Proteomics of Brucella

Brucella spp. are Gram negative intracellular bacteria responsible for brucellosis, a worldwide distributed zoonosis. A prominent aspect of the Brucella life cycle is its ability to invade, survive and multiply within host cells. Comprehensive approaches, such as proteomics, have aided in unravelling the molecular mechanisms underlying Brucella pathogenesis. Technological and methodological advancements such as increased instrument performance and multiplexed quantification have broadened the range of proteome studies, enabling new and improved analyses, providing deeper and more accurate proteome coverage. Indeed, proteomics has demonstrated its contribution to key research questions in Brucella biology, i.e., immunodominant proteins, host-cell interaction, stress response, antibiotic targets and resistance, protein secretion. Here, we review the proteomics of Brucella with a focus on more recent works and novel findings, ranging from reconfiguration of the intracellular bacterial proteome and studies on proteomic profiles of Brucella infected tissues, to the identification of Brucella extracellular proteins with putative roles in cell signaling and pathogenesis. In conclusion, proteomics has yielded copious new candidates and hypotheses that require future verification. It is expected that proteomics will continue to be an invaluable tool for Brucella and applications will further extend to the currently ill-explored aspects including, among others, protein processing and post-translational modification.Fil: Poetsch, Ansgar. Center For Marine And Molecular Biotechnology; China. Ocean University of China; ChinaFil: Marchesini, Maria Ines. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas. - Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Biotecnológicas; Argentin

A proteomic study of Corynebacterium glutamicum AAA+ protease FtsH

BACKGROUND: The influence of the membrane-bound AAA+ protease FtsH on membrane and cytoplasmic proteins of Corynebacterium glutamicum was investigated in this study. For the analysis of the membrane fraction, anion exchange chromatography was combined with SDS-PAGE, while the cytoplasmic protein fraction was studied by conventional two-dimensional gel electrophoresis. RESULTS: In contrast to the situation in other bacteria, deletion of C. glutamicum ftsH has no significant effect on growth in standard minimal medium or response to heat or osmotic stress. On the proteome level, deletion of the ftsH gene resulted in a strong increase of ten cytoplasmic and membrane proteins, namely biotin carboxylase/biotin carboxyl carrier protein (accBC), glyceraldehyde-3-phosphate dehydrogenase (gap), homocysteine methyltransferase (metE), malate synthase (aceB), isocitrate lyase (aceA), a conserved hypothetical protein (NCgl1985), succinate dehydrogenase A (sdhA), succinate dehydrogenase B (sdhB), succinate dehydrogenase CD (sdhCD), and glutamate binding protein (gluB), while 38 cytoplasmic and membrane-associated proteins showed a decreased abundance. The decreasing amount of succinate dehydrogenase A (sdhA) in the cytoplasmic fraction of the ftsH mutant compared to the wild type and its increasing abundance in the membrane fraction indicates that FtsH might be involved in the cleavage of a membrane anchor of this membrane-associated protein and by this changes its localization. CONCLUSION: The data obtained hint to an involvement of C. glutamicum FtsH protease mainly in regulation of energy and carbon metabolism, while the protease is not involved in stress response, as found in other bacteria

Proteomics Reveal Enhanced Oxidative Stress Responses and Metabolic Adaptation in Acidithiobacillus ferrooxidans Biofilm Cells on Pyrite

