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Transcriptional profiling of Klebsiella pneumoniae defines signatures for planktonic, sessile and biofilm-dispersed cells.

ABSTRACT: Surface-associated communities of bacteria, known as biofilms, play a critical role in the persistence and dissemination of bacteria in various environments. Biofilm development is a sequential dynamic process from an initial bacterial adhesion to a three-dimensional structure formation, and a subsequent bacterial dispersion. Transitions between these different modes of growth are governed by complex and partially known molecular pathways.Using RNA-seq technology, our work provided an exhaustive overview of the transcriptomic behavior of the opportunistic pathogen Klebsiella pneumoniae derived from free-living, biofilm and biofilm-dispersed states. For each of these conditions, the combined use of Z-scores and principal component analysis provided a clear illustration of distinct expression profiles. In particular, biofilm-dispersed cells appeared as a unique stage in the bacteria lifecycle, different from both planktonic and sessile states. The K-means cluster analysis showed clusters of Coding DNA Sequences (CDS) and non-coding RNA (ncRNA) genes differentially transcribed between conditions. Most of them included dominant functional classes, emphasizing the transcriptional changes occurring in the course of K. pneumoniae lifestyle transitions. Furthermore, analysis of the whole transcriptome allowed the selection of an overall of 40 transcriptional signature genes for the five bacterial physiological states.This transcriptional study provides additional clues to understand the key molecular mechanisms involved in the transition between biofilm and the free-living lifestyles, which represents an important challenge to control both beneficial and harmful biofilm. Moreover, this exhaustive study identified physiological state specific transcriptomic reference dataset useful for the research community.

SUBMITTER: Guilhen C

PROVIDER: S-EPMC4791964 | biostudies-literature | 2016 Mar

REPOSITORIES: biostudies-literature

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Transcriptional profiling of Klebsiella pneumoniae defines signatures for planktonic, sessile and biofilm-dispersed cells.

Guilhen Cyril C Charbonnel Nicolas N Parisot Nicolas N Gueguen Nathalie N Iltis Agnès A Forestier Christiane C Balestrino Damien D

BMC genomics 20160315

<h4>Background</h4>Surface-associated communities of bacteria, known as biofilms, play a critical role in the persistence and dissemination of bacteria in various environments. Biofilm development is a sequential dynamic process from an initial bacterial adhesion to a three-dimensional structure formation, and a subsequent bacterial dispersion. Transitions between these different modes of growth are governed by complex and partially known molecular pathways.<h4>Results</h4>Using RNA-seq technolo ...[more]

PMID: 26979871

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Project description:Purpose: The goal of this study was to use RNA-seq to define the Klebsiella pneumoniae transcriptome recorded under 5 different experimental conditions, and to identify signature genes of each condition by comparing global transcriptional profiles. Methods: mRNA profiles were generated for Klebsiella pneumoniae CH1034 clinical isolate, in triplicate, by deep sequencing. Total RNAs were harvested from bacteria cultured at 37Â°C in M63B1 minimal media under different conditions: (i) planktonic aerobic condition at OD 620nm=0.250 (exponential growth-phase), (ii) overnight planktonic aerobic condition (stationnary growth-phase), (iii) biofilm in a flow-cell chamber after 7 hours of incubation (7-hours old biofilm), (iv) biofilm in a flow-cell chamber after 13 hours of incubation (13-hours old biofilm), (v) bacteria self-dispersed from biofilm recovered in the flow-cell effluent (biofilm-dispersed bacteria). Ribosomal RNAs were removed using the Bacteria Ribo-Zero Magnetic kit (Epicentre Biotechnologies). Libraries were prepared using the TruSeq Stranded mRNA Sample Preparation kit (Illumina), and 50bp single-reads were obtained by HiSeq 2000 (Illumina).The sequence reads that passed FastQC quality filters were mapped to the CH1034 genome using BurrowsâWheeler Aligner (BWA) (0.7.12-r1039 version). The transcript levels were determined using HTSeq-count (0.6.1p1 version) with union mode followed by DESeq (1.16.0 version) analysis. qRTâPCR validation was performed using SYBR Green assays. Results: We found that each condition has a specific transcriptional profile, and we identify 4 robust signature genes for each. Conclusion: Our study represents the first detailed analysis of K. pneumoniae transcriptomes under different experimental conditions generated by RNA-seq technology. The data reported here should permit the dissection of complex biologic functions involved in the transition between the sessile and planktonic modes of growth. Determination of the transcriptional profiling of Klebsiella pneumoniae under 5 different experimental conditions. mRNA profiles were generated for bacteria under exponential planktonic growth-phase, stationary planktonic growth-phase, 7 hours-old biofilm, 13 hours-old biofilm and biofilm-dispersed modes, each in three biological replicates, by deep sequencing using Illumina HiSeq

Project description:Klebsiella pneumoniae causes significant morbidity and mortality worldwide, particularly amongst hospitalized individuals. The principle mechanism for pathogenesis in hospital environments involves the formation of biofilms, primarily on implanted medical devices. In this study, we constructed a transposon mutant library in a clinical isolate, K. pneumoniae AJ218, to identify the genes and pathways implicated in biofilm formation. Three mutants severely defective in biofilm formation contained insertions within the mrkABCDF genes encoding the main structural subunit and assembly machinery for type 3 fimbriae. Two other mutants carried insertions within the yfiN and mrkJ genes, which encode GGDEF domain- and EAL domain-containing c-di-GMP turnover enzymes, respectively. The remaining two isolates contained insertions that inactivated the mrkH and mrkI genes, which encode for novel proteins with a c-di-GMP-binding PilZ domain and a LuxR-type transcriptional regulator, respectively. Biochemical and functional assays indicated that the effects of these factors on biofilm formation accompany concomitant changes in type 3 fimbriae expression. We mapped the transcriptional start site of mrkA, demonstrated that MrkH directly activates transcription of the mrkA promoter and showed that MrkH binds strongly to the mrkA regulatory region only in the presence of c-di-GMP. Furthermore, a point mutation in the putative c-di-GMP-binding domain of MrkH completely abolished its function as a transcriptional activator. In vivo analysis of the yfiN and mrkJ genes strongly indicated their c-di-GMP-specific function as diguanylate cyclase and phosphodiesterase, respectively. In addition, in vitro assays showed that purified MrkJ protein has strong c-di-GMP phosphodiesterase activity. These results demonstrate for the first time that c-di-GMP can function as an effector to stimulate the activity of a transcriptional activator, and explain how type 3 fimbriae expression is coordinated with other gene expression programs in K. pneumoniae to promote biofilm formation to implanted medical devices.

Dataset Information

Transcriptional profiling of Klebsiella pneumoniae defines signatures for planktonic, sessile and biofilm-dispersed cells.

Publications

Transcriptional profiling of Klebsiella pneumoniae defines signatures for planktonic, sessile and biofilm-dispersed cells.

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