Quantitative Proteomic Analysis Reveals Changes in the Benchmark Corynebacterium pseudotuberculosis Biovar Equi Exoproteome after Passage in a Murine Host

Corynebacterium pseudotuberculosis biovar equi is the etiologic agent of ulcerative lymphangitis. To investigate proteins that could be related to the virulence of this pathogen, we combined an experimental passage process using a murine model and high-throughput proteomics with a mass spectrometry, data-independent acquisition (LC-MSE) approach to identify and quantify the proteins released into the supernatants of strain 258_equi. To our knowledge, this approach allowed characterization of the exoproteome of a C. pseudotuberculosis equi strain for the first time. Interestingly, the recovery of this strain from infected mouse spleens induced a change in its virulence potential, and it became more virulent in a second infection challenge. Proteomic screening performed from culture supernatant of the control and recovered conditions revealed 104 proteins that were differentially expressed between the two conditions. In this context, proteomic analysis of the recovered condition detected the induction of proteins involved in bacterial pathogenesis, mainly related to iron uptake. In addition, KEGG enrichment analysis showed that ABC transporters, bacterial secretion systems and protein export pathways were significantly altered in the recovered condition. These findings show that secretion and secreted proteins are key elements in the virulence and adaptation of C. pseudotuberculosis. Collectively, bacterial pathogenesis-related proteins were identified that contribute to the processes of adherence, intracellular growth and evasion of the immune system. Moreover, this study enhances our understanding of the factors that may influence the pathogenesis of C. pseudotuberculosis.Fil: Marques Da Silva, Wanderson. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; Brasil. Institut National de la Recherche Agronomique; Francia. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Carvalho, Rodrigo D. De Oliveira. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Dorella, Fernanda A.. Universidade Federal de Minas Gerais; BrasilFil: Folador, Edson L.. Universidade Federal da Paraíba. Centro de Biotecnologia; BrasilFil: Souza, Gustavo H. M. F.. Waters Corporation; BrasilFil: Pimenta, Adriano M. C.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; BrasilFil: Figueiredo, Henrique C. P.. Universidade Federal de Minas Gerais; BrasilFil: Le Loir, Yves. Institut National de la Recherche Agronomique; FranciaFil: Silva, Artur. Universidade Federal do Pará; BrasilFil: Azevedo, Vasco. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; Brasi

Proteomic analysis of Chromobacterium violaceum and its adaptability to stress

Chromobacterium violaceum (C. violaceum) occurs abundantly in a variety of ecosystems, including ecosystems that place the bacterium under stress. This study assessed the adaptability of C. violaceum by submitting it to nutritional and pH stresses and then analyzing protein expression using bi-dimensional electrophoresis (2-DE) and Maldi mass spectrometry. Chromobacterium violaceum grew best in pH neutral, nutrient-rich medium (reference conditions); however, the total protein mass recovered from stressed bacteria cultures was always higher than the total protein mass recovered from our reference culture. The diversity of proteins expressed (repressed by the number of identifiable 2-DE spots) was seen to be highest in the reference cultures, suggesting that stress reduces the overall range of proteins expressed by C. violaceum. Database comparisons allowed 43 of the 55 spots subjected to Maldi mass spectrometry to be characterized as containing a single identifiable protein. Stress-related expression changes were noted for C. violaceum proteins related to the previously characterized bacterial proteins: DnaK, GroEL-2, Rhs, EF-Tu, EF-P; MCP, homogentisate 1,2-dioxygenase, Arginine deiminase and the ATP synthase β-subunit protein as well as for the ribosomal protein subunits L1, L3, L5 and L6. The ability of C. violaceum to adapt its cellular mechanics to sub-optimal growth and protein production conditions was well illustrated by its regulation of ribosomal protein subunits. With the exception of the ribosomal subunit L3, which plays a role in protein folding and maybe therefore be more useful in stressful conditions, all the other ribosomal subunit proteins were seen to have reduced expression in stressed cultures. Curiously, C. violeaceum cultures were also observed to lose their violet color under stress, which suggests that the violacein pigment biosynthetic pathway is affected by stress. Analysis of the proteomic signatures of stressed C. violaceum indicates that nutrient-starvation and pH stress can cause changes in the expression of the C. violaceum receptors, transporters, and proteins involved with biosynthetic pathways, molecule recycling, energy production. Our findings complement the recent publication of the C. violeaceum genome sequence and could help with the future commercial exploitation of C. violeaceum

A sportomics soccer investigation unveils an exercise-induced shift in tyrosine metabolism leading to hawkinsinuria

Tyrosine metabolism has an intense role in the synthesis of neurotransmitters. Our study used an untargeted, sportomics-based analysis of urine samples to investigate changes in metabolism during a soccer match in 30 male junior professional soccer players. Samples were collected before and after the match and analyzed using liquid chromatography and mass spectrometry. Results showed significant changes in tyrosine metabolism. Exercise caused a downregulation of the homogentisate metabolites 4-maleylacetoacetate and succinylacetone to 20% (p = 4.69E−5) and 16% (p = 4.25E−14), respectively. 4-Hydroxyphenylpyruvate, a homogentisate precursor, was found to be upregulated by 26% (p = 7.20E−3). The concentration of hawkinsin and its metabolite 4-hydroxycyclohexyl acetate increased ~six-fold (p = 1.49E−6 and p = 9.81E−6, respectively). Different DOPA metabolism pathways were also affected by exercise. DOPA and dopaquinone increased four-to six-fold (p = 5.62E−14 and p = 4.98E−13, respectively). 3-Methoxytyrosine, indole-5,6-quinone, and melanin were downregulated from 1 to 25%, as were dopamine and tyramine (decreasing to up to 5% or 80%; p= 5.62E−14 and p = 2.47E−2, respectively). Blood TCO2 decreased as well as urinary glutathione and glutamate (40% and 10% respectively) associated with a two-fold increase in pyroglutamate. Our study found unexpected similarities between exercise-induced changes in metabolism and the inherited disorder Hawkinsinuria, suggesting a possible transient condition called exercise-induced hawkinsinuria (EIh). Additionally, our research suggests changes in DOPA pathways may be involved. Our findings suggest that soccer exercise could be used as a model to search for potential countermeasures in Hawkinsinuria and other tyrosine metabolism disorders