Reactive oxygen species (ROS) cause oxidative stress and growth inhibition by inactivation of essential enzymes, DNA and lipid damage in microbial cells. Acid mine drainage (AMD) ecosystems are characterized by low pH values, enhanced levels of metal ions and low species abundance. Furthermore, metal sulfides, such as pyrite and chalcopyrite, generate extracellular ROS upon exposure to acidic water. Consequently, oxidative stress management is especially important in acidophilic leaching microorganisms present in industrial biomining operations, especially when forming biofilms on metal sulfides. Several adaptive mechanisms have been described, but the molecular repertoire of responses upon exposure to pyrite and the presence of ROS are not thoroughly understood in acidophiles. In this study the impact of the addition of H2O2 on iron oxidation activity in Acidithiobacillus ferrooxidans DSM 14882T was investigated. Iron(II)- or sulfur-grown cells showed a higher sensitivity toward H2O2 than pyrite-grown ones. In order to elucidate which molecular responses may be involved, we used shot-gun proteomics and compared proteomes of cells grown with iron(II)-ions against biofilm cells, grown for 5 days in presence of pyrite as sole energy source. In total 1157 proteins were identified. 213 and 207 ones were found to have increased levels in iron(II) ion-grown or pyrite-biofilm cells, respectively. Proteins associated with inorganic sulfur compound (ISC) oxidation were among the latter. In total, 80 proteins involved in ROS degradation, thiol redox regulation, macromolecule repair mechanisms, biosynthesis of antioxidants, as well as metal and oxygen homeostasis were found. 42 of these proteins had no significant changes in abundance, while 30 proteins had increased levels in pyrite-biofilm cells. New insights in ROS mitigation strategies, such as importance of globins for oxygen homeostasis and prevention of unspecific reactions of free oxygen that generate ROS are presented for A. ferrooxidans biofilm cells. Furthermore, proteomic analyses provide insights in adaptations of carbon fixation and oxidative phosphorylation pathways under these two growth conditions

Response of the cytoplasmic and membrane proteome of Corynebacterium glutamicum ATCC 13032 to pH changes

<p>Abstract</p> <p>Background</p> <p><it>C. glutamicum </it>has traditionally been grown in neutral-pH media for amino acid production, but in a previous article we reported that this microorganism is a moderate alkaliphile since it grows optimally at pH 7.0–9.0, as shown in fermentor studies under tightly controlled pH conditions. We determined the best pH values to study differential expression of several genes after acidic or basic pH conditions (pH 6.0 for acidic expression and pH 9.0 for alkaline expression). Thus, it was interesting to perform a detailed analysis of the pH-adaptation response of the proteome of <it>C. glutamicum </it>ATCC 13032 to clarify the circuits involved in stress responses in this bacterium. In this paper we used the above indicated pH conditions, based on transcriptional studies, to confirm that pH adaptation results in significant changes in cytoplasmatic and membrane proteins.</p> <p>Results</p> <p>The cytoplasmatic and membrane proteome of <it>Corynebacterium glutamicum </it>ATCC 13032 at different pH conditions (6.0, 7.0 and 9.0) was analyzed by classical 2D-electrophoresis, and by anion exchange chromatography followed by SDS-PAGE (AIEC/SDS-PAGE). A few cytoplasmatic proteins showed differential expression at the three pH values with the classical 2D-technique including a hypothetical protein <it>cg</it>2797, L-2.3-butanediol dehydrogenase (ButA), and catalase (KatA). The AIEC/SDS-PAGE technique revealed several membrane proteins that respond to pH changes, including the succinate dehydrogenase complex (SdhABCD), F₀F₁-ATP synthase complex subunits b, α and δ (AtpF, AtpH and AtpA), the nitrate reductase II α subunit (NarG), and a hypothetical secreted/membrane protein <it>cg</it>0752. Induction of the F₀F₁-ATP synthase complex β subunit (AtpD) at pH 9.0 was evidenced by Western analysis. By contrast, L-2.3-butanediol dehydrogenase (ButA), an ATPase with chaperone activity, the ATP-binding subunit (ClpC) of an ATP-dependent protease complex, a 7 TMHs hypothetical protein <it>cg</it>0896, a conserved hypothetical protein <it>cg</it>1556, and the dihydrolipoamide acyltransferase SucB, were clearly up-regulated at pH 6.0.</p> <p>Conclusion</p> <p>The observed protein changes explain the effect of the extracellular pH on the growth and physiology of <it>C. glutamicum</it>. Some of the proteins up-regulated at alkaline pH respond also to other stress factors suggesting that they serve to integrate the cell response to different stressing conditions.</p

