Selection of Salmonella enterica Serovar Typhi Genes Involved during Interaction with Human Macrophages by Screening of a Transposon Mutant Library

Abstract

The human-adapted Salmonella enterica serovar Typhi (S. Typhi) causes a systemic infection known as typhoid fever. This disease relies on the ability of the bacterium to survive within macrophages. In order to identify genes involved during interaction with macrophages, a pool of approximately 105 transposon mutants of S. Typhi was subjected to three serial passages of 24 hours through human macrophages. Mutants recovered from infected macrophages (output) were compared to the initial pool (input) and those significantly underrepresented resulted in the identification of 130 genes encoding for cell membrane components, fimbriae, flagella, regulatory processes, pathogenesis, and many genes of unknown function. Defined deletions in 28 genes or gene clusters were created and mutants were evaluated in competitive and individual infection assays for uptake and intracellular survival during interaction with human macrophages. Overall, 26 mutants had defects in the competitive assay and 14 mutants had defects in the individual assay. Twelve mutants had defects in both assays, including acrA, exbDB, flhCD, fliC, gppA, mlc, pgtE, typA, waaQGP, SPI-4, STY1867-68, and STY2346. The complementation of several mutants by expression of plasmid-borne wild-type genes or gene clusters reversed defects, confirming that the phenotypic impairments within macrophages were gene-specific. In this study, 35 novel phenotypes of either uptake or intracellular survival in macrophages were associated with Salmonella genes. Moreover, these results reveal several genes encoding molecular mechanisms not previously known to be involved in systemic infection by human-adapted typhoidal Salmonella that will need to be elucidated.

TyphoidalSalmonella: Distinctive virulence factors and pathogenesis

“…In S. Typhi, the SPI-1 T3SS is also required for invasion of nonphagocytic cells (Bishop et al, 2008), but the importance of the SPI-2 T3SS is less clear. Disruption of the SPI-2 T3SS did not influence the survival of S. Typhi in THP-1 and human monocyte-derived macrophages (Forest, Ferraro, Sabbagh, & Daigle, 2010); however, S. Typhi strains with transposon insertions in the SPI-2 components ssaQ, ssaP, or ssaN were negatively selected against during competitive growth in human macrophages (Sabbagh, Lepage, McClelland, & Daigle, 2012). The role of SPI-2 during the intracellular lifestyle of typhoidal serovars therefore warrants further investigation.…”

Characterization of the Molecular Interplay between Moraxella catarrhalis and Human Respiratory Tract Epithelial Cells

“…Mutants of trmB , MCR_0609, and MCR_0837, however, displayed wild-type adherence levels, while deletion of MCR_1742, encoding an uncharacterized outer membrane protein, even appeared to result in enhanced adhesion to both cell lines (Figure 2AB). This apparent discrepancy may be explained by the difference in experimental setup between the Tn-seq screen and the validation experiments: adherence of transposon mutants during the screen was assessed in a competition setup (i.e., between a particular mutant and the remainder of the library), whereas directed deletion mutants were assayed individually for their adherence ability in validation experiments, which is a less stringent approach as has also been shown by others [27,28]. Furthermore, differences in inter-bacterial interaction dynamics may have resulted in the increased adherence of the MCR_1742 deletion mutant.…”

New roles for two-component system response regulators ofSalmonella entericaserovar Typhi during host cell interactions

“…For each deletion, this assay was performed at least three times. Motility assays on plates were performed as described previously (82).…”