Signature-Tagged Mutagenesis of Klebsiella pneumoniae To Identify Genes That Influence Biofilm Formation on Extracellular Matrix Material

Abstract

Klebsiella pneumoniae causes urinary tract infections, respiratory tract infections, and septicemia in susceptible individuals. Strains of Klebsiella frequently produce extended-spectrum beta-lactamases, and infections with these strains can lead to relatively high mortality rates (approximately 15%). Other virulence factors include production of an antiphagocytic capsule and outer membrane lipopolysaccharide (LPS), which mediates serum resistance, as well as fimbriae on the surface of the bacteria. Type 1 fimbriae mediate adherence to many types of epithelial cells and may facilitate adherence of the bacteria to the bladder epithelium. Type 3 fimbriae can bind in vitro to the extracellular matrix of urinary and respiratory tissues, suggesting that they mediate binding to damaged epithelial surfaces. In addition, type 3 fimbriae are required for biofilm formation by Klebsiella pneumoniae on plastics and human extracellular matrix; thus, they may facilitate the formation of treatment-resistant biofilm on indwelling plastic devices, such as catheters and endotracheal tubing. The presence of these devices may cause tissue damage, allowing Klebsiella to grow as a biofilm on exposed tissue basement membrane components. Though in vivo biofilm growth may be an important step in the infection process, little is known about the genetic factors required for biofilm formation by Klebsiella pneumoniae. Thus, we performed signature-tagged mutagenesis to identify factors produced by K. pneumoniae strain 43816 that are required for biofilm formation. We identified mutations in the cps capsule gene cluster, previously unidentified transcriptional regulators, fimbrial, and sugar phosphotransferase homologues, as well as genetic loci of unknown function, that affect biofilm formation.

Klebsiella pneumoniae: Going on the Offense with a Strong Defense

“…Two of these mutants lack genes encoding homologues of proteins involved in sugar transport (phosphotransferases EIIB and EIIC), and one mutant lacks a gene predicted to encode a protein of unknown function (YPTB1848). Combined, the results of this study and that by Lavender et al suggest that biofilm production most likely does not play an essential role during K. pneumoniae pneumonia after instillation of bacteria into the lungs (369,370). Rather, the virulence defects observed in the pneumonic model with certain mutants with biofilm defects may be due to pleiotropic functions of these genes.…”

“…In (370). Interestingly, five of the eight confirmed mutants were attenuated in a pneumonic mouse model.…”

“…Two other studies used genomic comparisons between K. pneumoniae strains, with one study focusing on classical K. pneumoniae and the other focusing on HV K. pneumoniae, to identify genes of interest and then characterized the roles of specific genes in K. pneumoniae in a mouse lung infection model (168,367). Finally, four large-scale screens, three with classical K. pneumoniae and one with HV K. pneumoniae strains, examined K. pneumoniae growth in in vitro assays and then assessed the importance of these genes in virulence in mouse models of infection (368)(369)(370)(371). Specifically, in these studies, genes important for in vitro biofilm production and genes contributing to the inhibition of in vitro NF-B signaling were identified, followed by interrogation with pneumonic and intestinal colonization models to assess the contribution of these genes to the virulence of K. pneumoniae.…”

“…At least two studies have searched for K. pneumoniae biofilm factors that play a role in a pneumonic mouse model (369,370). In (370).…”

Role of MrkJ, a Phosphodiesterase, in Type 3 Fimbrial Expression and Biofilm Formation in Klebsiella pneumoniae

“…Plasmids LM2449 and LM2796 (kindly supplied by Linda McCarter, University of Iowa) were introduced into Klebsiella strains by conjugation using the donor strain E. coli S17-1pir carrying the appropriate plasmid (7). The construction and characterization of LM2449 and LM2796 has been described in detail elsewhere (15).…”