BacA: a possible regulator that contributes to the biofilm formation of Pseudomonas aeruginosa.

Previously, we pointed out in P. aeruginosa PAO1 biofilm cells the accumulation of a hypothetical protein named PA3731 and showed that the deletion of the corresponding gene impacted its biofilm formation capacity. PA3731 belongs to a cluster of 4 genes (pa3732 to pa3729) that we named bac for "Biofilm Associated Cluster." The present study focuses on the PA14_16140 protein, i.e., the PA3732 (BacA) homolog in the PA14 strain. The role of BacA in rhamnolipid secretion, biofilm formation and virulence, was confirmed by phenotypic experiments with a bacA mutant. Additional investigations allow to advance that the bac system involves in fact 6 genes organized in operon, i.e., bacA to bacF. At a molecular level, quantitative proteomic studies revealed an accumulation of the BAC cognate partners by the bacA sessile mutant, suggesting a negative control of BacA toward the bac operon. Finally, a first crystallographic structure of BacA was obtained revealing a structure homologous to chaperones or/and regulatory proteins.

Lysine Trimethylation in Planktonic and Pellicle Modes of Growth in Acinetobacter baumannii.

Over the past 30 years, Acinetobacter baumannii has been described as an important nosocomial pathogen due to frequent ventilator-associated infections. Many biological processes of A. baumannii remain elusive, such as the formation of an air-liquid biofilm (pellicle). Several studies demonstrated the importance of post-translational modifications (PTMs) in A. baumannii physiology. Here, we investigated K-trimethylation in A. baumannii ATCC 17978 in planktonic and pellicle modes using proteomic analysis. To identify the most high-confidence K-trimethylated peptides, we compared different sample preparation methods (i.e., strong cation exchange, antibody-capture) and processing software (i.e., different database search engines). We identified, for the first time, 84 K-trimethylated proteins, many of which are involved in DNA and protein synthesis (HupB, RplK), transporters (Ata, AdeB), or lipid metabolism processes (FadB, FadD). In comparison with previous studies, several identical lysine residues were observed acetylated or trimethylated, indicating the presence of proteoforms and potential PTM cross-talks. This is the first large-scale proteomic study of trimethylation in A. baumannii and will be an important resource for the scientific community (availability in Pride repository under accession PXD035239).

Hepatoprotective Effect of Grape Seed and Skin Extract Against Lithium Exposure Examined by the Window of Proteomics.

Context: The liver is the organ by which the majority of substances are metabolized, including psychotropic drugs. Lithium (Li) used as drug for many neurological disorders such as bipolar disorders. Objective: This study aims to assess lithium toxicity and to evaluate the hepatic-protective properties of a grape skin seed and extract (GSSE). Materials and methods: Twenty-four male Wistar rats were exposed for 30 days to either various lithium concentrations, GSSE alone, or lithium supplemented with GSSE. The proteomic analysis revealed alterations of liver protein profiles after lithium treatments that were successfully identified by mass spectrometry. Results: Lithium treatment induced an oxidative damage by the alteration of antioxidant enzymes activities such as superoxide dismutase, CAT, and Gpx. The regulated proteins are mainly involved in the respiratory electron transport chain, detoxification processes, ribosomal stress pathway, glycolysis, and cytoskeleton. Proteins were differentially expressed in a dose-dependent manner. Interestingly, GSSE reversed the situation and restored the level of liver proteins whose abundance was modified after lithium treatment, arguing for its protective activity. Conclusion: Our data demonstrated the ability of proteomic analysis to underline the toxicity mechanisms of lithium in animal models. Based on these results, GSSE may be envisaged as a nutritional supplement to weaken the liver toxicity of lithium.

Escherichia coli Biofilm Formation, Motion and Protein Patterns on Hyaluronic Acid and Polydimethylsiloxane Depend on Surface Stiffness.

The surface stiffness of the microenvironment is a mechanical signal regulating biofilm growth without the risks associated with the use of bioactive agents. However, the mechanisms determining the expansion or prevention of biofilm growth on soft and stiff substrates are largely unknown. To answer this question, we used PDMS (polydimethylsiloxane, 9-574 kPa) and HA (hyaluronic acid gels, 44 Pa-2 kPa) differing in their hydration. We showed that the softest HA inhibited Escherichia coli biofilm growth, while the stiffest PDMS activated it. The bacterial mechanical environment significantly regulated the MscS mechanosensitive channel in higher abundance on the least colonized HA-44Pa, while Type-1 pili (FimA) showed regulation in higher abundance on the most colonized PDMS-9kPa. Type-1 pili regulated the free motion (the capacity of bacteria to move far from their initial position) necessary for biofilm growth independent of the substrate surface stiffness. In contrast, the total length travelled by the bacteria (diffusion coefficient) varied positively with the surface stiffness but not with the biofilm growth. The softest, hydrated HA, the least colonized surface, revealed the least diffusive and the least free-moving bacteria. Finally, this shows that customizing the surface elasticity and hydration, together, is an efficient means of affecting the bacteria's mobility and attachment to the surface and thus designing biomedical surfaces to prevent biofilm growth.

