Cell Wall Stress Response Research Articles

Membrane-Interactive Compounds From Pistacia lentiscus L. Thwart Pseudomonas aeruginosa Virulence.

Pseudomonas aeruginosa is capable to deploy a collection of virulence factors that are not only essential for host infection and persistence, but also to escape from the host immune system and to become more resistant to drug therapies. Thus, developing anti-virulence agents that may directly counteract with specific virulence factors or disturb higher regulatory pathways controlling the production of virulence armories are urgently needed. In this regard, this study reports that Pistacia lentiscus L. fruit cyclohexane extract (PLFE1) thwarts P. aeruginosa virulence by targeting mainly the pyocyanin pigment production by interfering with 4-hydroxy-2-alkylquinolines molecules production. Importantly, the anti-virulence activity of PLFE1 appears to be associated with membrane homeostasis alteration through the modulation of SigX, an extracytoplasmic function sigma factor involved in cell wall stress response. A thorough chemical analysis of PLFE1 allowed us to identify the ginkgolic acid (C17:1) and hydroginkgolic acid (C15:0) as the main bioactive membrane-interactive compounds responsible for the observed increased membrane stiffness and anti-virulence activity against P. aeruginosa. This study delivers a promising perspective for the potential future use of PLFE1 or ginkgolic acid molecules as an adjuvant therapy to fight against P. aeruginosa infections.

Intracellular Staphylococcus aureus persisters upon antibiotic exposure

Bacterial persister cells are phenotypic variants that exhibit a transient non-growing state and antibiotic tolerance. Here, we provide in vitro evidence of Staphylococcus aureus persisters within infected host cells. We show that the bacteria surviving antibiotic treatment within host cells are persisters, displaying biphasic killing and reaching a uniformly non-responsive, non-dividing state when followed at the single-cell level. This phenotype is stable but reversible upon antibiotic removal. Intracellular S. aureus persisters remain metabolically active but display an altered transcriptomic profile consistent with activation of stress responses, including the stringent response as well as cell wall stress, SOS and heat shock responses. These changes are associated with multidrug tolerance after exposure to a single antibiotic. We hypothesize that intracellular S. aureus persisters may constitute a reservoir for relapsing infection and could contribute to therapeutic failures.

Dynamic Surface Proteomes of Allergenic Fungal Conidia.

Fungal spores and hyphal fragments play an important role as allergens in respiratory diseases. In this study, we performed trypsin shaving and secretome analyses to identify the surface-exposed proteins and secreted/shed proteins of Aspergillus fumigatus conidia, respectively. We investigated the surface proteome under different conditions, including temperature variation and germination. We found that the surface proteome of resting A. fumigatus conidia is not static but instead unexpectedly dynamic, as evidenced by drastically different surface proteomes under different growth conditions. Knockouts of two abundant A. fumigatus surface proteins, ScwA and CweA, were found to function only in fine-tuning the cell wall stress response, implying that the conidial surface is very robust against perturbations. We then compared the surface proteome of A. fumigatus to other allergy-inducing molds, including Alternaria alternata, Penicillium rubens, and Cladosporium herbarum, and performed comparative proteomics on resting and swollen conidia, as well as secreted proteins from germinating conidia. We detected 125 protein ortholog groups, including 80 with putative catalytic activity, in the extracellular region of all four molds, and 42 nonorthologous proteins produced solely by A. fumigatus. Ultimately, this study highlights the dynamic nature of the A. fumigatus conidial surface and provides targets for future diagnostics and immunotherapy.

Interrogation of the cell wall integrity pathway in Aspergillus niger identifies a putative negative regulator of transcription involved in chitin deposition.

