Utility of In Vivo Transcription Profiling for Identifying Pseudomonas aeruginosa Genes Needed for Gastrointestinal Colonization and Dissemination

Andrew Y Koh,Akinobu Kamei,Kathleen T Coggshall,Gerald B Pier,Per J Mikkelsen,Michael Givskov,Stephen Lory,Roger S Smith,Olivier Neyrolles

doi:10.1371/journal.pone.0015131

Abstract

Microarray analysis of Pseudomonas aeruginosa mRNA transcripts expressed in vivo during animal infection has not been previously used to investigate potential virulence factors needed in this setting. We compared mRNA expression in bacterial cells recovered from the gastrointestinal (GI) tracts of P. aeruginosa-colonized mice to that of P. aeruginosa in the drinking water used to colonize the mice. Genes associated with biofilm formation and type III secretion (T3SS) had markedly increased expression in the GI tract. A non-redundant transposon library in P. aeruginosa strain PA14 was used to test mutants in genes identified as having increased transcription during in vivo colonization. All of the Tn-library mutants in biofilm-associated genes had an attenuated ability to form biofilms in vitro, but there were no significant differences in GI colonization and dissemination between these mutants and WT P. aeruginosa PA14. To evaluate T3SS factors, we tested GI colonization and neutropenia-induced dissemination of both deletional (PAO1 and PAK) and insertional (PA14) mutants in four genes in the P. aeruginosa T3SS, exoS or exoU, exoT, and popB. There were no significant differences in GI colonization among these mutant strains and their WT counterparts, whereas rates of survival following dissemination were significantly decreased in mice infected by the T3SS mutant strains. However, there was a variable, strain-dependent effect on overall survival between parental and T3SS mutants. Thus, increased transcription of genes during in vivo murine GI colonization is not predictive of an essential role for the gene product in either colonization or overall survival following induction of neutropenia.

Highlights

Modern molecular tools allow for analysis of levels of mRNA in bacterial cells living in different environments and many such studies have been applied to Pseudomonas aeruginosa [1,2,3,4,5]
We initially identified P. aeruginosa genes that were differentially transcribed in the GI tract of eight colonized C3H mice in comparison to genes expressed in sterile water containing 1500 U penicillin G/mL
Secretion Mutants in the murine GI tract Given that the uniform high expression levels of the genes involved in T3SS detected during GI colonization, we investigated the colonization and dissemination phenotypes of P. aeruginosa strains wherein the exoS, exoT or popB genes were deleted in strains PAO1 and PAK as well as the transposon insertional mutants in T3SS genes including exoU in the PA14 background

Summary

Introduction

Modern molecular tools allow for analysis of levels of mRNA in bacterial cells living in different environments and many such studies have been applied to Pseudomonas aeruginosa [1,2,3,4,5]. In a previously described model of murine GI colonization and dissemination following induction of neutropenia [7] we found that high levels of P. aeruginosa could be recovered from the mouse cecum, potentially identifying a source of in vivo bacterial mRNA sufficient for microarray analysis This model mimics the morbidity and mortality of immunocompromised hosts such as patients with leukemia, severe burn wounds or recipients of organ transplants [8]. In many patients at-risk for P. aeruginosa infection (i.e. surgical patients, cancer patients receiving chemotherapy) the gastrointestinal (GI) tract is believed to be the main tissue initially colonized by this organism, often times allowing for translocation to extra-gastrointestinal sites and, in the worst cases, development of life-threatening sepsis [9,10] In this patient group, P. aeruginosa has the highest case-fatality rate among all gram-negative pathogens [11]. The mere presence of P. aeruginosa in the GI tract of critically-ill surgical patients is associated with a 70% mortality rate, a three-fold increase over physiologically matched critically-ill patients not infected with P. aeruginosa [12]

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