Dataset Information

Quorum sensing-dependent post-secretional activation of extracellular proteases in Pseudomonas aeruginosa.

ABSTRACT: Pseudomonas aeruginosa secretes multiple proteases that are implicated in its pathogenesis, and most of them are regulated by quorum sensing (QS). In this study, we found that the activities of three major extracellular proteases, protease IV (PIV), elastase A (LasA), and elastase B (LasB), are reduced considerably when expressed in a QS mutant (MW1). PIV and LasA expressed in MW1 exhibited little activity, even when purified, and their activities were inhibited by noncleavage or binding of their propeptides. LasB was activated by a QS-dependent factor, indicating that, unlike what has been proposed previously, LasB is not autoactivated. When LasB was relieved from inhibition, it activated PIV, which then sequentially processed pro-LasA to mature LasA. When activated, LasB was not inhibited by exogenous addition of its propeptide, but LasA and PIV were inhibited by their propeptides, even after prior activation. These differences may be explained by the fact that LasB can degrade its own propeptide but PIV and LasA cannot. We also found that, although PIV is the preferred LasA-activating factor, LasB can also partially activate LasA. Overall, LasB, PIV, and LasA were activated postsecretionally in a cascading manner in which the initial activation of LasB was controlled tightly by QS at the protein level in addition to the well-known transcriptional control of these proteases by QS. Interestingly, human elastase also activated LasA, indicating that the activation cascade is triggered by host factors during infection. In summary, a QS-induced proteolytic cascade activates secreted proteases from P. aeruginosa.

SUBMITTER: Li XH

PROVIDER: S-EPMC6926461 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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Quorum sensing-dependent post-secretional activation of extracellular proteases in <i>Pseudomonas aeruginosa</i>.

Li Xi-Hui XH Lee Joon-Hee JH

The Journal of biological chemistry 20191114 51

<i>Pseudomonas aeruginosa</i> secretes multiple proteases that are implicated in its pathogenesis, and most of them are regulated by quorum sensing (QS). In this study, we found that the activities of three major extracellular proteases, protease IV (PIV), elastase A (LasA), and elastase B (LasB), are reduced considerably when expressed in a QS mutant (MW1). PIV and LasA expressed in MW1 exhibited little activity, even when purified, and their activities were inhibited by noncleavage or binding ...[more]

PMID: 31727738

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Project description:The bacterium Pseudomonas aeruginosa has emerged as a central threat in health care settings and can cause a large variety of infections. It expresses an arsenal of virulence factors and a diversity of survival functions, many of which are finely and tightly regulated by an intricate circuitry of three quorum sensing (QS) systems. The las system is considered at the top of the QS hierarchy and activates the rhl and pqs systems. It is composed of the LasR transcriptional regulator and the LasI autoinducer synthase, which produces 3-oxo-C12-homoserine lactone (3-oxo-C12-HSL), the ligand of LasR. RhlR is the transcriptional regulator for the rhl system and is associated with RhlI, which produces its cognate autoinducer C4-HSL. The third QS system is composed of the pqsABCDE operon and the MvfR (PqsR) regulator. PqsABCD synthetize 4-hydroxy-2-alkylquinolines (HAQs), which include ligands activating MvfR. PqsE is not required for HAQ production and instead is associated with the expression of genes controlled by the rhl system. While RhlR is often considered the main regulator of rhlI, we confirmed that LasR is in fact the principal regulator of C4-HSL production and that RhlR regulates rhlI and production of C4-HSL essentially only in the absence of LasR by using liquid chromatography-mass spectrometry quantifications and gene expression reporters. Investigating the expression of RhlR targets also clarified that activation of RhlR-dependent QS relies on PqsE, especially when LasR is not functional. This work positions RhlR as the key QS regulator and points to PqsE as an essential effector for full activation of this regulation.IMPORTANCE Pseudomonas aeruginosa is a versatile bacterium found in various environments. It can cause severe infections in immunocompromised patients and naturally resists many antibiotics. The World Health Organization listed it among the top priority pathogens for research and development of new antimicrobial compounds. Quorum sensing (QS) is a cell-cell communication mechanism, which is important for P. aeruginosa adaptation and pathogenesis. Here, we validate the central role of the PqsE protein in QS particularly by its impact on the regulator RhlR. This study challenges the traditional dogmas of QS regulation in P. aeruginosa and ties loose ends in our understanding of the traditional QS circuit by confirming RhlR to be the main QS regulator in P. aeruginosa PqsE could represent an ideal target for the development of new control methods against the virulence of P. aeruginosa This is especially important when considering that LasR-defective mutants frequently arise, e.g., in chronic infections.

Project description:N-Acyl homoserine lactone (AHL) quorum sensing (QS) controls expression of over 200 genes in Pseudomonas aeruginosa. There are two AHL regulatory systems: the LasR-LasI circuit and the RhlR-RhlI system. We mapped transcription termination sites affected by AHL QS in P. aeruginosa, and in doing so we identified AHL-regulated small RNAs (sRNAs). Of interest, we noted that one particular sRNA was located within the rhlI locus. We found that rhlI, which encodes the enzyme that produces the AHL N-butanoyl-homoserine lactone (C4-HSL), is controlled by a 5' untranslated region (UTR)-derived sRNA we name RhlS. We also identified an antisense RNA encoded opposite the beginning of the rhlI open reading frame, which we name asRhlS. RhlS accumulates as wild-type cells enter stationary phase and is required for the production of normal levels of C4-HSL through activation of rhlI translation. RhlS also directly posttranscriptionally regulates at least one other unlinked gene, fpvA. The asRhlS appears to be expressed at maximal levels during logarithmic growth, and we suggest RhlS may act antagonistically to the asRhlS to regulate rhlI translation. The rhlI-encoded sRNAs represent a novel aspect of RNA-mediated tuning of P. aeruginosa QS.IMPORTANCE The opportunistic human pathogen Pseudomonas aeruginosa possesses multiple quorum sensing systems that regulate and coordinate production of virulence factors and adaptation to different environments. Despite extensive research, the regulatory elements that play a role in this complex network are still not fully understood. By using several RNA sequencing techniques, we were able to identify a small regulatory RNA we named RhlS. RhlS increases translation of RhlI, a key enzyme in the quorum sensing pathway, and represses the fpvA mRNA encoding one of the siderophore pyoverdine receptors. Our results highlight a new regulatory layer of P. aeruginosa quorum sensing and contribute to the growing understanding of the role regulatory RNAs play in bacterial physiology.

Dataset Information

Quorum sensing-dependent post-secretional activation of extracellular proteases in Pseudomonas aeruginosa.

Publications

Quorum sensing-dependent post-secretional activation of extracellular proteases in <i>Pseudomonas aeruginosa</i>.

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