Post-fermentation fungal biomass waste provides a viable source for chitin. Cell wall chitin of filamentous fungi, and in particular its de-N-acetylated derivative chitosan, has a wide range of commercial applications. Although the cell wall of filamentous fungi comprises 10–30% chitin, these yields are too low for cost-effective production. Therefore, we aimed to identify the genes involved in increased chitin deposition by screening a collection of UV-derived cell wall mutants in Aspergillus niger. This screen revealed a mutant strain (RD15.4#55) that showed a 30–40% increase in cell wall chitin compared to the wild type. In addition to the cell wall chitin phenotype, this strain also exhibited sensitivity to SDS and produces an unknown yellow pigment. Genome sequencing combined with classical genetic linkage analysis identified two mutated genes on chromosome VII that were linked with the mutant phenotype. Single gene knockouts and subsequent complementation analysis revealed that an 8 bp deletion in NRRL3_09595 is solely responsible for the associated phenotypes of RD15.4#55. The mutated gene, which was named cwcA (cell wall chitin A), encodes an orthologue of Saccharomyces cerevisiae Bypass of ESS1 (BYE1), a negative regulator of transcription elongation. We propose that this conserved fungal protein is involved in preventing cell wall integrity signaling under non-inducing conditions, where loss of function results in constitutive activation of the cell wall stress response pathway, and consequently leads to increased chitin content in the mutant cell wall.

The Arabidopsis receptor kinase STRUBBELIG regulates the response to cellulose deficiency.

Plant cells are encased in a semi-rigid cell wall of complex build. As a consequence, cell wall remodeling is essential for the control of growth and development as well as the regulation of abiotic and biotic stress responses. Plant cells actively sense physico-chemical changes in the cell wall and initiate corresponding cellular responses. However, the underlying cell wall monitoring mechanisms remain poorly understood. In Arabidopsis the atypical receptor kinase STRUBBELIG (SUB) mediates tissue morphogenesis. Here, we show that SUB-mediated signal transduction also regulates the cellular response to a reduction in the biosynthesis of cellulose, a central carbohydrate component of the cell wall. SUB signaling affects early increase of intracellular reactive oxygen species, stress gene induction as well as ectopic lignin and callose accumulation upon exogenous application of the cellulose biosynthesis inhibitor isoxaben. Moreover, our data reveal that SUB signaling is required for maintaining cell size and shape of root epidermal cells and the recovery of root growth after transient exposure to isoxaben. SUB is also required for root growth arrest in mutants with defective cellulose biosynthesis. Genetic data further indicate that SUB controls the isoxaben-induced cell wall stress response independently from other known receptor kinase genes mediating this response, such as THESEUS1 or MIK2. We propose that SUB functions in a least two distinct biological processes: the control of tissue morphogenesis and the response to cell wall damage. Taken together, our results reveal a novel signal transduction pathway that contributes to the molecular framework underlying cell wall integrity signaling.

Transcriptome changes in reciprocal grafts involving watermelon and bottle gourd reveal molecular mechanisms involved in increase of the fruit size, rind toughness and soluble solids.

Transcriptome landscape reveals the molecular mechanisms involved in the improvement of fruit traits by the grafting of watermelon and bottle gourd. Grafting has been used as a sustainable alternative for watermelon breeding to control soil-borne pathogens and to increase tolerance to various abiotic stresses. However, some reports have shown that grafting can negatively affect the quality of fruits. Despite several field studies on the effects of grafting on fruit quality, the regulation of this process at the molecular level has not been revealed. The aim of this study was to elucidate various molecular mechanisms involved in different tissues of heterografted watermelon and bottle gourd plants. Grafting with bottle gourd rootstock increased the size and rind thickness of watermelon fruits, whereas that with watermelon rootstock produced bottle gourd fruits with higher total soluble solid content and thinner rinds. Correspondingly, genes related to ripening, softening, cell wall strengthening, stress response and disease resistance were differentially expressed in watermelon fruits. Moreover, genes associated mainly with sugar metabolism were differentially expressed in bottle gourd fruits. RNA-seq revealed more than 400 mobile transcripts across the heterografted sets. More than half of these were validated from PlaMoM, a database for plant mobile macromolecules. In addition, some of these mobile transcripts contained a transfer RNA-like structure. Other RNA motifs were also enriched in these transcripts, most with a biological role based on GO analysis. This transcriptome study provided a comprehensive understanding of various molecular mechanisms underlying grafted tissues in watermelon.

Multi-Omics Analysis of Fatty Alcohol Production in Engineered Yeasts Saccharomyces cerevisiae and Yarrowia lipolytica.

