Establishment of persistent infection requires <i>arcA, fnr, frdA</i>, and <i>wrbA</i>.

<p>(A) Infection profile 42 dpi for FVB/N mice infected orally with10⁷ CFUs of wt <i>Y. pseudotuberculosis</i> (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 <i>p</i>-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 <i>p</i>-values from 1 to 0. <i>p</i>-values were calculated with 2×2 contingency table by Fisher’s Exact Test, see also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004600#ppat.1004600.s013" target="_blank">S6 Table</a>. (C) Motility profile of wt <i>Y. pseudotuberculosis</i> 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 <i>Y. pseudotuberculosis</i> YPIII/pIBX.</p

Hypothetical model of <i>Y. pseudotuberculosis</i> reprogramming for persistent infection in cecum.

<p>Upon initial infection, <i>Y. pseudotuberculosis</i> 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), <i>Y. pseudotuberculosis</i> 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.</p

Summary of RNA-seq reads.

<p>Asterisks indicate samples without rRNA depletion.</p><p>Summary of RNA-seq reads.</p

Up-regulated genes indicative of different environmental cues.

<p>Up-regulated genes indicative of different environmental cues.</p

<i>Y. pseudotuberculosis</i> infection alters the bacterial composition of the cecum.

<p>(A) Representative Bioanalyzer 2100 electrographs and associated gel pictures for replicates of <i>in vitro</i>-derived RNA samples (grown at 26°C and 37°C), <i>in vivo</i>-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 <i>in vivo</i>-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.</p

<i>Y. pseudotuberculosis</i> undergoes transcriptional reprogramming for adaption to persistence.

<p>(A) Comparison of genes up-regulated in <i>Y. pseudotuberculosis in vitro</i> at 26°C and 37°C compared to <i>in vivo</i> 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 <i>Y. pseudotuberculosis</i> genes up-regulated during early and persistent infection (KEGG pathway mapping tool). (C) Comparison of the <i>in vivo</i> gene expression profiles and the expression profiles of bacteria grown under anaerobic conditions <i>in vitro</i>. 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.</p