Biology subjects

Mendoza, A. G.

Publications and source records attributed to Mendoza, A. G..

6 recordsLinked to original sources

bioRxiv2026

Proteolytic feedback of quorum sensing promotes infection by Pseudomonas aeruginosa

Quorum sensing (QS) enables bacteria to coordinate collective behaviors in response to population density. However, the mechanisms by which QS circuits are reset or attenuated remain poorly understood. In Pseudomonas aeruginosa, the LuxR-type transcription factor RhlR controls virulence gene expression at high cell density, requiring both its cognate acyl-homoserine lactone signal and the accessory protein PqsE for full activity. Here, we show that RhlR abundance is actively controlled by ATP-dependent proteolysis through a self-limiting negative feedback loop. We identify an N-terminal degron within RhlR that is protected from protease recognition by PqsE binding and is required for degradation by both the Lon and the ClpP1-ClpP2 protease complex. Removal of this degron stabilizes RhlR, advancing the timing of QS-regulated gene expression, enhancing biofilm formation, imposes a competitive fitness cost in vitro, and reduced virulence in an acute mouse lung infection. Mechanistically, RhlR directly activates transcription of clpP2, encoding a subunit of the protease complex required for its own degradation, establishing a self-limiting negative feedback loop. Disruption of RhlR DNA binding stabilizes the receptor through dual effects on reduced clpP2 expression and increased PqsE availability via the loss of RhlR-dependent repression of pqsE. Together, these findings reveal proteolytic turnover as an underappreciated regulatory layer of QS-activation and establish a mechanism for how receptor turnover may facilitate the transition away from high-cell density states.

microbiology↗

bioRxiv2026

PhoPQ is an upstream regulator of quorum sensing in small colony variant subpopulations of Pseudomonas aeruginosa

Pseudomonas aeruginosa is an opportunistic human pathogen that is hospital-endemic, forming biofilms on medical equipment and causing thousands of hospital-acquired infections each year. The success of P. aeruginosa as an opportunistic pathogen is linked to its phenotypic and genotypic adaptability. Relatedly, P. aeruginosa forms a small colony subpopulation in response to stresses like oxygen limitation and antibiotic exposure. Additionally, P. aeruginosa coordinates population-level decisions using a mechanism of cell-cell communication called quorum sensing. P. aeruginosa uses these signaling pathways to control virulence factor production and biofilm formation in the host. We show that certain quorum-sensing mutations promote phenotypic variation; specifically, deletion of lasR and autoinducer modulating mutations in rhlI enhanced small colony formation in a time course-dependent manner. Using transcriptome analyses of isogenic small and large colony variants, we show that small colony formation is driven in part by the PhoPQ two-component signal transduction system in quorum-sensing mutant backgrounds. Specifically, our data show that unphosphorylated PhoP represses rhlR gene expression, and that subsequent de-repression of quorum sensing contributes to the production of virulence factors and the small colony phenotype. In total, these findings provide insight on how mutations evolved by clinical strains might serve as a bet-hedging strategy to promote the formation of a small colony phenotype and alter quorum-sensing signaling within a subpopulation of a community.

microbiology↗

bioRxiv2025

RhlR quorum-sensing receptor ligand sensitivity regulates the differential expression of phenazine genes in Pseudomonas aeruginosa

Bacteria control individualistic and group behaviors using a form of cell-cell communication called quorum sensing. Quorum sensing relies on the production of chemical signals called autoinducers and the subsequent detection of those signals by a cognate receptor. Many Gram-negative bacteria use the LuxR-type family of transcription factor receptors that bind to acyl-homoserine lactone autoinducer signals to regulate their function as DNA-binding proteins. A subclass of this family of transcription factor receptors requires their cognate autoinducer to fold and dimerize to bind DNA to regulate gene expression and, thus, traits associated with quorum sensing, such as biofilm formation and virulence factor production. Here, we use a chemical-genetic approach to determine the structural basis for ligand selection by the quorum-sensing receptor RhlR from Pseudomonas aeruginosa. The native ligand for RhlR is N-butyryl-L-homoserine lactone, and this protein-ligand interaction is important for initiating gene expression in P. aeruginosa. We determine key residues that drive ligand specificity and selectivity of RhlR to define the role of ligand-driven RhlR-dependent gene regulation of quorum-sensing traits, namely the differential expression of the phenazine genes, which encode the enzymes responsible for the synthesis of the redox-sensitive virulence factor pyocyanin, among other phenazines. Furthermore, we provide a chemical-genetic framework for future studies aimed at disrupting the RhlR-ligand interaction to suppress virulence in P. aeruginosa, an important nosocomial pathogen with widespread antimicrobial resistance.

microbiology↗

bioRxiv2025

Pseudomonas aeruginosa uses kinases NahK and RetS to control the motile-biofilm switch