Fatty alcohols are widely used in various applications within a diverse set of industries, such as the soap and detergent industry, the personal care, and cosmetics industry, as well as the food industry. The total world production of fatty alcohols is over 2 million tons with approximately equal parts derived from fossil oil and from plant oils or animal fats. Due to the environmental impact of these production methods, there is an interest in alternative methods for fatty alcohol production via microbial fermentation using cheap renewable feedstocks. In this study, we aimed to obtain a better understanding of how fatty alcohol biosynthesis impacts the host organism, baker’s yeast Saccharomyces cerevisiae or oleaginous yeast Yarrowia lipolytica. Producing and non-producing strains were compared in growth and nitrogen-depletion cultivation phases. The multi-omics analysis included physiological characterization, transcriptome analysis by RNAseq, 13Cmetabolic flux analysis, and intracellular metabolomics. Both species accumulated fatty alcohols under nitrogen-depletion conditions but not during growth. The fatty alcohol–producing Y. lipolytica strain had a higher fatty alcohol production rate than an analogous S. cerevisiae strain. Nitrogen-depletion phase was associated with lower glucose uptake rates and a decrease in the intracellular concentration of acetyl–CoA in both yeast species, as well as increased organic acid secretion rates in Y. lipolytica. Expression of the fatty alcohol–producing enzyme fatty acyl–CoA reductase alleviated the growth defect caused by deletion of hexadecenal dehydrogenase encoding genes (HFD1 and HFD4) in Y. lipolytica. RNAseq analysis showed that fatty alcohol production triggered a cell wall stress response in S. cerevisiae. RNAseq analysis also showed that both nitrogen-depletion and fatty alcohol production have substantial effects on the expression of transporter encoding genes in Y. lipolytica. In conclusion, through this multi-omics study, we uncovered some effects of fatty alcohol production on the host metabolism. This knowledge can be used as guidance for further strain improvement towards the production of fatty alcohols.

Cell wall proteome analysis of banana fruit softening using iTRAQ technology

Softening is important for quality formation in some climacteric fruits. The molecular mechanism underlying fruit softening is, however, not well understood. In this study, we performed a comparative cell wall proteomics analysis on pre-climacteric (P1) and post-climacteric (P2) banana fruit, corresponding to fruit at the mature green stage and softening ripening stage, respectively, using isobaric tags for relative and absolute quantitation (iTRAQ) technology. A total of 5230 proteins were identified in both sample groups, of which 928 were predicted to be secreted proteins. Of the secreted proteins, 162 were differentially expressed in P2 versus P1. The majority of these proteins had catalytic activity, binding activity, electron carrier activity and antioxidant activity. Compared with P1, P2 had 105 and 57 up- and down-regulated proteins, respectively. GO and KEGG-pathway analysis of these differentially expressed secreted proteins revealed that most were implicated in cell wall metabolism, stress and defense response, signaling, and protein metabolism and modification. Quantitative RT-PCR further validated some key differentially expressed secreted proteins associated with cell wall metabolism, stress and defense response, signaling and protein destination. Our results represent the first cell wall proteome of banana fruit and comprehensive proteomic study of banana fruit softening. SignificanceSoftening, which is the consequence of cell wall and turgor modification, is one of the most important factors determining banana fruit quality. The molecular mechanism regulating fruit softening in harvested banana is not currently well understood. In this study, we performed a comparative cell wall proteome analysis for pre-climacteric (P1) and post-climacteric (P2) banana fruit, corresponding to the mature green stage and softening ripening stage, respectively, using iTRAQ technology. We found 162 differentially expressed secreted proteins that were mainly implicated in cell wall metabolism, stress response and defense, signaling, and protein metabolism and modification. We have presented the first cell wall proteome of banana fruit and conducted a comprehensive proteomic study of banana fruit softening that will help to develop strategies to improve the sensorial quality and reduce post-harvest fruit losses.

Fusarium verticillioides SNARE protein FvSyn1 harbours two key functional motifs that play selective roles in fungal development and virulence.

