Biology subjects

Dubern, J.-F.

Publications and source records attributed to Dubern, J.-F..

7 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↗

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↗

bioRxiv2020

NirA is an alternative nitrite reductase from Pseudomonas aeruginosa with potential as an anti-virulence target

The opportunistic pathogen Pseudomonas aeruginosa produces an arsenal of virulence factors causing a wide range of diseases in multiple hosts and is difficult to eradicate due to its intrinsic resistance to antibiotics. With the antibacterial pipeline drying up, anti-virulence therapy has become an attractive alternative strategy to the traditional use of antibiotics to treat P. aeruginosa infections. To identify P. aeruginosa genes required for virulence in multiple hosts, a random library of Tn5 mutants in PAO1-L was previously screened in vitro for those showing pleiotropic effects in the production of virulence phenotypes. Using this strategy, we have identified a Tn5 mutant with an insertion in PA4130 showing reduced levels in a number of virulence traits in vitro. Construction of an isogenic mutant in this gene presented similar results as those from the Tn5 mutant. Furthermore, the PA4130 isogenic mutant showed substantial attenuation in disease models of Drosophila melanogaster, Caenorhabditis elegans as well as decreased toxicity in human cell lines. This mutant also presented an 80% increased survival in murine acute and agar-bead lung infection models. PA4130 codes for a protein with homology to nitrite and sulphite reductases. Overexpression of PA4130 in the presence of the siroheme synthase CysG enabled its purification as a soluble protein. Methyl viologen oxidation assays with purified PA4130 showed that this protein is a nitrite reductase operating in a siroheme and 4Fe-4S dependant manner. The preference for nitrite and the production of ammonium revealed that PA4130 is an ammonia:ferredoxin nitrite reductase and hence was named as NirA.

microbiology↗

bioRxiv2020

AbaM Regulates Quorum Sensing, Biofilm Formation and Virulence in Acinetobacter baumannii

Acinetobacter baumannii possesses a single divergent luxR/luxI-type quorum sensing (QS) locus named abaR/abaI. This locus also contains a third gene located between abaR and abaI which we term abaM that codes for an uncharacterized member of the RsaM protein family known to regulate N-acylhomoserine lactone (AHL) dependent QS in other {beta}- and {gamma}-proteobacteria. Here we show that disruption of abaM via a T26 insertion in A. baumannii strain AB5075 resulted in increased production of N-(3-hydroxydodecanoyl)-L-homoserine lactone (OHC12) and enhanced surface motility and biofilm formation. In contrast to the wild type and abaI::T26 mutant, the virulence of the abaM::T26 mutant was completely attenuated in a Galleria mellonella infection model. Transcriptomic analysis of the abaM::T26 mutant revealed that abaM differentially regulates at least 76 genes including the csu pilus operon and the acinetin 505 lipopeptide biosynthetic operon, that are involved in surface adherence, biofilm formation and virulence. A comparison of the wild type, abaM::T26 and abaI::T26 transcriptomes, indicates that abaM regulates ~21% of the QS regulon including the csu operon. Moreover, the QS genes (abaI/abaR) were among the most upregulated in the abaM::T26 mutant. A. baumannii lux-based abaM reporter gene fusions revealed that abaM expression is positively regulated by QS but negatively auto-regulated. Overall, the data presented in this work demonstrates that abaM plays a central role in regulating A. baumannii QS, virulence, surface motility and biofilm formation. IMPORTANCEAcinetobacter baumanni is a multi-antibiotic resistant pathogen of global healthcare importance. Understanding Acinetobacter virulence gene regulation could aid the development of novel anti-infective strategies. In A. baumannii, the abaR and abaI genes that code for the receptor and synthase components of an N-acylhomoserine (AHL) lactone-dependent quorum sensing system (QS) are separated by abaM. Here we show that although mutation of abaM increased AHL production, surface motility and biofilm development, it resulted in the attenuation of virulence. abaM was found to control both QS-dependent and QS-independent genes. The significance of this work lies in the identification of abaM, an RsaM ortholog known to control virulence in plant pathogens, as a modulator of virulence in a human pathogen.

microbiology↗

bioRxiv2020

Topographical biomaterials instruct bacterial surface attachment and the in vivo host-pathogen response

Bio-instructive materials that prevent bacterial biofilm formation and drive an appropriate host immune response have the potential to significantly reduce the burden of medical device-associated infections. Since bacterial surface attachment is known to be sensitive to surface topography, we experimentally survey 2,176 combinatorially generated shapes using an unbiased high-throughput micro topographical screen on polystyrene. This identifies topographies that reduce colonization in vitro by up to 15-fold compared with a flat surface for both motile and non-motile bacterial pathogens. Equivalent reductions are achieved on polyurethane, a polymer commonly used in medical devices. Using machine learning methods, a set of design rules based on generalisable descriptors is established for predicting bacteria-resistant micro topographies. In a murine foreign body infection model, anti-attachment topographies are shown to be refractory to P. aeruginosa and to recruit a productive host response, highlighting the potential of simple topographical patterning of non-eluting implants for preventing medical device associated infections.

microbiology↗

bioRxiv2020

Inkjet based 3D Printing of bespoke medical devices that resist bacterial biofilm formation

We demonstrate the formulation of advanced functional 3D printing inks that prevent the formation of bacterial biofilms in vivo. Starting from polymer libraries, we show that a biofilm resistant object can be 3D printed with the potential for shape and cell instructive function to be selected independently. When tested in vivo, the candidate materials not only resisted bacterial attachment but drove the recruitment of host defences in order to clear infection. To exemplify our approach, we manufacture a finger prosthetic and demonstrate that it resists biofilm formation – a cell instructive function that can prevent the development of infection during surgical implantation. More widely, cell instructive behaviours can be ‘dialled up’ from available libraries and may include in the future such diverse functions as the modulation of immune response and the direction of stem cell fate.Competing Interest StatementThe authors have declared no competing interest.View Full Text

bioengineering↗

bioRxiv2019

Simultaneous tracking of cell motility in liquid and at the solid-liquid interface

1.To better understand key behaviors of living cells, such as bacterial biofilm formation, they must be observed above surfaces and at the interface between the surface and liquid medium. We have established a methodology for label-free imaging and tracking of individual cells simultaneously at both the solid-liquid interface and within the bulk, utilizing imaging modes of digital holographic microscopy (DHM) in 3D, differential interference contrast (DIC) and total internal reflectance microscopy (TIRM) in 2D as well as analysis protocols using a bespoke software package. We illustrate the power of this method by making detailed single cell measurements of Pseudomonas aeruginosa in the first minutes of their interaction with a glass surface, focusing on the role of the flagella stators, motAB and motCD. Using this new method we have determined their relative contributions to bulk and near surface motion for populations of cells at the single cell level.

biophysics↗