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Legionella pneumophila zinc metalloprotease is structurally and functionally homologous to Pseudomonas aeruginosa elastase.

ABSTRACT: The sequence of the structural gene encoding the Legionella pneumophila extracellular zinc metalloprotease has been determined and was found to possess a single large open reading frame (ORF) of 1,629 nucleotides (nt). This ORF was preceded by consensus promoter (TTAACT . . . 17 nt . . . TATAAC) and ribosome-binding (TAAGGAG) sequences. The deduced polypeptide contained a putative signal sequence and a total of 543 amino acid residues with a computed molecular size of 60,775 daltons, substantially larger than the observed 38,000 daltons of the native and recombinant proteins. A homology search revealed extensive amino acid identity with Pseudomonas aeruginosa elastase, a protein that is also encoded by an ORF substantially larger than that predicted for the mature size of the protein. The structural identity between the L. pneumophila protease and P. aeruginosa elastase was most pronounced in the regions forming the enzymatic active site of elastase. Amino acid residues constituting the active-site cleft of elastase were greater than 75% conserved. Elastase residues that interact with and mediate proteolysis of substrate peptides were 100% conserved. Competitive inhibitors of elastase and the structurally and functionally related thermolysin (phosphoramidon and a phosphoramidate analog, Z-GlyP(O)Leu-Ala), were shown to be equally potent at inhibiting the proteolytic activity of the L. pneumophila protease. These inhibitor studies along with the amino acid sequence similarities provide strong evidence that the L. pneumophila protease and P. aeruginosa elastase share a similar molecular mechanism of proteolysis.

SUBMITTER: Black WJ

PROVIDER: S-EPMC208904 | biostudies-other | 1990 May

REPOSITORIES: biostudies-other

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Legionella pneumophila zinc metalloprotease is structurally and functionally homologous to Pseudomonas aeruginosa elastase.

Black W J WJ Quinn F D FD Tompkins L S LS

Journal of bacteriology 19900501 5

The sequence of the structural gene encoding the Legionella pneumophila extracellular zinc metalloprotease has been determined and was found to possess a single large open reading frame (ORF) of 1,629 nucleotides (nt). This ORF was preceded by consensus promoter (TTAACT . . . 17 nt . . . TATAAC) and ribosome-binding (TAAGGAG) sequences. The deduced polypeptide contained a putative signal sequence and a total of 543 amino acid residues with a computed molecular size of 60,775 daltons, substantial ...[more]

PMID: 2110146

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Project description:Legionella pneumophila is an environmental bacterium and the causative agent of Legionnaires' disease. Previous genomic studies have shown that recombination accounts for a high proportion (>96%) of diversity within several major disease-associated sequence types (STs) of L. pneumophila. This suggests that recombination represents a potentially important force shaping adaptation and virulence. Despite this, little is known about the biological effects of recombination in L. pneumophila, particularly with regards to homologous recombination (whereby genes are replaced with alternative allelic variants). Using newly available population genomic data, we have disentangled events arising from homologous and non-homologous recombination in six major disease-associated STs of L. pneumophila (subsp. pneumophila), and subsequently performed a detailed characterisation of the dynamics and impact of homologous recombination. We identified genomic "hotspots" of homologous recombination that include regions containing outer membrane proteins, the lipopolysaccharide (LPS) region and Dot/Icm effectors, which provide interesting clues to the selection pressures faced by L. pneumophila. Inference of the origin of the recombined regions showed that isolates have most frequently imported DNA from isolates belonging to their own clade, but also occasionally from other major clades of the same subspecies. This supports the hypothesis that the possibility for horizontal exchange of new adaptations between major clades of the subspecies may have been a critical factor in the recent emergence of several clinically important STs from diverse genomic backgrounds. However, acquisition of recombined regions from another subspecies, L. pneumophila subsp. fraseri, was rarely observed, suggesting the existence of a recombination barrier and/or the possibility of ongoing speciation between the two subspecies. Finally, we suggest that multi-fragment recombination may occur in L. pneumophila, whereby multiple non-contiguous segments that originate from the same molecule of donor DNA are imported into a recipient genome during a single episode of recombination.

