Cold atmospheric plasma (CAP) has been shown to be effective against a variety of microorganisms. In this study, we described effects on an Escherichia coli strain isolated from hospital wastewater caused by a treatment with physical plasma. E. coli GW-AmxH19 was incubated in artificial wastewater and treated for 15 minutes with CAP. Transcriptomes and proteomes were monitored at different timepoints within a 24 h period to differentiate between immediate physiological responses and adaptations in the recovery phase. Reduction of viable cells was on average at 90%. The short-term response of the surviving cells to physical plasma aims at repairing and protecting cellular structures from plasma-induced damages, mainly provoked by reactive oxygen and nitrogen species. Notably, CAP induced a temporary transcription of genes from a conjugative plasmid carrying antibiotic resistance determinants. The late response during recovery phase is dominated by a massive activation of two prophages turning cold plasma treatment into a novel possible strategy to induce the lytic cycle of prophages. This study is the first report on the combined analysis of transcriptional and translational effects of CAP on an environmental bacterial isolate in a time-resolved manner. Chances and risks of considering CAP as an additional purification step in wastewater treatment plants are depicted and discussed. ### Competing Interest Statement The authors have declared no competing interest. Bundesministerium für Bildung und Forschung (BMBF) in the ANTIRES2.0 project, BMBF grant 03ZZ0815A/B

Climate change with increasing ambient temperatures negatively influences the biology of dairy cows and their milk production in the mammary gland (MG). This study aimed to elucidate the MG proteome, differences in milk composition, and ruminal short-chain fatty acid concentrations of dairy cows experiencing 7 days of heat stress [HS, 28 °C, temperature humidity index (THI) = 76], pair-feeding (PF), or ad libitum feeding (CON) at thermoneutrality (16 °C, THI = 60). Ruminal acetate, acetate/propionate ratio, and milk urea concentrations were greater, whereas milk protein and lactose were lower in HS than in control cows. Proteome analysis revealed an induced bacterial invasion of epithelial cells, leukocyte transendothelial migration, reduction of the pyruvate and carbon metabolism, and platelet activation in the MG of HS compared to CON or PF cows. These results highlight adaptive metabolic and immune responses to mitigate the negative effects of ambient heat in the MG.

Heat stress causes barrier dysfunction and inflammation of the small intestine of several species. However, less is known about the molecular and cellular mechanisms underlying the response of the bovine large intestine to hyperthermia. We aimed to identify changes in the colon of dairy cows in response to constant heat stress using a proteomic approach. Eighteen lactating Holstein dairy cows were kept under constant thermoneutral conditions (16°C and 68% relative humidity (RH); temperature-humidity-index (THI) = 60) for 6 d (period 1) with free access to feed and water. Thereafter, 6 cows were equally allocated to: 1) thermoneutral condition with ad libitum feeding (TNAL; 16°C, RH = 68%, THI = 60), 2) heat-stressed condition (HS; 28°C, RH = 50%, THI = 76) with ad libitum feeding, or 3) pair-feeding at thermoneutrality (TNPF; 16°C, RH = 68%, THI = 60) for another 7 d (period 2). Rectal temperature, milk yield, dry matter and water intake were monitored daily. Then, cows were sacrificed and colon mucosa samples were taken for proteomic analysis. Physiological data were analyzed by ANOVA and colon proteome data was processed using DESeq2 package in R. Rectal temperature was significantly higher in HS than in TNPF and TNAL cows in period 2. Proteomic analysis revealed an enrichment of activated pathways related to colonic barrier function and inflammation, heat shock proteins, amino acid metabolism, reduced overall protein synthesis rate, and post-transcriptional regulation induced by heat stress. Further regulations were found for enzymes of the tricarboxylic acid cycle and components of the mitochondrial electron transport chain, presumably to reduce the generation of reactive oxygen species, maintain cellular ATP levels, and prevent apoptosis in the colon of HS cows. These results highlight the cellular, extracellular, and mitochondrial adaptations of the colon during heat stress and suggest a dysfunction of the hindgut barrier integrity potentially resulting in a ‘leaky’ colon.

