Transcriptomic and Phenotypic Analysis Reveals New Functions for the Tat Pathway in Yersinia pseudotuberculosis

Abstract

The twin-arginine translocation (Tat) system mediates the secretion of folded proteins that are identified via an N-terminal signal peptide in bacteria, plants, and archaea. Tat systems are associated with virulence in many bacterial pathogens, and our previous studies revealed that Tat-deficient Yersinia pseudotuberculosis was severely attenuated for virulence. Aiming to identify Tat-dependent pathways and phenotypes of relevance for in vivo infection, we analyzed the global transcriptome of parental and ⌬tatC mutant strains of Y. pseudotuberculosis during exponential and stationary growth at 26°C and 37°C. The most significant changes in the transcriptome of the ⌬tatC mutant were seen at 26°C during stationary-phase growth, and these included the altered expression of genes related to virulence, stress responses, and metabolism. Subsequent phenotypic analysis based on these transcriptome changes revealed several novel Tat-dependent phenotypes, including decreased YadA expression, impaired growth under iron-limited and high-copper conditions, as well as acidic pH and SDS. Several functionally related Tat substrates were also verified to contribute to these phenotypes. Interestingly, the phenotypic defects observed in the Tat-deficient strain were generally more pronounced than those in mutants lacking the Tat substrate predicted to contribute to that specific function. Altogether, this provides new insight into the impact of Tat deficiency on in vivo fitness and survival/replication of Y. pseudotuberculosis during infection. IMPORTANCEIn addition to its established role in mediating the secretion of housekeeping enzymes, the Tat system has been recognized as being involved in infection. In some clinically relevant bacteria, such as Pseudomonas spp., several key virulence determinants can readily be identified among the Tat substrates. In enteropathogens, such as Yersinia spp., there are no obvious virulence determinants among the Tat substrates. Tat mutants show no growth defect in vitro but are highly attenuated in in vivo. This makes Tat an attractive target for the development of novel antimicrobials. Therefore, it is important to establish the causes of the attenuation. Here, we show that the attenuation is likely due to synergistic effects of different Tat-dependent phenotypes that each contributes to lowered in vivo fitness. Bacteria have evolved several specialized secretion systems for protein export as part of their successful strategies to colonize niches where they encounter various environmental conditions. Gram-negative bacteria possess different mechanisms to transport/export proteins, including virulence-related factors from the cytoplasm across the inner membrane and/or the outer membrane, either in a single step or acting together with other secretion systems (1). Two pathways, the secretory (Sec) and the twin-arginine translocation (Tat) pathways, are involved in the translocation of proteins from the cytoplasm to the periplasm. In Gramnegative bacteria, they can cooperate with other s...

The Tat Substrate SufI Is Critical for the Ability of Yersinia pseudotuberculosis To Cause Systemic Infection

“…In these bacteria, ΔtatC or ΔamiA ΔamiC mutants have been shown to be sensitive to SDS, bile, and antimicrobial peptides (35,43). However, according to our findings in Y. pseudotuberculosis, only AmiC and SufI are potential Tat substrates, but neither of them contributes to envelope stress and only the ΔtatC mutant is important for cell envelope maintenance (23). Even though the specific role of these cell division proteins and their contribution to virulence differ, these findings indicate a central role for cell division proteins in the virulence of enteric pathogens.…”

“…In our previous studies, we showed that a functional Tat system is required for establishment of systemic infection and colonization of the spleen (22) in mice and that the loss of Tat function leads to a growth defect in the presence of low-iron medium, increasing copper concentrations, acidic medium, and SDS (23). However, the Tat substrates functionally related to these phenotypes only partly contributed to the respective phenotypes.…”

“…For a more detailed investigation of the early colonization of the different strains, the bacterial loads in the stomach, cecum, small intestine, cecal contents, and feces were determined at 6, 12, and 24 hpi. We included the stomach in the samples, as we have previously shown that the ΔtatC mutant has a lower rate of survival under in vitro acidic pH conditions (23). Indeed, the ΔtatCL mutant showed a bacterial load in the stomach 200 times lower than that of the wtL strain at 6 hpi (3.5 ϫ 10 3 versus 8.5 ϫ 10 5 CFU/g) (Fig.…”

“…We recently reported that the loss of Tat function leads to drastic changes in the transcriptome, including genes involved in metabolism, stress responses, and virulence, especially at 26°C during stationary-phase growth. We could also correlate the changes in transcription to phenotypic changes, including defects in iron acquisition, downregulation of the expression of the adhesin YadA, sensitivity to copper, and sensitivity to the detergent sodium dodecyl sulfate (SDS) (23). However, none of the strains with mutations in Tat substrates predicted to be functionally related to these phenotypes showed the same level of phenotypic defect as the Tat-deficient strain (23).…”

“…We could also correlate the changes in transcription to phenotypic changes, including defects in iron acquisition, downregulation of the expression of the adhesin YadA, sensitivity to copper, and sensitivity to the detergent sodium dodecyl sulfate (SDS) (23). However, none of the strains with mutations in Tat substrates predicted to be functionally related to these phenotypes showed the same level of phenotypic defect as the Tat-deficient strain (23). Therefore, in this study, we sought to investigate the specific role of the putative Tat substrates encoded by the Y. pseudotuberculosis genome in in vivo virulence.…”