Persistent Salmonella infections in humans are associated with mutations in the BarA/SirA regulatory pathway

Abstract

Several bacterial pathogens, including Salmonella enterica, can cause persistent infections in humans by mechanisms that are poorly understood. By comparing genomes of isolates longitudinally collected from 256 prolonged salmonellosis patients, we identified repeated mutations in global regulators, including the barA/sirA two-component regulatory system, across multiple patients and Salmonella serovars. Comparative RNA-seq analysis revealed that distinct mutations in barA/sirA led to diminished expression of Salmonella pathogenicity islands 1 and 4 genes, which are required for Salmonella invasion and enteritis. Moreover, barA/sirA mutants were attenuated in an acute salmonellosis mouse model and induced weaker transcription of host immune responses. In contrast, in a persistent infection mouse model, these mutants exhibited long-term colonization and prolonged shedding. Taken together, these findings suggest that selection of mutations in global virulence regulators facilitates persistent Salmonella infection in humans, by attenuating Salmonella virulence and inducing a weaker host inflammatory response.

Keywords: BarA/SirA; Salmonella enterica; chronic infections; genomics; host immune response; persistence; salmonellosis; transcriptomics; two-component regulatory system; virulence regulation.

Figure 1.. Genetic diversity of S. enterica isolates responsible for persistent infections

(A) Phylogeny of 562 S. enterica isolate genomes from human persistent infections. The colored ring represents serovar information as determined by serotyping according to the White-Kauffmann-Le Minor scheme. (B) Distribution of SNP distances between 545 isolate pairs obtained from the same patient at different time points. (C) Variants, including SNPs, insertions, and deletions, in all genes, and in genes with the GO term for ‘‘Regulation of Biological Process’’ (GO:0050789), between early and late same-patient isolates. Bars are colored by serovar, with light tone representing all variants and dark tone representing variants in genes with GO:0050789. The star indicates the serovars presenting variants in barA and/or sirA.

Figure 2.. Most commonly mutated genes do not have high nucleotide diversity

(A) The number of distinct variants between early and late same-patient isolates per gene for all genes that contain variants, plotted by gene start position. (B) Average nucleotide diversity, π, per gene in the reference genome, plotted by gene start position. barA in purple, sirA in green, shdA in black, common genes (genes with variants in at least two patients) in light blue, and all other genes in gray.

Figure 3.. Differentially expressed Salmonella genes in patients with barA/sirA variants

(A) Diagram of the BarA and SirA proteins with domains indicated by boxes: TM, transmembrane domain; HAMP, histidine kinase, adenyl cyclase, methyl-binding protein, phosphatase domain; HisKa, dimerization and phosphoacceptor domain; HATpase, histidine kinase-like ATPase; REC, primary receiver domain; HPT, secondary transmitter domain; 2^nd REC, secondary receiver domain; HTH, helix-turn-helix domain. SNPs are indicated by circles and deletions by rectangles, the four colors indicate the SNPs and deletion that were tested in the mouse model. (B) MA plots showing log fold change (logFC) vs. log average expression (logCPM) for four different patients: two with barA mutations (patients 106 and 140) and two with sirA mutations (patients 124 and 177). Genes of interest that were found to be differentially expressed in either both barA variant strains, both sirA variant strains or in all four variant strains are colored based on annotation: propionate degradation (blue), porin (green), nitrate reduction (light blue), stress response (purple), host adhesion (light purple), SPI-1 (red), SPI-4 (orange), and SPI-5 (yellow), virulence (maroon). The colored box around the location matches the color of the SNP or deletion in (A). The complete list of DEGs is shown in Table S6.

Figure 4.. Mutations in barA and sirA acquired during persistence corresponds to attenuated Salmonella virulence

(A) Competitive index (CI) of late vs. early isolates with variants in barA or sirA after co-infection of C57BL/6 mice. CI was calculated for the spleen, liver, cecum, and colon of the mice and is plotted on the log scale. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant; nc, no colonization was identified. The Salmonella serovar and location of the SNP in barA or sirA is shown at the top of each graph. (B) Competitive infection in the acute mouse model using reconstructed barA/sirA mutations in S. Typhimurium SL1344. C57BL/6 mice were pre-treated with streptomycin and infected with a mixture of S. Typhimurium SL1344 WT strain (Amp^r) and each one of the four barA/sirA mutants (Kan^r) identified in patients 106, 124, 140, and 177. At day 4 p.i., mice were euthanized and bacterial loads in the cecum, colon, liver, and spleen were determined by plating their homogenates on selective plates supplemented with ampicillin or kanamycin. Competitive index (CI) was calculated as (mutant/WT) _output/(mutant/WT) _input and is plotted on the log scale. Each dot represents the CI in one mouse and the geometrical mean is shown by a horizontal bar. A competitive value of one indicates no significant difference in colonization between both strains.

Figure 5.. BMDMs infected with barA/sirA mutants provoke a weaker immune response

Volcano plots of DEGs between C57BL/6 BMDMs infected with either S. Typhimurium WT strain and isogenic barA/sirA mutants at MOI of 2.5. Larger point size indicates the gene is differentially expressed in both mutants; color indicates gene annotation with an enriched GO term.

Figure 6.. barA/sirA mutants can establish a long-term infection and prolonged shedding in a persistent mouse model

7-week-old CBA/CA mice were orally infected with S. Typhimurium WT or four isogenic barA/sirA mutant strains, named after the mutations found in isolates from patients (106, 140, 124, and 177). (A) Mice feces were collected at days 6, 9, 12, and 16 p.i., and the number of CFUs per pellet is shown. (B) Salmonella colonization at systemic (spleen and liver) and intestinal (cecum and colon) organs was determined at day 21 p.i. by plating tissue homogenates onto selective plates. Each dot represents the number of CFUs from one sample in one mouse and the geometrical mean is indicated by a horizontal bar. (C) The expression level of interleukin 22 in the spleen of the infected mice was measured by RT-qPCR and normalized to the expression of the hprt housekeeping gene. Each dot shows the average of three RT-qPCR reactions from one mouse spleen sample, and the geometric mean of each sample is indicated by the horizontal line. One-way ANOVA was used to determine statistical significance. ****, p < 0.0001.