The Escherichia coli TetR-related transcriptional regulator RutR is involved in the coordination of pyrimidine and purine metabolism. Here we report that lysine acetylation modulates RutR function. Applying the genetic code expansion concept, we produced site-specifically lysine-acetylated RutR proteins. The crystal structure of lysine-acetylated RutR reveals how acetylation switches off RutR-DNA-binding. We apply the genetic code expansion concept in E. coli in vivo revealing the consequences of RutR acetylation on the transcriptional level. We propose a model in which RutR acetylation follows different kinetic profiles either reacting non-enzymatically with acetyl-phosphate or enzymatically catalysed by the lysine acetyltransferases PatZ/YfiQ and YiaC. The NAD + -dependent sirtuin deacetylase CobB reverses enzymatic and non-enzymatic acetylation of RutR playing a dual regulatory and detoxifying role. By detecting cellular acetyl-CoA, NAD + and acetyl-phosphate, bacteria apply lysine acetylation of transcriptional regulators to sense the cellular metabolic state directly adjusting gene expression to changing environmental conditions.

Classical Zn2+-dependent deac(et)ylases play fundamental regulatory roles in life and are well characterized in eukaryotes regarding their structures, substrates and physiological roles. In bacteria, however, classical deacylases are less well understood. We construct a Generalized Profile (GP) and identify thousands of uncharacterized classical deacylases in bacteria, which are grouped into five clusters. Systematic structural and functional characterization of representative enzymes from each cluster reveal high functional diversity, including polyamine deacylases and protein deacylases with various acyl-chain type preferences. These data are supported by multiple crystal structures of enzymes from different clusters. Through this extensive analysis, we define the structural requirements of substrate selectivity, and discovered bacterial de-d-/l-lactylases and long-chain deacylases. Importantly, bacterial deacylases are inhibited by archetypal HDAC inhibitors, as supported by co-crystal structures with the inhibitors SAHA and TSA, and setting the ground for drug repurposing strategies to fight bacterial infections. Thus, we provide a systematic structure-function analysis of classical deacylases in bacteria and reveal the basis of substrate specificity, acyl-chain preference and inhibition. In bacteria Zn2+-dependent deacylases are underexplored. Here, the authors identify bacterial deacylases, providing systemic structure-function analyses to reveal the basis of substrate specificity, acyl-chain preference and inhibition.

IntroductionThe environmental bacterium Burkholderia pseudomallei causes the often fatal and massively underreported infectious disease melioidosis. Antigens inducing protective immunity in experimental models have recently been identified and serodiagnostic tools have been improved. However, further elucidation of the antigenic repertoire of B. pseudomallei during human infection for diagnostic and vaccine purposes is required. The adaptation of B. pseudomallei to very different habitats is reflected by a huge genome and a selective transcriptional response to a variety of conditions. We, therefore, hypothesized that exposure of B. pseudomallei to culture conditions mimicking habitats encountered in the human host might unravel novel antigens that are recognized by melioidosis patients.Methods and resultsIn this study, B. pseudomallei was exposed to various stress and growth conditions, including anaerobiosis, acid stress, oxidative stress, iron starvation and osmotic stress. Immunogenic proteins were identified by probing two-dimensional Western blots of B. pseudomallei intracellular and extracellular protein extracts with sera from melioidosis patients and controls and subsequent MALDI-TOF MS. Among B. pseudomallei specific immunogenic signals, 90 % (55/61) of extracellular immunogenic proteins were identified by acid, osmotic or oxidative stress. A total of 84 % (44/52) of intracellular antigens originated from the stationary growth phase, acidic, oxidative and anaerobic conditions. The majority of the extracellular and intracellular protein antigens were identified in only one of the various stress conditions. Sixty-three immunoreactive proteins and an additional 38 candidates from a literature screening were heterologously expressed and subjected to dot blot analysis using melioidosis sera and controls. Our experiments confirmed melioidosis-specific signals in 58 of our immunoproteome candidates. These include 15 antigens with average signal ratios (melioidosis:controls) greater than 10 and another 26 with average ratios greater than 5, including new promising serodiagnostic candidates with a very high signal-to-noise ratio.ConclusionOur study shows that a comprehensive B. pseudomallei immunoproteomics approach, using conditions which are likely to be encountered during infection, can identify novel antibody targets previously unrecognized in human melioidosis.

