MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression

Abstract

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. Biofilms are surface-associated communities of microorganisms. Biofilm-associated bacteria are protected from host defenses and antibiotics and are the cause of many infections. Klebsiella pneumoniae is primarily a hospital-acquired bacterial pathogen that causes pneumonia, urinary tract infections and septicemia. Its success is related to its ability to form biofilms on medical devices, such as catheters. In K. pneumoniae, biofilm formation is mediated by type 3 fimbriae – hair-like, protein appendages extending out from the cell surface that adhere to surfaces. This study investigated how K. pneumoniae regulates the expression of these fimbriae. We identified a protein, MrkH, which behaves as a “biofilm switch” that turns on the expression of genes responsible for producing type 3 fimbriae. MrkH works by binding to regulatory regions of DNA nearby to these genes and initiates their expression. Importantly, MrkH binds to DNA strongly only when the protein is stimulated by a small molecule, c-di-GMP. Furthermore, we identified bacterial enzymes that either produce or break down c-di-GMP to control its concentration within the cell, and thus modulate MrkH activity. Understanding the molecular basis for these processes may lead to the development of therapeutic compounds, possibly for incorporation into medical device materials to inhibit biofilm formation and pathogenesis.