Biology subjects

Parsek, M. R.

Publications and source records attributed to Parsek, M. R..

7 recordsLinked to original sources

bioRxiv2026

Analysis of the DNA-binding domain of the Pseudomonas aeruginosa quorum sensing transcription factor LasR

Many bacteria engage in quorum sensing (QS), a cell-cell communication system used to coordinate group behaviors. In one type of QS, acyl-homoserine lactone signals generated by LuxI homologs bind to LuxR homolog transcription factors, usually resulting in gene activation. The genome of Pseudomonas aeruginosa encodes three such LuxR homologs: LasR, RhlR, and QscR. Of these, LasR regulates the most genes, including that encoding RhlR. There is strong evidence that, during chronic infections, lasR and other genes encoding LuxR-type regulators are under strong selective pressure for mutations that both inactivate and modulate their function. Thus, we wondered if some mutations in the lasR gene might result in a protein with affinity for promoters usually regulated by the other LuxR homologs; to do so, we investigated the DNA-binding domain (DBD) of LasR through alanine substitution. As expected, we found that most alanine substitutions across the LasR DBD led to loss of function, as did previously identified clinical LasR DBD variants. Additionally, some alanine mutants were indistinguishable from the wild type. We describe a handful of variant LasR polypeptides that unexpectedly exhibit enhanced regulation on a RhlR-regulated gene, which conferred a fitness defect when competed against the wild type. Most other LasR variants had a competitive advantage. Our results suggest a pathway for expansion of the regulon of LuxR-homolog transcription factors, but also that such mutations may be disfavored due to the incurred metabolic burden.

microbiology↗

bioRxiv2026

A metabolic checkpoint coordinates bacterial cell envelope biosynthesis and c-di-GMP signaling

The bacterial second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) drives the transition from motile to biofilm lifestyles, yet the mechanisms by which bacteria couple cell envelope stress to c-di-GMP signaling remain poorly understood. Here, we show that sub-inhibitory concentrations of antibiotics targeting early cytoplasmic steps of peptidoglycan (PG) biosynthesis, but not inhibitors of PG polymerization, membrane integrity, or other intracellular processes, specifically elevate intracellular c-di-GMP levels in Pseudomonas aeruginosa. Using live-cell imaging and in vitro enzymatic assays, we demonstrate that this elevation results from reduced phosphodiesterase (PDE) activity rather than increased diguanylate cyclase (DGC) activity, with multiple PDEs contributing to the response. A screen of the complete P. aeruginosa DGC/PDE mutant library identified DipA, BifA, RocR, and RmcA as the primary PDEs mediating this effect. Strikingly, we find that acetyl-CoA, a central metabolite consumed during cell envelope precursor biosynthesis, directly inhibits PDE activity by competing for the conserved EAL domain active site, as supported by biochemical assays with purified RocR and molecular docking analysis. Because EAL domain residues that contact acetyl-CoA are broadly conserved across bacterial species, this mechanism may represent a widespread strategy for sensing metabolic perturbations of cell envelope synthesis. Together, these findings reveal that P. aeruginosa monitors the metabolic status of cell envelope biogenesis through acetyl-CoA-mediated allosteric inhibition of c-di-GMP PDEs, linking envelope biosynthetic flux to adaptive biofilm formation.

microbiology↗

bioRxiv2025

Extracellular matrix chemistry tunes bacterial biofilm metabolism and optimizes fitness

