Pyrosequencing-Based Comparative Genome Analysis of Vibrio vulnificus Environmental Isolates

Between 1996 and 2006, the US Centers for Disease Control reported that the only category of food-borne infections increasing in frequency were those caused by members of the genus Vibrio. The Gram-negative bacterium Vibrio vulnificus is a ubiquitous inhabitant of estuarine waters, and is the number one cause of seafood-related deaths in the US. Many V. vulnificus isolates have been studied, and it has been shown that two genetically distinct subtypes, distinguished by 16S rDNA and other gene polymorphisms, are associated predominantly with either environmental or clinical isolation. While local genetic differences between the subtypes have been probed, only the genomes of clinical isolates have so far been completely sequenced. In order to better understand V. vulnificus as an agent of disease and to identify the molecular components of its virulence mechanisms, we have completed whole genome shotgun sequencing of three diverse environmental genotypes using a pyrosequencing approach. V. vulnificus strain JY1305 was sequenced to a depth of 33×, and strains E64MW and JY1701 were sequenced to lesser depth, covering approximately 99.9% of each genome. We have performed a comparative analysis of these sequences against the previously published sequences of three V. vulnificus clinical isolates. We find that the genome of V. vulnificus is dynamic, with 1.27% of genes in the C-genotype genomes not found in the E- genotype genomes. We identified key genes that differentiate between the genomes of the clinical and environmental genotypes. 167 genes were found to be specifically associated with environmental genotypes and 278 genes with clinical genotypes. Genes specific to the clinical strains include components of sialic acid catabolism, mannitol fermentation, and a component of a Type IV secretory pathway VirB4, as well as several other genes with potential significance for human virulence. Genes specific to environmental strains included several that may have implications for the balance between self-preservation under stress and nutritional competence

Genomic Analysis Reveals Novel Diversity among the 1976 Philadelphia Legionnaires’ Disease Outbreak Isolates and Additional ST36 Strains

<div><p><i>Legionella pneumophila</i> was first recognized as a cause of severe and potentially fatal pneumonia during a large-scale outbreak of Legionnaires’ disease (LD) at a Pennsylvania veterans’ convention in Philadelphia, 1976. The ensuing investigation and recovery of four clinical isolates launched the fields of <i>Legionella</i> epidemiology and scientific research. Only one of the original isolates, “Philadelphia-1”, has been widely distributed or extensively studied. Here we describe the whole-genome sequencing (WGS), complete assembly, and comparative analysis of all Philadelphia LD strains recovered from that investigation, along with <i>L</i>. <i>pneumophila</i> isolates sharing the Philadelphia sequence type (ST36). Analyses revealed that the 1976 outbreak was due to multiple serogroup 1 strains within the same genetic lineage, differentiated by an actively mobilized, self-replicating episome that is shared with <i>L</i>. <i>pneumophila</i> str. Paris, and two large, horizontally-transferred genomic loci, among other polymorphisms. We also found a completely unassociated ST36 strain that displayed remarkable genetic similarity to the historical Philadelphia isolates. This similar strain implies the presence of a potential clonal population, and suggests important implications may exist for considering epidemiological context when interpreting phylogenetic relationships among outbreak-associated isolates. Additional extensive archival research identified the Philadelphia isolate associated with a non-Legionnaire case of “Broad Street pneumonia”, and provided new historical and genetic insights into the 1976 epidemic. This retrospective analysis has underscored the utility of fully-assembled WGS data for <i>Legionella</i> outbreak investigations, highlighting the increased resolution that comes from long-read sequencing and a sequence type-matched genomic data set.</p></div

Nucleotide polymorphisms unique to the CDC Philadelphia strains relative to the NCBI Philadelphia-1 reference sequence.

