Biology subjects

Heeb, S.

Publications and source records attributed to Heeb, S..

4 recordsLinked to original sources

bioRxiv2025

SadB acts as a master regulator modulating Pseudomonas aeruginosa pathogenicity

Pseudomonas aeruginosa sadB mutants exhibit a hyper-swarming, biofilm defective phenotype. To acquire insights into the in vivo contribution of sadB to virulence in a mouse soft tissue infection model, we employed bioluminescence imaging and histopathology. Compared with the parent PAO1 strain, the {Delta}sadB mutant was highly attenuated and rapidly cleared from the infection site whereas genetic complementation conferring constitutive expression of sadB resulted in a much more persistent phenotype. Transcriptome analysis of exponential and stationary phase planktonic cells revealed that SadB modulates expression of diverse genes involved in biofilm development, quorum sensing (QS), secondary metabolite production, iron acquisition, virulence, protein secretion and anaerobiosis. In {Delta}sadB, we observed log phase induction of the rhl and pqs QS systems, increased production of siderophores and pyocyanin, differential regulation of genes involved in c-di-GMP signalling and a growth defect under static conditions. Since {Delta}sadB is a hyperswarmer and as swarming requires rhamnolipids, this suggested that deletion of sadB may impact on the timing and level of rhamnolipids produced. In {Delta}sadB, the rhlA and rhlB genes were induced in log rather than stationary phase resulting in overproduction of rhamnolipids. Since they also contribute to biofilm maturation and dispersal and can act as anti-adhesives, we deleted the rhlA in {Delta}sadB and observed that biofilm formation was restored offering mechanistic insight into the biofilm defective phenotype of {Delta}sadB. SadB clearly has a more global role than previously appreciated and acts as a master regulator of diverse genes involved in environmental adaptation, biofilm formation and virulence. IMPORTANCEBiofilms are characterised by their intrinsic tolerance to antibiotics, host immune defences and ability to cause persistent infections. In Pseudomonas aeruginosa, mutation of the surface attachment defect gene, sadB results in cells that are biofilm, defective, hyperswarmers. Here we describe an investigation of the contribution of SadB to virulence. Our study indicates that in P. aeruginosa a sadB deletion mutant is greatly attenuated in an acute mouse skin infection model. We further demonstrate that SadB acts as a pleiotropic regulator of many genes involved in biofilm development, quorum sensing, iron acquisition, protein secretion and anaerobiosis, uncovering a wider role in pathogenesis than formerly recognized, and that it has considerable potential as a novel protein target for antibacterial drug discovery.

microbiology↗

bioRxiv2024

The Quorum Sensing regulated sRNA Lrs1 is involved in the adaptation to low iron in Pseudomonas aeruginosa

Iron is an essential nutrient for microbial growth. The opportunistic pathogen Pseudomonas aeruginosa can survive under diverse conditions, including iron-depleted environments with the aid of small non-coding RNAs (sRNAs). P. aeruginosa also uses three quorum sensing (QS) systems Las, Rhl, and Pqs to coordinate virulence and infection establishment at the population level. This study links the sRNA Lrs1, which is located within the promoter of the Pqs biosynthetic operon pqsABCDE, to iron uptake regulation in the P. aeruginosa strain PAO1-L. Transcriptomics and phenotypic assays indicate that Lrs1 downregulates the production of the siderophore pyochelin but not pyoverdine, and that lrs1 regulation itself is dependent on iron availability. Although Lrs1 has been implicated in a positive feedback loop with the transcriptional regulator LasR in the strain PA14, the present findings indicate that this is not the case in PAO1-L in the tested conditions. Transcription of Lrs1 is dependent on quorum sensing, predominantly on RhlR with an auxiliary effect by PqsE. Furthermore, the Pqs system and phenazine production are modulated by Lrs1 only under iron limitation. This study identifies Lrs1 as a new QS-dependent post-transcriptional regulator of iron uptake and virulence highlighting its importance in environmental adaptation in P. aeruginosa. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/627364v1_ufig1.gif" ALT="Figure 1"> View larger version (12K): org.highwire.dtl.DTLVardef@1585ca5org.highwire.dtl.DTLVardef@2a7d55org.highwire.dtl.DTLVardef@18a712eorg.highwire.dtl.DTLVardef@c84d2_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Polymer-directed inhibition of reversible to irreversible attachment prevents Pseudomonas aeruginosa biofilm formation

