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 ΔtatC 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 ΔtatC 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.

Importance: In 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.

FIG 1

Functional clustering of differentially regulated genes in the ΔtatC mutant. (A) Functional clustering was performed by using the KEGG pathway mapping, KEGG database, and BLAST search. Inf., information. (B) Venn scheme of differentially regulated genes under all conditions tested. Exp., exponential phase; Stat., stationary phase.

FIG 2

Comparison of in vitro growth under iron-limited conditions. Growth of parental (wt), ΔtatC mutant, ΔfhuD mutant, and trans-complemented tatC mutant (ptatC) strains in iron-depleted TMH medium at 26°C (A) and of their pYV⁻ variants at 37°C (B). (C) Growth of parental (wtc), ΔtatC mutant, ΔfhuD mutant, ΔybtP mutant, and trans-complemented tatC mutant (ptatC) strains in iron-depleted TMH medium supplemented with 10 μM ferrichrome (Ferchr). The results are representative of three independent experiments, with the error bars representing the standard deviation. OD₆₀₀, optical density at 600 nm.

FIG 3

YadA expression is downregulated in the ΔtatC mutant. Parental (wt), ΔtatC mutant, and trans-complemented tatC mutant (ptatC) strains were incubated in LB medium supplemented with or depleted of Ca²⁺ with the addition of 5 mM EGTA. Whole-cell extracts were separated on SDS-polyacrylamide gel and analyzed by Western blotting using rabbit polyclonal anti-YadA antibody (top). The membrane was also incubated with anti-DnaK antibody as a loading control (bottom). The signal intensities of each of the YadA bands were normalized with DnaK signals in each strain. The relative signal levels of YadA were calculated with comparison to the wild type (set as 100%). The analyses were performed with ImageJ. Molecular masses (Spectra multicolor high-range protein ladder; Thermo Scientific) are shown on the left.

FIG 4

(A) Genes involved in different metabolic functions that are differentially regulated in the ΔtatC mutant at 26°C in stationary phase are shown in the pie graph. Degr., degradation; Met., metabolism. (B) Genes involved in central carbon metabolism differentially regulated in the ΔtatC mutant at 26°C in stationary phase. (Upregulated genes are indicated in red; downregulated genes are indicated in green). diP, diphosphate; CoA, coenzyme A. (C) Growth of parental (wt), ΔtatC mutant, and trans-complemented tatC mutant (ptatC) strains on LB–1.5% agar supplemented with phenol red and 0.2% glucose at 26°C for 24 h. (D) Intracellular concentrations of fumarate in parental (wt), ΔtatC mutant, and trans-complemented tatC mutant (ptatC) strains. Data represent the mean and standard deviation of the results from four independent experiments that were analyzed by Student's t test. Asterisks indicate results that were significantly different from those for the parental strain. ***, 0.0001 ≤ P < 0.001.

FIG 5

Survival of the ΔtatC mutant in CuCl₂ and in acidic pH. (A) Survival of WT, ΔtatC mutant, ΔcueO mutant, and trans-complemented tatC mutant (ptatC) strains in increasing CuCl₂ concentrations at 26°C. (B) Growth of pYV⁻ variant WTc, ΔtatCc mutant, ΔcueOc mutant, and trans-complemented tatC mutant (ptatCc) strains in increasing CuCl₂ concentrations at 37°C. Data represent the mean and standard deviation of the results from four independent experiments that were analyzed by Student's t test. (C) Survival of pYV⁻ variant WTc, ΔtatCc mutant, ΔcynTc mutant, and trans-complemented tatC mutant (ptatCc) strains in LB medium with pH 3.0 at 37°C. Percent survival was calculated from 15 min of growth of each strain in LB medium with pH 7.0 at 37°C. Data represent the mean and standard deviation of the results from five independent experiments that were analyzed by Student's t test. Asterisks indicate results that were significantly different from those for the parental strain. ****, P < 0.0001; ***, 0.0001 ≤ P < 0.001; **, 0.001 ≤ P < 0.05; *, P < 0.05.

FIG 6

Growth of the WT and the ΔtatC mutant in SDS. (A) Growth of WT, ΔtatC mutant, ΔsufI mutant, ΔamiC mutant, and trans-complemented tatC mutant (ptatC) strains in 0.0125% SDS at 26°C. (B) Growth of pVY⁻ variant WTc, ΔtatCc mutant, ΔsufIc mutant, ΔamiCc mutant, and trans-complemented tatC mutant (ptatCc) strains in 0.00625% SDS at 37°C. Data represent the mean and standard deviation of the results from three independent experiments that were analyzed by Student's t test. Asterisks indicate results that were significantly different from those for the parental strain. *, P < 0.05.