Fusarium verticillioides is one of the key fungal pathogens responsible for maize stalk rot. While stalk rot pathogens are prevalent worldwide, our understanding of the stalk rot virulence mechanism in pathogenic fungi is still very limited. We previously identified the F. verticillioides FvSYN1 gene, which was demonstrated to play an important role in maize stalk rot virulence. FvSyn1 belongs to a family of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins that play critical roles in a variety of developmental processes. In this study, we further characterized the cellular features of the FvSyn1 protein, namely how different motifs contribute to development and virulence in F. verticillioides by generating motif-specific deletion mutants. Microscopic observation showed that the ∆Fvsyn1 mutant exhibits rough and hyper-branched hyphae when compared to the wild-type progenitor. Moreover, the ∆Fvsyn1 mutant was sensitive to cell wall stress agents, resulting in vegetative growth reduction. We showed that the FvSyn1::GFP protein is associated with the endomembrane, but this did not clarify why the deletion of FvSyn1 led to stress sensitivity and aberrant hyphal development. Characterization of the FvSyn1 domains indicated that both the syntaxin N-terminus (SynN) domain and the SNARE C-terminus domain play distinct roles in fungal development, but also function collectively in the context of virulence. We also determined that two domains in FvSyn1 are not required for fumonisin production. Interestingly, these two domains were involved in carbon nutrient utilization, including pectin, starch and sorbitol. This study further characterized the role of FvSyn1 domains in hyphal growth, cell wall stress response and virulence in F. verticillioides.

Progress in brewer's yeast cell wall stress response

Yeast cell wall plays an important role in the establishment and maintenance of cell morphology upon the cell wall stress. The cell wall of yeast consists of β-glucans, mannoproteins and chitin. The composition and structure remodel due to cell wall stress. Brewer's yeast cell wall exhibits stress response during long-term acclimation in order to adapt to environmental changes. This paper reviews the composition and structure of yeast cell wall and the molecular mechanisms of cell wall remodeling and signal pathway regulation.

The dimerization interface in VraR is essential for induction of the cell wall stress response in Staphylococcus aureus: a potential druggable target

BackgroundStaphylococcus aureus remains a medical challenge in the treatment of bacterial infections. It has acquired resistance to commonly used antibiotics, and to those considered to be the last weapons in treating staphylococcal infections, such as vancomycin. Studies have revealed that S. aureus is capable of mounting a rapid response to antibiotics that target cell wall peptidoglycan biosynthesis, such as β-lactams and vancomycin. The two-component system VraSR has been linked to the coordination of this response. VraS is a histidine kinase that undergoes autophosphorylation in the presence of signals elicited upon cell wall damage and it then transfers its phosphoryl group to VraR. VraR is a response regulator protein that functions as a transcription factor. Phosphorylation of VraR leads to its dimerization, which is required for optimum binding to its target promoters. Two-component systems have been targeted for the development of antibacterial agents. Deletion of the vraS or vraR gene has been shown to re-sensitize S. aureus to β-lactams and vancomycin.ResultsIn this study, we explored perturbation of the VraR phosphorylation-induced activation as a means to inhibit the VraSR-mediated signal transduction pathway. We show that dimerization of VraR is essential for the phosphorylation-induced activation of VraR. A single point mutation in the dimerization interface of VraR, in which Met13 was replaced by Ala, led to the inability of VraR to dimerize and to bind optimally to the target promoter. The consequences of these in vitro molecular deficiencies are equally dramatic in vivo. Complementation of a vraR deletion S. aureus strain with the vraRM13Ala mutant gene failed to induce the cell wall stress response.ConclusionsThis study highlights the potential of targeting the phosphorylation-induced dimerization of VraR to disrupt the S. aureus cell wall stress response and in turn to re-sensitize S. aureus to β-lactams and vancomycin.

Transcriptome Kinetics of Saccharomyces cerevisiae in Response to Viral Killer Toxin K1.