The multidrug-resistant bacterium Pseudomonas aeruginosa (Pa) poses a significant threat to public health. This Gram-negative bacterium establishes pathogenicity through formation of multicellular communities, known as biofilms, that result in significant resistance to antibiotics and host immune systems. In Pa, the motile-to-biofilm transition is regulated through an interconnected signaling network known as the Gac Multikinase Network (Gac-MKN). This network comprises two regulatory branches: the HptB signaling network and GacS/A signaling network. In the Gac-MKN, several histidine kinases converge to regulate the activity of the post-transcriptional regulator protein, RsmA. Although previous studies have assessed the role of individual kinases in this network, the role of each Gac-MKN kinase in regulating RsmA activity has not been quantitatively characterized and compared in side-by-side experiments in the same reference strain, which is presented here. In this study, we show that kinases NahK and RetS are the predominant regulators of the Gac-MKN. Through controlled testing of RsmA-dependent phenotypes, we demonstrate loss of nahK or retS leads to complete inactivation of RsmA, triggering biofilm formation. Our results support previous findings that RetS regulates RsmA through the GacS/A network but present the new finding that NahK is the central kinase involved in HptB phosphorylation; previous studies have attributed HptB phosphorylation to PA1611 and SagS. Our findings demonstrate that NahK signaling controls RsmA activity to rapidly transition between the motile and biofilm states. We anticipate the results of this study will facilitate the use of targeting the Gac-MKN to trigger biofilm dispersal for improved antibiotic treatment.

microbiology↗

bioRxiv2023

The histidine kinase NahK regulates pyocyanin production through the PQS system

Many bacterial histidine kinases work in two-component systems that combine into larger multi-kinase networks. NahK is one of the kinases in the GacS Multi-Kinase Network (MKN), which is the MKN that controls biofilm regulation in the opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa). This network has also been associated with regulating many virulence factors P. aeruginosa secretes to cause disease. However, the individual role of each kinase is unknown. In this study, we identify NahK as a novel regulator of the phenazine pyocyanin (PYO). Deletion of nahK led to a four-fold increase in PYO production, almost exclusively through upregulation of phenazine operon two (phz2). We determined that this upregulation is due to mis-regulation of all P. aeruginosa quorum sensing systems, with a large upregulation of the Pseudomonas quinolone signal (PQS) system and a decrease in production of the acyl-homoserine lactone-producing system, las. In addition, we see differences in expression of quorum sensing inhibitor proteins that align with these changes. Together, this data contributes to understanding how the GacS MKN modulates QS and virulence. ImportancePseudomonas aeruginosa is a Gram-negative bacterium that establishes biofilms as part of its pathogenicity. P. aeruginosa infections are associated with nosocomial infections. As the prevalence of multi-drug resistant P. aeruginosa increases, it is essential to understand underlying virulence molecular mechanisms. Histidine kinase NahK is one of several kinases in P. aeruginosa implicated in biofilm formation and dispersal. Previous work has shown that the nitric oxide sensor, NosP, triggers biofilm dispersal by inhibiting NahK. The data presented here demonstrates that NahK plays additional important roles in the P. aeruginosa lifestyle, including regulating bacterial communication mechanisms such as quorum sensing. These effects have larger implications in infection as they affect toxin production and virulence.

microbiology↗

bioRxiv2019

Co-immunoprecipitation with MYR1 identifies three additional proteins within the Toxoplasma parasitophorous vacuole required for translocation of dense granule effectors into host cells

Toxoplasma gondii is a ubiquitous, intracellular protozoan that extensively modifies infected host cells through secreted effector proteins. Many such effectors must be translocated across the parasitophorous vacuole (PV) in which the parasites replicate, ultimately ending up in the host cytosol or nucleus. This translocation has previously been shown to be dependent on five parasite proteins: MYR1, MYR2, MYR3, ROP17, and ASP5. We report here the identification of several MYR1-interacting and novel PV-localized proteins via affinity purification of MYR1, including TGGT1_211460 (dubbed MYR4), TGGT1_204340 (dubbed GRA54) and TGGT1_270320 (PPM3C). Further, we show that three of the MYR1-interacting proteins, GRA44, GRA45, and MYR4, are essential for the translocation of the Toxoplasma effector protein GRA16, and for the upregulation of human c-Myc and cyclin E1 in infected cells. GRA44 and GRA45 contain ASP5-processing motifs, but like MYR1, processing at these sites appears to be nonessential for their role in protein translocation. These results expand our understanding of the mechanism of effector translocation in Toxoplasma and indicate that the process is highly complex and dependent on at least eight discrete proteins. ImportanceToxoplasma is an extremely successful intracellular parasite and important human pathogen. Upon infection of a new cell, Toxoplasma establishes a replicative vacuole and translocates parasite effectors across this vacuole to function from the host cytosol and nucleus. These effectors play a key role in parasite virulence. The work reported here newly identifies three parasite proteins that are necessary for protein translocation into the host cell. These results significantly increase our knowledge of the molecular players involved in protein translocation in Toxoplasma-infected cells, and provide additional potential drug targets.

microbiology↗