Non-toxic, biocompatible materials that inhibit bacterial biofilm formation on implanted medical devices and so prevent infection are urgently required. Weakly amphiphilic acrylate polymers with rigid hydrocarbon pendant groups resist bacterial biofilm formation in vitro and in vivo but the biological mechanism involved is not known. By comparing biofilm formation on polymers with the same acrylate backbone but with different pendant groups, we show that poly(ethylene glycol dicyclopentenyl ether acrylate; pEGdPEA) but not neopentyl glycol propoxylate diacrylate (pNGPDA) inhibited the transition from reversible to irreversible attachment. By using single-cell tracking algorithms and controlled flow microscopy we observed that fewer Pseudomonas aeruginosa PAO1 cells accumulated on pEGdPEA compared with pNGPDA. Bacteria reaching the pEGdPEA surface exhibited shorter residence times and greater asymmetric division with more cells departing from the surface post-cell division, characteristic of reversible attachment. Migrating cells on pEGdPEA deposited fewer exopolysaccharide trails and were unable top adhere strongly. Discrimination between the polymers required type IV pili and flagella. On pEGdPEA, the lack of accumulation of cyclic diguanylate or expression of sadB were consistent with the failure to transit from reversible to irreversible attachment. Constitutive expression of sadB increased surface adhesion sufficient to enable P. aeruginosa to form biofilms in a Mot flagellar stator dependent manner. These findings were extendable to other biofilm resistant acrylates highlighting their unique ability to inhibit reversible to irreversible attachment as a mechanism for preventing biofilm-associated infections. SignificanceBacteria readily attach to surfaces forming biofilms. These are commonly associated with medical device-associated infections and highly refractory to antibiotics. Biocompatible, weakly amphiphilic acrylate polymers with large hydrophobic pendant groups that inhibit biofilm formation and can prevent such infections have been described. However, the biological mechanism involved is not understood. By comparing a biofilm-inhibiting with a biofilm-supporting acrylate, we showed that Pseudomonas aeruginosa PAO1 cells responded differentially to the two polymers and were unable to accumulate and adhere strongly, activate cyclic diguanylate signalling or transit from reversible to irreversible attachment on the inhibitory polymer. Constitutive expression of sadB increased surface adhesion sufficient to enable P. aeruginosa to form biofilms in a flagellar stator dependent manner overcoming the biofilm inhibitory properties of the polymer.

microbiology↗

bioRxiv2021

Disruption of the Pseudomonas aeruginosa Tat system perturbs PQS-dependent quorum sensing and biofilm maturation through loss of the Rieske cytochrome bc1 sub-unit

Extracellular DNA (eDNA) is a major constituent of the extracellular matrix of Pseudomonas aeruginosa biofilms and its release is regulated via the pseudomonas quinolone signal (PQS) dependent quorum sensing (QS). By screening a P. aeruginosa transposon library to identify factors required for DNA release, mutants with insertions in the twin-arginine translocation (Tat) pathway were identified as exhibiting reduced eDNA release, and defective biofilm architecture with enhanced susceptibility to tobramycin. P. aeruginosa tat mutants showed substantial reductions in pyocyanin, rhamnolipid and membrane vesicle (MV) production consistent with perturbation of 2-heptyl-3-hydroxy-4-quinolone (PQS) dependent QS as demonstrated by changes in pqsA expression and 2-alkyl-4-quinolone (AQ) production. Provision of exogenous PQS to the tat mutants did not return pqsA, rhlA or phzA1 expression or pyocyanin production to wild type levels. However, transformation of the tat mutants with the AQ-independent pqs effector pqsE restored phzA1 expression and pyocyanin production. Since mutation or inhibition of Tat prevented PQS-driven auto-induction, we sought to identify the Tat secretion substrate responsible. A pqsA::lux fusion was introduced into each of 34 validated P. aeruginosa Tat substrate deletion mutants. Analysis of each mutant for reduced bioluminescence revealed that the signalling defect was associated with the Rieske iron-sulfur subunit of the cytochrome bc1 complex. In common with the parent strain, a Rieske mutant exhibited defective PQS signalling, AQ production, rhlA expression and eDNA release that could be restored by genetic complementation. Thus, lack of the Rieske sub-unit export is clearly responsible for the Tat-mediated perturbation of PQS-dependent QS, the loss of virulence factor production, biofilm eDNA and the tobramycin tolerance of P. aeruginosa biofilms. Author SummaryPseudomonas aeruginosa is a highly adaptable human pathogen responsible for causing chronic biofilm-associated infections. Biofilms are highly refractory to host defences and antibiotics and thus difficult to eradicate. The biofilm extracellular matrix incorporates extracellular DNA (eDNA). This stabilizes biofilm architecture and helps confer tolerance to antibiotics. Since mechanisms that control eDNA release are not well understood, we screened a P. aeruginosa mutant bank for strains with defects in eDNA release and discovered a role for the twin-arginine translocation (Tat) pathway that exports folded proteins across the cytoplasmic membrane. Perturbation of the Tat pathway resulted in defective biofilms susceptible to antibiotic treatment as a consequence of perturbed pseudomonas quinolone (PQS) signalling. This resulted in the failure to produce or release biofilm components including eDNA, phenazines and rhamnolipids as well as microvesicles. Furthermore, we discovered that perturbation of PQS signalling was a consequence of the inability of tat mutants to translocate the Rieske subunit of the cytochrome bc1 complex involved in electron transfer and energy transduction. Given the importance of PQS signalling and the Tat system to virulence and biofilm maturation in P. aeruginosa, our findings underline the potential of the Tat system as a drug target for novel antimicrobial agents.

microbiology↗