Membrane fluidity homeostasis is required for tobramycin-enhanced biofilm in Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic pathogen, which causes chronic infections, especially in cystic fibrosis (CF) patients where it colonizes the lungs via the build-up of biofilms. Tobramycin, an aminoglycoside, is often used to treat P. aeruginosa infections in CF patients. Tobramycin at sub-minimal inhibitory concentrations enhances both biofilm biomass and thickness in vitro; however, the mechanism(s) involved are still unknown. Herein, we show that tobramycin increases the expression and activity of SigX, an extracytoplasmic sigma factor known to be involved in the biosynthesis of membrane lipids and membrane fluidity homeostasis. The biofilm enhancement by tobramycin is not observed in a sigX mutant, and the sigX mutant displays increased membrane stiffness. Remarkably, the addition of polysorbate 80 increases membrane fluidity of sigX-mutant cells in biofilm, restoring the tobramycin-enhanced biofilm formation. Our results suggest the involvement of membrane fluidity homeostasis in biofilm development upon tobramycin exposure.IMPORTANCEPrevious studies have shown that sub-lethal concentrations of tobramycin led to an increase biofilm formation in the case of infections with the opportunistic pathogen Pseudomonas aeruginosa. We show that the mechanism involved in this phenotype relies on the cell envelope stress response, triggered by the extracytoplasmic sigma factor SigX. This phenotype was abolished in a sigX-mutant strain. Remarkably, we show that increasing the membrane fluidity of the mutant strain is sufficient to restore the effect of tobramycin. Altogether, our data suggest the involvement of membrane fluidity homeostasis in biofilm development upon tobramycin exposure.

The Temperature-Regulation of Pseudomonas aeruginosa cmaX-cfrX-cmpX Operon Reveals an Intriguing Molecular Network Involving the Sigma Factors AlgU and SigX.

Pseudomonas aeruginosa is a highly adaptable Gram-negative opportunistic pathogen, notably due to its large number of transcription regulators. The extracytoplasmic sigma factor (ECFσ) AlgU, responsible for alginate biosynthesis, is also involved in responses to cell wall stress and heat shock via the RpoH alternative σ factor. The SigX ECFσ emerged as a major regulator involved in the envelope stress response via membrane remodeling, virulence and biofilm formation. However, their functional interactions to coordinate the envelope homeostasis in response to environmental variations remain to be determined. The regulation of the putative cmaX-cfrX-cmpX operon located directly upstream sigX was investigated by applying sudden temperature shifts from 37°C. We identified a SigX- and an AlgU- dependent promoter region upstream of cfrX and cmaX, respectively. We show that cmaX expression is increased upon heat shock through an AlgU-dependent but RpoH independent mechanism. In addition, the ECFσ SigX is activated in response to valinomycin, an agent altering the membrane structure, and up-regulates cfrX-cmpX transcription in response to cold shock. Altogether, these data provide new insights into the regulation exerted by SigX and networks that are involved in maintaining envelope homeostasis.

Extracellular DNA release, quorum sensing, and PrrF1/F2 small RNAs are key players in Pseudomonas aeruginosa tobramycin-enhanced biofilm formation.

Biofilms are structured microbial communities that are the leading cause of numerous chronic infections which are difficult to eradicate. Within the lungs of individuals with cystic fibrosis (CF), Pseudomonas aeruginosa causes persistent biofilm infection that is commonly treated with aminoglycoside antibiotics such as tobramycin. However, sublethal concentrations of this aminoglycoside were previously shown to increase biofilm formation by P. aeruginosa, but the underlying adaptive mechanisms still remain elusive. Herein, we combined confocal laser scanning microscope analyses, proteomics profiling, gene expression assays and phenotypic studies to unravel P. aeruginosa potential adaptive mechanisms in response to tobramycin exposure during biofilm growth. Under this condition, we show that the modified biofilm architecture is related at least in part to increased extracellular DNA (eDNA) release, most likely as a result of biofilm cell death. Furthermore, the activity of quorum sensing (QS) systems was increased, leading to higher production of QS signaling molecules. We also demonstrate upon tobramycin exposure an increase in expression of the PrrF small regulatory RNAs, as well as expression of iron uptake systems. Remarkably, biofilm biovolumes and eDNA relative abundances in pqs and prrF mutant strains decrease in the presence of tobramycin. Overall, our findings offer experimental evidences for a potential adaptive mechanism linking PrrF sRNAs, QS signaling, biofilm cell death, eDNA release, and tobramycin-enhanced biofilm formation in P. aeruginosa. These specific adaptive mechanisms should be considered to improve treatment strategies against P. aeruginosa biofilm establishment in CF patients' lungs.