The K1 A/B toxin secreted by virus-infected Saccharomyces cerevisiae strains kills sensitive cells via disturbance of cytoplasmic membrane functions. Despite decades of research, the mechanisms underlying K1 toxicity and immunity have not been elucidated yet. In a novel approach, this study aimed to characterize transcriptome changes in K1-treated sensitive yeast cells in a time-dependent manner. Global transcriptional profiling revealed substantial cellular adaptations in target cells resulting in 1,189 differentially expressed genes in total. Killer toxin K1 induced oxidative, cell wall and hyperosmotic stress responses as well as rapid down-regulation of transcription and translation. Essential pathways regulating energy metabolism were also significantly affected by the toxin. Remarkably, a futile cycle of the osmolytes trehalose and glycogen was identified probably representing a critical feature of K1 intoxication. In silico analysis suggested several transcription factors involved in toxin-triggered signal transduction. The identified transcriptome changes provide valuable hints to illuminate the still unknown molecular events leading to K1 toxicity and immunity implicating an evolutionarily conserved response at least initially counteracting ionophoric toxin action.

Endosidin2-14 Targets the Exocyst Complex in Plants and Fungal Pathogens to Inhibit Exocytosis.

The evolutionarily conserved octameric exocyst complex tethers secretory vesicles to the site of membrane fusion during exocytosis. The plant exocyst complex functions in cell wall biosynthesis, polarized growth, stress responses, and hormone signaling. In fungal pathogens, the exocyst complex is required for growth, development, and pathogenesis. Endosidin2 (ES2) is known to inhibit exocytosis in plant and mammalian cells by targeting the EXO70 subunit of the exocyst complex. Here we show that an analog of ES2, ES2-14, targets plant and two fungal EXO70s. A lower dosage of ES2-14 than of ES2 is required to inhibit plant growth, plant exocytic trafficking, and fungal growth. ES2-14 treatments inhibit appressorium formation and reduce lesion sizes caused by Magnaporthe oryzae Inhibition of EXO70 by ES2-14 in Botrytis cinerea also reduces its virulence in Arabidopsis (Arabidopsis thaliana). Interestingly, ES2-14 did not affect EXO70 localization or transferrin recycling in mammalian cells. Overall, our results indicate that a minor change in ES2 affects its specificity in targeting EXO70s in different organisms and they demonstrate the potential of using ES2-14 to study the mechanisms of plant and fungal exocytosis and the roles of exocytosis in fungus-plant interactions.

Silicon supply affects the root transcriptome of Brassica napus L.

Modulation of gene expression in roots of Brassica napus by silicon (Si) supply could allow plants to cope with future stresses. The origin of the beneficial effects of silicon (Si) in plants, especially when they are subject to stress, remains poorly understood. Some authors have shown that Si alleviates plant stress and consider that this is mainly due to a mechanical effect on the cell wall. In addition, the other studies have shown that Si can also affect gene expression and modulate a number of metabolic pathways, especially in plants cultivated under stress conditions. Previously, Haddad et al. (Front Plant Sci 9:5-16, 2018) showed that a pretreatment of Brassica napus plants with Si (1.7mM) for 1week alleviated the stress induced by N privation. These results suggest that this improved resistance in Si-treated plants might be due to the establishment of defense mechanisms prior to exposure to the N stress. The aim of the current work was to test this assumption in Brassica napus roots (where Si is mainly stored) using a transcriptomic approach via the RNA sequencing. Our results indicated that the Si supply leads to a modulation of the expression of genes in Brassica napus roots. Functional categorization of the differentially expressed genes demonstrated that numerous genes are involved in different metabolic pathways and especially in cell wall synthesis, phytohormone metabolism, and stress responses. All these results show that Si modifies the root metabolism of B. napus, which could allow a better adaptation to future stresses.

Carbohydrate-binding property of a cell wall integrity and stress response component (WSC) domain of an alcohol oxidase from the rice blast pathogen Pyricularia oryzae

