Reprogramming of Yersinia from virulent to persistent mode revealed by complex in vivo RNA-seq analysis

Abstract

We recently found that Yersinia pseudotuberculosis can be used as a model of persistent bacterial infections. We performed in vivo RNA-seq of bacteria in small cecal tissue biopsies at early and persistent stages of infection to determine strategies associated with persistence. Comprehensive analysis of mixed RNA populations from infected tissues revealed that Y. pseudotuberculosis undergoes transcriptional reprogramming with drastic down-regulation of T3SS virulence genes during persistence when the pathogen resides within the cecum. At the persistent stage, the expression pattern in many respects resembles the pattern seen in vitro at 26oC, with for example, up-regulation of flagellar genes and invA. These findings are expected to have impact on future rationales to identify suitable bacterial targets for new antibiotics. Other genes that are up-regulated during persistence are genes involved in anaerobiosis, chemotaxis, and protection against oxidative and acidic stress, which indicates the influence of different environmental cues. We found that the Crp/CsrA/RovA regulatory cascades influence the pattern of bacterial gene expression during persistence. Furthermore, arcA, fnr, frdA, and wrbA play critical roles in persistence. Our findings suggest a model for the life cycle of this enteropathogen with reprogramming from a virulent to an adapted phenotype capable of persisting and spreading by fecal shedding.

Figure 1. Persistent Y. pseudotuberculosis resides in cecal tissue in the presence of an immune response.

(A-B) Immunofluorescent staining of Y. pseudotuberculosis in cecum from a mouse with persistent asymptomatic infection (35 dpi) using anti-Yersiniae rabbit polyclonal serum detected by anti-rabbit Al488 (green). Nuclei were stained with DAPI (blue); (A) 4× magnification, scale bar 500 μm, (B) 40× magnification, scale bar 50 μm. (C) Immunohistochemical staining of PMNs with anti-Ly6G6C in cecal tissue from a persistently infected asymptomatic mouse (35 dpi). Positive cells are brown (DAB) and the background is green. (methyl green). 4× magnification, scale bar 500 μm. (D) Hematoxylin-eosin staining of persistently infected cecal tissue (42 dpi). 60× magnification, scale bar 20 μm.

Figure 2. Y. pseudotuberculosis infection alters the bacterial composition of the cecum.

(A) Representative Bioanalyzer 2100 electrographs and associated gel pictures for replicates of in vitro-derived RNA samples (grown at 26°C and 37°C), in vivo-derived samples of early (isolated from mouse cecal tissue 2 dpi) and persistent infection (isolated from mouse cecal tissue 42 dpi), and uninfected samples (isolated from uninfected mouse cecal tissue). (B) The number of reads mapping to 16S rRNA from different bacteria in non-depleted in vivo-derived samples. Data represent the mean ± SD of the two replicates for each sample group. (C) Relative abundance of different bacterial phyla in samples according to reads mapped to the 16SMicrobial database. The proportions are given as the percent of bacterial phyla identified in specific samples.

Figure 3. In vivo Y. pseudotuberculosis gene expression revealed by RNA-seq.

(A) Gene expression data for Y. pseudotuberculosis from two biological replicates of in vitro and in vivo-derived samples. From outside to inside, the 10 circles in the plot correspond to: (1) a histogram of RPKMO values for each gene expressed at 26°C; (2) genes expressed only at 26°C under in vitro conditions; (3) heat map (combined gray and red Brewer palettes) of the log2 difference in RPKMO values for genes expressed at both 26°C and 37°C; (4) genes expressed only at 37°C under in vitro conditions; (5) histogram of RPKMO values for each gene expressed at 37°C; (6) histogram of RPKMO values for each gene expressed during the early phase of infection (2 dpi) in FVB/N cecum; (7) genes expressed only during the early phase of infection in FVB/N cecum; (8) heat map (combined blue and orange Brewer palettes) of the log2 difference in RPKMO values for genes expressed during both the early phase of infection and persistent infection (42 dpi) in the cecum; (9) genes expressed only during persistent infection in the FVB/N cecum; and (10) a histogram of RPKMO values for each gene expressed during persistent infection in FVB/N cecum. Regions outlined with green borders indicate the locations of genes encoding T3SS components and effectors of the virulence plasmid and genes involved in flagellar assembly on the chromosome. Regions outlined with gray borders indicate the locations of transcriptionally active regions. The asterisk indicates a chromosomal region shown in Fig. 3B. The plots were created using Circos [62]. (B) Distributions of reads mapped to a specific transcriptionally active region on the Y. pseudotuberculosis YPIII chromosome (from 1,191 Mb to 1,230 Mb) in one replicate of each sample group. The height of each peak corresponds to the relative number of reads mapped to the region. The tracks were created using CLC Genomic Workbench. (C). Expression of indicated genes during early (blue; 2 dpi) and persistent infections (orange; 42 dpi) determined by qPCR of cDNAs from two biological and three technical replicates for each gene. lpp, ompF, fliC, hdeB, ftn, ompA, yhbH, aspA and pal gene expressions were 1.1, 6.8, 9.2, 4.8, 9.7, 1.7, 2.3, 1.1 and 1.1 log2fold upregulated respectively during persistent infection in RNA-seq analysis.