In vitro antimicrobial susceptibility of equine clinical isolates from France, 2006-2016.

OBJECTIVES: This study aimed to analyse antimicrobial susceptibility evolution of equine pathogens isolated from clinical samples from 2006-2016. METHODS: A collection of 25 813 bacterial isolates was studied, clustered according to their origins (respiratory tract, cutaneous, genital and other), and analysed for their antimicrobial susceptibility using the disk diffusion method. RESULTS: The most frequently isolated pathogens were group C Streptococci (27.6%), Escherichia coli (20.0%), Staphylococcus aureus (7.8%), Pseudomonas aeruginosa (4.0%), Enterobacter spp. (3.4%), Klebsiella pneumoniae (2.4%), and Rhodococcus equi (1.8%). Of the isolates, 9512 were from respiratory samples (36.8%), 7689 from genital origin (29.8%), and 4083 from cutaneous samples (15.8%). Over the 11-year period, the frequency of multidrug-resistant (MDR) strains fluctuated between 6.4-20.4% for group C Streptococci and 17-37.7% for Klebsiella pneumoniae. From 2006-2009, 24.5-43.0% of Staphylococcus aureus isolates were MDR; after 2009 the level did not exceeded 27.6%. For Escherichia coli and Enterobacter spp., these levels were mostly >30.0% until 2012, but significantly decreased thereafter (22.5-26.3%). CONCLUSIONS: This study is the first large-scale analysis of equine pathogens, by the number of samples and duration of study. The results showed high levels of MDR strains and the need to support veterinary antimicrobial stewardship to encourage proper use of antibiotics.

Extracytoplasmic function sigma factors in Pseudomonas aeruginosa.

The opportunistic pathogen Pseudomonas aeruginosa, like all members of the genus Pseudomonas, has the capacity to thrive in very different environments, ranging from water, plant roots, to animals, including humans to whom it can cause severe infections. This remarkable adaptability is reflected in the number of transcriptional regulators, including sigma factors in this bacterium. Among those, the 19 to 21 extracytoplasmic sigma factors (ECFσ) are endowed with different regulons and functions, including the iron starvation σ (PvdS, FpvI, HasI, FecI, FecI2 and others), the cell wall stress ECFσ AlgU, SigX and SbrI, and the unorthodox σVreI involved in the expression of virulence. Recently published data show that these ECFσ have separate regulons although presenting some cross-talk. We will present evidence that these different ECFσ are involved in the expression of different phenotypes, ranging from cell-wall stress response, production of extracellular polysaccharides, formation of biofilms, to iron acquisition.

The absence of SigX results in impaired carbon metabolism and membrane fluidity in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, SigX is an extra-cytoplasmic function σ factor that belongs to the cell wall stress response network. In previous studies, we made the puzzling observation that sigX mutant growth was severely affected in rich lysogeny broth (LB) but not in minimal medium. Here, through comparative transcriptomic and proteomic analysis, we show that the absence of SigX results in dysregulation of genes, whose products are mainly involved in transport, carbon and energy metabolisms. Production of most of these genes is controlled by carbon catabolite repression (CCR), a key regulatory system than ensures preferential carbon source uptake and utilization, substrate prioritization and metabolism. The strong CCR response elicited in LB was lowered in a sigX mutant, suggesting altered nutrient uptake. Since the absence of SigX affects membrane composition and fluidity, we suspected membrane changes to cause such phenotype. The detergent polysorbate 80 (PS80) can moderately destabilize the envelope resulting in non-specific increased nutrient intake. Remarkably, growth, membrane fluidity and expression of dysregulated genes in the sigX mutant strain were restored in LB supplemented with PS80. Altogether, these data suggest that SigX is indirectly involved in CCR regulation, possibly via its effects on membrane integrity and fluidity.

