstructed ancient genome of Yersinia pestis at 30-fold average coverage from Black Death victims securely dated to episodes of pestilence-associated mortality in London, England, 1348-1350. Genetic architecture and phylogenetic analysis indicate that the ancient organism is ancestral to most extant strains and sits very close to the ancestral node of all Y. pestis commonly associated with human infection. Temporal estimates suggest that the Black Death of 1347-1351 was the main historical event responsible for the introduction and widespread dissemination of the ancestor to all currently circulating Y. pestis strains pathogenic to humans, and further indicates that contemporary Y.pestis epidemics have their origins in the medieval era. Comparisons against modern genomes reveal no unique derived positions in the medieval organism, indicating that the perceived increased virulence of the disease during the Black Death may not have been due to bacterial phenotype. These findings support the notion that factors other than microbial genetics, such as environment, vector dynamics and host susceptibility, should be at the forefront of epidemiological discussions regarding emerging Y.pestis infections. The Black Death of 1347-1351, caused by the bacterium Yersinia pestis (2,3), provides one of the best historical examples of an emerging infection with rapid dissemination and high mortality, claiming an estimated 30-50% of the European population in only a five-year period (4). Discrepancies in epidemiological trends between the medieval disease and modern Y. pestis infections have ignited controversy over the pandemic's aetiologic agent (5,6). Although ancient DNA investigations have strongly implicated Y. pestis (2,3) in the ancient pandemic, genetic changes in the bacterium may be partially responsible for differences in disease manifestation and severity. To understand the organism's evolution it is necessary to characterize the genetic changes involved in its transformation from a sylvatic pathogen to one capable of pandemic human infection on the scale of the Black Death, and to determine its relationship with currently circulating strains. Here we begin this discussion by presenting the first draft genome sequence of the ancient pathogen., Y. pestis is a recently evolved descendent of the soil-dwelling bacillus Yersinia pseudotuberculosis (7), which in the course of its evolution acquired two additional plasmids (pMT1 and pPCP1) that provide [...]

The Hha/YmoA family of nucleoid-associated proteins is involved in gene regulation in enterobacteria. In Salmonella enterica serovar Typhimurium, virulence genes required for intracellular growth are induced following host cell invasion but the proteins responsible for repressing these genes prior to host cell entry have not been fully identified. We demonstrate here that Hha is the major repressor responsible for silencing virulence genes carried in Salmonella pathogenicity island 2 prior to bacteria sensing an intracellular environmental cue. doi:10.1128/JB.00120-07

Salmonella enterica serovar Typhimurium is a facultative intracellular pathogen causing disease in several hosts. These bacteria use two distinct type III secretion systems that inject effector proteins into the host cell for invasion and to alter maturation of the Salmonella-containing vacuole. Members of the Salmonella translocated effector (STE) family contain a conserved N-terminal translocation signal of approximately 140 aa. In this study, the STE family member SifA was examined using deletion strategies. Small deletions (approx. 20 residues long) throughout SifA were sufficient to block its secretion and/or translocation into host cells. Transfection of HeLa cells with a GFP-SifA fusion was previously shown to be sufficient to induce formation of Sif-like tubules resembling structures present in Salmonella-infected cells. The present study showed that both N- and C-terminal domains of SifA are required for this phenotype. Furthermore, both domains could induce aggregation of Lamp1-positive compartments, provided they were coupled to the minimal C-terminal membrane-anchoring motif of SifA. Mutation or deletion of the conserved STE N-terminal WEK(I/M)xxFF translocation motif of SopD2 disrupted its association with Lamp1-positive compartments, implicating these residues in both effector translocation and subcellular localization. Interestingly, one GFP-SifA deletion mutant lacking residues 42-101, but retaining the WEK(I/M)xxFF motif, targeted the Golgi apparatus. In addition, short peptides containing the signature WEK(I/M)xxFF motif derived from the N-termini of Salmonella effectors SopD2, SseJ and SspH2 were sufficient to localize GFP to the Golgi. These studies suggest that Salmonella effectors contain multifunctional motifs or domains that regulate several effector traits, including protein secretion/translocation, localization and subversion of host cell systems. Conditions that perturb the tertiary structure of effectors can influence their localization in host cells by liberating cryptic intracellular targeting motifs.