A guide through the computational analysis of isotope-labeled mass spectrometry-based quantitative proteomics data: an application study

Albaum S, Hahne H, Otto A, et al. A guide through the computational analysis of isotope-labeled mass spectrometry-based quantitative proteomics data: an application study. Proteome Science. 2011;9(1): 30.Background: Mass spectrometry-based proteomics has reached a stage where it is possible to comprehensively analyze the whole proteome of a cell in one experiment. Here, the employment of stable isotopes has become a standard technique to yield relative abundance values of proteins. In recent times, more and more experiments are conducted that depict not only a static image of the up- or down-regulated proteins at a distinct time point but instead compare developmental stages of an organism or varying experimental conditions. Results: Although the scientific questions behind these experiments are of course manifold, there are, nevertheless, two questions that commonly arise: 1) which proteins are differentially regulated regarding the selected experimental conditions, and 2) are there groups of proteins that show similar abundance ratios, indicating that they have a similar turnover? We give advice on how these two questions can be answered and comprehensively compare a variety of commonly applied computational methods and their outcomes. Conclusions: This work provides guidance through the jungle of computational methods to analyze mass spectrometry-based isotope-labeled datasets and recommends an effective and easy-to-use evaluation strategy. We demonstrate our approach with three recently published datasets on Bacillus subtilis [1,2] and Corynebacterium glutamicum [3]. Special focus is placed on the application and validation of cluster analysis methods. All applied methods were implemented within the rich internet application QuPE [4]. Results can be found at http://qupe.cebitec.uni-bielefeld.de webcite

Absolute proteomic quantification reveals design principles of sperm flagellar chemosensation

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Troetschel, C., Hamzeh, H., Alvarez, L., Pascal, R., Lavryk, F., Boenigk, W., Koerschen, H. G., Mueller, A., Poetsch, A., Rennhack, A., Gui, L., Nicastro, D., Struenker, T., Seifert, R., & Kaupp, U. B. Absolute proteomic quantification reveals design principles of sperm flagellar chemosensation. Embo Journal, 39(4), (2020): e102723, doi:10.15252/embj.2019102723.Cilia serve as cellular antennae that translate sensory information into physiological responses. In the sperm flagellum, a single chemoattractant molecule can trigger a Ca2+ rise that controls motility. The mechanisms underlying such ultra‐sensitivity are ill‐defined. Here, we determine by mass spectrometry the copy number of nineteen chemosensory signaling proteins in sperm flagella from the sea urchin Arbacia punctulata. Proteins are up to 1,000‐fold more abundant than the free cellular messengers cAMP, cGMP, H+, and Ca2+. Opto‐chemical techniques show that high protein concentrations kinetically compartmentalize the flagellum: Within milliseconds, cGMP is relayed from the receptor guanylate cyclase to a cGMP‐gated channel that serves as a perfect chemo‐electrical transducer. cGMP is rapidly hydrolyzed, possibly via “substrate channeling” from the channel to the phosphodiesterase PDE5. The channel/PDE5 tandem encodes cGMP turnover rates rather than concentrations. The rate‐detection mechanism allows continuous stimulus sampling over a wide dynamic range. The textbook notion of signal amplification—few enzyme molecules process many messenger molecules—does not hold for sperm flagella. Instead, high protein concentrations ascertain messenger detection. Similar mechanisms may occur in other small compartments like primary cilia or dendritic spines.We thank Heike Krause for preparing the manuscript. Financial support by the Deutsche Forschungsgemeinschaft (DFG) via the priority program SPP 1726 “Microswimmers” and the Cluster of Excellence 1023 “ImmunoSensation” is gratefully acknowledged. We thank D. Stoddard for management of the UTSW cryo‐electron microscope facility, which is funded in part by a Cancer Prevention and Research Institute of Texas (CPRIT) Core Facility Award (RP170644). This study was supported by HHS|National Institutes of Health (NIH) grant R01 GM083122 and by CPRIT grant RR140082 to D. Nicastro

