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Table S1. Memory and CPU time requirement of multiple PanACEA runs on a 2.3GHz Linux VM. Figure S1. PanACEA HTML page flowchart. (DOCX 22 kb

Pathogenomic Inference of Virulence-Associated Genes in <i>Leptospira interrogans</i>

<div><p>Leptospirosis is a globally important, neglected zoonotic infection caused by spirochetes of the genus <i>Leptospira</i>. Since genetic transformation remains technically limited for pathogenic <i>Leptospira</i>, a systems biology pathogenomic approach was used to infer leptospiral virulence genes by whole genome comparison of culture-attenuated <i>Leptospira interrogans</i> serovar Lai with its virulent, isogenic parent. Among the 11 pathogen-specific protein-coding genes in which non-synonymous mutations were found, a putative soluble adenylate cyclase with host cell cAMP-elevating activity, and two members of a previously unstudied ∼15 member paralogous gene family of unknown function were identified. This gene family was also uniquely found in the alpha-proteobacteria <i>Bartonella bacilliformis</i> and <i>Bartonella australis</i> that are geographically restricted to the Andes and Australia, respectively. How the pathogenic <i>Leptospira</i> and these two <i>Bartonella</i> species came to share this expanded gene family remains an evolutionary mystery. <i>In vivo</i> expression analyses demonstrated up-regulation of 10/11 <i>Leptospira</i> genes identified in the attenuation screen, and profound <i>in vivo</i>, tissue-specific up-regulation by members of the paralogous gene family, suggesting a direct role in virulence and host-pathogen interactions. The pathogenomic experimental design here is generalizable as a functional systems biology approach to studying bacterial pathogenesis and virulence and should encourage similar experimental studies of other pathogens.</p></div

Phylogenetic and in vivo gene expression analysis of the PF07598 paralogous gene family shared by pathogenic <i>Leptospira</i> and <i>Bartonella bacilliformis</i>.

<p>(<b>A</b>) Distribution of the paralogous gene family shared by <i>Leptospira</i> and <i>Bartonella bacilliformis</i> in the genus <i>Leptospira</i>. P, pathogen; I, intermediate; S, saprophyte. (<b>B</b>) An unrooted phylogenetic tree was constructed of protein sequences from all identifiable homologs of the DUF1561 protein family found in GenBank and the PATRIC databases, which included predicted sequences from the following bacteria (<i>Helicobacter</i> spp. and <i>B. bacilliformis</i> genome locus tags and protein sequences used for constructing the tree are listed in Table S4 in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002468#pntd.0002468.s001" target="_blank">Text S1</a>): <i>L. interrogans</i> Lai, <i>L. borgpeterseni</i> Hardjo; <i>Helicobacter cetorum</i>, <i>H. hepaticus</i> and <i>H. mustelae</i>; and <i>B. bacilliformis</i> full-length sequences were aligned using MAFFT. Node labels represent support from 500 bootstrap replicates. Tree drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated. Analyses were conducted in MEGA5. (<b>C–E</b>) <i>In vivo</i> relevance of the leptospiral paralogous gene family was assessed in the acute hamster infection model as described in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002468#pntd-0002468-g001" target="_blank">Fig. 1</a>. Transcript levels of the genes were assessed by real time, reverse transcriptase quantitative PCR of blood, liver and kidney 4 days after hamster infection and compared to log phase <i>in vitro</i> cultured <i>Leptospira</i>. Leptospiral gene expression levels in infected tissue vs. EMJH medium alone were expressed logarithmically as the log₂ of the fold change between the two conditions. Solid bars indicate proteins containing predicted signal peptides that suggest extracellular presence, i.e. secretion or cell-surface, of the protein, consistent with bacterial interaction with the host. Data represented are the mean ± SEM of 3 independent experiments (n = 7 animals).</p

Pathogenomic analysis of <i>Leptospira interrogans</i> serovar Lai strain 556021 to identify virulence related genes.

<p>(<b>A</b>) Schematic of phylogenetic relatedness of “Pathogenic” (P), “Intermediate” (I) and “Saprophytic” (S) members of the genus <i>Leptospira</i>. (<b>B</b>) Workflow to identify putative virulence-associated genes. Asterisk denotes a hypothetical position in which a SNV has been identified (<b>C</b>) Genomic Locations of SNPs and PF07598 paralogs in the reference genome of <i>L. interrogans</i> serovar Lai strain 56601. Each concentric circle represents genomic data and is numbered from the outermost to the innermost circle. The outermost circles represent the predicted CDS on the + and − strands, respectively, colored by functional role categories (see key). The following circle descriptions apply to chromosome I. The third circle notes the location of predicted prophage regions (olive) and the LPS region (slate). The fourth circle indicates those CDS found to have non-synonymous amino acid substitutions (black) as well as the location of CDS annotated as “transposase” in Genbank (salmon). The fifth circle represents the location of the 12 PF07598 family members (blue). The innermost circle denotes atypical regions (χ² value). For chromosome II, the outermost and innermost circles are the same as for chromosome I; however, the third circle notes the location of transposases (salmon), while the fourth circle indicates the location of the CDS found to have non-synonymous amino acid substitutions (black).</p

<i>In vivo</i> transcriptional analysis of putative virulence-associated genes.

