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

Brucella abortus depends on L-serine biosynthesis for intracellular proliferation

L-Serine is a nonessential amino acid and a key intermediate in several relevant metabolic pathways. In bacteria, the major source of L-serine is the phosphorylated pathway, which comprises three enzymes: D-3-phosphoglycerate dehydrogenase (PGDH; SerA), phosphoserine amino transferase (PSAT; SerC), and L-phosphoserine phosphatase (PSP; SerB). The Brucella abortus genome encodes two PGDHs (SerA-1 and SerA-2), involved in the first step in L-serine biosynthesis, and one PSAT and one PSP, responsible for the second and third steps, respectively. In this study, we demonstrate that the serA1 serA2 double mutant and the serC and serB single mutants are auxotrophic for L-serine. These auxotrophic mutants can be internalized but are unable to replicate in HeLa cells and in J774A.1 macrophage-like cells. Replication defects of auxotrophic mutants can be reverted by cell medium supplementation with L-serine at early times postinfection. In addition, the serB mutant is attenuated in the murine intraperitoneal infection model and has an altered lipid composition, since the lack of L-serine abrogates phosphatidylethanolamine synthesis in this strain. Taken together, these results reveal that limited availability of L-serine within the host cell impairs proliferation of the auxotrophic strains, highlighting the relevance of this biosynthetic pathway in Brucella pathogenicity.Fil: Revora, Virginia. 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; ArgentinaFil: 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; ArgentinaFil: Comerci, Diego José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentina. Comisión Nacional de Energía Atómica. Centro Atómico Ezeiza; Argentin

Brucella abortus Encodes an Active Rhomboid Protease: Proteome Response after Rhomboid Gene Deletion

Rhomboids are intramembrane serine proteases highly conserved in the three domains of life. Their key roles in eukaryotes are well understood but their contribution to bacterial physiology is still poorly characterized. Here we demonstrate that Brucella abortus, the etiological agent of the zoonosis called brucellosis, encodes an active rhomboid protease capable of cleaving model heterologous substrates like Drosophila melanogaster Gurken and Providencia stuartii TatA. To address the impact of rhomboid deletion on B. abortus physiology, the proteomes of mutant and parental strains were compared by shotgun proteomics. About 50% of the B. abortus predicted proteome was identified by quantitative proteomics under two experimental conditions and 108 differentially represented proteins were detected. Membrane associated proteins that showed variations in concentration in the mutant were considered as potential rhomboid targets. This class included nitric oxide reductase sub-unit C NorC (Q2YJT6) and periplasmic protein LptC involved in LPS transport to the outer membrane (Q2YP16). Differences in secretory proteins were also addressed. Differentially represented proteins included a putative lytic murein transglycosylase (Q2YIT4), nitrous-oxide reductase NosZ (Q2YJW2) and high oxygen affinity Cbb3-type cytochrome c oxidase subunit (Q2YM85). Deletion of rhomboid had no obvious effect in B. abortus virulence. However, rhomboid overexpression had a negative impact on growth under static conditions, suggesting an effect on denitrification enzymes and/or high oxygen affinity cytochrome c oxidase required for growth in low oxygen tension conditions.Fil: 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; ArgentinaFil: Poetsch, Ansgar. Ruhr-universität Bochum. Fakultät Für Biologie & Biotechnologie. Lehrstuhl Für Biochemie Der Pflanzen; AlemaniaFil: Guidolin, Leticia Soledad. 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; ArgentinaFil: Comerci, Diego José. 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

VirJ Is a Brucella Virulence Factor Involved in the Secretion of Type IV Secreted Substrates

The VirB secretion apparatus in Brucella belongs to the type IV secretion systems present in many pathogenic bacteria and is absolutely necessary for the efficient evasion of the Brucellacontaining vacuole from the phagocytic route in professional phagocytes. This system is responsible for the secretion of a plethora of effector proteins that alter the biology of the host cell and promote the intracellular replication process. Although many VirB substrates have been identified in Brucella, we still know very little about the secretion mechanism that mediates their translocation across the two membranes and the periplasmic space. In this manuscript, we describe the identification of a gene, virJ, that codes for a protein with periplasmic localization that is involved in the intracellular replication process and virulence in mice. Our analysis revealed that this protein is necessary for the secretion of at least two VirB substrates that have a periplasmic intermediate and that it directly interacts with them. We additionally show that VirJ also associates with the apparatus per se and that its absence affects the assembly of the complex. We hypothesize that VirJ is part of a secretion platform composed of the translocon and several secretion substrates and that it probably coordinates the proper assembly of this macromolecular complex.Fil: del Giudice, Mariela Giselda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Dohmer Pisani, Peter Hans. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Spera, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Laporte, Fernando Tomás. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Marchesini, Maria Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Czibener, Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; ArgentinaFil: Ugalde, Juan Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Universidad Nacional de San Martín. Instituto de Investigaciones Biotecnológicas; Argentin