Cytoskeletal components can turn wall-less spherical bacteria into kinking helices.

Bacterial cell shape is generally determined through an interplay between the peptidoglycan cell wall and cytoplasmic filaments made of polymerized MreB. Indeed, some bacteria (e.g., Mycoplasma) that lack both a cell wall and mreB genes consist of non-motile cells that are spherical or pleomorphic. However, other members of the same class Mollicutes (e.g., Spiroplasma, also lacking a cell wall) display a helical cell shape and kink-based motility, which is thought to rely on the presence of five MreB isoforms and a specific fibril protein. Here, we show that heterologous expression of Spiroplasma fibril and MreB proteins confers helical shape and kinking ability to Mycoplasma capricolum cells. Isoform MreB5 is sufficient to confer helicity and kink propagation to mycoplasma cells. Cryoelectron microscopy confirms the association of cytoplasmic MreB filaments with the plasma membrane, suggesting a direct effect on membrane curvature. However, in our experiments, the heterologous expression of MreBs and fibril did not result in efficient motility in culture broth, indicating that additional, unknown Spiroplasma components are required for swimming.

Detoxification Response of Pseudomonas fluorescens MFAF76a to Gaseous Pollutants NO2 and NO.

Bacteria are often exposed to nitrosative stress from their environment, from atmospheric pollution or from the defense mechanisms of other organisms. Reactive nitrogen species (RNS), which mediate nitrosative stress, are notably involved in the mammalian immune response through the production of nitric oxide (NO) by the inducible NO synthase iNOS. RNS are highly reactive and can alter various biomolecules such as lipids, proteins and DNA, making them toxic for biological organisms. Resistance to RNS is therefore important for the survival of bacteria in various environments, and notably to successfully infect their host. The fuel combustion processes used in industries and transports are responsible for the emission of important quantities of two major RNS, NO and the more toxic nitrogen dioxide (NO2). Human exposure to NO2 is notably linked to increases in lung infections. While the response of bacteria to NO in liquid medium is well-studied, few data are available on their exposure to gaseous NO and NO2. This study showed that NO2 is much more toxic than NO at similar concentrations for the airborne bacterial strain Pseudomonas fluorescens MFAF76a. The response to NO2 involves a wide array of effectors, while the response to NO seemingly focuses on the Hmp flavohemoprotein. Results showed that NO2 induces the production of other RNS, unlike NO, which could explain the differences between the effects of these two molecules.

Gaseous NO2 induces various envelope alterations in Pseudomonas fluorescens MFAF76a.

Anthropogenic atmospheric pollution and immune response regularly expose bacteria to toxic nitrogen oxides such as NO• and NO2. These reactive molecules can damage a wide variety of biomolecules such as DNA, proteins and lipids. Several components of the bacterial envelope are susceptible to be damaged by reactive nitrogen species. Furthermore, the hydrophobic core of the membranes favors the reactivity of nitrogen oxides with other molecules, making membranes an important factor in the chemistry of nitrosative stress. Since bacteria are often exposed to endogenous or exogenous nitrogen oxides, they have acquired protection mechanisms against the deleterious effects of these molecules. By exposing bacteria to gaseous NO2, this work aims to analyze the physiological effects of NO2 on the cell envelope of the airborne bacterium Pseudomonas fluorescens MFAF76a and its potential adaptive responses. Electron microscopy showed that exposure to NO2 leads to morphological alterations of the cell envelope. Furthermore, the proteomic profiling data revealed that these cell envelope alterations might be partly explained by modifications of the synthesis pathways of multiple cell envelope components, such as peptidoglycan, lipid A, and phospholipids. Together these results provide important insights into the potential adaptive responses to NO2 exposure in P. fluorescens MFAF76a needing further investigations.

