Crosstalk between the serine/threonine kinase StkP and the response regulator ComE controls the stress response and intracellular survival of Streptococcus pneumoniae

Streptococcus pneumoniae is an opportunistic human bacterial pathogen that usually colonizes the upper respiratory tract, but the invasion and survival mechanism in respiratory epithelial cells remains elusive. Previously, we described that acidic stress-induced lysis (ASIL) and intracellular survival are controlled by ComE through a yet unknown activation mechanism under acidic conditions, which is independent of the ComD histidine kinase that activates this response regulator for competence development at pH 7.8. Here, we demonstrate that the serine/threonine kinase StkP is essential for ASIL, and show that StkP phosphorylates ComE at Thr128. Molecular dynamic simulations predicted that Thr128-phosphorylation induces conformational changes on ComE’s DNA-binding domain. Using nonphosphorylatable (ComET128A) and phosphomimetic (ComET128E) proteins, we confirmed that Thr128-phosphorylation increased the DNA-binding affinity of ComE. The non-phosphorylated form of ComE interacted more strongly with StkP than the phosphomimetic form at acidic pH, suggesting that pH facilitated crosstalk. To identify the ComE-regulated genes under acidic conditions, a comparative transcriptomic analysis was performed between the comET128Aand wt strains, and differential expression of 104 genes involved in different cellular processes was detected, suggesting that the StkP/ComE pathway induced global changes in response to acidic stress. In the comET128Amutant, the repression of spxB and sodA correlated with decreased H2O2production, whereas the reduced expression of murN correlated with an increased resistance to cell wall antibiotic-induced lysis, compatible with cell wall alterations. In the comET128Amutant, ASIL was blocked and acid tolerance response was higher compared to the wt strain. These phenotypes, accompanied with low H2O2production,are likely responsible for the increased survival in pneumocytes of the comET128Amutant. We propose that the StkP/ComE pathway controls the stress response, thus affecting the intracellular survival of S. pneumoniae in pneumocytes, one of the first barriers that this pathogen must cross to establish an infection.Fil: Piñas, German Eduardo. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Reinoso Vizcaino, Nicolas Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Yandar Barahona, Nubia Yadira. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Cortes, Paulo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Duran, Rosario. Instituto Pasteur de Montevideo; Uruguay. Instituto de Investigaciones Biológicas "Clemente Estable"; UruguayFil: Badapanda, Chandan. Xcelris Lab Limited; IndiaFil: Rathore, Ankita. Xcelris Lab Limited; IndiaFil: Bichara, Darío Román. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Cian, Melina Beatriz. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; ArgentinaFil: Olivero, Nadia Belén. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; ArgentinaFil: Perez, Daniel R.. University of Georgia; Estados UnidosFil: Echenique, Jose Ricardo. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Córdoba. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentin

The pneumococcal two-component system SirRH is linked to enhanced intracellular survival of Streptococcus pneumoniae in influenza-infected pulmonary cells

Artículo científico de investigación en acceso abierto.The virus-bacterial synergism implicated in secondary bacterial infections caused by Streptococcus pneumoniae following infection with epidemic or pandemic influenza A virus (IAV) is well documented. However, the molecular mechanisms behind such synergism remain largely ill-defined. In pneumocytes infected with influenza A virus, subsequent infection with S. pneumoniae leads to enhanced pneumococcal intracellular survival. The pneumococcal two-component system SirRH appears essential for such enhanced survival. Through comparative transcriptomic analysis between the ΔsirR and wt strains, a list of 179 differentially expressed genes was defined. Among those, the clpL protein chaperone gene and the psaB Mn+2 transporter gene, which are involved in the stress response, are important in enhancing S. pneumoniae survival in influenza-infected cells. The ΔsirR, ΔclpL and ΔpsaB deletion mutants display increased susceptibility to acidic and oxidative stress and no enhancement of intracellular survival in IAV-infected pneumocyte cells. These results suggest that the SirRH two-component system senses IAV-induced stress conditions and controls adaptive responses that allow survival of S. pneumoniae in IAV-infected pneumocytes.publishedVersionFil: Cortes, Paulo R. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.Fil: Cian, Melina Beatriz. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.Fil: Olivero, Nadia B. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.Fil: Hernández-Morfa, Mirelys. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.Fil: Piñas, Germán. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.Fil: Badapanda, Chandan. Bioinformatics Division. Xcelris Labs Limited; Ahmedabad; India.Fil: Rathore, Ankita. Bioinformatics Division. Xcelris Labs Limited; Ahmedabad; India.Fil: Echenique, José Ricardo. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.Fil: Reinoso-Vizcaíno, Nicolás Martín. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Departamento de Bioquímica Clínica; Argentina.Fil: Cortes, Paulo R. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Fil: Cian, Melina Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Fil: Olivero, Nadia B. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Fil: Hernández-Morfa, Mirelys. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Fil: Piñas, Germán. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Fil: Echenique, José Ricardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Fil: Reinoso-Vizcaíno, Nicolás Martín. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro de Investigaciones en Bioquímica Clínica e Inmunología; Argentina.Fil: Perez, Daniel R. University of Georgia. Department of Population Health. College of Veterinary Medicine; Athens; Georgia; United States of America.Fil: Cian, Melina Beatriz. University of Oklahoma. Departament of Microbiology and Inmunology. Health Sciences Center; Oklahoma, United States of America.Fil: Piñas, Germán. University of Utah. School of Biological Sciences; Salt Lake City; Utah; United States of America

