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Structural and functional characterization of Pseudomonas aeruginosa CupB chaperones.

ABSTRACT: Pseudomonas aeruginosa, an important human pathogen, is estimated to be responsible for ?10% of nosocomial infections worldwide. The pathogenesis of P. aeruginosa starts from its colonization in the damaged tissue or medical devices (e.g. catheters, prothesis and implanted heart valve etc.) facilitated by several extracellular adhesive factors including fimbrial pili. Several clusters containing fimbrial genes have been previously identified on the P. aeruginosa chromosome and named cup[1]. The assembly of the CupB pili is thought to be coordinated by two chaperones, CupB2 and CupB4. However, due to the lack of structural and biochemical data, their chaperone activities remain speculative. In this study, we report the 2.5 Å crystal structure of P. aeruginosa CupB2. Based on the structure, we further tested the binding specificity of CupB2 and CupB4 towards CupB1 (the presumed major pilus subunit) and CupB6 (the putative adhesin) using limited trypsin digestion and strep-tactin pull-down assay. The structural and biochemical data suggest that CupB2 and CupB4 might play different, but not redundant, roles in CupB secretion. CupB2 is likely to be the chaperone of CupB1, and CupB4 could be the chaperone of CupB4:CupB5:CupB6, in which the interaction of CupB4 and CupB6 might be mediated via CupB5.

SUBMITTER: Cai X

PROVIDER: S-EPMC3031594 | biostudies-literature | 2011 Jan

REPOSITORIES: biostudies-literature

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Structural and functional characterization of Pseudomonas aeruginosa CupB chaperones.

Cai Xun X Wang Rui R Filloux Alain A Waksman Gabriel G Meng Guoyu G

PloS one 20110131 1

Pseudomonas aeruginosa, an important human pathogen, is estimated to be responsible for ∼10% of nosocomial infections worldwide. The pathogenesis of P. aeruginosa starts from its colonization in the damaged tissue or medical devices (e.g. catheters, prothesis and implanted heart valve etc.) facilitated by several extracellular adhesive factors including fimbrial pili. Several clusters containing fimbrial genes have been previously identified on the P. aeruginosa chromosome and named cup[1]. The ...[more]

PMID: 21304995

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Project description:Hibernation-promoting factor (HPF) is a ribosomal accessory protein that inactivates ribosomes during bacterial starvation. In Pseudomonas aeruginosa, HPF protects ribosome integrity while the cells are dormant. The sequence of HPF has diverged among bacteria but contains conserved charged amino acids in its two alpha helices that interact with the rRNA. Here, we characterized the function of HPF in P. aeruginosa by performing mutagenesis of the conserved residues and then assaying mutant HPF alleles for their ability to protect ribosome integrity of starved P. aeruginosa cells. The results show that HPF functionally tolerates point mutations in charged residues and in the conserved Y71 residue as well as a C-terminal truncation. Double and triple mutations of charged residues in helix 1 in combination with a Y71F substitution reduce HPF activity. Screening for single point mutations that caused impaired HPF activity identified additional substitutions in the two HPF alpha helices. However, alanine substitutions in equivalent positions restored HPF activity, indicating that HPF is tolerant to mutations that do not disrupt the protein structure. Surprisingly, heterologous HPFs from Gram-positive bacteria that have long C-terminal domains functionally complement the P. aeruginosa Δhpf mutant, suggesting that HPF may play a similar role in ribosome protection in other bacterial species. Collectively, the results show that HPF has diverged among bacteria and is tolerant to most single amino acid substitutions. The Y71 residue in combination with helix 1 is important for the functional role of HPF in ribosome protection during bacterial starvation and resuscitation of the bacteria from dormancy.IMPORTANCE In most environments, bacteria experience conditions where nutrients may be readily abundant or where nutrients are limited. Under nutrient limitation conditions, even non-spore-forming bacteria may enter a dormant state. Dormancy is accompanied by a variety of cellular physiological changes that are required for the cells to remain viable during dormancy and to resuscitate when nutrients become available. Among the physiological changes that occur in dormant bacteria is the inactivation and preservation of ribosomes by the dormancy protein, hibernation-promoting factor (HPF). In this study, we characterized the activity of HPF of Pseudomonas aeruginosa, an opportunistic pathogen that causes persistent infections, and analyzed the role of HPF in ribosome protection and bacterial survival during dormancy.

Dataset Information

Structural and functional characterization of Pseudomonas aeruginosa CupB chaperones.

Publications

Structural and functional characterization of Pseudomonas aeruginosa CupB chaperones.

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