Comparative Proteomic Analysis of the Diatom Phaeodactylum tricornutum Reveals New Insights Into Intra- and Extra-Cellular Protein Contents of Its Oval, Fusiform, and Triradiate Morphotypes.

Phaeodactylum tricornutum is an atypical diatom since it can display three main morphotypes: fusiform, triradiate, and oval. Such pleomorphism is possible thanks to an original metabolism, which is tightly regulated in order to acclimate to environmental conditions. Currently, studies dedicated to the comparison of each morphotype issued from one specific strain are scarce and little information is available regarding the physiological significance of this morphogenesis. In this study, we performed a comparative proteomic analysis of the three morphotypes from P. tricornutum. Cultures highly enriched in one dominant morphotype (fusiform, triradiate, or oval) of P. tricornutum Pt3 strain were used. Pairwise comparisons highlighted biological processes, which are up- and down-regulated in the oval (e.g., purine and cellular amino acid metabolism) and triradiate morphotypes (e.g., oxido-reduction and glycolytic processes) compared to the fusiform one used as a reference. Intersection analysis allowed us to identify the specific features of the oval morphotype. Results from this study confirmed previous transcriptomic RNA sequencing observation showing that the oval cells present a distinct metabolism with specific protein enrichment compared to fusiform and triradiate cells. Finally, the analysis of the secretome of each morphotype was also performed.

Impact of Carbon Source Supplementations on Pseudomonas aeruginosa Physiology.

Pseudomonas aeruginosa is an opportunistic pathogen highly resistant to a wide range of antimicrobial agents, making its infections very difficult to treat. Since microorganisms need to perpetually adapt to their surrounding environment, understanding the effect of carbon sources on P. aeruginosa physiology is therefore essential to avoid increasing drug-resistance and better fight this pathogen. By a global proteomic approach and phenotypic assays, we investigated the impact of various carbon source supplementations (glucose, glutamate, succinate, and citrate) on the physiology of the P. aeruginosa PA14 strain. A total of 581 proteins were identified as differentially expressed in the 4 conditions. Most of them were more abundant in citrate supplementation and were involved in virulence, motility, biofilm development, and antibiotic resistance. Phenotypic assays were performed to check these hypotheses. By coupling all this data, we highlight the importance of the environment in which the bacterium evolves on its metabolism, and thus the necessity to better understand the metabolic pathways implied in its adaptative response according to the nutrient availability.

Validation in the ESPOIR cohort of vitamin K-dependent protein S (PROS) as a potential biomarker capable of predicting response to the methotrexate/etanercept combination.

BACKGROUND: To validate the ability of PROS (vitamin K-dependent protein S) and CO7 (complement component C7) to predict response to the methotrexate (MTX)/etanercept (ETA) combination in rheumatoid arthritis (RA) patients who received this therapeutic combination in a well-documented cohort. METHOD: From the ESPOIR cohort, RA patients having received the MTX/ETA or MTX/adalimumab (ADA) combination as a first-line biologic treatment were included. Serum concentrations of PROS and CO7 were measured by ELISA prior to the initiation of ETA or ADA, at a time where the disease was active (DAS28 ESR > 3.2). The clinical efficacy (response/non-response) of both combinations has been evaluated after at least 6 months of treatment, according to the EULAR response criteria with some modifications. RESULTS: Thirty-two were treated by MTX/ETA; the numbers of responders and non-responders were 24 and 8, respectively. Thirty-three patients received the MTX/ADA combination; 27 and 5 patients were respectively responders and non-responders. While there were no differences for demographic, clinical, biological, and X-rays data, as well as for CO7, serum levels of PROS tended to be significantly higher in responders to the MTX/ETA combination (p = 0.08) while no difference was observed in the group receiving MTX/ADA. For PROS, the best concentration threshold to differentiate both groups was calculated at 40 µg/ml using ROC curve. The theranostic performances of PROS appeared better for the ETA/MTX combination. When considering the response to this combination, analysis of pooled data from ESPOIR and SATRAPE (initially used to validate PROS and CO7 as potential theranostic biomarkers) cohorts led to a higher theranostic value of PROS that became significant (p = 0.009). CONCLUSION: PROS might be one candidate of a combination of biomarkers capable of predicting the response to MTX/ETA combination in RA patients refractory to MTX. TRIAL REGISTRATION: ClinicalTrials.gov identifiers: NCT03666091 and NCT00234234 .

Phosphorylation of Extracellular Proteins in Acinetobacter baumannii in Sessile Mode of Growth.