Functional genomics of pH homeostasis in Corynebacterium glutamicum revealed novel links between pH response, oxidative stress, iron homeostasis and methionine synthesis

Follmann M, Ochrombel I, Kraemer R, et al. Functional genomics of pH homeostasis in Corynebacterium glutamicum revealed novel links between pH response, oxidative stress, iron homeostasis and methionine synthesis. BMC Genomics. 2009;10(1):621.Background: The maintenance of internal pH in bacterial cells is challenged by natural stress conditions, during host infection or in biotechnological production processes. Comprehensive transcriptomic and proteomic analyses has been conducted in several bacterial model systems, yet questions remain as to the mechanisms of pH homeostasis. Results: Here we present the comprehensive analysis of pH homeostasis in C. glutamicum, a bacterium of industrial importance. At pH values between 6 and 9 effective maintenance of the internal pH at 7.5 +/- 0.5 pH units was found. By DNA microarray analyses differential mRNA patterns were identified. The expression profiles were validated and extended by 1D-LC-ESI-MS/MS based quantification of soluble and membrane proteins. Regulators involved were identified and thereby participation of numerous signaling modules in pH response was found. The functional analysis revealed for the first time the occurrence of oxidative stress in C. glutamicum cells at neutral and low pH conditions accompanied by activation of the iron starvation response. Intracellular metabolite pool analysis unraveled inhibition of the TCA and other pathways at low pH. Methionine and cysteine synthesis were found to be activated via the McbR regulator, cysteine accumulation was observed and addition of cysteine was shown to be toxic under acidic conditions. Conclusions: Novel limitations for C. glutamicum at non-optimal pH values were identified by a comprehensive analysis on the level of the transcriptome, proteome, and metabolome indicating a functional link between pH acclimatization, oxidative stress, iron homeostasis, and metabolic alterations. The results offer new insights into bacterial stress physiology and new starting points for bacterial strain design or pathogen defense

How, where and when is SPINK3 bound and removed from mouse sperm?

Sperm capacitation in mammals is a fundamental requirement to acquire their fertilizing capacity. Little is known about the action mechanism of the molecules that prevent capacitation from occurring prematurely. These molecules are known as decapacitation factors (DFs) and they must be removed from the sperm surface for capacitation to occur successfully. Serine protease inhibitor Kazal type 3 (SPINK3) has been proposed as one of these DFs. Here, we evaluate how this protein binds to mouse sperm and its removal kinetics. We describe that SPINK3 is capable of binding to the membrane of mature epididymal sperm through protein-lipid interactions, specifically to lipid rafts subcellular fraction. Moreover, cholera toxin subunit b (CTB) avoids SPINK3 binding. We observe that SPINK3 is removed from the sperm under in vitro capacitating conditions and by the uterine fluid from estrus females. Our ex vivo studies show the removal kinetics of this protein within the female tract, losing SPINK3 formerly from the apical region of the sperm in the uterus and later from the flagellar region within the oviduct. The presence of acrosome-reacted sperm in the female duct concurs with the absence of SPINK3 over its surface.Fil: Nicolli, Anabella Rita. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Alonso, Carlos A. I.. McGill University; CanadáFil: Otamendi, Catalina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Cerletti, Micaela. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Poetsch, Ansgar. Ruhr Universität Bochum; AlemaniaFil: Sharma, Vikram. University of Plymouth; Reino UnidoFil: Zalazar, Lucia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; ArgentinaFil: Perez Martinez, Silvina Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Centro de Estudios Farmacológicos y Botánicos. Universidad de Buenos Aires. Facultad de Medicina. Centro de Estudios Farmacológicos y Botánicos; ArgentinaFil: Cesari, Andreina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mar del Plata. Instituto de Investigaciones Biológicas. Universidad Nacional de Mar del Plata. Facultad de Ciencias Exactas y Naturales. Instituto de Investigaciones Biológicas; Argentin

