Article

Transcriptional Priming of Salmonella Pathogenicity Island-2 Precedes Cellular Invasion

Department of Biochemistry and Biomedical Sciences, Michael G. DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, Ontario, Canada.
PLoS ONE (Impact Factor: 3.53). 06/2011; 6(6):e21648. DOI: 10.1371/journal.pone.0021648
Source: PubMed

ABSTRACT Invasive salmonellosis caused by Salmonella enterica involves an enteric stage of infection where the bacteria colonize mucosal epithelial cells, followed by systemic infection with intracellular replication in immune cells. The type III secretion system encoded in Salmonella Pathogenicity Island (SPI)-2 is essential for intracellular replication and the regulators governing high-level expression of SPI-2 genes within the macrophage phagosome and in inducing media thought to mimic this environment have been well characterized. However, low-level expression of SPI-2 genes is detectable in media thought to mimic the extracellular environment suggesting that additional regulatory pathways are involved in SPI-2 gene expression prior to cellular invasion. The regulators involved in this activity are not known and the extracellular transcriptional activity of the entire SPI-2 island in vivo has not been studied. We show that low-level, SsrB-independent promoter activity for the ssrA-ssrB two-component regulatory system and the ssaG structural operon encoded in SPI-2 is dependent on transcriptional input by OmpR and Fis under non-inducing conditions. Monitoring the activity of all SPI-2 promoters in real-time following oral infection of mice revealed invasion-independent transcriptional activity of the SPI2 T3SS in the lumen of the gut, which we suggest is a priming activity with functional relevance for the subsequent intracellular host-pathogen interaction.

Download full-text

Full-text

Available from: Brian Coombes, Jan 20, 2015
0 Followers
 · 
138 Views
  • Source
    • "The regulon governed by the PhoP–PhoQ TCS includes genes that are critical for Mg 2+ homeostasis (Soncini et al., 1996) and those that provoke modifications of the lipopolysaccharide (LPS), which determine bacterial susceptibility to cationic anti-microbial peptides (Soncini et al., 1996; Guo et al., 1997; Groisman, 1998; Gunn et al., 1998). This TCS controls the expression of essential virulence factors (García Véscovi et al., 1996; Soncini et al., 1996; Blanc-Potard and Groisman, 1997; Guo et al., 1997, 1998), which are critical for the bacterial entry mechanism into the host cell (Aguirre et al., 2006; Deiwick et al., 1999; Osborne and Coombes, 2011). Expression of the PhoP–PhoQ regulon is also necessary for intramacrophage survival, resistance to acid pH, modification of antigen presentation , formation of intracellular spacious vacuoles and alteration of macrophage cell death (Groisman, 2001). "
    [Show abstract] [Hide abstract]
    ABSTRACT: The PhoP-PhoQ system from Salmonella enterica serovar Typhimurium controls the expression of factors that are critical for the bacterial entry into host cells and the bacterial intramacrophage survival. Therefore it constitutes an interesting target to search for compounds that would control Salmonella virulence. Localisation of such compounds in complex matrixes could be facilitated by thin-layer chromatography (TLC) bioautography. To develop a TLC bioautography to detect inhibitors of the PhoP-PhoQ regulatory system in complex matrixes. The TLC plates were covered by a staining solution containing agar, Luria-Bertani medium, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal), kanamycin and a S. typhimurium strain that harbours a reporter transcriptional lacZ-fusion to an archetypal PhoP-activated gene virK. After solidification, the plate was incubated at 37°C for 16 h. A bioautographic assay suitable for the localisation of inhibitors of the PhoP-PhoQ system activity in S. enterica serovar Typhimurium present in a complex matrix is described. The assay was used to analyse a series of hydrolysed extracts prepared by alkaline treatment of crude plant extracts. Bioassay-guided analysis of the fractions by NMR spectroscopy and MS led to the identification of linolenic and linoleic acids as inhibitory input signals of the PhoP-PhoQ system. A practical tool is introduced that facilitates detection of inhibitors of the Salmonella PhoP-PhoQ regulatory system. The assay convenience is illustrated with the identification of the first naturally occurring organic compounds that down-regulate a PhoP-PhoQ regulatory system from a hydrolysed extract. Copyright © 2013 John Wiley & Sons, Ltd.
    Phytochemical Analysis 03/2014; 25(2). DOI:10.1002/pca.2482 · 2.45 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Many Gram-negative pathogens possess virulence-related type III secretion systems. Salmonella enterica uses two of these systems, encoded on the pathogenicity islands SPI-1 and SPI-2, respectively, to translocate more than 30 effector proteins into eukaryotic host cells. SteA is one of the few effectors that can be translocated by both systems. We investigated the conditions affecting the synthesis of this effector, its secretion to culture media and its translocation into host cells. Whereas steA was expressed under a wide range of conditions, some factors, including low and high osmolarity, and presence of butyrate, decreased expression. SteA was efficiently secreted to the culture media under both SPI-1 and SPI-2 inducing conditions. The kinetics of translocation into murine macrophages and human epithelial cells was studied using fusions with the 3xFLAG tag, and fusions with CyaA from Bordetella pertussis. Translocation into macrophages under non-invasive conditions was mainly dependent on the SPI-2-encoded type III secretion system but some participation of the SPI-1 system was also detected 6 hours post-infection. Interestingly, both type III secretion systems had a relevant role in the translocation of SteA into epithelial cells. Finally, a deletion approach allowed the identification of the N-terminal signal necessary for translocation of this effector. The amino acid residues 1-10 were sufficient to direct translocation into host cells through both type III secretion systems. Our results provide new examples of functional overlapping between the two type III secretion systems of Salmonella.
    PLoS ONE 10/2011; 6(10):e26930. DOI:10.1371/journal.pone.0026930 · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: DNA topology has fundamental control over the ability of transcription factors to access their target DNA sites at gene promoters. However, the influence of DNA topology on protein-DNA and protein-protein interactions is poorly understood. For example, relaxation of DNA supercoiling strongly induces the well-studied pathogenicity gene ssrA (also called spiR) in Salmonella enterica, but neither the mechanism nor the proteins involved are known. We have found that relaxation of DNA supercoiling induces expression of the Salmonella pathogenicity island (SPI)-2 regulator ssrA as well as the SPI-1 regulator hilC through a mechanism that requires the two-component regulator OmpR-EnvZ. Additionally, the ompR promoter is autoregulated in the same fashion. Conversely, the SPI-1 regulator hilD is induced by DNA relaxation but is repressed by OmpR. Relaxation of DNA supercoiling caused an increase in OmpR binding to DNA and a concomitant decrease in binding by the nucleoid-associated protein FIS. The reciprocal occupancy of DNA by OmpR and FIS was not due to antagonism between these transcription factors, but was instead a more intrinsic response to altered DNA topology. Surprisingly, DNA relaxation had no detectable effect on the binding of the global repressor H-NS. These results reveal the underlying molecular mechanism that primes SPI genes for rapid induction at the onset of host invasion. Additionally, our results reveal novel features of the archetypal two-component regulator OmpR. OmpR binding to relaxed DNA appears to generate a locally supercoiled state, which may assist promoter activation by relocating supercoiling stress-induced destabilization of DNA strands. Much has been made of the mechanisms that have evolved to regulate horizontally-acquired genes such as SPIs, but parallels among the ssrA, hilC, and ompR promoters illustrate that a fundamental form of regulation based on DNA topology coordinates the expression of these genes regardless of their origins.
    PLoS Genetics 03/2012; 8(3):e1002615. DOI:10.1371/journal.pgen.1002615 · 8.17 Impact Factor
Show more