Chemically complex extracellular matrices define cellular microenvironments and shape cell behavior. We hypothesized a composition-properties-function relationship in these natural living materials, where interactions among matrix components govern material properties and cellular physiology. Using Pseudomonas aeruginosa biofilms as a model system, we show that electrostatic interactions between the cationic polysaccharide Pel and extracellular DNA (eDNA) regulate retention of pyocyanin (PYO), a redox-active metabolite that supports anaerobic metabolism via extracellular electron transfer (EET). Biofilm-mimetic hydrogels and natural biofilms revealed that altering Pels charge via pH adjustment or chemical acetylation, or tuning the Pel:eDNA ratio, predictably modulates PYO retention and EET efficiency. Functionally, a lower Pel:eDNA ratio enhances metabolism under oxygen limitation, whereas a higher ratio promotes survival under antibiotic stress. These findings highlight how matrix chemistry encodes tunable material properties that confer biofilm fitness advantages and establish a materials-based framework for understanding extracellular matrices in multicellular communities.

microbiology↗

bioRxiv2020

PelX is a UDP-N-acetylglucosamine C4-epimerase involved in Pel polysaccharide-dependent biofilm formation

Pel is an N-acetylgalactosamine rich polysaccharide that contributes to the structure and function of Pseudomonas aeruginosa biofilms. The pelABCDEFG operon is highly conserved among diverse bacterial species, and thus Pel may be a widespread biofilm determinant. Previous annotation of pel gene clusters led us to identify an additional gene, pelX, that is found adjacent to pelABCDEFG in over 100 different bacterial species. The pelX gene is predicted to encode a member of the short-chain dehydrogenase/reductase (SDR) superfamily of enzymes, but its potential role in Pel-dependent biofilm formation is unknown. Herein, we have used Pseudomonas protegens Pf-5 as a model to understand PelX function as P. aeruginosa lacks a pelX homologue in its pel gene cluster. We find that P. protegens forms Pel-dependent biofilms, however, despite expression of pelX under these conditions, biofilm formation was unaffected in a {Delta}pelX strain. This observation led to our identification of the pelX paralogue, PFL_5533, which we designate pgnE, that appears to be functionally redundant to pelX. In line with this, a {Delta}pelX {Delta}pgnE double mutant was substantially impaired in its ability to form Pel-dependent biofilms. To understand the molecular basis for this observation, we determined the structure of PelX to 2.1[A] resolution. The structure revealed that PelX resembles UDP-N-acetylglucosamine (UDP-GlcNAc) C4-epimerases and, using 1H NMR analysis, we show that PelX catalyzes the epimerization between UDP-GlcNAc and UDP-GalNAc. Taken together, our results demonstrate that Pel-dependent biofilm formation requires a UDP-GlcNAc C4-epimerase that generates the UDP-GalNAc precursors required by the Pel synthase machinery for polymer production.

microbiology↗

bioRxiv2019

Phenotypic differences in reversible attachment behavior reveal distinct P. aeruginosa surface colonization strategies

Despite possessing the machinery to sense, adhere to, and proliferate on surfaces, it is commonly observed that bacteria initially have a difficult time attaching to a surface. Before forming a bacterial biofilm, planktonic bacteria exhibit a random period of transient surface attachment known as \"reversible attachment\" which is poorly understood. Using community tracking methods at single-cell resolution, we examine how reversible attachment progresses during initial stages of surface sensing. Pseudomonas aeruginosa strains PAO1 and PA14, which exhibit similar exponential trends of surface cell population increase, show unanticipated differences when the behavior of each cell was considered at the full lineage level and interpreted using the unifying quantitative framework of an exactly solvable stochastic model. Reversible attachment comprises two regimes of behavior, processive and nonprocessive, corresponding to whether cells of the lineage stay on the surface long enough to divide, or not, before detaching. Stark differences between PAO1 and PA14 in the processive regime of reversible attachment suggest the existence of two complementary surface colonization strategies, which are roughly analogous to \"immediate-\" vs \"deferred-gratification\" in a prototypical cognitive-affective processing system. PAO1 lineages commit relatively quickly to a surface compared to PA14 lineages. PA14 lineages allow detaching cells to retain memory of the surface so that they are primed for improved subsequent surface attachment. In fact, it is possible to identify motility suppression events in PA14 lineages in the process of surface commitment. We hypothesize that these contrasting strategies are rooted in downstream differences between Wsp-based and Pil-Chp-based surface sensing systems.