Die Neue Rechte in der Sozialen Arbeit in NRW: Exemplarische Analysen

Die Neue Rechte in der Sozialen Arbeit in NRW - exemplarische Analysen Soziale Arbeit ist eng mit gesellschaftlichen Entwicklungen verbunden. Auch wenn Selbstverständnis und Positionierungen von Profession und Disziplin häufig den Anspruch erheben, neurechten Entwicklungen entgegen zu treten, können sie sich von den aktuellen gesellschaftlichen Verschiebungen in Richtung einer Normalisierung von rechtsnationalen Positionen nicht frei machen. Auf der Basis einer Fragebogenerhebung, einer Expert_innenbefragung und einer Dokumentenanalyse werden das Vorkommen, die Formen und die Strategien der Neuen Rechten in der Sozialen Arbeit in NRW untersucht. Dabei werden sowohl alle Regionen als auch alle Arbeitsbereiche der Sozialen Arbeit berücksichtigt und exemplarische Analysen zum Vorkommen vorgenommen. Auf der Basis der dadurch gewonnenen Daten können sowohl eigene Angebote als auch Einflussnahmen von außerhalb wie innerhalb etablierter Angebote Sozialer Arbeit durch extrem rechte Akteure ausgemacht werden. Dabei wird häufig an Themen angeknüpft, die Anschluss an ethnopluralistische und rassistische, antifeministische oder völkisch-nationale ideologische Narrative ermöglichen. Einfluss- und Landnahmeversuche finden sich in allen Regionen von NRW und vielen verschiedenen Arbeitsfeldern.The Far Right in Social Work of North-Rhine Westphalia - exemplary analyses Social Work is strongly interconnected with societal developments. Even though self-positioning of profession and discipline frequently claim to fight against racist or illiberal ideas in society, they cannot assert to be free of the current normalization of nationalistic and authoritarian positions. On the basis of a questionnaire survey, structured expert interviews and a document analysis, occurrence, forms and strategies of the far right in the social sector of North-Rhine Westphalia are examined. All the regions of that German federal state as well as different fields of social work are incorporated, on which exemplary analyses are realized. On that base, own “social” activities by the far right can be detected as well as exertions of influence from outside and inside of the established social work institutions. These activities often tie on topics which can be connected to ethno-pluralist, racist, anti-feminist or nationalistic narratives. Exertions of influence take place over all regions and fields of social work

ceRNA crosstalk mediated by ncRNAs is a novel regulatory mechanism in fish sex determination and differentiation

Competing endogenous RNAs (ceRNAs) are vital regulators of gene networks in mammals. The involvement of noncoding RNAs (ncRNAs) as ceRNA in genotypic sex determination (GSD) and environmental sex determination (ESD) in fish is unknown. The Chinese tongue sole, which has both GSD and ESD mechanisms, was used to map the dynamic expression pattern of ncRNAs and mRNA in gonads during sex determination and differentiation. Transcript expression patterns shift during the sex differentiation phase, and ceRNA modulation occurs through crosstalk of differentially expressed long ncRNAs (lncRNAs), circular RNAs (circRNAs), microRNAs (miRNAs), and sex-related genes in fish. Of note was the significant up-regulation of a circRNA from the sex-determining gene dmrt1 (circular RNA dmrt1) and a lncRNA, called AMSDT (which stands for associated with male sex differentiation of tongue sole) in Chinese tongue sole testis. These two ncRNAs both share the same miRNA response elements with gsdf, which has an up-regulated expression when they bind to miRNA cse-miR-196 and concurrent down-regulated female sex-related genes to facilitate testis differentiation. This is the first demonstration in fish that ceRNA crosstalk mediated by ncRNAs modulates sexual development and unveils a novel regulatory mechanism for sex determination and differentiation.info:eu-repo/semantics/publishedVersio