The concept of donor-unrestricted T cells (DURTs) comprises a heterogeneity of lymphoid cells that respond to an abundance of unconventional epitopes in a non-MHC-restricted manner. Vaccinologists strive to harness this so far underexplored branch of the immune system for new vaccines against tuberculosis. A particular division of DURTs are T cells that recognize their cognate lipid antigen in the context of CD1-molecules. Mycobacteria are characterized by a particular lipid-rich cell wall. Several of these lipids have been shown to be presented to T cells via CD1b-molecules. Guinea pigs functionally express CD1b and are hence an appropriate small animal model to study the role of CD1b-restricted, lipid-specific immune responses. In the current study, guinea pigs were vaccinated with BCG or highly-purified, liposome-formulated phosphatidylinositol-hexa-mannoside (PIM6) to assess the effect of CD1-restricted DURTs on the course of infection after virulent Mycobacterium tuberculosis (Mtb) challenge. Robust PIM6-specific T cell-responses were observed both after BCG- and PIM6-vaccination. The cellular response was significantly reduced in the presence of monoclonal, CD1b-blocking antibodies, indicating that a predominant part of this reactivity was CD1b-restricted. When animals were challenged with Mtb, BCG- and PIM6-vaccinated animals showed significantly reduced pathology, smaller necrotic granulomas in lymph node and spleen and reduced bacterial loads. While BCG conferred an almost sterile protection in this setting, compared to control animals' lesions were reduced roughly by two thirds in PIM6-vaccinated. Comprehensive histological and transcriptional analyses in the draining lymph node revealed that protected animals showed reduced transcription-levels of inflammatory cyto- and chemokines and higher levels of CD1b-expression on professional antigen cells compared to controls. Although BCG as a comparator induced by far stronger effects, our observations in the guinea pig model suggest that CD1b-restricted, PIM6-reactive DURTs contribute to immune-mediated containment of virulent Mtb.

Milk urea concentration is an indicator for dietary nitrogen (N)-supply and urinary N-excretion. Dairy cows with high (HMU) compared to low milk urea (LMU) concentration have greater plasma urea, creatinine and uric acid concentrations, but if the liver metabolism accounts for these differences is unknown. Eighteen HMU and 18 LMU cows were fed a diet with a low (LP) or normal (NP) crude protein concentration. A N balance study was performed and a 13C-urea bolus was administered to measure urea pool size. Liver samples were analyzed by 2D-gel-based proteomics and RT-qPCR. Although HMU cows had a greater urea pool, plasma urea, uric acid, and hippuric acid concentrations, these differences were not associated with altered expressions of genes related to urea cycling or N-metabolism. Instead, HMU cows had higher oxidative stress levels. Conclusively, other factors than hepatic urea metabolism account for milk urea concentrations. Despite higher plasma urea concentrations and argininosuccinate synthase 1 protein expression on the LP diet, urea cycle mRNA expressions were not affected, indicating that its activity is not controlled at transcriptional level. Feeding the LP diet resulted in increased expressions of enzymes catabolizing fatty acids, but the reason remains to be investigated in future studies.

The biotechnologically interesting bacterium Cupriavidus necator H16 was thoroughly investigated. Fifteen years ago, it was sequenced entirely and annotated (A.