microbiology↗

bioRxiv2019

Heterogeneity in surface sensing produces a division of labor in Pseudomonas aeruginosa populations

The second messenger signaling molecule cyclic diguanylate monophosphate (c-di-GMP) drives the transition from planktonic to biofilm growth in many bacterial species. Pseudomonas aeruginosa has two surface sensing systems that produce c-di-GMP in response to surface adherence. The current thinking in the field is that once cells attach to a surface, they uniformly respond with elevated c-di-GMP. Here, we describe how the Wsp system generates heterogeneity in surface sensing, resulting in two physiologically distinct subpopulations of cells. One subpopulation has elevated c-di-GMP and produces biofilm matrix, serving as the founders of initial microcolonies. The other subpopulation has low c-di-GMP and engages in surface motility, allowing for exploration of the surface. We also show that this heterogeneity strongly correlates to surface behavior for descendent cells. Together, our results suggest that after surface attachment, P. aeruginosa engages in a division of labor that persists across generations, accelerating early biofilm formation and surface exploration.

microbiology↗

bioRxiv2017

Microbial glycoside hydrolases as antibiofilm agents with cross-kingdom activity

Galactosaminogalactan and Pel are cationic heteropolysaccharides produced by the opportunistic pathogens, Aspergillus fumigatus and Pseudomonas aeruginosa, respectively. These exopolysaccharides both contain 1,4-linked N-acetyl-D-galactosamine and play an important role in biofilm formation by these organisms. Proteins containing glycoside hydrolase domains have recently been identified within the biosynthetic pathway of each exopolysaccharide. Recombinant hydrolase domains from these proteins (Sph3h from A. fumigatus and PelAh from P. aeruginosa) were found to degrade their respective polysaccharides in vitro. We therefore hypothesized that these glycoside hydrolases could exhibit anti-biofilm activity, and further, given the chemical similarity between galactosaminogalactan and Pel, that they might display cross-species activity. Treatment of A. fumigatus with Sph3h disrupted A. fumigatus biofilms with an EC50 of 0.4 nM. PelAh treatment also disrupted pre-formed A. fumigatus biofilms with EC50 values similar to those obtained for Sph3h. In contrast, Sph3h was unable to disrupt P. aeruginosa Pel-based biofilms, despite being able to bind to the exopolysaccharide. Treatment of A. fumigatus hyphae with either Sph3h or PelAh significantly enhanced the activity of the antifungals posaconazole, amphotericin B and caspofungin, likely through increasing antifungal penetration of hyphae. Both enzymes were non-cytotoxic and protected A549 pulmonary epithelial cells from A. fumigatus-induced cell damage for up to 24 hours. Intratracheal administration of Sph3h was well tolerated, and reduced pulmonary fungal burden in a neutropenic mouse model of invasive aspergillosis. These findings suggest that glycoside hydrolases can exhibit activity against diverse microorganisms and may be useful as therapeutic agents by degrading biofilms and attenuating virulence.\n\nSignificanceThe production of biofilms is an important strategy used by both bacteria and fungi to colonize surfaces and to enhance resistance to killing by immune cells and antimicrobial agents. We demonstrate that glycoside hydrolases derived from the opportunistic fungus Aspergillus fumigatus and Gram-negative bacterium Pseudomonas aeruginosa can be exploited to disrupt pre-formed fungal biofilms and reduce virulence. Additionally, these glycoside hydrolases can be utilized to potentiate antifungal drugs by increasing their hyphal penetration, to protect human cells from fungal-induced injury and to attenuate virulence of A. fumigatus in a mouse model of invasive aspergillosis. The findings of this study identify recombinant microbial glycoside hydrolases as promising therapeutics with the potential for anti-biofilm activity against pathogens across different taxonomic kingdoms.

microbiology↗