Salmonella enterica serovar Typhi uses two type 3 secretion systems to replicate in human macrophages and colonize humanized mice

Abstract

Salmonella enterica serovar Typhi (S. Typhi) is a human-restricted pathogen that replicates in macrophages. In this study, we investigated the roles of the S. Typhi type 3 secretion systems (T3SSs) encoded on Salmonella pathogenicity islands (SPI)-1 (T3SS-1) and SPI-2 (T3SS-2) during human macrophage infection. We found that mutants of S. Typhi deficient for both T3SSs were defective for intramacrophage replication as measured by flow cytometry, viable bacterial counts, and live time-lapse microscopy. T3SS-secreted proteins PipB2 and SifA contributed to S. Typhi replication and were translocated into the cytosol of human macrophages through both T3SS-1 and T3SS-2, demonstrating functional redundancy for these secretion systems. Importantly, an S. Typhi mutant strain that is deficient for both T3SS-1 and T3SS-2 was severely attenuated in the ability to colonize systemic tissues in a humanized mouse model of typhoid fever. Overall, this study establishes a critical role for S. Typhi T3SSs during its replication within human macrophages and during systemic infection of humanized mice. IMPORTANCE Salmonella enterica serovar Typhi is a human-restricted pathogen that causes typhoid fever. Understanding the key virulence mechanisms that facilitate S. Typhi replication in human phagocytes will enable rational vaccine and antibiotic development to limit the spread of this pathogen. While S. Typhimurium replication in murine models has been studied extensively, there is limited information available about S. Typhi replication in human macrophages, some of which directly conflict with findings from S. Typhimurium murine models. This study establishes that both of S. Typhi's two type 3 secretion systems (T3SS-1 and T3SS-2) contribute to intramacrophage replication and virulence.

Keywords: T3SS-1; T3SS-2; pathogenesis; typhoid fever; typhoidal.

Fig 1

S. Typhi uses both T3SS-1 and T3SS-2 to replicate in human macrophages. (A) Replication of S. Typhi in THP-1 macrophages by fluorescence dilution (pFCcGi) at 2 and 24 h.p.i. Statistical significance by analysis of variance (ANOVA). One biological replicate representative of three replicates. Dots: technical replicates. Bars: mean. Error: SD. (B) Replication of S. Typhi in hMDMs measured by fluorescence dilution (pFCcGi) by time-lapse microscopy during 24-h infection. Statistical significance compared with WT at 24 h.p.i. by ANOVA. Dots: mean of six biological replicates. Error: SEM. (C) Replication of S. Typhi in hMDMs measured by fluorescence dilution (pFCcGi) at 20 h.p.i. by time-lapse microscopy. Statistical significance compared with WT by ANOVA. Bars: mean of four biological replicates, three technical replicates each. Error: SD. (D) Number of bacteria per cell in fixed macrophages at 16 h.p.i. Statistical significance by ANOVA. Dots: number of bacteria counted in one cell. Violin outline: population distribution. Thick line: median. Thin line: upper and lower quartiles. (E) WT-infected hMDMs, 63X. Maximum intensity projection of Z-stack images taken with confocal microscope. Blue, nuclei; green, LAMP-1; white, actin; red, Salmonella. Scale: top right. (F) ΔinvA ΔssaV-infected hMDMs, 63X. Maximum intensity projection of Z-stack images taken with confocal microscope. Blue, nuclei; green, LAMP-1; white, actin; red, Salmonella. Scale: top right. For all, non-significant (ns) = P-value > 0.05, *≤ 0.05, **≤ 0.01, ***≤ 0.001, ****≤ 0.0001.

Fig 2

T3SS-dependent effectors contribute to intracellular replication. (A) Replication index of each S. Typhi strain in THP-1 macrophages at 24 h postinfection. T3SS effectors in ascending order of replication at 24 h.p.i. Statistical significance compared with hypothetical mean of 1.0 by the Wilcoxon test. Each dot: mean of three to five biological replicates, each with an average of three technical replicates. N = number of biological replicates performed for each strain. Bar: mean or biological replicates. Colored by category of the gene knocked out. Gray, control strain, expected outcome; purple, T3SS effector knockout; white, T3SS effectors pseudogenized in S. Typhi strain Ty2. Error: SEM of biological replicates. (B) Replication of S. Typhi in THP-1 macrophages by CFU/well at 2 and 24 h.p.i. Statistical significance by ANOVA. Dots: biological replicates, each with an average of three technical replicates. Bars: mean. Bars filled with color indicate a disrupted gene, whereas bars filled with white indicate that the corresponding gene has been complimented. Error: SD. For all, ns or blank = P-value > 0.05, *≤ 0.05, **≤ 0.01, ***≤ 0.001, ****≤ 0.0001. (C) Replication of S. Typhi in hMDMs measured by fluorescence dilution (pFCcGi) at throughout infection by time-lapse microscopy. Statistical significance compared with WT at 8 and 24 h.p.i. by ANOVA. Dots: mean of three to five biological replicates. Error: SEM. For all, ns = P-value > 0.05, *≤ 0.05, **≤ 0.01, ***≤ 0.001, ****≤ 0.0001.

Fig 3

Translocation of SifA by 8 h.p.i. is T3SS-1 dependent, whereas translocation of SifA by 16 h.p.i. is dependent on both T3SS-1 and T3SS-2. (A) Representative flow cytometric analysis of THP-1 macrophages infected with S. Typhi expressing effector-BlaM constructs at 8 h.p.i. Columns = GST-BlaM, PipB2-BlaM, SifA-BlaM translocation at 8 h.p.i. detection in hMDMs. Rows = strain of Ty2 used to infect, carrying BlaM construct. (B) Quantification of tranlocation assay in samples fixed at 8 h.p.i., statistical significance by two-way ANOVA. Dots: technical replicates. Bars: mean of technical replicates. Representative of three biological replicates. ns = P-value > 0.05, ***≤ 0.001, ****≤ 0.0001. (C) Quantification of tranlocation assay in samples fixed at 16 h.p.i., statistical significance by two-way ANOVA. Dots: technical replicates. Bars: mean of technical replicates. Representative of three biological replicates. ns = P-value > 0.05, ****≤ 0.0001.

Fig 4

S.Typhi T3SS-1 and T3SS-2 contribute to virulence in a humanized mouse model of typhoid fever. (A) Humanized mice infected with 2 × 10⁵ bacteria intraperitoneally. Competitive index of viable bacteria recovered from the spleen and the liver 2 days p.i. Statistical significance by one-sample t-test compared with a theoretical mean of 1.0. S, spleens; L, livers. Dots: individual mice. ns = P-value > 0.05. (B) Humanized mice infected with 2 × 10⁴ bacteria intraperitoneally. Competitive index of viable bacteria recovered from the spleen and the liver 5 days p.i. Statistical significance by one-sample t-test compared with a theoretical mean of 1.0. S, spleens; L, livers. Dots: individual mice. ns = P-value > 0.05, *≤ 0.05, ***≤ 0.001.