Salmonella enterica relies on a type III secretion system encoded in Salmonella pathogenicity island-2 (SPI-2) to survive and replicate within macrophages at systemic sites during typhoid. SPI-2 virulence is induced upon entry into macrophages, but the mechanisms of SPI-2 gene control in vivo remain unclear, particularly with regard to negative regulators that control the contextual activation of SPI-2. Here, we identified and characterized YdgT as a negative modulator of the SPI-2 pathogenicity island and established that this negative regulation is central to systemic pathogenesis because ydgT mutants overexpressing typhoid virulence genes were ultimately attenuated during infection, ydgT mutants displayed a biphasic virulence phenotype during in vivo competitive infections that consisted of an early 'gain-of-virulence' dependent on SPI-2 activation, followed by attenuation later in infection indicating that proper contextual regulation of SPI-2 by YdgT is necessary for full virulence during systemic colonization. These data suggest that overexpression of virulence-associated type III secretion genes can have an adverse effect on bacterial pathogenesis in vivo. in vivo | type III secretion | YdgT | pathogenesis

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 Adherent and invasive Escherichia coli (AIEC) are commonly found in ileal lesions of Crohn's Disease (CD) patients, where they adhere to intestinal epithelial cells and invade into and survive in epithelial cells and macrophages, thereby gaining access to a typically restricted host niche. Colonization leads to strong inflammatory responses in the gut suggesting that AIEC could play a role in CD immunopathology. Despite extensive investigation, the genetic determinants accounting for the AIEC phenotype remain poorly defined. To address this, we present the complete genome sequence of an AIEC, revealing the genetic blueprint for this disease-associated E. coli pathotype. Results We sequenced the complete genome of E. coli NRG857c (O83:H1), a clinical isolate of AIEC from the ileum of a Crohn's Disease patient. Our sequence data confirmed a phylogenetic linkage between AIEC and extraintestinal pathogenic E. coli causing urinary tract infections and neonatal meningitis. The comparison of the NRG857c AIEC genome with other pathogenic and commensal E. coli allowed for the identification of unique genetic features of the AIEC pathotype, including 41 genomic islands, and unique genes that are found only in strains exhibiting the adherent and invasive phenotype. Conclusions Up to now, the virulence-like features associated with AIEC are detectable only phenotypically. AIEC genome sequence data will facilitate the identification of genetic determinants implicated in invasion and intracellular growth, as well as enable functional genomic studies of AIEC gene expression during health and disease.

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.

Abstract Background Salmonella meningitis is a rare and serious infection of the central nervous system following acute Salmonella enterica sepsis. For this pathogen, no appropriate model has been reported in which to examine infection kinetics and natural dissemination to the brain. Methods Five mouse lines including C57BL/6, Balb/c, 129S6-Slc11a1tm1Mcg, 129S1/SvImJ, B6.129-Inpp5dtm1Rkh were used in the murine typhoid model to examine the dissemination of systemic Salmonella enterica serovar Typhimurium following oral infection. Results We report data on spontaneous meningitis and brain infection following oral infection of mice with Salmonella enterica serovar Typhimurium. Conclusion This model may provide a system in which dissemination of bacteria through the central nervous system and the influence of host and bacterial genetics can be queried.

The identification of three genomic islands encoding non-LEE effector (nle) genes and 14 individual nle genes in non-O157 shiga toxin-producing Escherichia coli (STEC) strains is reported. A molecular risk assessment method to rapidly recognize and respond to non-O157 STEC strains is presented.

Bacterial pathogens promote their survival by deploying virulence factors that modify the host environment. But these virulence proteins are energetically costly to produce, and so avirulent defector mutants that grow [...]