Fig 1.
Invasion of epithelial cells by S. Typhimurium is impaired in strains deficient for the Gre factors.
Cultures of the WT (SV5015) and the ΔgreA, ΔgreB and ΔgreAΔgreB derivatives were assessed for invasion of HT-29 (A) and Caco-2 (B) cell lines. As a control, cultures of the invasion impaired mutants ΔhilA and ΔmotA were used. A bar shows the arithmetic mean of experimental results and the error bar indicates the standard deviation. Significance was tested by an unpaired two–sided Student’s t-test. Statistical significance is indicated by *p<0.05, **p<0.01, ns: non-significant.
Table 1.
Competition assay of the ΔgreAΔgreB mutant strain versus WT.
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 ΔgreAΔgreB strain at a 1:1 ratio was administered orally to 5 mice.
Fig 2.
The Gre factors affect the expression of SPI-1 effector proteins.
(A) Contact haemolysis assays with cell-free supernatants from cultures of the WT and ΔgreAΔgreB derivative strains. The haemolytic activity of LB bacterial cultures of WT and ΔgreAΔgreB strains was monitored as an increase in the OD550nm of the supernatant of a blood suspension with serial dilutions of cell-free supernatants. U: undiluted supernatant. Three independent bacterial cultures were tested. (B) Cell-free supernatants of two independent LB cultures of WT (SV5015) and its ΔgreA, ΔgreB and ΔgreAΔgreB derivatives. Extracts were analyzed by Coomassie blue stained 12.5% SDS-PAGE. Lane M: molecular mass markers (size in kDa indicated). The bands labelled were identified as SipA (1), FliD (2) and SipC (3) by LC-MS/MS. (C) Immunodetection (lower panel) of the SPI-1 encoded SipA-FLAG protein in whole culture extracts from two independent cultures of WT and ΔgreAΔgreB derivative strains. The upper panel is a section of a Coomassie stained gel as a loading control. (D) Immunodetection of SopE protein was performed in extracts from WT and ΔgreAΔgreB strains obtained from cell-free supernatants of two independent LB cultures. (E) Semiquantitative RT-PCR of sipA in total RNA samples from LB cultures of the WT (SV5015) and ΔgreAΔgreB strains. 16S RNA was used as endogenous control to confirm that equivalent quantities of templates were used. (F) sipC transcriptional expression was tested in cultures of WT and ΔgreAΔgreB derivative strains carrying a chromosomal sipC::lacZ fusion and either pBR322 or pBRgreAB. A bar shows the arithmetic mean of experimental results and the error bar indicates the standard deviation from three biological replicates. All cultures were grown in LB at 37°C with vigorous shaking (200 rpm) up to an OD600nm of 2.0.
Fig 3.
Gre factors are essential for HilA expression.
(A) Transcriptional expression of hilA in WT and ΔgreAΔgreB derivative strains. β-galactosidase activity from a hilA::lacZ fusion was assessed in LB cultures grown at 37°C up to logarithmic (OD600nm 0.4) and stationary growth phase (OD600nm 2.0). (B) Relative hilA mRNA quantification by qPCR in WT and ΔgreAΔgreB derivative strains. Results are normalized with gapA (GAPDH) as an endogenous control. RNA samples were extracted from cultures of WT and ΔgreAΔgreB derivative strains grown in LB at 37°C up to an OD600nm 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 ΔgreAΔgreB 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 OD600nm of 2.0, of WT and ΔgreAΔgreB 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.
Fig 4.
Gre factors-mediated regulation of S. Typhimurium virulence is focused in the regulation of the master regulator HilD.
(A) Relative hilC, hilD and rtsA mRNA quantification by qPCR in WT and ΔgreAΔgreB derivative strains. Results are normalized after detection of gapA (GAPDH) that was used as an endogenous control. Same RNA samples as in Fig 3B. (B) Transcriptional expression of hilA in WT, hilC, rtsA and hilD derivative strains either proficient (grey bars) or deficient (black bars) in the Gre factors was monitored by β-galactosidase activity determination from a hilA::lacZ 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 ΔgreAΔgreB derivative strains in a hilD+ and hilD- 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 OD600nm of 2.0.
