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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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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.
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ppat.1004600.g001: 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.

Mentions: To identify mechanisms promoting Y. pseudotuberculosis persistence, RNA-seq was employed to determine the differential gene expression profiles of bacteria in the cecum during the early phase of infection and during persistence. To obtain infected tissue for the isolation of Y. pseudotuberculosis RNA, FVB/N mice were infected orally with bioluminescent wild-type (wt) bacteria at an infection dose of ∼2×107 CFUs. The infection was monitored in real time by an in vivo imaging system (IVIS) at certain intervals for 42 days. In agreement with that reported earlier [6], we found bacterial foci associated with the cecal lymphoid tissue (Fig. 1A–B), massive infiltration of PMNs surrounding the bacterial foci (Fig. 1C–D) as well as superficial destruction of the epithelial lining and mixed inflammatory infiltrates.


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)

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.
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getmorefigures.php?uid=PMC4295882&req=5

ppat.1004600.g001: 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.
Mentions: To identify mechanisms promoting Y. pseudotuberculosis persistence, RNA-seq was employed to determine the differential gene expression profiles of bacteria in the cecum during the early phase of infection and during persistence. To obtain infected tissue for the isolation of Y. pseudotuberculosis RNA, FVB/N mice were infected orally with bioluminescent wild-type (wt) bacteria at an infection dose of ∼2×107 CFUs. The infection was monitored in real time by an in vivo imaging system (IVIS) at certain intervals for 42 days. In agreement with that reported earlier [6], we found bacterial foci associated with the cecal lymphoid tissue (Fig. 1A–B), massive infiltration of PMNs surrounding the bacterial foci (Fig. 1C–D) as well as superficial destruction of the epithelial lining and mixed inflammatory infiltrates.

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