The absence of the Pseudomonas aeruginosa OprF protein leads to increased biofilm formation through variation in c-di-GMP level.

OprF is the major outer membrane porin in bacteria belonging to the Pseudomonas genus. In previous studies, we have shown that OprF is required for full virulence expression of the opportunistic pathogen Pseudomonas aeruginosa. Here, we describe molecular insights on the nature of this relationship and report that the absence of OprF leads to increased biofilm formation and production of the Pel exopolysaccharide. Accordingly, the level of c-di-GMP, a key second messenger in biofilm control, is elevated in an oprF mutant. By decreasing c-di-GMP levels in this mutant, both biofilm formation and pel gene expression phenotypes were restored to wild-type levels. We further investigated the impact on two small RNAs, which are associated with the biofilm lifestyle, and found that expression of rsmZ but not of rsmY was increased in the oprF mutant and this occurs in a c-di-GMP-dependent manner. Finally, the extracytoplasmic function (ECF) sigma factors AlgU and SigX displayed higher activity levels in the oprF mutant. Two genes of the SigX regulon involved in c-di-GMP metabolism, PA1181 and adcA (PA4843), were up-regulated in the oprF mutant, partly explaining the increased c-di-GMP level. We hypothesized that the absence of OprF leads to a cell envelope stress that activates SigX and results in a c-di-GMP elevated level due to higher expression of adcA and PA1181. The c-di-GMP level can in turn stimulate Pel synthesis via increased rsmZ sRNA levels and pel mRNA, thus affecting Pel-dependent phenotypes such as cell aggregation and biofilm formation. This work highlights the connection between OprF and c-di-GMP regulatory networks, likely via SigX (ECF), on the regulation of biofilm phenotypes.

Sucrose favors Pseudomonas aeruginosa pellicle production through the extracytoplasmic function sigma factor SigX.

Pseudomonas aeruginosa biofilm formation was increased by addition of sucrose to Luria-Bertani medium, whereas addition of NaCl to a final similar osmolarity and use of maltose instead of sucrose, were ineffective. In a previous study, we showed that the extracytoplasmic sigma factor SigX is activated in the presence of sucrose. The sucrose-mediated pellicle increase was abolished in a sigX mutant strain. Sucrose addition led to an increase in pel expression and cyclic-diguanylate (c-di-GMP) pool level production. Interestingly, these two phenotypes were strongly decreased in a sigX mutant. Since pel is not known as a SigX-target, we suspect SigX to be involved in the c-di-GMP production. We found that expression of the diguanylate cyclase PA4843 gene was increased in the presence of sucrose at least partly through SigX activity. Our study shows that sucrose itself rather than osmolarity favours the biofilm mode of P. aeruginosa through the activation of SigX.

A proteomic approach of SigX function in Pseudomonas aeruginosa outer membrane composition.

SigX is one of the 19 extracytoplasmic function sigma factors that have been predicted in the human opportunistic pathogen Pseudomonas aeruginosa genome. SigX is involved in the transcription of oprF, encoding the major outer membrane protein OprF, a pleiotropic porin that contributes to the maintaining of the wall structure, and is essential to P. aeruginosa virulence. This study aimed to get further insights into the functions of SigX. We performed here an outer membrane subproteome of a sigX mutant. Proteomic investigations revealed lower production of 8 porins among which 4 gated channels involved in iron or hem uptake, OprF, and the three substrate-specific proteins OprD, OprQ and OprE. On the other side, the glucose-specific porin OprB and the lipid A 3-O-deacylase that is involved in LPS modification were up-regulated. Our results indicate that SigX may be involved in the control and/or regulation of the outer membrane composition. BIOLOGICAL SIGNIFICANCE: A proteomic approach was used herein to get further insights into SigX functions in P. aeruginosa. The data presented here suggest that SigX is involved in the outer membrane protein composition, and could be linked to a regulatory network involved in OM homeostasis.