Impact of Hfq on Global Gene Expression and Virulence in Klebsiella pneumoniae

Citation: Chiang M-K, Lu M-C, Liu L-C, Lin C-T, Lai Y-C. ( Impact of Hfq on Global Gene Expression and Virulence in Klebsiella pneumoniae Ming-Ko Chiang 0 Min-Chi Lu 0 Li-Cheng Liu 0 Ching-Ting Lin 0 Yi-Chyi Lai 0 Jean-Pierre Gorvel, Universite de la Mediterranee, France 0 1 Department of Life Science, National Chung-Cheng University , Chia-Yi, Taiwan , 2 Division of Infectious Diseases, Department of Internal Medicine, Chung-Shan Medical University Hospital , Taichung, Taiwan , 3 Department of Microbiology and Immunology, Chung-Shan Medical University , Taichung, Taiwan , 4 Institute of Medicine, Chung- Shan Medical University , Taichung, Taiwan , 5 School of Chinese Medicine, China Medical University , Taichung , Taiwan Klebsiella pneumoniae is responsible for a wide range of clinical symptoms. How this bacterium adapts itself to everchanging host milieu is still a mystery. Recently, small non-coding RNAs (sRNAs) have received considerable attention for their functions in fine-tuning gene expression at a post-transcriptional level to promote bacterial adaptation. Here we demonstrate that Hfq, an RNA-binding protein, which facilitates interactions between sRNAs and their mRNA targets, is critical for K. pneumoniae virulence. A K. pneumoniae mutant lacking hfq (Dhfq) failed to disseminate into extra-intestinal organs and was attenuated on induction of a systemic infection in a mouse model. The absence of Hfq was associated with alteration in composition of envelope proteins, increased production of capsular polysaccharides, and decreased resistance to H2O2, heat shock, and UV irradiation. Microarray-based transcriptome analyses revealed that 897 genes involved in numerous cellular processes were deregulated in the Dhfq strain. Interestingly, Hfq appeared to govern expression of many genes indirectly by affecting sigma factor RpoS and RpoE, since 19.5% (175/897) and 17.3% (155/897) of Hfq-dependent genes belong to the RpoE- and RpoS-regulon, respectively. These results indicate that Hfq regulates global gene expression at multiple levels to modulate the physiological fitness and virulence potential of K. pneumoniae. - Funding: This work was supported by the National Science Council and Chung-Shan Medical University of Taiwan R.O.C. (NSC 98-2311-B-194-001-MY3 to MingKo Chiang, NSC98-2320-B-040-013 and CSMU-TSMH-099-005 to Yi-Chyi Lai). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Since Klebsiella was first identified as a pathogen of pneumonia in 1882, the remarkable ability of K. pneumoniae to cause a wild range of human diseases, from urinary tract infections to lifethreatening systemic infections [1], has attracted increasing attention to the pathogenesis of this bacterium. Not solely confined inside the human host, K. pneumoniae has a great capacity for adaptation to diverse environments, including the surface water, sewage, soil, intestinal tracts of mammals [1], and even the interior of plants [2]. How K. pneumoniae responds to environmental changes and thus adapts itself to a specific niche becomes an interesting question. Nevertheless, our knowledge with regard to the regulatory mechanisms which this bacterium utilizes to ensure its survival upon different conditions is very limited. An ever-increasing number and variety of small non-coding RNAs (sRNAs) are being identified to serve regulatory functions in bacteria. Numerous cellular processes, such as iron homeostasis [3], outer membrane proteins (OMPs) biogenesis [4], sugar metabolism [5], quorum sensing [6] and various stress responses [7], are subject to the post-transcriptional control exerted by sRNAs. At present, most characterized sRNAs regulate gene expression by basepairing with mRNAs. While some sRNAs are cis-encoded having the potential to basepair mRNAs with long stretches, the majority of regulatory sRNAs in Gram-negative bacteria are trans-encoded and share limited complementarity with their target mRNAs [8]. The trans-acting sRNAs are functionally analogous to eukaryotic miRNAs that usually exert negative regulation by repress protein levels through translation inhibition, mRNA degradation, or both [9]. In many cases, because of the limited complementarity, the trans-acting sRNAs mediated regulation requires the chaperone protein Hfq to facilitate RNA-RNA interactions. Hfq assembles into homohexameric rings which are structurally similar to those formed by Sm and Sm-like proteins in eukaryotic cells [10]. Besides enhancing the formation of sRNA-mRNA duplex, Hfq contributes to RNA regulation through interacting with RNA turnover enzymes, including RNase E, polynucleotide phosphorylase, and poly (A) polymerase [11]. Hfq has a broad and diverse impact on bacterial physiology and virulence beyond its original role as a host factor required for replication of Qb RNA bacteriophage [12]. Defects including reduced growth, impaired resistance to various stresses, and altered virulence are detected in E. coli lacking hfq [13]. It has also been shown that virulence of several pathogenic bacteria, including Brucella abortus [14], Francisella tularensis [15], Vibrio cholera [16], Listeria monocytogenes [17], Legionella pneumophila [18], Pseudomonas aeruginosa [19], Yersinia [20,21], Salmonella Typhimurium [22], and uropathogenic E. coli [23], were significantly attenuated by hfq mutations. Recently, by sequence analysis, we have identified an hfq homologue and sRNAs from K. pneumoniae genomes. The presence of these homologues in various strains of K. pneumoniae, including NTUH-K2044 (NC_012731), MGH78578 (NC_009648), and 342 (NC_011283) suggests that this pathogen also utilizes the posttranscriptional regulation mediated by Hfq to control various cellular processes. However, the role of Hfq-sRNA mediated regulation in K. pneumoniae is yet to be defined. In this study, we aimed to understand how Hfq contributed to the control of gene expression and the pathogenesis in K. pneumoniae. An hfq deletion mutant was generated in K. pneumoniae CG43S. The loss of hfq attenuated K. pneumoniae virulence in a mouse model, as well as altered physiological characteristics of K. pneumoniae, including production of capsular polysaccharides, stress tolerance, and homeostasis of envelope. The microarray data demonstrated that the expression of almost a fifth of K. pneumoniae genes was drastically deregulated. It also suggested that beside directly regulating individual genes expression at the post-transcriptional level, by affecting sigma factor RpoS and RpoE, Hfq positioned itself at the upper level of the gene regulatory hierarchy that control the physiological fitness and virulence potential of K. pneumoniae. Deletion of hfq attenuated K. pneumoniae virulence The hfq gene is located in clockwise orientation at bps 446148 446456 in the genome of K. pneumo (...truncated)