Modified intracellular-associated phenotypes in a recombinant Salmonella Typhi expressing S. Typhimurium SPI-3 sequences

Abstract

A bioinformatics comparison of Salmonella Pathogenicity Island 3 sequences from S. Typhi and S. Typhimurium serovars showed that ten genes are highly conserved. However three of them are pseudogenes in S. Typhi. Our aim was to understand what functions are lost in S. Typhi due to pseudogenes by constructing a S. Typhi genetic hybrid carrying the SPI-3 region of S. Typhimurium instead of its own SPI-3. We observed that under stressful conditions the hybrid strain showed a clear impairment in resistance to hydrogen peroxide and decreased survival within U937 culture monocytes. We hypothesized that the marT-fidL operon, encoded in SPI-3, was responsible for the new phenotypes because marT is a pseudogen in S. Typhi and has a demonstrated role as a transcriptional regulator in S. Typhimurium. Therefore we cloned and transferred the S. Typhimurium marT-fidL operon into S. Typhi and confirmed that invasion of monocytes was dramatically decreased. Finally, our findings suggest that the genomic and functional differences between SPI-3 sequences have implications in the host specificity of Typhi and Typhimurium serovars.

Figure 1. Genomic organization of Salmonella pathogenicity island 3.

A, SPI-3 in S. Typhimurium. B, SPI-3 in S. Typhi. ORFs are depicted by arrows and tRNAs appear as closed triangles. ORFs with no orthologs between these serovars and pseudogenes are depicted by white and segmented arrows respectively. The percentage (%) of nucleotide identity of the SPI3 loci is shown in between. The aligning sites of the PCR primers are shown by black dots.

Figure 2. Phenotypic assays with a S. Typhi genetic hybrid strain.

A, Percentage (%) of survival following 30 min exposure to hydrogen peroxide (3 mM). B, Growth at pH 5, based on the optical density at 600 nm (OD600) at different times (h). The strains used were a S. Typhi STH2370 containing the SPI-3 region of S. Typhimurium (SPI-3 STm) and the STH2370 wild-type strain (WT).Values represent the mean of at least three independent experiments ± SD (*p<0.05).

Figure 3. Infection assays in human cells.

A, early survival (2 h). B, late survival (24 h). Percentage of colony forming units (CFU%) inside U937 human cells. The strains used were the STH2370 wild-type strain (WT), a S. Typhi STH2370 expressing the SPI-3 of S. Typhimurium (SPI-3 STm), the SPI-3 STm strain containing deletions of marT (ΔmarT), misL (ΔmisL) and cigR (ΔcigR) sequences, and a S. Typhi STH2370 t3766 mutant strain (Δt3766). The values represent the mean of at least three independent experiments ± SD. Different numbers of asterisks represent significant differences between results (p<0.05).

Figure 4. Reverse-transcription PCR assays.

A, Amplification of the marT-fidL internal sequence with RNA extracted from S. Typhimurium. B, Amplification of the marT-fidL internal sequence. C, Amplification of the t3766 sequence. In B and C the RNA was extracted from strains STH2370 wild-type (WT), STH2370/pBBR-5 (WT/pBBR5) and STH2370/pBmarT (WT/pBmarT). All strains were grown up to the stationary phase, and the 16S rRNA amplification was used as the control.

Figure 5. The S. Typhimurium marT-fidL operon modifies the invasiveness of S. Typhi to U937 human cells.

Percentage of colony forming units (CFU%) after invasion (2 h) and proliferation (24 h) inside U937 human cells. The STH2370 wild-type strain was transformed with either the pBBR-5 (WT/pBBR5) or pBmarT (WT/pBmarT) plasmids. Values represent the mean of at least three independent experiments ± SD (*p<0.05).