<p>Nucleotide polymorphisms unique to the CDC Philadelphia strains relative to the NCBI Philadelphia-1 reference sequence.</p

Transcriptome Sequencing Reveals the Virulence and Environmental Genetic Programs of <i>Vibrio vulnificus</i> Exposed to Host and Estuarine Conditions

<div><p><i>Vibrio vulnificus</i> is a natural inhabitant of estuarine waters worldwide and is of medical relevance due to its ability to cause grievous wound infections and/or fatal septicemia. Genetic polymorphisms within the virulence-correlated gene (<i>vcg</i>) serve as a primary feature to distinguish clinical (C-) genotypes from environmental (E-) genotypes. C-genotypes demonstrate superior survival in human serum relative to E-genotypes, and genome comparisons have allowed for the identification of several putative virulence factors that could potentially aid C-genotypes in disease progression. We used RNA sequencing to analyze the transcriptome of C-genotypes exposed to human serum relative to seawater, which revealed two divergent genetic programs under these two conditions. In human serum, cells displayed a distinct “virulence profile” in which a number of putative virulence factors were upregulated, including genes involved in intracellular signaling, substrate binding and transport, toxin and exoenzyme production, and the heat shock response. Conversely, the “environmental profile” exhibited by cells in seawater revealed upregulation of transcription factors such as <i>rpoS, rpoN</i>, and <i>iscR</i>, as well as genes involved in intracellular signaling, chemotaxis, adherence, and biofilm formation. This dichotomous genetic switch appears to be largely governed by cyclic-di-GMP signaling, and remarkably resembles the dual life-style of <i>V. cholerae</i> as it transitions from host to environment. Furthermore, we found a “general stress response” module, known as the stressosome, to be upregulated in seawater. This signaling system has been well characterized in Gram-positive bacteria, however its role in <i>V. vulnificus</i> is not clear. We examined temporal gene expression patterns of the stressosome and found it to be upregulated in natural estuarine waters indicating that this system plays a role in sensing and responding to the environment. This study advances our understanding of gene regulation in <i>V. vulnificus</i>, and brings to the forefront a number of previously overlooked genetic networks.</p></div

Summary of differentially expressed genes (p<0.0001).

a<p>N/A – not applicable.</p>b<p>Chromosomal percentages were calculated as number of DE genes divided by total number of genes on each chromosome.</p><p>Summary of differentially expressed genes (p<0.0001).</p

Gubbins-based recombinational analysis and phylogenetic tree reconstruction.

<p>Regions of elevated SNP density, representing potential horizontal gene transfer events, were identified by the Gubbins algorithm and software package, masked in the original multiple sequence alignment, and then core SNPs were identified with kSNP v3, as detailed in the Methods. The maximum-likelihood tree shown was constructed using RAxML v8 and 307 core, non-recombinant SNPs with 1000 bootstrappings. Red blocks represent regions of elevated SNP density conserved in multiple strains and blue blocks represent elevated SNP density only found in a single strain. <i>L</i>. <i>pneumophila</i> str. Philadelphia-4 was used as the reference/outgroup, and VGR-1 and -2 are labeled. Gubbins was run with default parameters. A Gubbins-generated tree with 480 non-recombining SNPs also exhibited similar topology.</p

Diagrammatic summary of genes upregulated in artificial seawater relative to human serum.

<p>Depicted in this figure are genes (or sets of genes) upregulated in ASW along with the inferred phenotypic outcomes. The cell expresses several transcription factors involved in stress response, motility, and biofilm formation (RpoS, RpoN and IscR). The cell also expresses genes involved in attachment and biofilm formation including Flp pilus (tad locus), MSHA pilus, and EPS (syp locus). Through the action of diguanylate cyclases (DGCs), the cell has high intracellular concentrations of c-di-GMP which enhances biofilm formation and possibly reduces virulence factor production. In response to chemoattractants and chemorepellents, the cell also engages in chemotactic sensing and signaling which may result in modulation of flagellar rotation and/or flagellum-independent motility. As a result of some unknown environmental cue, the cell also expresses genes involved in the stressosome module (RsbRSTU) which is likely involved in chemotaxis and/or stress response. Note, this diagram is developed based solely on transcriptomic data and further investigation is necessary to establish the proposed model.</p

Nucleotide polymorphisms shared by all CDC Philadelphia strains relative to the NCBI Philadelphia-1 reference sequence.

<p>Nucleotide polymorphisms shared by all CDC Philadelphia strains relative to the NCBI Philadelphia-1 reference sequence.</p

Nucleotide polymorphisms shared by strains CDC Philadelphia-2, -3, -4, and ATCC Philadelphia-1 relative to the NCBI Philadelphia-1 reference sequence.

<p>Nucleotide polymorphisms shared by strains CDC Philadelphia-2, -3, -4, and ATCC Philadelphia-1 relative to the NCBI Philadelphia-1 reference sequence.</p