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1.
bioRxiv ; 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38405947

RESUMO

In Vibrio species, quorum sensing signaling culminates in the production of a TetR-type master transcription factor collectively called the LuxR/HapR family, which regulates genes required for colonization and infection of host organisms. These proteins possess a solvent accessible putative ligand binding pocket. However, a native ligand has not been identified, and the role of ligand binding in LuxR/HapR function in Vibrionaceae is unknown. To probe the role of the ligand binding pocket, we utilize the small molecule thiophenesulfonamide inhibitor PTSP (3- p henyl-1-( t hiophen-2-yl s ulfonyl)-1 H - p yrazole) that we previously showed targets LuxR/HapR proteins. Amino acid conservation in the ligand binding pocket determines the specificity and efficacy of PTSP inhibition across Vibrio species. Here, we used structure-function analyses to identify PTSP-interacting residues in the ligand binding pocket of SmcR - the Vibrio vulnificus LuxR/HapR homolog - that are required for PTSP inhibition of SmcR activity in vivo . Forward genetic screening combined with X-ray crystallography structural determination of SmcR bound to PTSP identified substitutions at eight residues that were sufficient to reduce or eliminate PTSP-mediated SmcR inhibition. Small-angle X-ray scattering and computational modeling determined that PTSP drives allosteric unfolding at the N-terminal DNA binding domain. We discovered that SmcR is degraded by the ClpAP protease in the presence of PTSP in vivo ; substitution of key PTSP-interacting residues stabilized or increased SmcR levels in the cell. This mechanism of inhibition is observed for all thiophenesulfonamide compounds tested and against other Vibrio species. We conclude that thiophenesulfonamides specifically bind in the ligand binding pocket of LuxR/HapR proteins, promoting protein degradation and thereby suppressing downstream gene expression, implicating ligand binding as a mediator of LuxR/HapR protein stability and function to govern virulence gene expression in Vibrio pathogens. SIGNIFICANCE: LuxR/HapR proteins were discovered in the 1990s as central regulators of quorum sensing gene expression and later discovered to be conserved in all studied Vibrio species. LuxR/HapR homologs regulate a wide range of genes involved in pathogenesis, including but not limited to genes involved in biofilm production and toxin secretion. As archetypal members of the broad class of TetR-type transcription factors, each LuxR/HapR protein has a predicted ligand binding pocket. However, no ligand has been identified for LuxR/HapR proteins that control their function as regulators. Here, we used LuxR/HapR-specific chemical inhibitors to determine that ligand binding drives proteolytic degradation in vivo , the first demonstration of LuxR/HapR function connected to ligand binding for this historical protein family.

2.
PLoS Genet ; 19(12): e1010900, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38064526

RESUMO

Quorum sensing is a mechanism of bacterial cell-cell communication that relies on the production and detection of small molecule autoinducers, which facilitate the synchronous expression of genes involved in group behaviors, such as virulence factor production and biofilm formation. The Pseudomonas aeruginosa quorum sensing network consists of multiple interconnected transcriptional regulators, with the transcription factor, RhlR, acting as one of the main drivers of quorum sensing behaviors. RhlR is a LuxR-type transcription factor that regulates its target genes when bound to its cognate autoinducer, C4-homoserine lactone, which is synthesized by RhlI. RhlR function is also regulated by the metallo-ß-hydrolase enzyme, PqsE. We recently showed that PqsE binds RhlR to alter its affinity for promoter DNA, a new mechanism of quorum-sensing receptor activation. Here, we perform ChIP-seq analyses of RhlR to map the binding of RhlR across the P. aeruginosa genome, and to determine the impact of C4-homoserine lactone and PqsE on RhlR binding to different sites across the P. aeruginosa genome. We identify 40 RhlR binding sites, all but three of which are associated with genes known to be regulated by RhlR. C4-homoserine lactone is required for maximal binding of RhlR to many of its DNA sites. Moreover, C4-homoserine lactone is required for maximal RhlR-dependent transcription activation from all sites, regardless of whether it impacts RhlR binding to DNA. PqsE is required for maximal binding of RhlR to many DNA sites, with similar effects on RhlR-dependent transcription activation from those sites. However, the effects of PqsE on RhlR specificity are distinct from those of C4-homoserine lactone, and PqsE is sufficient for RhlR binding to some DNA sites in the absence of C4-homoserine lactone. Together, C4-homoserine lactone and PqsE are required for RhlR binding at the large majority of its DNA sites. Thus, our work reveals three distinct modes of activation by RhlR: i) when RhlR is unbound by autoinducer but bound by PqsE, ii) when RhlR is bound by autoinducer but not bound by PqsE, and iii) when RhlR is bound by both autoinducer and PqsE, establishing a stepwise mechanism for the progression of the RhlR-RhlI-PqsE quorum sensing pathway in P. aeruginosa.


Assuntos
Percepção de Quorum , Fatores de Transcrição , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Percepção de Quorum/genética , Pseudomonas aeruginosa/metabolismo , Regulação Bacteriana da Expressão Gênica , DNA/metabolismo , Proteínas de Bactérias/metabolismo
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