Acinetobacter baumannii is a problematic nosocomial pathogen owing to its increasing resistance to antibiotics and its great ability to survive in the hospital environment, which is linked to its capacity to form biofilms. Structural and functional investigations of post-translational modifications, such as phosphorylations, may lead to identification of candidates for therapeutic targets against this pathogen. Here, we present the first S/T/Y phosphosecretome of two A. baumannii strains, the reference strain ATCC 17978 and the virulent multi-drug resistant strain AB0057, cultured in two modes of growth (planktonic and biofilm) using TiO2 chromatography followed by high resolution mass spectrometry. In ATCC 17978, we detected a total of 137 (97 phosphoproteins) and 52 (33 phosphoproteins) phosphosites in biofilm and planktonic modes of growth, respectively. Similarly, in AB0057, 155 (119 phosphoproteins) and 102 (74 phosphoproteins) phosphosites in biofilm and planktonic modes of growth were identified, respectively. Both strains in the biofilm mode of growth showed a higher number of phosphosites and phosphoproteins compared to planktonic growth. Several phosphorylated sites are localized in key regions of proteins involved in either drug resistance (ß-lactamases), adhesion to host tissues (pilins), or protein secretion (Hcp). Site-directed mutagenesis of the Hcp protein, essential for type VI secretion system-mediated interbacterial competition, showed that four of the modified residues are essential for type VI secretion system activity.

Variability of the response of human vaginal Lactobacillus crispatus to 17ß-estradiol.

We previously showed that the physiological concentration of 17ß-estradiol in the vaginal environment is sufficient to affect the membrane dynamics and adhesion phenotype of the Lactobacillus crispatus strain CIP104459. However, L. crispatus is a heterogeneous species. Here, we investigated the effect of 17ß-estradiol on the recently isolated L. crispatus vaginal strain V4, related to a cluster distant from CIP104459 and at the limit of being a different subspecies. Grown in the same medium, the two strains expressed a highly similar pool of proteins. However, in contrast to CIP104459, L. crispatus V4 showed high aggregation potential and 17ß-estradiol promoted this phenotype. This effect was associated with large changes in cell-surface polarity and Lewis acid/base properties. In addition, we observed no effect on the membrane dynamics, contrary to CIP104459. These results can be explained by differences in the properties and organization of the S layer between the two strains. However, as for CIP104459, 17ß-estradiol increased biosurfactant production of L. crispatus V4 and their adhesion to vaginal cells. This suggests that 17ß-estradiol agonists would be valuable tools to favor a stable re-implantation of L. crispatus in the vaginal mucosa.

MacAB-TolC Contributes to the Development of Acinetobacter baumannii Biofilm at the Solid-Liquid Interface.

Acinetobacter baumannii has emerged as one of the most problematic bacterial pathogens responsible for hospital-acquired and community infections worldwide. Besides its high capacity to acquire antibiotic resistance mechanisms, it also presents high adhesion abilities on inert and living surfaces leading to biofilm development. This lifestyle confers additional protection against various treatments and allows it to persist for long periods in various hospital niches. Due to their remarkable antimicrobial tolerance, A. baumannii biofilms are difficult to control and ultimately eradicate. Further insights into the mechanism of biofilm development will help to overcome this challenge and to develop novel antibiofilm strategies. To unravel critical determinants of this sessile lifestyle, the proteomic profiles of two A. baumannii strains (ATTC17978 and SDF) grown in planktonic stationary phase or in mature solid-liquid (S-L) biofilm were compared using a semiquantitative proteomic study. Of interest, among the 69 common proteins determinants accumulated in the two strains at the S-L interface, we sorted out the MacAB-TolC system. This tripartite efflux pump played a role in A. baumannii biofilm formation as demonstrated by using ΔmacAB-tolC deletion mutant. Complementary approaches allowed us to get an overview of the impact of macAB-tolC deletion in A. baumannii physiology. Indeed, this efflux pump appeared to be involved in the envelope stress response occurring in mature biofilm. It contributes to maintain wild type (WT) membrane rigidity and provides tolerance to high osmolarity conditions. In addition, this system is probably involved in the maintenance of iron and sulfur homeostasis. MacAB-TolC might help this pathogen face and adapt to deleterious conditions occurring in mature biofilms. Increasing our knowledge of A. baumannii biofilm formation will undoubtedly help us develop new therapeutic strategies to tackle this emerging threat to human health.

Exoproteomics for Better Understanding Pseudomonas aeruginosa Virulence.