Identification of metabolic pathways influenced by the G-protein coupled receptors GprB and GprD in Aspergillus nidulans

Heterotrimeric G-protein-mediated signaling pathways play a pivotal role in transmembrane signaling in eukaryotes. Our main aim was to identify signaling pathways regulated by A. nidulans GprB and GprD G-protein coupled receptors (GPCRs). When these two null mutant strains were compared to the wild-type strain, the DeltagprB mutant showed an increased protein kinase A (PKA) activity while growing in glucose 1% and during starvation. In contrast, the DeltagprD has a much lower PKA activity upon starvation. Transcriptomics and (1)H NMR-based metabolomics were performed on two single null mutants grown on glucose. We noted modulation in the expression of 11 secondary metabolism gene clusters when the DeltagprB and DeltagprD mutant strains were grown in 1% glucose. Several members of the sterigmatocystin-aflatoxin gene cluster presented down-regulation in both mutant strains. The genes of the NR-PKS monodictyphenone biosynthesis cluster had overall increased mRNA accumulation in DeltagprB, while in the DeltagprD mutant strain the genes had decreased mRNA accumulation. Principal component analysis of the metabolomic data demonstrated that there was a significant metabolite shift in the DeltagprD strain. The (1)H NMR analysis revealed significant expression of essential amino acids with elevated levels in the DeltagprD strain, compared to the wild-type and DeltagprB strains. With the results, we demonstrated the differential expression of a variety of genes related mainly to secondary metabolism, sexual development, stress signaling, and amino acid metabolism. We propose that the absence of GPCRs triggered stress responses at the genetic level. The data suggested an intimate relationship among different G-protein coupled receptors, fine-tune regulation of secondary and amino acid metabolisms, and fungal development

SARS-CoV-2 uses CD4 to infect T helper lymphocytes

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent of a major global outbreak of respiratory tract disease known as Coronavirus Disease 2019 (COVID-19). SARS-CoV-2 infects mainly lungs and may cause several immune-related complications, such as lymphocytopenia and cytokine storm, which are associated with the severity of the disease and predict mortality. The mechanism by which SARS-CoV-2 infection may result in immune system dysfunction is still not fully understood. Here, we show that SARS-CoV-2 infects human CD4+ T helper cells, but not CD8+ T cells, and is present in blood and bronchoalveolar lavage T helper cells of severe COVID-19 patients. We demonstrated that SARS-CoV-2 spike glycoprotein (S) directly binds to the CD4 molecule, which in turn mediates the entry of SARS-CoV-2 in T helper cells. This leads to impaired CD4 T cell function and may cause cell death. SARS-CoV-2-infected T helper cells express higher levels of IL-10, which is associated with viral persistence and disease severity. Thus, CD4-mediated SARS-CoV-2 infection of T helper cells may contribute to a poor immune response in COVID-19 patients.</p

SARS-CoV-2 uses CD4 to infect T helper lymphocytes

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the agent of a major global outbreak of respiratory tract disease known as Coronavirus Disease 2019 (COVID-19). SARS-CoV-2 infects mainly lungs and may cause several immune-related complications, such as lymphocytopenia and cytokine storm, which are associated with the severity of the disease and predict mortality. The mechanism by which SARS-CoV-2 infection may result in immune system dysfunction is still not fully understood. Here, we show that SARS-CoV-2 infects human CD4+ T helper cells, but not CD8+ T cells, and is present in blood and bronchoalveolar lavage T helper cells of severe COVID-19 patients. We demonstrated that SARS-CoV-2 spike glycoprotein (S) directly binds to the CD4 molecule, which in turn mediates the entry of SARS-CoV-2 in T helper cells. This leads to impaired CD4 T cell function and may cause cell death. SARS-CoV-2-infected T helper cells express higher levels of IL-10, which is associated with viral persistence and disease severity. Thus, CD4-mediated SARS-CoV-2 infection of T helper cells may contribute to a poor immune response in COVID-19 patients.</p

A list of land plants of Parque Nacional do Caparaó, Brazil, highlights the presence of sampling gaps within this protected area

Brazilian protected areas are essential for plant conservation in the Atlantic Forest domain, one of the 36 global biodiversity hotspots. A major challenge for improving conservation actions is to know the plant richness, protected by these areas. Online databases offer an accessible way to build plant species lists and to provide relevant information about biodiversity. A list of land plants of “Parque Nacional do Caparaó” (PNC) was previously built using online databases and published on the website "Catálogo de Plantas das Unidades de Conservação do Brasil." Here, we provide and discuss additional information about plant species richness, endemism and conservation in the PNC that could not be included in the List. We documented 1,791 species of land plants as occurring in PNC, of which 63 are cited as threatened (CR, EN or VU) by the Brazilian National Red List, seven as data deficient (DD) and five as priorities for conservation. Fifity-one species were possible new ocurrences for ES and MG states