The cell wall integrity and stress response component (WSC) domain was first described in the Wsc-family protein of the yeast Saccharomyces cerevisiae, and later found in diverse eukaryotic organisms. Due solely to their presence in the Wsc-family proteins working as a plasma membrane sensor for surface stress and in a fungal β-1,3-exoglucanse, WSC domains have been presumed to possess carbohydrate-binding property without any experimental evidence. Aiming at elucidation of function(s) of WSC domains, we characterized a WSC domain-containing alcohol oxidase from the rice blast pathogen Pyricularia oryzae (PoAlcOX). Recombinant PoAlcOX produced with Pichia pastoris showed alcohol oxidase activity toward a wide range of substrates including two aliphatic alcohols, a branched-chain alcohol, a diol, and a polyol. Deletion of the WSC domain virtually unaffected oxidation of these substrates by PoAlcOX, indicating that the domain makes no contribution to the catalytic activity. In analogy to some carbohydrate-binding modules, we inferred that the WSC domain plays a role in protein anchoring, and evaluated binding capability of PoAlcOX to a set of polysaccharide components of fungal and plant cell walls. This revealed that PoAlcOX binds to xylans and fungal chitin/β-1,3-glucan in the WSC domain-dependent manner, demonstrating for the first time the carbohydrate-binding property of the domain. Additionally, we provide evidence that PoAlcOX immobilized on birch wood xylan retains the catalytic activity. Overall, the data we collected suggest that the role of the WSC domain of PoAlcOX is not recognition of substrates but attaching the enzyme to plant and/or fungal cell wall.

Hexokinase and Glucokinases Are Essential for Fitness and Virulence in the Pathogenic Yeast Candida albicans.

The pathogenic yeast Candida albicans is both a powerful commensal and a pathogen of humans that can infect wide range of organs and body sites. Metabolic flexibility promotes infection and commensal colonization by this opportunistic pathogen. Yeast cell survival depends upon assimilation of fermentable and non-fermentable locally available carbon sources. Physiologically relevant sugars like glucose and fructose are present at low levels in host niches. However, because glucose is the preferred substrate for energy and biosynthesis of structural components, its efficient detection and metabolism are fundamental for the metabolic adaptation of the pathogen. We explored and characterized the C. albicans hexose kinase system composed of one hexokinase (CaHxk2) and two glucokinases (CaGlk1 and CaGlk4). Using a set of mutant strains, we found that hexose phosphorylation is mostly performed by CaHxk2, which sustains growth on hexoses. Our data on hexokinase and glucokinase expression point out an absence of cross regulation mechanisms at the transcription level and different regulatory pathways. In the presence of glucose, CaHxk2 migrates in the nucleus and contributes to the glucose repression signaling pathway. In addition, CaHxk2 participates in oxidative, osmotic and cell wall stress responses, while glucokinases are overexpressed under hypoxia. Hexose phosphorylation is a key step necessary for filamentation that is affected in the hexokinase mutant. Virulence of this mutant is clearly impacted in the Galleria mellonella and macrophage models. Filamentation, glucose phosphorylation and stress response defects of the hexokinase mutant prevent host killing by C. albicans. By contributing to metabolic flexibility, stress response and morphogenesis, hexose kinase enzymes play an essential role in the virulence of C. albicans.

An active β-lactamase is a part of an orchestrated cell wall stress resistance network of Bacillus subtilis and related rhizosphere species.

A hallmark of the Gram-positive bacteria, such as the soil-dwelling bacterium Bacillus subtilis, is their cell wall. Here, we report that d-leucine and flavomycin, biofilm inhibitors targeting the cell wall, activate the β-lactamase PenP. This β-lactamase contributes to ampicillin resistance in B. subtilis under all conditions tested. In contrast, both Spo0A, a master regulator of nutritional stress, and the general cell wall stress response, differentially contribute to β-lactam resistance under different conditions. To test whether β-lactam resistance and β-lactamase genes are widespread in other Bacilli, we isolated Bacillus species from undisturbed soils, and found that their genomes can encode up to five β-lactamases with differentiated activity spectra. Surprisingly, the activity of environmental β-lactamases and PenP, as well as the general stress response, resulted in a similarly reduced lag phase of the culture in the presence of β-lactam antibiotics, with little or no impact on the logarithmic growth rate. The length of the lag phase may determine the outcome of the competition between β-lactams and β-lactamases producers. Overall, our work suggests that antibiotic resistance genes in B. subtilis and related species are ancient and widespread, and could be selected by interspecies competition in undisturbed soils.

Combined Transcriptome and Proteome Analysis of RpoS Regulon Reveals Its Role in Spoilage Potential of Pseudomonas fluorescens.