prepare_taxa_charts.py: A Python program to automate generation of publication ready taxonomic pie chart images from QIIME

QIIME (Quantitative Insights Into Microbial Ecology) is one of the most popular open-source bioinformatics suite for performing metagenome, 16S rRNA amplicon and Internal Transcribed Spacer (ITS) data analysis. Although, it is very comprehensive and powerful tool, it lacks a method to provide publication ready taxonomic pie charts. The script plot_taxa_summary.py bundled with QIIME generate a html file and a folder containing taxonomic pie chart and legend as separate images. The images have randomly generated alphanumeric names. Therefore, it is difficult to associate the pie chart with the legend and the corresponding sample identifier. Even if the option to have the legend within the html file is selected while executing plot_taxa_summary.py, it is very tedious to crop a complete image (having both the pie chart and the legend) due to unequal image sizes. It requires a lot of time to manually prepare the pie charts for multiple samples for publication purpose. Moreover, there are chances of error while identifying the pie chart and legend pair due to random alphanumeric names of the images. To bypass all these bottlenecks and make this process efficient, we have developed a python based program, prepare_taxa_charts.py, to automate the renaming, cropping and merging of taxonomic pie chart and corresponding legend image into a single, good quality publication ready image. This program not only augments the functionality of plot_taxa_summary.py but is also very fast in terms of CPU time and user friendly

Apophysomyces variabilis: draft genome sequence and comparison of predictive virulence determinants with other medically important Mucorales

Abstract Background Apophysomyces species are prevalent in tropical countries and A. variabilis is the second most frequent agent causing mucormycosis in India. Among Apophysomyces species, A. elegans, A. trapeziformis and A. variabilis are commonly incriminated in human infections. The genome sequences of A. elegans and A. trapeziformis are available in public database, but not A. variabilis. We, therefore, performed the whole genome sequence of A. variabilis to explore its genomic structure and possible genes determining the virulence of the organism. Results The whole genome of A. variabilis NCCPF 102052 was sequenced and the genomic structure of A. variabilis was compared with already available genome structures of A. elegans, A. trapeziformis and other medically important Mucorales. The total size of genome assembly of A. variabilis was 39.38 Mb with 12,764 protein-coding genes. The transposable elements (TEs) were low in Apophysomyces genome and the retrotransposon Ty3-gypsy was the common TE. Phylogenetically, Apophysomyces species were grouped closely with Phycomyces blakesleeanus. OrthoMCL analysis revealed 3025 orthologues proteins, which were common in those three pathogenic Apophysomyces species. Expansion of multiple gene families/duplication was observed in Apophysomyces genomes. Approximately 6% of Apophysomyces genes were predicted to be associated with virulence on PHIbase analysis. The virulence determinants included the protein families of CotH proteins (invasins), proteases, iron utilisation pathways, siderophores and signal transduction pathways. Serine proteases were the major group of proteases found in all Apophysomyces genomes. The carbohydrate active enzymes (CAZymes) constitute the majority of the secretory proteins. Conclusion The present study is the maiden attempt to sequence and analyze the genomic structure of A. variabilis. Together with available genome sequence of A. elegans and A. trapeziformis, the study helped to indicate the possible virulence determinants of pathogenic Apophysomyces species. The presence of unique CAZymes in cell wall might be exploited in future for antifungal drug development

Additional file 2: Table S1. of Apophysomyces variabilis: draft genome sequence and comparison of predictive virulence determinants with other medically important Mucorales

