CRP-cAMP mediates silencing of Salmonella virulence at the post-transcriptional level

Invasion of epithelial cells by Salmonella enterica requires expression of genes located in the pathogenicity island I (SPI-1). The expression of SPI-1 genes is very tightly regulated and activated only under specific conditions. Most studies have focused on the regulatory pathways that induce SPI-1 expression. Here, we describe a new regulatory circuit involving CRP-cAMP, a widely established metabolic regulator, in silencing of SPI-1 genes under non-permissive conditions. In CRP-cAMP-deficient strains we detected a strong upregulation of SPI-1 genes in the mid-logarithmic growth phase. Genetic analyses revealed that CRP-cAMP modulates the level of HilD, the master regulator of Salmonella invasion. This regulation occurs at the post-transcriptional level and requires the presence of a newly identified regulatory motif within the hilD 3’UTR. We further demonstrate that in Salmonella the Hfq-dependent sRNA Spot 42 is under the transcriptional repression of CRP-cAMP and, when this transcriptional repression is relieved, Spot 42 exerts a positive effect on hilD expression. In vivo and in vitro assays indicate that Spot 42 targets, through its unstructured region III, the 3’UTR of the hilD transcript. Together, our results highlight the biological relevance of the hilD 3’UTR as a hub for post-transcriptional control of Salmonella invasion gene expression.Spanish Ministry of Economy and Competitiveness BIO2010-15417 BIO2013-44220-R AGL2013-45339-RRecerCaixa program 2012/ACUP/00048Catalonian government 2017SGR49

Mutaciones en ppGpp Y DksA afectan la secreción de proteínas efectoras de la isla de patogenicidad 1 de Salmonella enterica serovar Typhimurium

PublishedEl género Salmonella, perteneciente a la familia Enterobacteriaceae, está compuesto por bacterias Gram negativas, no esporuladas, en forma de bacilo. Salmonella tiene importante relevancia a nivel de salud pública ya que es uno de los principales patógenos entéricos tanto en países desarrollados como en vías de desarrollo. Diferentes serovares pueden cau-sar dos patologías claramente diferenciadas, salmonelosis y fiebres tifoideas. La salmonelosis es una gastroenteritis cuyos síntomas son diarrea, fiebre, vómito y dolor abdominal, causada por toda una serie de serovares no tifoideos, que también pueden infectar un amplio rango de animales. Los serovares más relevantes son Typhimurium y Enteritidis, (Ohl & Miller, 2001; Gordon, 2008). En Colombia, Salmonella enterica se-rovarTyphimurium es el serovar más prevalente. El último reporte del Instituto Nacional de Salud mostró que, de los 23 serovares incidentes en el país, el serovar Typhimurium representa el 30% del total de los aislamientos que se realizaron entre los años 1997 y 2016

Gre factors are required fot biofilm formation in Salmonella enterica serovar Typhimurium by targeting transcription of the cgsD gene

Rdar biofilm formation of Salmonella typhimurium and Escherichia coli is a common ancient multicellular behavior relevant in cell-cell and inter-organism interactions equally, as in interaction with biotic and abiotic surfaces. With the expression of the characteristic extracellular matrix components amyloid curli fimbriae and the exopolysaccharide cellulose, the central hub for the delicate regulation of rdar morphotype expression is the orphan transcriptional regulator CsgD. Gre factors are ubiquitously interacting with RNA polymerase to selectively overcome transcriptional pausing. In this work, we found that GreA/GreB are required for expression of the csgD operon and consequently the rdar morphotype. The ability of the Gre factors to suppress transcriptional pausing and the 147 bp 5′-UTR of csgD are required for the stimulatory effect of the Gre factors on csgD expression. These novel mechanism(s) of regulation for the csgD operon might be relevant under specific stress conditions

ppGpp, the general stress response alarmone, is required for the expression of the alpha-hemolysin toxin in the uropathogenic EScherichia coli isolate, J96.

ppGpp is an intracellular sensor that, in response to different types of stress, coordinates the rearrangement of the gene expression pattern of bacteria to promote adaptation and survival to new environmental conditions. First described to modulate metabolic adaptive responses, ppGpp modulates the expression of genes belonging to very diverse functional categories. In Escherichia coli, ppGpp regulates the expression of cellular factors that are important during urinary tract infections. Here, we characterize the role of this alarmone in the regulation of the hlyCABDII operon of the UPEC isolate J96, encoding the toxin α-hemolysin that induces cytotoxicity during infection of bladder epithelial cells. ppGpp is required for the expression of the α-hemolysin encoded in hlyCABDII by stimulating its transcriptional expression. Prototrophy suppressor mutations in a ppGpp-deficient strain restore the α-hemolysin expression from this operon to wild-type levels, confirming the requirement of ppGpp for its expression. ppGpp stimulates hlyCABDII expression independently of RpoS, RfaH, Zur, and H-NS. The expression of hlyCABDII is promoted at 37 °C and at low osmolarity. ppGpp is required for the thermoregulation but not for the osmoregulation of the hlyCABDII operon. Studies in both commensal and UPEC isolates demonstrate that no UPEC specific factor is strictly required for the ppGpp-mediated regulation described. Our data further support the role of ppGpp participating in the coordinated regulation of the expression of bacterial factors required during infection

