Characterization of the yehUT two-component regulatory system of Salmonella enterica Serovar Typhi and Typhimurium

Abstract

Proteins exhibiting hyper-variable sequences within a bacterial pathogen may be associated with host adaptation. Several lineages of the monophyletic pathogen Salmonella enterica serovar Typhi (S. Typhi) have accumulated non-synonymous mutations in the putative two-component regulatory system yehUT. Consequently we evaluated the function of yehUT in S. Typhi BRD948 and S. Typhimurium ST4/74. Transcriptome analysis identified the cstA gene, encoding a carbon starvation protein as the predominantly yehUT regulated gene in both these serovars. Deletion of yehUT had no detectable effect on the ability of these mutant Salmonella to invade cultured epithelial cells (S. Typhi and S. Typhimurium) or induce colitis in a murine model (S. Typhimurium only). Growth, metabolic and antimicrobial susceptibility tests identified no obvious influences of yehUT on these phenotypes.

Figure 1. A The yehUT operon with surrounding genes.

The operon is 2349bp in length and located between 782210 to 784611 bp in S. Typhi Ty2 (Accession number AE014613) and between c2251460 to c253861 bp in S. Typhimurium SL1344 (Accession number FQ312003). The surrounding genes include yehV, t0694, yehW, yehX (upstream of yehU) and yehS, t0699 and yehR (downstream from yehT). B The domain organisation of the YehU and YehT proteins. YehU protein comprises of five transmembrane (TM) alpha helical segments predicted using TMHMM webserver [17]. The intracellular signalling component consists of a GAF domain, a DHp (dimerization/histidine-containing phosphotransfer) domain that is connected to an ATP (adenosine triphosphate)-dependent Kinase domain. YehT protein consists of a Receiver domain and a LyTR-homologous DNA binding domain [16]. The phosphorylation sites are indicated (H, Histidine; D, Aspartate). The SNP positions identified in yehU and yehT genes are indicated below the proteins by grey boxes [10].

Figure 2. SDS-PAGE and western blot analysis of epitope-tagged YehT protein from S.

Typhi BRD948. Proteins from whole cell bacterial lysates were transferred after electrophoretic separation in a 12% SDS-PAGE gel (Invitrogen) onto a nitrocelluose membrane (Invitrogen) and probed with anti-FLAG M2 monoclonal antibodies (Sigma). Lanes: (M) protein molecular weight markers (SeeBlue Plus2 Pre-stained standard; Invitrogen); (-) proteins extracted from S. Typhi BRD948 (negative control); (+) proteins extracted from S. Typhimurium SL1344 ΔfliA (positive control, 27.4kDa); FLAG proteins extracted from S. Typhi ΔyehT-tagged (27.4kDa). Bacteria was grown in low salt LB; normal salt LB; SPI-2 inducing; SPI-2 + 0.5% glucose; SPI-2 + 0.25% acetate overnight, shaking at 37°C.

Figure 3. SDS-PAGE and western blot analysis of epitope-tagged YehT protein from S.

Typhimurium ST4/74. Proteins from whole cell bacterial lysates were transferred after electrophoretic separation in a 12% SDS-PAGE gel (Invitrogen) onto a nitrocelluose membrane (Invitrogen) and probed with anti-FLAG M2 monoclonal antibodies (Sigma). Lanes: (M) protein molecular weight markers (SeeBlue Plus2 Pre-stained standard; Invitrogen); (-) proteins extracted from S. Typhimurium ST4/74 (negative control); (+) proteins extracted from S. Typhimurium SL1344 ΔfliA (positive control; 27.4kDa); FLAG proteins extracted from S. Typhimurium ΔyehT-tagged (27.4kDa). Bacteria was grown in low salt LB; normal salt LB; SPI-2 inducing; SPI-2 + 0.5% glucose; SPI-2 + 0.25% acetate overnight, shaking at 37°C.

Figure 4. qRT-PCR analyses of differentially expressed genes of the S.

Typhi ΔyehUT mutant. A The relative expression levels of the 15 genes found to be differentially expressed on microarray analysis of the S. Typhi ΔyehUT mutant when compared to S. Typhi in low salt LB, at mid-log phase, at 37°C (Table S6 in File S1). The 15 genes examined are listed on the y-axis. Grey boxes < 1.0 indicates down regulated genes and grey boxes >1.0 indicates up regulated genes relative to S. Typhi. B qRT-PCR analysis showing relative expression levels of genes after complementation of the ΔyehUT mutant compared to S. Typhi (Table S6 in File S1).

Figure 5. qRT-PCR analyses of differentially expressed genes of the S.

Typhimurium ΔyehUT mutant. A The relative expression levels of the 4 genes found to be differentially expressed on microarray analysis of the S. Typhimurium ΔyehUT mutant when compared to S. Typhimurium. in low salt LB, at mid-log phase, at 37°C (Table S6 in File S1). The 4 genes examined are listed on the y-axis. Grey boxes < 1.0 indicates down regulated genes and grey boxes >1.0 indicates up regulated genes relative to S. Typhimurium. B qRT-PCR analysis showing relative expression levels of genes after complementation of the ΔyehUT mutant compared to S. Typhimurium.(Table S6 in File S1).