Respiratory tract infections are a worldwide health problem for humans and animals. Different cell types produce lipid mediators in response to infections, which consist of eicosanoids like hydroxyeicosatetraenoic acids (HETEs) or oxylipins like hydroxydocosahexaenoic acids (HDHAs). Both substance classes possess immunomodulatory functions. However, little is known about their role in respiratory infections. Here, we aimed to analyze the lipid mediator imprint of different organs of C57BL/6J mice after intranasal mono-infections with Streptococcus pneumoniae (pneumococcus), Staphylococcus aureus or Influenza A virus (IAV) as wells as pneumococcal-IAV co-infection. C57BL/6J mice were infected with different pathogens and lungs, spleen, and plasma were collected. Lipid mediators were analyzed using HPLC-MS/MS. In addition, spatial-distribution of sphingosine 1-phosphate (S1P) and ceramide 1-phosphates (C1P) in tissue samples was examined using MALDI-MS-Imaging. The presence of bacterial pathogens in the lung was confirmed via immunofluorescence staining. We found IAV specific changes for different HDHAs and HETEs in mouse lungs as well as enhanced levels of 20-HETE in severe S. aureus infection. Moreover, MALDI-MS-Imaging analysis showed an accumulation of C1P and a decrease of S1P during co-infection in lung and spleen. Long chain C1P was enriched in the red and not in the white pulp of the spleen. Lipid mediator analysis showed that host synthesis of bioactive lipids is in part specific for a certain pathogen, in particular for IAV infection. Furthermore, MS-Imaging displayed great potential to study infections and revealed changes of S1P and C1P in lungs and spleen of co-infected animals, which was not described before.

The neritid snail Theodoxus fluviatilis has formed regional subgroups in northern Europe, where it appears in both freshwater (FW) and brackish water (BW) in coastal areas of the Baltic Sea. These ecotypes show clear differences in osmotolerance and in the modes of accumulating organic osmolytes under hyperosmotic stress. We reasoned that the expression patterns of soluble proteins in the two ecotypes may differ as well. BW snails have to deal with a higher salinity (up to 20 parts per thousand) than FW snails (0.5 parts per thousand) and also cope with frequent fluctuations in environmental salinity that occur after heavy rains or evaporation caused by extended periods of intense sunshine. Therefore, the protein expression patterns of specimens collected at five different FW and BW sites were analyzed using 2D SDS-PAGE, mass spectrometry, and sequence comparisons based on a transcriptome database for Theodoxus fluviatilis. We identified 89 differentially expressed proteins. The differences in the expression between FW and BW snails may be due to phenotypic plasticity, but may also be determined by local genetic adaptations. Among the differentially expressed proteins, 19 proteins seem to be of special interest as they may be involved in mediating the higher tolerance of BW animals towards environmental change compared with FW animals.

Climate change affects the duration and intensity of heat waves during summer months and jeopardizes animal health and welfare. High ambient temperatures cause heat stress in dairy cows resulting in a reduction of milk yield, feed intake, and alterations in gut barrier function. The objectives of this study were to investigate the mucosal amino acid, glucose and lactate metabolism, as well as the proteomic response of the small intestine in heat stressed (HS) Holstein dairy cows. Cows of the HS group (n = 5) were exposed for 4 days to 28 °C (THI = 76) in a climate chamber. Percentage decrease in daily ad libitum intake of HS cows was calculated to provide isocaloric energy intake to pair-fed control cows kept at 15 °C (THI = 60) for 4 days. The metabolite, mRNA and proteomic analyses revealed that HS induced incorrect protein folding, cellular destabilization, increased proteolytic degradation and protein kinase inhibitor activity, reduced glycolysis, and activation of NF-κB signaling, uronate cycling, pentose phosphate pathway, fatty acid and amino acid catabolism, mitochondrial respiration, ATPase activity and the antioxidative defence system. Our results highlight adaptive metabolic and immune mechanisms attempting to maintain the biological function in the small intestine of heat-stressed dairy cows.

