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

Avican K, Fahlgren A, Huss M, Heroven AK, Beckstette M, Dersch P, Fällman M - PLoS Pathog. (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Umeå Centre for Microbial Research (UCMR), Umeå University, Umeå, Sweden; Department of Molecular Biology, Laboratory for Molecular Infection Medicine Sweden (MIMS), Umea University, Umeå, Sweden.

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.

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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.
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ppat.1004600.g002: 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.

Mentions: For sample preparation, isolated cecal tissues from 2 and 42 days post-infection (dpi) were analyzed by IVIS to verify that they contained Yersinia. The bioluminescent signal from Y. pseudotuberculosis allowed identification of the precise location of the bacteria in the tissue. Small biopsies (3 mm Ø) of cecal tissue containing bioluminescent bacteria were isolated using a hole punch. Total RNAs were extracted from biopsies from two mice infected for 2 days and two asymptomatic mice infected for 42 days. As a control, we extracted total RNAs from the cecal tissue of two un-infected mice. We also included RNA samples from bacteria grown in Luria broth (LB) in vitro at 26°C and in Ca2+-depleted LB at 37°C, a condition known to induce T3SS [22], hereafter referred to as T3SS-inducing conditions. The quality and quantity of all RNA samples were determined using an Agilent Bioanalyzer 2100, and all total RNA preparations had RIN values >7. This analysis revealed pure bacterial RNA in the in vitro samples (16S and 23S rRNAs) and mouse RNA (18S and 26S rRNAs) in samples from un-infected cecal tissue. As expected, both eukaryotic and prokaryotic RNAs were detected in the samples from infected cecal tissue and appeared as four distinct bands: 16S, 18S, 23S, and 26S rRNAs (Fig. 2A). However, as we previously recovered only 1×105 to 2×106 CFUs Y. pseudotuberculosis from cecal tissues [6], the amount of prokaryotic RNA was unexpectedly high in the infected tissues. Therefore, we performed qPCR to determine the Yersinia RNA abundance in infected tissues by comparing ymoA expression as an indication of Yersinia RNAs and GAPDH expression as an indication of host RNAs, finding ∼0.2% Yersinia RNA in the total RNA preparations. Therefore, the remarkably higher amount of prokaryotic RNA compared to that predicted for Yersinia RNA was assumed to reflect the presence of other microbial inhabitants in the samples.


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

Avican K, Fahlgren A, Huss M, Heroven AK, Beckstette M, Dersch P, Fällman M - PLoS Pathog. (2015)

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.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4295882&req=5

ppat.1004600.g002: 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.
Mentions: For sample preparation, isolated cecal tissues from 2 and 42 days post-infection (dpi) were analyzed by IVIS to verify that they contained Yersinia. The bioluminescent signal from Y. pseudotuberculosis allowed identification of the precise location of the bacteria in the tissue. Small biopsies (3 mm Ø) of cecal tissue containing bioluminescent bacteria were isolated using a hole punch. Total RNAs were extracted from biopsies from two mice infected for 2 days and two asymptomatic mice infected for 42 days. As a control, we extracted total RNAs from the cecal tissue of two un-infected mice. We also included RNA samples from bacteria grown in Luria broth (LB) in vitro at 26°C and in Ca2+-depleted LB at 37°C, a condition known to induce T3SS [22], hereafter referred to as T3SS-inducing conditions. The quality and quantity of all RNA samples were determined using an Agilent Bioanalyzer 2100, and all total RNA preparations had RIN values >7. This analysis revealed pure bacterial RNA in the in vitro samples (16S and 23S rRNAs) and mouse RNA (18S and 26S rRNAs) in samples from un-infected cecal tissue. As expected, both eukaryotic and prokaryotic RNAs were detected in the samples from infected cecal tissue and appeared as four distinct bands: 16S, 18S, 23S, and 26S rRNAs (Fig. 2A). However, as we previously recovered only 1×105 to 2×106 CFUs Y. pseudotuberculosis from cecal tissues [6], the amount of prokaryotic RNA was unexpectedly high in the infected tissues. Therefore, we performed qPCR to determine the Yersinia RNA abundance in infected tissues by comparing ymoA expression as an indication of Yersinia RNAs and GAPDH expression as an indication of host RNAs, finding ∼0.2% Yersinia RNA in the total RNA preparations. Therefore, the remarkably higher amount of prokaryotic RNA compared to that predicted for Yersinia RNA was assumed to reflect the presence of other microbial inhabitants in the samples.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology, Umeå Centre for Microbial Research (UCMR), Umeå University, Umeå, Sweden; Department of Molecular Biology, Laboratory for Molecular Infection Medicine Sweden (MIMS), Umea University, Umeå, Sweden.

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.

Show MeSH
Related in: MedlinePlus