Fig 5.
Overexpression of HilD restores HilA expression and epithelial cell invasiveness in ΔgreAΔgreB strains.
(A) In lower panels, immunodetection of the HilA-FLAG protein in whole cell extracts from cultures of WT and ΔgreAΔgreB derivative strains carrying either pBAD18 or pBADHilD grown in LB at 37°C up to an OD600nm of 2.0, arabinose (0.02%) was added in all cultures. The upper panels are sections of Coomassie stained gels as loading controls. (B) Invasion assays using WT, ΔmotA and ΔgreAΔgreB strains carrying the indicated plasmids. Bacterial cultures were grown as in Fig 1. A bar shows the arithmetic mean of experimental results and the error bar indicates the standard deviation.
Fig 6.
The 3’UTR of hilD is required for the Gre-mediated regulation.
(A) Transcriptional expression from hilD::lacZ chromosomal fusions at positions +76, +965, +1065 and +1235 (relative location in the hilD gene is indicated in the upper panel). β-galactosidase activity was monitored in LB cultures grown at 37°C up to an OD600nm of 2.0.of both WT and ΔgreAΔgreB strains carrying the indicated fusions. (B) hilD1235::lacZ transcriptional expression was tested in cultures of WT and ΔgreAΔgreB derivative strains carrying the indicated plasmids. β-galactosidase activity was determined as in A. (C) Ratio in the levels of hilD and hilA transcripts between WT and ΔgreAΔgreB strains in hilD 3’UTR+ and hilD 3’UTR- genetic backgrounds. mRNA levels were monitored by qPCR using detection of gapA (GAPDH) as an endogenous control. Total RNA was isolated from cultures grown as in A. (D) Transcriptional expression from a sipC::lacZ chromosomal fusion. β-galactosidase activity was monitored as in A in cultures of both WT and ΔgreAΔgreB strains in both hilD3’UTR+ and hilD3’UTR- genetic backgrounds. In all panels, a bar shows the arithmetic mean of experimental results and the error bar indicates the standard deviation from three biological replicates. In B and C, to assess differences in the values, an unpaired, two-sided Student’s test was performed. Statistical significance is indicated by ***p<0.001, ns: non-significant.
Fig 7.
The anti-backtracking activity of the Gre factors is required for the expression of SPI-1 genes by alleviating a transcriptional pause located within the 3’-UTR of hilD.
(A) Transcriptional expression from hilD:: hilA:: and sipC::lacZ chromosomal fusions. β-galactosidase activity was monitored in cultures of both WT and ΔgreAΔgreB strains carrying the following plasmids pHM1883 (pVC), pHM1873 (pGreA) and pHM1854 (pGreA*). (B) Immunodetection of GreA protein was performed in extracts from the ΔgreAΔgreB strain with plasmids as in A. (C) lacZ expression from plasmids constructs carrying the indicated hilD fragments (-UTR, +UTR and +UTR+T) cloned in pTT68 vector downstream of a PBAD promoter and upstream of a promoter less lacZ gene in both WT and ΔgreAΔgreB strains. (D) Expression from the +UTR lacZ fusion, as denoted in panel C, in WT and ΔgreAΔgreB strains carrying the plasmids pHM1883, pHM1873 and pHM1854. (E) Effect of the Gre factors on expression of the -UTR and +UTR lacZ fusions, as denoted in panel C, in WT and its hfq and rne537 mutants. In all cases, cultures were grown in LB at 37°C up to an OD600nm of 2.0. In B, D and E, 0.02% arabinose was added to the LB medium. In A and C-E panels, a bar shows the arithmetic mean of experimental results and the error bar indicates the standard deviation from three biological replicates.
Fig 8.
Summary of the effects of Gre factor deficiency in the expression of virulence in S. Typhimurium.