Global warming and accompanying high ambient temperatures reduce feed intake of dairy cows and shift the blood flow from the core of the body to the periphery. As a result, hypoxia may occur in the digestive tract accompanied by disruption of the intestinal barrier, local endotoxemia and inflammation, and altered nutrient absorption. However, whether the barrier of the rumen, like the intestine, is affected by ambient heat has not been studied so far. Lactating Holstein dairy cows were subjected to heat stress at 28°C (temperature-humidity index = 76; n = 5) with ad libitum feed intake or to thermoneutral conditions at 15°C (temperature-humidity index = 60; n = 5) and pair-feeding to heat-stressed animals for a total of 4 d. Gas exchange and feed intake behavior were measured in a respiration chamber, and rumen epithelia were taken after slaughter. Heat stress significantly reduced meal size and whole-body fat oxidation but increased meal frequency and carbohydrate oxidation. The mRNA expression of toll-like receptor 4 (TLR4) and tight junction proteins and the phosphorylation of TLR4 downstream targets (interleukin-1 receptor-associated kinase 4, stress-activated protein kinase, p38 mitogen-activated protein kinase, and nuclear factor k-B) in the rumen epithelium were not affected by heat. The proteomics approach revealed increased expression of rumen epithelium proteins involved in the AMP-activated protein kinase (AMPK) and insulin signaling pathways in heat-stressed cows. Also, proteins involved in chaperone-mediated folding of proteins were upregulated, whereas those involved in antioxidant defense system were downregulated. Further, we found evidence for increased carbohydrate phosphorylation accompanied with an increased flux of carbohydrates through the hexosamine biosynthetic pathway, providing substrates for protein glycosylation. In conclusion, the mild heat stress did not induce barrier dysfunction or inflammatory responses in the rumen epithelium of dairy cows, probably because of adaptations in feed intake behavior and defense mechanisms at the tissue level.

In food chain, Pseudomonas spp. cause spoilage by reducing shelf life of fresh products, especially during cold storage, with a high economic burden for industries. However, recent studies have shed new light on health risks occurring when they colonize immunocompromised patient tissues. Likewise to P. aeruginosa, they exhibit antibiotic resistance and biofilm formation, responsible for their spread and persistence in the environment. Biofilm formation might be induced by environmental stresses, such as temperature fluctuations causing physiological and metabolic changes exacerbating food spoilage (by protease and pigment synthesis), and the production of adhesion molecules, chemotactic or underestimated virulence factors. In order to provide a new insight into phenotypic biodiversity of Pseudomonas spoilers isolated from cold stored cheese, in this work 19 Pseudomonas spp. were investigated for biofilm, pigments, exopolysaccharide production and motility at low temperature. Only nine strains showed these phenotypic traits and the blue pigmenting cheese strain P. fluorescens ITEM 17298 was the most distinctive. In addition, this strain decreased the survival probability of infected Galleria mellonella larvae, showing, for the first time, a pathogenic potential. Genomic and proteomic analyses performed on the ITEM 17298 planktonic cells treated or not with lactoferrin derived antibiofilm peptides allowed to reveal specific biofilm related-pathways as well as proteins involved in pathogenesis. Indeed, several genes were found related to signaling system by cGMP-dependent protein kinases, cellulose, rhamnolipid and alginate synthesis, antibiotic resistance, adhesion and virulence factors. The proteome of the untreated ITEM 17298, growing at low temperature, showed that most of the proteins associated with biofilm regulation, pigmentation motility, antibiotic resistance and pathogenecity were repressed, or decreased their levels in comparison to that of the untreated cultures. Thus, the results of this work shed light on the complex pathways network allowing psychrotrophic pseudomonads to adapt themselves to food-refrigerated conditions and enhance their spoilage. In addition, the discovery of virulence factors and antibiotic resistance determinants raises some questions about the need to deeper investigate these underestimated bacteria in order to increase awareness and provide input to update legislation on their detection limits in foods.

This study aimed to establish a robust and reliable metaproteomics protocol for an in-depth characterization of marine particle-associated (PA) bacteria. To this end, we compared six well-established protein extraction protocols together with different MS-sample preparation techniques using particles sampled during a North Sea spring algae bloom in 2009. In the final optimized workflow, proteins are extracted using a combination of SDS-containing lysis buffer and cell disruption by bead-beating, separated by SDS-PAGE, in-gel digested and analysed by LC-MS/MS, before MASCOT search against a metagenome-based database and data processing/visualization with the in-house-developed bioinformatics tools Prophane and Paver. As an application example, free-living (FL) and particulate communities sampled in April 2009 were analysed, resulting in an as yet unprecedented number of 9354 and 5034 identified protein groups for FL and PA bacteria, respectively. Our data suggest that FL and PA communities appeared similar in their taxonomic distribution, with notable exceptions: eukaryotic proteins and proteins assigned to Flavobacteriia, Cyanobacteria, and some proteobacterial genera were found more abundant on particles, whilst overall proteins belonging to Proteobacteria were more dominant in the FL fraction. Furthermore, our data points to functional differences including proteins involved in polysaccharide degradation, sugar- and phosphorus uptake, adhesion, motility, and stress response.