Incidence of severe critical events in paediatric anaesthesia (APRICOT): a prospective multicentre observational study in 261 hospitals in Europe

Background Little is known about the incidence of severe critical events in children undergoing general anaesthesia in Europe. We aimed to identify the incidence, nature, and outcome of severe critical events in children undergoing anaesthesia, and the associated potential risk factors. Methods The APRICOT study was a prospective observational multicentre cohort study of children from birth to 15 years of age undergoing elective or urgent anaesthesia for diagnostic or surgical procedures. Children were eligible for inclusion during a 2-week period determined prospectively by each centre. There were 261 participating centres across 33 European countries. The primary endpoint was the occurence of perioperative severe critical events requiring immediate intervention. A severe critical event was defined as the occurrence of respiratory, cardiac, allergic, or neurological complications requiring immediate intervention and that led (or could have led) to major disability or death. This study is registered with ClinicalTrials.gov, number NCT01878760. Findings Between April 1, 2014, and Jan 31, 2015, 31â127 anaesthetic procedures in 30â874 children with a mean age of 6·35 years (SD 4·50) were included. The incidence of perioperative severe critical events was 5·2% (95% CI 5·0â5·5) with an incidence of respiratory critical events of 3·1% (2·9â3·3). Cardiovascular instability occurred in 1·9% (1·7â2·1), with an immediate poor outcome in 5·4% (3·7â7·5) of these cases. The all-cause 30-day in-hospital mortality rate was 10 in 10â000. This was independent of type of anaesthesia. Age (relative risk 0·88, 95% CI 0·86â0·90; p<0·0001), medical history, and physical condition (1·60, 1·40â1·82; p<0·0001) were the major risk factors for a serious critical event. Multivariate analysis revealed evidence for the beneficial effect of years of experience of the most senior anaesthesia team member (0·99, 0·981â0·997; p<0·0048 for respiratory critical events, and 0·98, 0·97â0·99; p=0·0039 for cardiovascular critical events), rather than the type of health institution or providers. Interpretation This study highlights a relatively high rate of severe critical events during the anaesthesia management of children for surgical or diagnostic procedures in Europe, and a large variability in the practice of paediatric anaesthesia. These findings are substantial enough to warrant attention from national, regional, and specialist societies to target education of anaesthesiologists and their teams and implement strategies for quality improvement in paediatric anaesthesia. Funding European Society of Anaesthesiology

Incidence of severe critical events in paediatric anaesthesia (APRICOT): a prospective multicentre observational study in 261 hospitals in Europe

Background Little is known about the incidence of severe critical events in children undergoing general anaesthesia in Europe. We aimed to identify the incidence, nature, and outcome of severe critical events in children undergoing anaesthesia, and the associated potential risk factors. Methods The APRICOT study was a prospective observational multicentre cohort study of children from birth to 15 years of age undergoing elective or urgent anaesthesia for diagnostic or surgical procedures. Children were eligible for inclusion during a 2-week period determined prospectively by each centre. There were 261 participating centres across 33 European countries. The primary endpoint was the occurence of perioperative severe critical events requiring immediate intervention. A severe critical event was defined as the occurrence of respiratory, cardiac, allergic, or neurological complications requiring immediate intervention and that led (or could have led) to major disability or death. This study is registered with ClinicalTrials.gov, number NCT01878760. Findings Between April 1, 2014, and Jan 31, 2015, 31 127 anaesthetic procedures in 30 874 children with a mean age of 6.35 years (SD 4.50) were included. The incidence of perioperative severe critical events was 5.2% (95% CI 5.0-5.5) with an incidence of respiratory critical events of 3.1% (2.9-3.3). Cardiovascular instability occurred in 1.9% (1.7-2.1), with an immediate poor outcome in 5.4% (3.7-7.5) of these cases. The all-cause 30-day in-hospital mortality rate was 10 in 10 000. This was independent of type of anaesthesia. Age (relative risk 0.88, 95% CI 0.86-0.90; p<0.0001), medical history, and physical condition (1.60, 1.40-1.82; p<0.0001) were the major risk factors for a serious critical event. Multivariate analysis revealed evidence for the beneficial effect of years of experience of the most senior anaesthesia team member (0.99, 0.981-0.997; p<0.0048 for respiratory critical events, and 0.98, 0.97-0.99; p=0.0039 for cardiovascular critical events), rather than the type of health institution or providers. Interpretation This study highlights a relatively high rate of severe critical events during the anaesthesia management of children for surgical or diagnostic procedures in Europe, and a large variability in the practice of paediatric anaesthesia. These findings are substantial enough to warrant attention from national, regional, and specialist societies to target education of anaesthesiologists and their teams and implement strategies for quality improvement in paediatric anaesthesia