Characterization of Type 2 Quorum Sensing in Klebsiella pneumoniae and Relationship with Biofilm Formation

Abstract

Quorum sensing is a process by which bacteria communicate by using secreted chemical signaling molecules called autoinducers. Many bacterial species modulate the expression of a wide variety of physiological functions in response to changes in population density by this mechanism. In this study, the opportunistic pathogen Klebsiella pneumoniae was observed to secrete type 2 signaling molecules. A homologue of luxS, the gene required for AI-2 synthesis in Vibrio harveyi, was isolated from the K. pneumoniae genome. A V. harveyi bioassay showed the luxS functionality in K. pneumoniae and its ability to complement the luxS-negative phenotype of Escherichia coli DH5␣. Autoinducer activity was detected in the supernatant, and maximum expression of specific messengers detected by quantitative reverse transcription-PCR analysis occurred during the late exponential phase. The highest levels of AI-2 were observed in minimal medium supplemented with glycerol. To determine the potential role of luxS in colonization processes, a K. pneumoniae luxS isogenic mutant was constructed and tested for its capacity to form biofilms in vitro on an abiotic surface and to colonize the intestinal tract in a murine model. No difference was observed in the level of intestinal colonization between the wild-type strain and the luxS mutant. Microscopic analysis of biofilm structures revealed that the luxS mutant was able to form a mature biofilm but with reduced capacities in the development of microcolonies, mostly in the early steps of biofilm formation. These data suggest that a LuxS-dependent signal plays a role in the early stages of biofilm formation by K. pneumoniae.Many species of bacteria regulate gene expression through an intercellular communication mechanism called quorum sensing (QS) (17). QS is a mechanism of cell density-dependent regulation of bacterial gene expression and was first described in the marine luminous bacteria Vibrio fischeri and Vibrio harveyi, which use them to control the expression of bioluminescence (28). Bacteria synthesize, release, and detect specific small signaling molecules, referred to as autoinducers (AI), which accumulate in the external environment in conjunction with the population growth. When a critical threshold concentration of autoinducer is reached within a population, a signal transduction cascade is triggered, which forms the basis for change in the expression of specific target genes and thereby modifies the bacterial phenotypes.At present, there are two major independent types of recognized quorum-sensing systems in bacteria. Type I quorum sensing is a highly specific system and is used for intraspecies communication. In gram-negative bacteria, the autoinducers of signal system I (AI-1) have been identified as derivates of an acyl homoserine lactone (acyl-HSL) backbone with speciesspecific substitutions. These molecules diffuse freely in and out of cells. Two genes are crucially involved in quorum sensing through acyl-HSL: the synthesis of acyl-HSL is dependent on luxI, while luxR encode...

1,3-Propanediol Dehydrogenase fromKlebsiella pneumoniae: Decameric Quaternary Structure and Possible Subunit Cooperativity

“…Glycerol can be used as an alternative source of carbon and energy in some nosocomial infections in which K. pneumoniae can survive in adverse conditions (6,34). Metabolic intermediates of glycerol fermentation are also used in some K. pneumoniae strains for the biosynthesis of quorum-sensing molecules involved in signal transmission between bacterial individuals (5,48).…”

Klebsiella pneumoniae yfiRNB operon affects biofilm formation, polysaccharide production and drug susceptibility

“…Deletions of yfiR, yfiN and yfiB genes were constructed in WT K. pneumoniae LM21gfp, which bears an egfp marker, by allelic replacement with a Km R gene using pKOBEG199 (Balestrino et al, 2005). Briefly, the Km R cassette was PCR-amplified from plasmid pKD4 with 60 nt forward and reverse primers specific for each target gene that included sequences homologous to the Km R gene and FLP recognition target (FRT) sites, as reported previously (Balestrino et al, 2008;Datsenko & Wanner, 2000).…”

“…Biofilm formation in K. pneumoniae is affected by mutations in genes responsible for capsule biosynthesis and surface EPSs (Balestrino et al, 2008;Boddicker et al, 2006), genes for the synthesis of cell surface type 3 fimbriae (Di Martino et al, 2003;Langstraat et al, 2001), by mutations that affect quorum sensing (Balestrino et al, 2005) and by the intracellular levels of the second messenger c-di-GMP (Johnson & Clegg, 2010;Johnson et al, 2011;Wilksch et al, 2011). The cellular levels of c-di-GMP are modulated by diguanylate cyclases (DGCs) and phosphodiesterases, which catalyse its synthesis and degradation, respectively (Boyd & O'Toole, 2012;Römling et al, 2013).…”

Potential Emergence of Multi-quorum Sensing Inhibitor Resistant (MQSIR) Bacteria

“…Gramnegative luxS based AI2 producer QSS have been reported to regulate virulence in Helicobacter, Klebsiella, Porphyromonas, and Shigella. Rhodopseudomonas, and Xanthomonas produce p-coumaroyl-HSL and DSF, as signal molecules, respectively [140][141][142][143][144][145].…”