Patógenos de importancia clínica

PublishedEl presente libro Patógenos de importancia clínica: Inves­tigaciones Recientes en el Valle del Cauca nace como una iniciativa para socializar los resultados obtenidos en los tra­bajos de investigación más recientes de los miembros del Grupo de Investigación en genética, fisiología y metabolis­mo –GEFIME– de la Universidad Santiago de Cali. Estas investigaciones involucran patógenos que son importantes agentes causales de infecciones y de alto impacto en la salud pública en la ciudad de Cali y en el Valle del Cauca. Este libro consta de cinco capítulos con trabajos de investiga­ción en bacterias resistentes a los antibióticos como Staphylo­coccus aureus y Mycobacterium tuberculosis y su comporta­miento epidemiológico en ambientes intrahospitalarios y en poblaciones, respectivamente. En el Capítulo 3 se presenta un estudio epidemiológico que establece la presencia del virus del papiloma humano (VPH) en mucosa oral y su relación con el desarrollando cáncer oral y en el Capítulo 4 se presenta una investigación realizada con moléculas efectoras (alarmona pp­Gpp y la proteína DksA) de Salmonella enterica serovar Typhi­murium y su influencia en la patogenicidad y la formación de biopelículas in-vitro

Gre factors-mediated control of hilD transcription is essential for the invasion of epithelial cells by Salmonella enterica serovar Typhimurium

The invasion of epithelial cells by Salmonella enterica serovar Typhimurium is a very tightly regulated process. Signaling cascades triggered by different environmental and physiological signals converge to control HilD, an AraC regulator that coordinates the expression of several virulence factors. The expression of hilD is modulated at several steps of the expression process. Here, we report that the invasion of epithelial cells by S. Typhimurium strains lacking the Gre factors, GreA and GreB, is impaired. By interacting with the RNA polymerase secondary channel, the Gre factors prevent backtracking of paused complexes to avoid arrest during transcriptional elongation. Our results indicate that the Gre factors are required for the expression of the bacterial factors needed for epithelial cell invasion by modulating expression of HilD. This regulation does not occur at transcription initiation and depends on the capacity of the Gre factors to prevent backtracking of the RNA polymerase. Remarkably, genetic analyses indicate that the 3'-untranslated region (UTR) of hilD is required for Gre-mediated regulation of hilD expression. Our data provide new insight into the complex regulation of S. Typhimurium virulence and highlight the role of the hilD 3'-UTR as a regulatory motif

Competition assay of the Δ<i>greA</i>Δ<i>greB</i> mutant strain versus WT.

<p>Amount of bacteria in liver and spleen was determined at 4 days post infection. A total of ~2E+7 colony forming units (cfu) of WT and the Δ<i>greA</i>Δ<i>greB</i> strain at a 1:1 ratio was administered orally to 5 mice.</p

Gre factors-mediated regulation of <i>S</i>. Typhimurium virulence is focused in the regulation of the master regulator HilD.

<p>(A) Relative <i>hilC</i>, <i>hilD and rtsA</i> mRNA quantification by qPCR in WT and Δ<i>greA</i>Δ<i>greB</i> derivative strains. Results are normalized after detection of <i>gapA</i> (GAPDH) that was used as an endogenous control. Same RNA samples as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006312#ppat.1006312.g003" target="_blank">Fig 3B</a>. (B) Transcriptional expression of <i>hilA</i> in WT, <i>hilC</i>, <i>rtsA</i> and <i>hilD</i> derivative strains either proficient (grey bars) or deficient (black bars) in the Gre factors was monitored by β-galactosidase activity determination from a <i>hilA</i>::<i>lacZ</i> fusion. In A and B, a bar shows the arithmetic mean of experimental results and the error bar indicates the standard deviation from three biological replicates. Immunodetection (lower panels) of HilA-FLAG protein (C) and the SPI-2 encoded SsrA-FLAG protein (D) in whole cell extracts from cultures of WT and Δ<i>greA</i>Δ<i>greB</i> derivative strains in a <i>hilD</i>⁺ and <i>hilD</i>^- genetic backgrounds. The upper panels are sections of Coomassie stained gels as loading controls. In all cases bacterial cultures were grown in LB at 37°C up to an OD_600nm of 2.0.</p

Gre factors are essential for HilA expression.

<p>(A) Transcriptional expression of <i>hilA</i> in WT and Δ<i>greA</i>Δ<i>greB</i> derivative strains. β-galactosidase activity from a <i>hilA</i>::<i>lacZ</i> fusion was assessed in LB cultures grown at 37°C up to logarithmic (OD_600nm 0.4) and stationary growth phase (OD_600nm 2.0). (B) Relative <i>hilA</i> mRNA quantification by qPCR in WT and Δ<i>greA</i>Δ<i>greB</i> derivative strains. Results are normalized with <i>gapA</i> (GAPDH) as an endogenous control. RNA samples were extracted from cultures of WT and Δ<i>greA</i>Δ<i>greB</i> derivative strains grown in LB at 37°C up to an OD_600nm 2.0. In A and B, a bar shows the arithmetic mean of experimental results and the error bar indicates the standard deviation from three biological replicates. (C) Immunodetection of HilA-FLAG (lower left panel) and InvF-FLAG (lower right panel) proteins in whole cell extracts from cultures of WT and Δ<i>greA</i>Δ<i>greB</i> derivative strains grown as in B. The upper panels are sections of Coomassie stained gels as loading controls. (D) Cell-free supernatants of LB cultures, grown at 37°C up to an OD_600nm of 2.0, of WT and Δ<i>greA</i>Δ<i>greB</i> derivative strains carrying either pBAD18 or pBADHilA. Arabinose (0.02%) was added in all cultures. Extracts were analyzed by Coomassie blue stained 12.5% SDS-PAGE.</p

Summary of the effects of Gre factor deficiency in the expression of virulence in <i>S</i>. Typhimurium.

<p>Summary of the effects of Gre factor deficiency in the expression of virulence in <i>S</i>. Typhimurium.</p