Figure 4. T3SS genes and flagellar genes are differentially regulated during persistent infection.

(A) Expression of indicated yop effectors in vitro (left) at 26°C (gray) and at 37°C inducing conditions (red), and in vivo (right) during early (2 dpi; blue) and persistent infections (42 dpi; orange) as determined by qPCR of cDNAs from two biological and three technical replicates for each gene. (B) Expression of indicated flagellar genes in vivo during early (2 dpi; blue) and persistent infection (42 dpi; orange) as determined by qPCR. All qPCR analyses were performed with cDNAs from two biological and three technical replicates. flhDC, fliA, fliE, flgL, fliK, flgH, flgG, flgB, and flgA genes expressions were 5.2, 8.3, 9.9, 3.6, 2.3, 3.8, 3.1, 5.2 and 3.6 log2fold upregulated respectively during persistent infection according to RNA-seq analysis.

Figure 5. Y. pseudotuberculosis undergoes transcriptional reprogramming for adaption to persistence.

(A) Comparison of genes up-regulated in Y. pseudotuberculosis in vitro at 26°C and 37°C compared to in vivo during early (2 dpi) and persistent (42 dpi) stages of infection. Similarities are shown with the number of genes up-regulated in both groups. (B) Functional annotation of Y. pseudotuberculosis genes up-regulated during early and persistent infection (KEGG pathway mapping tool). (C) Comparison of the in vivo gene expression profiles and the expression profiles of bacteria grown under anaerobic conditions in vitro. The analysis included genes up-regulated (>1.8-fold) during anaerobic or aerobic growth in both the exponential and stationary growth phase compared to genes up-regulated during early and persistent infection. Similarities are shown with the number of genes up-regulated in both groups.

Figure 6. Establishment of persistent infection requires arcA, fnr, frdA, and wrbA.

(A) Infection profile 42 dpi for FVB/N mice infected orally with10⁷ CFUs of wt Y. pseudotuberculosis (n = 20) and indicated mutant strains (each group n = 16). The infections were monitored by IVIS at certain intervals up to 42 dpi. (B) Heatmap showing differences in clearance (by p-value) between wt and indicated mutant strains at different time points during the 42 day infection period. Heatmap color scale, from green to yellow, was adjusted according to p-values from 1 to 0. p-values were calculated with 2×2 contingency table by Fisher’s Exact Test, see also S6 Table. (C) Motility profile of wt Y. pseudotuberculosis and indicated mutant strains under anaerobic conditions at 26°C. Images were captured by the ChemiDoc XRS System (Bio-Rad), showing the bioluminescent signal produced by Y. pseudotuberculosis YPIII/pIBX.

Figure 7. Hypothetical model of Y. pseudotuberculosis reprogramming for persistent infection in cecum.

Upon initial infection, Y. pseudotuberculosis is still flagellated and expresses T3SS virulence genes. At the early stage of infection (2 dpi) the T3SS is important for colonization of tissue, including breaking the epithelial barrier and resisting the attack from arriving PMNs. At the persistent stage of infection (42 dpi), Y. pseudotuberculosis had reprogrammed its transcriptome by reducing the expression of T3SS components and increasing the expression of genes important for survival in the cecal lymphoid compartment. At this stage the bacteria are flagellated and can spread to other hosts by shedding into the feces, possibly through motility.