Functional Characterization of the RNA Chaperone Hfq in the Opportunistic Human Pathogen Stenotrophomonas maltophilia

Abstract

Hfq is an RNA-binding protein known to regulate a variety of cellular processes by interacting with small RNAs (sRNAs) and mRNAs in prokaryotes. Stenotrophomonas maltophilia is an important opportunistic pathogen affecting primarily hospitalized and immunocompromised hosts. We constructed an hfq deletion mutant (⌬hfq) of S. maltophilia and compared the behaviors of wild-type and ⌬hfq S. maltophilia cells in a variety of assays. This revealed that S. maltophilia Hfq plays a role in biofilm formation and cell motility, as well as susceptibility to antimicrobial agents. Moreover, Hfq is crucial for adhesion to bronchial epithelial cells and is required for the replication of S. maltophilia in macrophages. Differential RNA sequencing analysis (dRNA-seq) of RNA isolated from S. maltophilia wild-type and ⌬hfq strains showed that Hfq regulates the expression of genes encoding flagellar and fimbrial components, transmembrane proteins, and enzymes involved in different metabolic pathways. Moreover, we analyzed the expression of several sRNAs identified by dRNA-seq in wild-type and ⌬hfq S. maltophilia cells grown in different conditions on Northern blots. The accumulation of two sRNAs was strongly reduced in the absence of Hfq. Furthermore, based on our dRNA-seq analysis we provide a genomewide map of transcriptional start sites in S. maltophilia. Stenotrophomonas maltophilia is a Gram-negative environmental gammaproteobacterium that has been recognized as an important opportunistic pathogen in the last decade, affecting primarily hospitalized and immunocompromised hosts (3). S. maltophilia is frequently isolated from the lungs of cystic fibrosis patients, and its multidrug-resistant phenotype complicates the eradication of S. maltophilia infections (35). Factors that may contribute to the pathogenicity of S. maltophilia include a wide range of extracellular enzymes potentially involved in the colonization process, such as fibrolysin, lipases, and proteases (6), the ability to adhere to and form biofilms on epithelial cells (31), and replication and persistence in the lung (34). Deterioration of lung function associated with S. maltophilia infections has been demonstrated by infection assays carried out in mice (10).Whole genome sequences of three S. maltophilia strains are available: (i) R551-3, a strain which was isolated from Populus trichocarpa (46); (ii) SKA14, which was isolated from the Baltic Sea (15); and (iii) K279a, a strain isolated from a cancer patient (6). Comparative genome-wide analyses revealed that the R551-3 and K279a genomes share a conserved backbone but contain different sets of genomic islands (GEIs) (33).Hfq (host factor I protein) is a highly conserved RNA chaperone that was first identified as a protein required for the replication of the RNA phage Q␤ in Escherichia coli (13). Hfq plays an important role in the fitness and virulence of many pathogenic bacteria, and hfq mutants often exhibit pleiotropic phenotypes, including defects in quorum sensing, growth rate, stress tolerance, and virulence ...

Stenotrophomonas maltophilia Encodes a Type II Protein Secretion System That Promotes Detrimental Effects on Lung Epithelial Cells

“…Although many advances have been made in our understanding of the genetic basis of antibiotic resistance in S. maltophilia (40), only a few previous studies have used mutants to define (potential) virulence factors of S. maltophilia (3). Key earlier studies focused on the role of flagella in bacterial adherence to bronchial epithelial cells in vitro, the impact of a diffusible, signal factor (cis-⌬2-11-metyl-dodecenoic acid) and a secreted cell-signaling protein (Ax21) in nematode and moth models of toxicity and virulence, the connection between phosphoglucomutase and lipopolysaccharide (LPS) and virulence in a rat lung model of infection, and the need for the RNA chaperone Hfq in adherence to bronchial cells (27,(41)(42)(43)(44). Thus, embarking upon a systematic assessment of protein secretion systems in S. maltophilia is significant when one considers the current state of the S. maltophilia pathogenesis field.…”

Genome-Wide Identification of Genes Necessary for Biofilm Formation by Nosocomial Pathogen Stenotrophomonas maltophilia Reveals that Orphan Response Regulator FsnR Is a Critical Modulator

“…Environmental factors, including phosphate, pH, temperature, metal ions, and antibiotics, were shown to have a profound effect on S. maltophilia biofilm formation (3). To date, however, only a small number of genes or biochemical cascades associated with S. maltophilia biofilm formation have been reported, and these include the polysaccharide synthesis genes rmlA, rmlC, and xanB (12), diffusible signal factor (DSF)-mediated cell-cell communication (13), the small RNA modulator Hfq (14), and the ABC-type efflux pump MacABC (15). Hence, it is critical to undertake a systematic approach to elucidate the molecular and regulatory properties of S. maltophilia biofilm development.…”

Small non-coding RNAs in plant-pathogenicXanthomonasspp

“…19,22 Phylogenetic analyses suggest that the majority of Xanthomonas sRNAs is exclusively conserved in members of the Xanthomonadaceae family. Exceptions include PtaRNA1 from Xcv, which exhibits an erratic phylogenetic distribution, and widely conserved sRNAs with housekeeping functions, e. g. 6S RNA, SRP RNA, tmRNA, and RNase P. 19,22,34 Homologs of some of the clade-specific sRNAs were identified by independent studies in different Xanthomonas spp. and in Smal ( Table 1).…”

“…45 Although the function of Hfq is obscure in Xanthomonas spp., the presumed role as RBP is supported by the Hfq-dependent accumulation of the Xcv sRNA sX14 and three Xoo sRNAs, including the sX14 homolog sRNA-Xoo3 (Table 1). 21,24 In Smal, two Hfq-dependent sRNAs were identified, one of which, SmsR36, 34 is homologous to the Hfq-dependent sRNA-Xoo1 ( Table 1). Hfq-independent Smal sRNAs include SmsR39, which is homologous to sX13 from Xcv.…”

“…and the related Smal, as an hfq mutation in Smal impacts on growth, motility, biofilm formation, tolerance against antimicrobial agents, and the bacterial interaction with eukaryotic cells. 34 Further research is needed to address physiological functions and the RNA-binding capability of Hfq in Xanthomonas spp.…”