<p><i>In vivo</i> relevance of the identified virulence-related genes, mRNA transcript levels of the genes identified by the pathogenomics approach was assessed by real time, reverse transcriptase quantitative PCR of blood, liver and kidney 4 d after hamster infection, compared to log phase <i>in vitro</i> cultured <i>Leptospira</i>. Leptospiral gene expression levels in infected tissue vs. EMJH were expressed logarithmically as the log₂ of the fold change between the two conditions (<b>A–J</b>). 16S rRNA transcript levels (previously validated <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002468#pntd.0002468-CarrilloCasas1" target="_blank">[61]</a>) were used to normalize gene expression in tissues and under the different conditions (Fig. S1 in <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002468#pntd.0002468.s001" target="_blank">Text S1</a>). Expression of 10/11 identified genes was detectable <i>in vivo</i> in all three tissues assayed; the exception was the hypothetical protein LA_0979. The remaining 10 genes were detected in all three tissues assayed. Expression varied between groups of animals, and interestingly, the highest levels of up-regulation were found in leptospires isolated from the blood of infected animals, with transcript levels also being up in bacteria from the liver. Virulence-associated genes were variably up-regulated in kidney. The data represented are the mean ± SEM of 3 independent experiments (n = 7 animals). (<b>K</b>) <i>Leptospira</i> species distribution of the 11 virulence-associated genes identified and associated single nucleotide variants found in coding sequences of the avirulent passage (P19) strain. Protein code is according to the annotated protein database; Accession is the GenBank code for the protein.</p

Confirmation of cAMP induction in target mammalian cells by LA_4008 activity in leptospiral culture supernatant.

<p>(<b>A</b>) THP-1 cell monolayers were treated with leptospire-free concentrated culture supernatant (CCS) from <i>L. interrogans</i> Lai or EMJH negative control. (<b>B</b>) THP-1 monolayers were treated with CCS from <i>L. interrogans</i> Lai or <i>L. licerasiae</i> Varillal, NT = not treated. (<b>C</b>) THP-1 cell monolayers were treated with CCS, CCS that was immunoprecipitated (IP) with specific anti-peptide antibody raised in rabbits and non-specific anti-LA 4008 antibody, and CCS that was digested with proteinase K. Values in all experiments are represented as the mean (n = 3) ± SD.</p

Ortholog sequence analysis of pathogenic <i>Leptospira</i> adenylate/guanylate cyclase compared to predatory environmental bacteria and the pathogen, <i>Mycobacterium tuberculosis</i>.

<p>Domain architecture comparison of LA_4008 with orthologs of <i>Myxococcus xanthus</i>, <i>Corallococcus coralloides</i>, <i>Stigmatella aurantiaca</i>, and <i>Mycobacterium tuberculosis</i> using NCBI CD Search, SMART, and TPRPred. Protein homology analysis was carried out using BLAST using the following reference sequences: LA_4008 (NP_714188.1), MXAN_4545 (YP_632713.1), COCOR_04748 (YP_005370712.1), STAUR_4866 (YP_003954471.1), Rv0386 (CCP43116). The coverage for the query sequence, statistical significance (E-value), and maximum amino acid identify (“Max Ident”) are indicated at right for each predicted primary sequence.</p

Standardized Metadata for Human Pathogen/Vector Genomic Sequences

<div><p>High throughput sequencing has accelerated the determination of genome sequences for thousands of human infectious disease pathogens and dozens of their vectors. The scale and scope of these data are enabling genotype-phenotype association studies to identify genetic determinants of pathogen virulence and drug/insecticide resistance, and phylogenetic studies to track the origin and spread of disease outbreaks. To maximize the utility of genomic sequences for these purposes, it is essential that metadata about the pathogen/vector isolate characteristics be collected and made available in organized, clear, and consistent formats. Here we report the development of the GSCID/BRC Project and Sample Application Standard, developed by representatives of the Genome Sequencing Centers for Infectious Diseases (GSCIDs), the Bioinformatics Resource Centers (BRCs) for Infectious Diseases, and the U.S. National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health (NIH), informed by interactions with numerous collaborating scientists. It includes mapping to terms from other data standards initiatives, including the Genomic Standards Consortium’s minimal information (MIxS) and NCBI’s BioSample/BioProjects checklists and the Ontology for Biomedical Investigations (OBI). The standard includes data fields about characteristics of the organism or environmental source of the specimen, spatial-temporal information about the specimen isolation event, phenotypic characteristics of the pathogen/vector isolated, and project leadership and support. By modeling metadata fields into an ontology-based semantic framework and reusing existing ontologies and minimum information checklists, the application standard can be extended to support additional project-specific data fields and integrated with other data represented with comparable standards. The use of this metadata standard by all ongoing and future GSCID sequencing projects will provide a consistent representation of these data in the BRC resources and other repositories that leverage these data, allowing investigators to identify relevant genomic sequences and perform comparative genomics analyses that are both statistically meaningful and biologically relevant.</p></div

Core Project Attributes.

<p>*Mandatory NCBI BioProject attributes.</p

Semantic Network of the Core Sample Data Fields.

<p>A semantic representation of the entities relevant to describe infectious disease samples based on the OBI and other OBO Foundry ontologies is shown. Distinctions are made between material entities (blue outlines), information entities and qualities (black outlines), and processes (red outlines). Entities are connected by standard semantic relations, in <i>italic</i>. The subset of entities selected as Core Sample fields are noted with ovals containing the respective Field ID. For example, the OBI:organism <i>has_quality</i> “Specimen Source Gender” (CS5), which is equivalent to the PATO:biological sex, and <i>has_quality</i> PATO:age, and <i>has_quality</i> “Specimen Source Health Status” (CS8), which is equivalent to PATO:organismal status. PATO:age <i>is_quality_measured_as</i> OBI:age since birth measurement datum, which <i>has_measurement_value</i> “Specimen Source Age – Value” (CS6) and <i>has_measurement_unit_label</i> “Specimen Source Age – Unit” (CS7).</p