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Structures of the activator of K. pneumonia biofilm formation, MrkH, indicates PilZ domains involved in c-di-GMP and DNA binding.

ABSTRACT: The pathogenesis of Klebsiella pneumonia is linked to the bacteria's ability to form biofilms. Mannose-resistant Klebsiella-like (Mrk) hemagglutinins are critical for K pneumonia biofilm development, and the expression of the genes encoding these proteins is activated by a 3',5'-cyclic diguanylic acid (c-di-GMP)-regulated transcription factor, MrkH. To gain insight into MrkH function, we performed structural and biochemical analyses. Data revealed MrkH to be a monomer with a two-domain architecture consisting of a PilZ C-domain connected to an N domain that unexpectedly also harbors a PilZ-like fold. Comparison of apo- and c-di-GMP-bound MrkH structures reveals a large 138° interdomain rotation that is induced by binding an intercalated c-di-GMP dimer. c-di-GMP interacts with PilZ C-domain motifs 1 and 2 (RxxxR and D/NxSxxG) and a newly described c-di-GMP-binding motif in the MrkH N domain. Strikingly, these c-di-GMP-binding motifs also stabilize an open state conformation in apo MrkH via contacts from the PilZ motif 1 to residues in the C-domain motif 2 and the c-di-GMP-binding N-domain motif. Use of the same regions in apo structure stabilization and c-di-GMP interaction allows distinction between the states. Indeed, domain reorientation by c-di-GMP complexation with MrkH, which leads to a highly compacted structure, suggests a mechanism by which the protein is activated to bind DNA. To our knowledge, MrkH represents the first instance of specific DNA binding mediated by PilZ domains. The MrkH structures also pave the way for the rational design of inhibitors that target K pneumonia biofilm formation.

SUBMITTER: Schumacher MA

PROVIDER: S-EPMC5018759 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Structures of the activator of K. pneumonia biofilm formation, MrkH, indicates PilZ domains involved in c-di-GMP and DNA binding.

Schumacher Maria A MA Zeng Wenjie W

Proceedings of the National Academy of Sciences of the United States of America 20160822 36

The pathogenesis of Klebsiella pneumonia is linked to the bacteria's ability to form biofilms. Mannose-resistant Klebsiella-like (Mrk) hemagglutinins are critical for K pneumonia biofilm development, and the expression of the genes encoding these proteins is activated by a 3',5'-cyclic diguanylic acid (c-di-GMP)-regulated transcription factor, MrkH. To gain insight into MrkH function, we performed structural and biochemical analyses. Data revealed MrkH to be a monomer with a two-domain architect ...[more]

PMID: 27551088

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Project description: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.

Project description:UnlabelledKlebsiella pneumoniae is an important cause of nosocomial infections, primarily through the formation of surface-associated biofilms to promote microbial colonization on host tissues. Expression of type 3 fimbriae by K. pneumoniae facilitates surface adherence, a process strongly activated by the cyclic di-GMP (c-di-GMP)-dependent transcriptional activator MrkH. In this study, we demonstrated the critical importance of MrkH in facilitating K. pneumoniae attachment on a variety of medically relevant materials and demonstrated the mechanism by which bacteria activate expression of type 3 fimbriae to colonize these materials. Sequence analysis revealed a putative MrkH recognition DNA sequence ("MrkH box"; TATCAA) located in the regulatory region of the mrkHI operon. Mutational analysis, electrophoretic mobility shift assay, and quantitative PCR experiments demonstrated that MrkH binds to the cognate DNA sequence to autoregulate mrkHI expression in a c-di-GMP-dependent manner. A half-turn deletion, but not a full-turn deletion, between the MrkH box and the -35 promoter element rendered MrkH ineffective in activating mrkHI expression, implying that a direct interaction between MrkH and RNA polymerase exists. In vivo analyses showed that residues L260, R265, N268, C269, E273, and I275 in the C-terminal domain of the RNA polymerase ? subunit are involved in the positive control of mrkHI expression by MrkH and revealed the regions of MrkH required for DNA binding and transcriptional activation. Taken together, the data suggest a model whereby c-di-GMP-dependent MrkH recruits RNA polymerase to the mrkHI promoter to autoactivate mrkH expression. Increased MrkH production subsequently drives mrkABCDF expression when activated by c-di-GMP, leading to biosynthesis of type 3 fimbriae and biofilm formation.ImportanceBacterial biofilms can cause persistent infections that are refractory to antimicrobial treatments. This study investigated how a commonly encountered hospital-acquired pathogen, Klebsiella pneumoniae, controls the expression of MrkH, the principal regulator of type 3 fimbriae and biofilm formation. We discovered a regulatory circuit whereby MrkH acts as a c-di-GMP-dependent transcriptional activator of both the gene cluster of type 3 fimbriae and the mrkHI operon. In this positive-feedback loop, whereby MrkH activates its own production, K. pneumoniae has evolved a mechanism to ensure rapid MrkH production, expression of type 3 fimbriae, and subsequent biofilm formation under favorable conditions. Deciphering the molecular mechanisms of biofilm formation by bacterial pathogens is important for the development of innovative treatment strategies for biofilm infections.

Project description:UnlabelledThe second messenger cyclic diguanylate (c-di-GMP) is an important regulator of motility in many bacterial species. In Pseudomonas aeruginosa, elevated levels of c-di-GMP promote biofilm formation and repress flagellum-driven swarming motility. The rotation of P. aeruginosa's polar flagellum is controlled by two distinct stator complexes, MotAB, which cannot support swarming motility, and MotCD, which promotes swarming motility. Here we show that when c-di-GMP levels are elevated, swarming motility is repressed by the PilZ domain-containing protein FlgZ and by Pel polysaccharide production. We demonstrate that FlgZ interacts specifically with the motility-promoting stator protein MotC in a c-di-GMP-dependent manner and that a functional green fluorescent protein (GFP)-FlgZ fusion protein shows significantly reduced polar localization in a strain lacking the MotCD stator. Our results establish FlgZ as a c-di-GMP receptor affecting swarming motility by P. aeruginosa and support a model wherein c-di-GMP-bound FlgZ impedes motility via its interaction with the MotCD stator.ImportanceThe regulation of surface-associated motility plays an important role in bacterial surface colonization and biofilm formation. c-di-GMP signaling is a widespread means of controlling bacterial motility, and yet the mechanism whereby this signal controls surface-associated motility in P. aeruginosa remains poorly understood. Here we identify a PilZ domain-containing c-di-GMP effector protein that contributes to c-di-GMP-mediated repression of swarming motility by P. aeruginosa We provide evidence that this effector, FlgZ, impacts swarming motility via its interactions with flagellar stator protein MotC. Thus, we propose a new mechanism for c-di-GMP-mediated regulation of motility for a bacterium with two flagellar stator sets, increasing our understanding of surface-associated behaviors, a key prerequisite to identifying ways to control the formation of biofilm communities.

Dataset Information

Structures of the activator of K. pneumonia biofilm formation, MrkH, indicates PilZ domains involved in c-di-GMP and DNA binding.

Publications

Structures of the activator of K. pneumonia biofilm formation, MrkH, indicates PilZ domains involved in c-di-GMP and DNA binding.

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