Project description:Legionella pneumophila, the causative agent of Legionnaires' disease, is mostly found in man-made water systems and is one of the most closely monitored waterborne pathogens. With the aim of finding natural ways to control waterborne pathogens and thus further reduce the impact of disinfection by-products on human health, some studies have demonstrated the ability of bacteria to kill Legionella through the production of secondary metabolites or antimicrobial compounds. Here, we describe an unexpected growth inhibition of L. pneumophila when exposed to a physically separated strain of Pseudomonas fluorescens, designated as MFE01. Most of the members of the Legionellaceae family are sensitive to the volatile substances emitted by MFE01, unlike other bacteria tested. Using headspace solid-phase microextraction GC-MS strategy, a volatilome comparison revealed that emission of 1-undecene, 2-undecanone, and 2-tridecanone were mainly reduced in a Tn5-transposon mutant unable to inhibit at distance the growth of L. pneumophila strain Lens. We showed that 1-undecene was mainly responsible for the inhibition at distance in vitro, and led to cell lysis in small amounts, as determined by gas chromatography-mass spectrometry (GC-MS). Collectively, our results provide new insights into the mode of action of bacterial volatiles and highlight them as potent anti-Legionella agents to focus research on novel strategies to fight legionellosis. IMPORTANCE Microbial volatile compounds are molecules whose activities are increasingly attracting the attention of researchers. Indeed, they can act as key compounds in long-distance intrakingdom and interkingdom communication, but also as antimicrobials in competition and predation. In fact, most studies to date have focused on their antifungal activities and only a few have reported on their antibacterial properties. Here, we describe that 1-undecene, naturally produced by P. fluorescens, is a volatile with potent activity against bacteria of the genus Legionella. In small amounts, it is capable of inducing cell lysis even when the producing strain is physically separated from the target. This is the first time that such activity is described. This molecule could therefore constitute an efficient compound to counter bacterial pathogens whose treatment may fail, particularly in pulmonary diseases. Indeed, inhalation of these volatiles should be considered as a possible route of therapy in addition to antibiotic treatment.

Project description:BackgroundPseudomonas aeruginosa (PA) is a common cause of respiratory infection and morbidity. Pseudomonas elastase is an important virulence factor regulated by the lasR gene. Whether PA elastase activity is associated with worse clinical outcomes in ICU patients is unknown.Research questionIs there an association between PA elastase activity and worse host outcomes in a cohort of ICU patients?MethodsPA respiratory isolates from 238 unique ICU patients from two tertiary-care centers within the University of Pittsburgh Medical Center health system were prospectively collected and screened for total protease and elastase activity, biofilm production, antimicrobial resistance, and polymicrobial status. The association between pathogen characteristics and 30-day and 90-day mortality was calculated using logistic regression. For subgroup analysis, two patterns of early (≤72 h) and late sample (>72 h) collection from the index ICU admission were distinguished using a finite mixture model. Lung inflammation and injury was evaluated in a mouse model using a PA high elastase vs low elastase producer.ResultsPA elastase activity was common in ICU respiratory isolates representing 75% of samples and was associated with increased 30-day mortality (adjusted OR [95% CI]: 1.39 [1.05-1.83]). Subgroup analysis demonstrated that elastase activity was a risk factor for 30- and 90-day mortality in the early sample group, whereas antimicrobial resistance was a risk factor for 90-day mortality in the late sample group. Whole genome sequencing of high and low elastase producers showed that predicted loss-of-function lasR genotypes were less common among high elastase producers. Mice infected with a high elastase producer showed increased lung bacterial burden and inflammatory profile compared with mice infected with a low elastase producer.InterpretationElastase activity is associated with 30-day ICU mortality. A high elastase producing clinical isolate confers increased lung tissue inflammation compared with a low elastase producer in vivo.

Dataset Information

Legionella pneumophila zinc metalloprotease is structurally and functionally homologous to Pseudomonas aeruginosa elastase.

Publications

Legionella pneumophila zinc metalloprotease is structurally and functionally homologous to Pseudomonas aeruginosa elastase.

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