Microbial contamination is considered the main cause of food spoilage. Pseudomonas fluorescens is a typical spoilage bacterium contributing to a large extent to the spoilage process of proteinaceous foods. RpoS is known as an alternative sigma factor controlling stress resistance and virulence in many pathogens. Our previous work revealed that RpoS contributes to the spoilage activities of P. fluorescens by regulating resistance to different stress conditions, extracellular acylated homoserine lactone (AHL) levels, extracellular protease and total volatile basic nitrogen (TVB-N) production. However, RpoS-dependent genes in P. fluorescens remained undefined. RNA-seq transcriptomics analysis combined with quantitative proteomics analysis based on multiplexed isobaric tandem mass tag (TMT) labeling was performed in the P. fluorescens wild-type strain UK4 and its derivative carrying an rpoS mutation. A total of 375 differentially expressed coding sequences (DECs) and 212 differentially expressed proteins (DEPs) were identified. The DECs were further verified by qRT-PCR. The combined transcriptome and proteome analyses revealed the involvement of this regulator in several cellular processes, mainly including polysaccharide metabolism, intracellular secretion, extracellular structures, cell wall biogenesis, stress responses, and amino acid and biogenic amine metabolism, which may contribute to the biofilm formation, stress resistance, and spoilage activities of P. fluorescens. Moreover, we indeed observed that RpoS contributed to the production of the macrocolony biofilm's matrix. Our results provide insights into the regulatory network of RpoS and expand the knowledge about the role of RpoS in the functioning of P. fluorescens in food spoilage.

Klebsiella pneumoniae prevents spore germination and hyphal development of Aspergillus species

Different bacteria and fungi live as commensal organisms as part of the human microbiota, but shifts to a pathogenic state potentially leading to septic infections commonly occur in immunocompromised individuals. Several studies have reported synergistic or antagonistic interactions between individual bacteria and fungi which might be of clinical relevance. Here, we present first evidence for the interaction between Klebsiella pneumoniae and several Aspergillus species including A. fumigatus, A. terreus, A. niger and A. flavus which cohabit in the lungs and the intestines. Microbiological and molecular methods were employed to investigate the interaction in vitro, and the results indicate that Klebsiella pneumoniae is able to prevent Aspergillus spp. spore germination and hyphal development. The inhibitory effect is reversible, as demonstrated by growth recovery of Aspergillus spp. upon inhibition or elimination of the bacteria, and is apparently dependent on the physical interaction with metabolically active bacteria. Molecular analysis of Klebsiella-Aspergillus interaction has shown upregulation of Aspergillus cell wall-related genes and downregulation of hyphae-related genes, suggesting that Klebsiella induces cell wall stress response mechanisms and suppresses filamentous growth. Characterization of polymicrobial interactions may provide the basis for improved clinical management of mixed infections by setting the stage for appropriate diagnostics and ultimately for optimized treatment strategies.

Redox Proteome Perturbation in Arabidopsis upon Pseudomonas syringae Infection

Oxidative burst is one of the earliest plant cellular responses triggered by pathogen infection. Reactive oxygen species can cause oxidative modifications of redox-sensitive proteins to mediate the defense responses. Identification and characterization of proteins that undergo oxidative modifications in these processes is an important step toward understanding molecular mechanisms of plant defense responses. In this study, an in vivo 15N metabolic labeling method combined with a cysteine-containing peptide enrichment technique was applied to identify and quantify proteins and their redox states in Arabidopsis in response to infection by Pseudomonas syringae pv tomato DC3000 (Pst). Changes of peptide redox states were compared and corrected with the changes of protein levels. A total of forty peptides representing thirty-six non-redundant proteins showed significantly redox state changes in response to the infection by the virulent Pst strain and the avirulent Pst strain (Pst avrRpm1), of which 23 had previously not been recognized to undergo oxidative PTMs. The differentially expressed redox-sensitive proteins are involved in cell wall organization, primary metabolism, photosynthesis and stress responses. Interestingly, proteins located at extracellular were more susceptible to be regulated on the redox PTMs level. These findings provide a foundation for further investigation into the redox signaling during plant defense responses.