Non coding RNAs in genomes of three Apophysomyces species. Table S2. Carbohydrate active enzymes (CAZymes) of Apophysomyces species. Table S3. GenBank accession numbers of whole genome sequences used in phylogenetic analysis of Apophysomyces species and orthoMCL analysis. (DOC 163 kb

Additional file 2: Table S1. of Apophysomyces variabilis: draft genome sequence and comparison of predictive virulence determinants with other medically important Mucorales

Non coding RNAs in genomes of three Apophysomyces species. Table S2. Carbohydrate active enzymes (CAZymes) of Apophysomyces species. Table S3. GenBank accession numbers of whole genome sequences used in phylogenetic analysis of Apophysomyces species and orthoMCL analysis. (DOC 163 kb

Additional file 1: Figure S1. of Apophysomyces variabilis: draft genome sequence and comparison of predictive virulence determinants with other medically important Mucorales

Gene ontology (GO) of Apophysomyces genomes. Figure S2. Phylogenetic analysis of Apophysomyces species with other Mucorales. (DOC 376 kb

Data_Sheet_2_De novo genome assembly of rice bean (Vigna umbellata) – A nominated nutritionally rich future crop reveals novel insights into flowering potential, habit, and palatability centric – traits for efficient domestication.docx

Rice bean is a less-known underutilized legume crop with a high nutritional value among members of the Vigna family. As an initiative to compose rice bean (Vigna umbellata) genomic resource, the size of 414 mega-base pairs with an estimated identification of 31,276 high confidence index genes via 15,521 scaffolds generated from Illumina and PacBio platform 30X coverage data has achieved 96.08% functional coverage data from Illumina and PacBio platform. Rice bean genome assembly was found to be exquisitely close to Vigna angularis (experimental control/outgroup), Vigna radiata, and Vigna unguiculata, however, Vigna angularis being the closest. The assembled genome was further aligned with 31 leguminous plants (13 complete genomes and 18 partial genomes), by collinearity block mapping. Further, we predicted similar discriminant results by complete coding sequence (CDS) alignment. In contrast, 17 medically influential genomes from the National Institute of General Medical Sciences-National Institutes of Health NIGMS-NIH, when compared to rice bean assembly for LCB clusters, led to the identification of more than 18,000 genes from the entire selected medicinal genomes. Empirical construction of all genome comparisons revealed symplesiomorphic character in turn uncovering the lineage of genetic and functional features of rice beans. Significantly, we found deserving late-flowering genes, palatably indexed uncommon genes that regulate various metabolite pathways, related to abiotic and biotic stress pathways and those that are specific to photoperiod and disease resistance and so on. Therefore, the findings from this report address the genomic value of rice bean to be escalated via breeding by allied and applied approaches.</p

Estimates of ComE binding affinities for StkP and StkP^K42M.

<p>Estimates of ComE binding affinities for StkP and StkP^K42M.</p

StkP phosphorylates ComE on the Thr¹²⁸ residue to control ASIL.

<p>(A) To identify the phosphorylation site, tryptic peptides obtained from ComE previously incubated with StkP were analyzed by nano-LC-MS/MS. The figure shows the MS/MS spectrum of the di-charged ion of m/z 618.8 corresponding to the phosphorylated sequence IEQNIFYTK. C-terminal <i>y i</i>ons are labeled in blue, while N-terminal <i>a</i> or <i>b</i> fragment ions are labeled in red. Ions containing pT residue present the phosphorylation characteristic neutral loss of 98 Da. Thr¹²⁸ is unequivocally identified as the phosphorylated residue (Xcorr 3, 45; pRS score 148). (B) StkP phosphorylates ComE at Thr¹²⁸. <i>In vitro</i> phosphorylation assays were performed with purified GST-StkP and His_x6-ComE^wt or His_x6-ComE^T128A proteins mixed in kinase buffer at a StkP/ComE ratio of 1:20. Phosphorylated proteins were detected with an anti-phospho-threonine polyclonal antibody. Lane 1: His_x6-ComE. Lane 2: His_x6-ComE + GST-StkP. Lane 3: His_x6-ComE^T128A. Lane 4: His_x6-ComE^T128A + GST-StkP. (C) ASIL requires the Thr¹²⁸ residue in ComE for lysis induction. Autolysis was determined as indicated in the legend of <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007118#ppat.1007118.g001" target="_blank">Fig 1</a>. Lytic curves corresponding to specific mutants are indicated, which data is representative of at least three independent experiments. References: ***<i>p</i>> 0.001.</p