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

Abstract

The twin arginine translocation (Tat) system targets folded proteins across the inner membrane and is crucial for virulence in many important human-pathogenic bacteria. Tat has been shown to be required for the virulence of Yersinia pseudotuberculosis, and we recently showed that the system is critical for different virulence-related stress responses as well as for iron uptake. In this study, we wanted to address the role of the Tat substrates in in vivo virulence. Therefore, 22 genes encoding potential Tat substrates were mutated, and each mutant was evaluated in a competitive oral infection of mice. Interestingly, a ΔsufI mutant was essentially as attenuated for virulence as the Tat-deficient strain. We also verified that SufI was Tat dependent for membrane/periplasmic localization in Y. pseudotuberculosisIn vivo bioluminescent imaging of orally infected mice revealed that both the ΔsufI and ΔtatC mutants were able to colonize the cecum and Peyer's patches (PPs) and could spread to the mesenteric lymph nodes (MLNs). Importantly, at this point, neither the ΔtatC mutant nor the ΔsufI mutant was able to spread systemically, and they were gradually cleared. Immunostaining of MLNs revealed that both the ΔtatC and ΔsufI mutants were unable to spread from the initial infection foci and appeared to be contained by neutrophils, while wild-type bacteria readily spread to establish multiple foci from day 3 postinfection. Our results show that SufI alone is required for the establishment of systemic infection and is the major cause of the attenuation of the ΔtatC mutant.

Keywords: SufI; Tat pathway; Yersinia pseudotuberculosis; mesenteric lymph nodes; neutrophils; virulence.

FIG 1

Tat dependency of SufI and YbtP in Y. pseudotuberculosis. (A) TatC-dependent export of SufIsp::mCherry in Y. pseudotuberculosis is shown by the use of fluorescence microscopy images of wild-type and ΔtatC strains carrying the plasmid pBAD24-sufIsp-mCherry after induction with 0.2% arabinose. (B) The expression and localization of YbtP-FLAG in the wild-type and ΔtatC strains were analyzed after induction with 0.4 mM IPTG. Cells were harvested as whole cells (WC) and fractionated into periplasmic (P), cytoplasmic (C), and membrane (M) fractions before immunoblotting with anti-FLAG and anti-FtsH antibodies, as indicated.

FIG 2

Characterization of bioluminescent Y. pseudotuberculosis IP32953 strains. (A) The bioluminescent Y. pseudotuberculosis IP32953 wtL, ΔtatCL, and ΔsufIL strains were incubated for 10 h, and the OD₆₀₀ and luminescence of each strain were measured. RLU, relative light units. (B) OD₆₀₀s and the numbers of CFU of the bioluminescent Y. pseudotuberculosis IP32953 wtL, ΔtatCL and ΔsufIL strains during 10 h of growth. (C) Survival curve for BALB/c mice infected with 5 × 10⁸ CFU/ml bioluminescent wtL and wt IP32953 strains. (D) Bacterial luminescence (in numbers of photons per second per square centimeter per steradian [p/sec/cm²/sr]) in the organs of a representative group of mice infected with the wtL strain, followed throughout the infection, and dissected at 9 dpi. The intensity of emission is represented with pseudo colors, with variations in color representing light intensity. Red represents the most intense light emission, while blue corresponds to the weakest signal. Min, minimum; Max, maximum.

FIG 3

Analysis and comparison of wtL, ΔtatCL, and ΔsufIL strains during early colonization. BALB/c mice were infected with bioluminescent Y. pseudotuberculosis IP32953 wtL, ΔtatCL, and ΔsufIL strains. The infection was monitored and analyzed by BLI, and enumeration of the bacterial load was performed at 6, 12, and 24 h. (A) Representative groups of mice infected with each strain were followed throughout the infection and are shown on a common scale. The intensity of emission is represented with pseudo colors, where red represents the highest light emission and blue corresponds to the weakest light emission. The overexposure of wtL strain-infected mice at 6 and 24 h of infection is due to the very low bioluminescent signal from the ΔtatCL and ΔsufIL mutants at those time points. (B) Measurement and comparison of the total flux (in number of photons per second [p/s]) in a defined region of interest over the abdominal side of the mouse. Data are presented as means ± SEMs. Significance compared to the results for the wtL strain was analyzed using the Mann-Whitney U test. *, P < 0.05. Each symbol represents an individual mouse. (C) Representation of bacterial luminescence at 24 hpi in organs from infected mice. (D) The stomachs of infected mice were collected, and the total number of CFU was determined. Each symbol represents an individual mouse. (E to G) At the time points indicated, the cecum (E), small intestine (F), and cecal contents (G) from infected mice were collected, homogenized, and plated for determination of the number of CFU. Each symbol represents an individual mouse. Data are presented as the means ± SEMs. Significance compared to the results for the wtL strain was analyzed using the Mann-Whitney U test. *, P < 0.05; ns, no significant difference.

FIG 4

Microscopic analysis of strains following growth. The wt, ΔtatC, and ΔsufI strains were grown for 10 h, and every 2 h, samples of bacteria were observed by phase-contrast microscopy. Bars, 5 μm. Magnifications, ×100.

FIG 5

Analysis and comparison of the wtL, ΔtatCL, and ΔsufIL mutants in late infection and during dissemination. BALB/c mice were infected with the bioluminescent Y. pseudotuberculosis IP32953 wtL, ΔtatCL, and ΔsufIL strains. The infection was monitored and analyzed by BLI for 18 dpi. (A) Representative groups of mice infected with each strain that were followed until the clearance of the bacteria are shown. The intensity of emission is represented with pseudo colors, where red represents the highest light emission and blue corresponds to the weakest light emission. (B) Measurement and comparison of the total flux (in numbers of photons per second [p/s]) in a defined region of interest over the abdominal side of mouse. Data are presented as means with SEMs. Significance compared to the results for the wtL strain was analyzed using the Mann-Whitney U test. **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. Each dot represents an individual mouse. (C) Representation of bacterial luminescence in the organs of mice that were infected with different strains and dissected at 3, 5, and 7 dpi. (D) Measurement and comparison of the total flux (in number of photons per second) in a defined region of interest in MLNs dissected from infected mice at the time points indicated. Significance compared to the results for the wtL strain was analyzed using the Mann-Whitney U test. *, P < 0.05; ns, no significant difference. Each dot represents an individual mouse.

FIG 6

The presence of neutrophils and bacteria in MLNs infected with the wtL, ΔtatCL, and ΔsufIL strains. Representative images of immunofluorescence staining of Y. pseudotuberculosis in sections of MLNs from mice infected with the indicated strains on days 3, 5, and 7 p.i. are shown. Tissue sections were stained for Y. pseudotuberculosis (anti-Yersinia antibody; green), neutrophils (anti-Ly6G; red), and nuclei (DAPI; blue). MLN tissues from three mice and 5 to 7 sections per mouse infected with each strain were analyzed.