As proof of principle, free-living (FL) and particulate communities sampled in April 2009 were analysed, resulting in an as yet unprecedented number of 9,354 and 5,034 identified protein groups for FL and PA bacteria, respectively. Our data revealed that FL and PA communities appeared similar in their taxonomic distribution, with notable exceptions: eukaryotic proteins and proteins assigned to , , and some proteobacterial genera were found more abundant on particles, whilst overall proteins belonging to were more dominant in the FL fraction. In contrast, significant functional differences including proteins involved in polysaccharide degradation, sugar- and phosphorus uptake, adhesion, motility, and stress response were detected.

Two-dimensional electrophoresis as a complementary approach to gel-free proteomic methods possesses the ability to separate physiologically important isoforms of proteins in an unbiased manner. Frequently, those isoforms are low-abundant regulators, and therefore, detection and identification of low-abundant proteins is highly necessary to exploit this advantage. We describe an experimental sequence of classical operations to process gels but optimized them, in order to identify each detectable protein spot on gel.

Purpose: Increasing incidence of onychomycosis and tinea pedis in humans of industrialized countries together with deep tissue infections are a therapeutic challenge in clinical mycology. For a better understanding of the pathology and immunology of infection, the authors analyze the exoproteomes of three reference strains of the most common clinical dermatophyte species (Trichophyton rubrum, Trichophyton interdigitale, Arthroderma benhamiae) and of Trichophyton strains isolated from affected patients. Experimental design: Extracellular proteins of those in vitro grown strains are separated via 2D High Performance Electrophoresis and identified by mass spectrometry to find proteins with provoked host immune reactivity. Results: More than 80 secreted proteins including virulence factors such as peptidases and other hydrolases are identified. By Western blotting with respective patient sera, up to 31 proteins with significant antigen-antibody reactions are detected in comparison with control sera, for example, peptidases as well as several oxidoreductases. One protein, beta-glucosidase F2SZI9 seems to be a commonly processed antigen in all Trichophyton infections. Conclusions and clinical relevance: These first global exoproteome data of three dermatophyte species can be a stepping stone on the way to further study the molecular mechanisms of Trichophyton pathogenicity-associated traits. Possible candidates for potential new diagnostic methods or vaccination have to be validated in further investigations.

The anaerobic pathogen Clostridium difficile is of growing significance for the health care system due to its increasing incidence and mortality. As C. difficile infection is both supported and treated by antibiotics, a deeper knowledge on how antimicrobial agents affect the physiology of this important pathogen may help to understand and prevent the development and spreading of antibiotic resistant strains. As the proteomic response of a cell to stress aims at counteracting the harmful effects of this stress, it can be expected that the pattern of a pathogen's responses to antibiotic treatment will be dependent on the antibiotic mechanism of action. Hence, every antibiotic treatment is expected to result in a specific proteomic signature characterizing its mode of action. In the study presented here, the proteomic response of C. difficile 630∆erm to vancomycin, metronidazole, and fidaxomicin stress was investigated on the level of protein abundance and protein synthesis based on 2D PAGE. The quantification of 425 proteins of C. difficile allowed the deduction of proteomic signatures specific for each drug treatment. Indeed, these proteomic signatures indicate very specific cellular responses to each antibiotic with only little overlap of the responses. Whereas signature proteins for vancomycin stress fulfil various cellular functions, the proteomic signature of metronidazole stress is characterized by alterations of proteins involved in protein biosynthesis and protein degradation as well as in DNA replication, recombination, and repair. In contrast, proteins differentially expressed after fidaxomicin treatment can be assigned to amino acid biosynthesis, transcription, cell motility, and the cell envelope functions. Notably, the data provided by this study hint also at so far unknown antibiotic detoxification mechanisms.