Pseudomonas aeruginosa is the most common human opportunistic pathogen associated with nosocomial diseases. In 2017, the World Health Organization has classified P. aeruginosa as a critical agent threatening human health, and for which the development of new treatments is urgently necessary. One interesting avenue is to target virulence factors to understand P. aeruginosa pathogenicity. Thus, characterising exoproteins of P. aeruginosa is a hot research topic and proteomics is a powerful approach that provides important information to gain insights on bacterial virulence. The aim of this review is to focus on the contribution of proteomics to the studies of P. aeruginosa exoproteins, highlighting its relevance in the discovery of virulence factors, post-translational modifications on exoproteins and host-pathogen relationships.

Crosstalk between the Type VI Secretion System and the Expression of Class IV Flagellar Genes in the Pseudomonas fluorescens MFE01 Strain.

Type VI secretion systems (T6SSs) are contractile bacterial multiprotein nanomachines that enable the injection of toxic effectors into prey cells. The Pseudomonas fluorescens MFE01 strain has T6SS antibacterial activity and can immobilise competitive bacteria through the T6SS. Hcp1 (hemolysin co-regulated protein 1), a constituent of the T6SS inner tube, is involved in such prey cell inhibition of motility. Paradoxically, disruption of the hcp1 or T6SS contractile tail tssC genes results in the loss of the mucoid and motile phenotypes in MFE01. Here, we focused on the relationship between T6SS and flagella-associated motility. Electron microscopy revealed the absence of flagellar filaments for MFE01Δhcp1 and MFE01ΔtssC mutants. Transcriptomic analysis showed a reduction in the transcription of class IV flagellar genes in these T6SS mutants. However, transcription of fliA, the gene encoding the class IV flagellar sigma factor, was unaffected. Over-expression of fliA restored the motile and mucoid phenotypes in both MFE01Δhcp1+fliA, and MFE01ΔtssC+fliA and a fliA mutant displayed the same phenotypes as MFE01Δhcp1 and MFE01ΔtssC. Moreover, the FliA anti-sigma factor FlgM was not secreted in the T6SS mutants, and flgM over-expression reduced both motility and mucoidy. This study provides arguments to unravel the crosstalk between T6SS and motility.

Epinephrine affects motility, and increases adhesion, biofilm and virulence of Pseudomonas aeruginosa H103.

Microbial endocrinology has demonstrated for more than two decades, that eukaryotic substances (hormones, neurotransmitters, molecules of the immune system) can modulate the physiological behavior of bacteria. Among them, the hormones/neurotransmitters, epinephrine (Epi) and norepinephrine (NE), released in case of stress, physical effort or used in medical treatment, were shown to be able to modify biofilm formation in various bacterial species. In the present study, we have evaluated the effect of Epi on motility, adhesion, biofilm formation and virulence of Pseudomonas aeruginosa, a bacterium linked to many hospital-acquired infections, and responsible for chronic infection in immunocompromised patients including persons suffering from cystic fibrosis. The results showed that Epi increased adhesion and biofilm formation of P. aeruginosa, as well as its virulence towards the Galleria mellonella larvae in vivo model. Deciphering the sensor of this molecule in P. aeruginosa and the molecular mechanisms involved may help to find new strategies of treatment to fight against this bacterium.

A versatile and recyclable molecularly imprinted polymer as an oxidative catalyst of sulfur derivatives: a new possible method for mustard gas and V nerve agent decontamination.

A molecularly imprinted polymer containing a porphyrin unit was developed as a biomimetic heterogenous catalyst for the oxidation of sulfur derivatives. Its catalytic efficiency under mild conditions and its easy recovery represent a great asset for the design of new decontamination tools for yperite and VX.

Protein post-translational modifications in bacteria.

Over the past decade the number and variety of protein post-translational modifications that have been detected and characterized in bacteria have rapidly increased. Most post-translational protein modifications occur in a relatively low number of bacterial proteins in comparison with eukaryotic proteins, and most of the modified proteins carry low, substoichiometric levels of modification; therefore, their structural and functional analysis is particularly challenging. The number of modifying enzymes differs greatly among bacterial species, and the extent of the modified proteome strongly depends on environmental conditions. Nevertheless, evidence is rapidly accumulating that protein post-translational modifications have vital roles in various cellular processes such as protein synthesis and turnover, nitrogen metabolism, the cell cycle, dormancy, sporulation, spore germination, persistence and virulence. Further research of protein post-translational modifications will fill current gaps in the understanding of bacterial physiology and open new avenues for treatment of infectious diseases.