Comparative genomic analysis of clinical and environmental strains provides insight into the pathogenicity and evolution of Vibrio parahaemolyticus

Background

Vibrio parahaemolyticus is a Gram-negative halophilic bacterium. Infections with the bacterium could become systemic and can be life-threatening to immunocompromised individuals. Genome sequences of a few clinical isolates of V. parahaemolyticus are currently available, but the genome dynamics across the species and virulence potential of environmental strains on a genome-scale have not been described before.

Results

Here we present genome sequences of four V. parahaemolyticus clinical strains from stool samples of patients and five environmental strains in Hong Kong. Phylogenomics analysis based on single nucleotide polymorphisms revealed a clear distinction between the clinical and environmental isolates. A new gene cluster belonging to the biofilm associated proteins of V. parahaemolyticus was found in clincial strains. In addition, a novel small genomic island frequently found among clinical isolates was reported. A few environmental strains were found harboring virulence genes and prophage elements, indicating their virulence potential. A unique biphenyl degradation pathway was also reported. A database for V. parahaemolyticus (http://kwanlab.bio.cuhk.edu.hk/vp) was constructed here as a platform to access and analyze genome sequences and annotations of the bacterium.

Conclusions

We have performed a comparative genomics analysis of clinical and environmental strains of V. parahaemolyticus. Our analyses could facilitate understanding of the phylogenetic diversity and niche adaptation of this bacterium.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1135) contains supplementary material, which is available to authorized users.     

A new insight into the pathogenesis of filarial disease

Filariasis is a major public health problem throughout many regions of the tropics. The disease is caused by several species of filarial nematode including Wuchereria bancrofti and Brugia malayi, the agents of lymphatic filariasis, and Onchocerca volvulus, the cause of 'riverblindness'. Disease caused by these worms varies depending on the tissue location of the parasite, and is associated with episodes of acute and chronic inflammation. These pathologies, including elephantiasis and blindness, rank among the most disabling in the world. Studies aimed at characterizing the molecular nature of the inflammatory stimuli derived from filarial nematodes uncovered a long forgotten secret, their symbiont Wolbachia. LPS-like molecules from these intracellular bacteria are responsible for potent inflammatory responses from macrophages and in animal models of filarial disease. Wolbachia has also been associated with severe inflammatory reactions to filarial chemotherapy, being released into the blood following the death of the parasite. Recent studies in animal models even implicate Wolbachia in the onset of lymphodema and blindness. Taken together these studies suggest a major role for Wolbachia in the pathogenesis of filarial disease. It may be possible, through the use of antibiotic therapy, to clear worms of their bacteria, in the hope that this will prevent the onset and development of filarial pathology.     

Conservation analysis of the CydX protein yields insights into small protein identification and evolution

Background

The reliable identification of proteins containing 50 or fewer amino acids is difficult due to the limited information content in short sequences. The 37 amino acid CydX protein in Escherichia coli is a member of the cytochrome bd oxidase complex, an enzyme found throughout Eubacteria. To investigate the extent of CydX conservation and prevalence and evaluate different methods of small protein homologue identification, we surveyed 1095 Eubacteria species for the presence of the small protein.

Results

Over 300 homologues were identified, including 80 unannotated genes. The ability of both closely-related and divergent homologues to complement the E. coli ΔcydX mutant supports our identification techniques, and suggests that CydX homologues retain similar function among divergent species. However, sequence analysis of these proteins shows a great degree of variability, with only a few highly-conserved residues. An analysis of the co-variation between CydX homologues and their corresponding cydA and cydB genes shows a close synteny of the small protein with the CydA long Q-loop. Phylogenetic analysis suggests that the cydABX operon has undergone horizontal gene transfer, although the cydX gene likely evolved in a progenitor of the Alpha, Beta, and Gammaproteobacteria. Further investigation of cydAB operons identified two additional conserved hypothetical small proteins: CydY encoded in CydA_Qlong operons that lack cydX, and CydZ encoded in more than 150 CydA_Qshort operons.

Conclusions

This study provides a systematic analysis of bioinformatics techniques required for the unique challenges present in small protein identification and phylogenetic analyses. These results elucidate the prevalence of CydX throughout the Proteobacteria, provide insight into the selection pressure and sequence requirements for CydX function, and suggest a potential functional interaction between the small protein and the CydA Q-loop, an enigmatic domain of the cytochrome bd oxidase complex. Finally, these results identify other conserved small proteins encoded in cytochrome bd oxidase operons, suggesting that small protein subunits may be a more common component of these enzymes than previously thought.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-946) contains supplementary material, which is available to authorized users.     

Serum proteome analysis of vivax malaria: An insight into the disease pathogenesis and host immune response

Vivax malaria is the most widely distributed human malaria resulting in 80-300 million clinical cases every year. It causes severe infection and mortality but is generally regarded as a benign disease and has not been investigated in detail. The present study aimed to perform human serum proteome analysis in a malaria endemic area in India to identify potential serum biomarkers for vivax malaria and understand host response. The proteomic analysis was performed on 16 age and gender matched subjects (vivax patients and control) in duplicate. Protein extraction protocols were optimized for large coverage of the serum proteome and to obtain high-resolution data. Identification of 67 differentially expressed and statistically significant (Student's t-test; p<0.05) protein spots was established by MALDI-TOF/TOF mass spectrometry. Many of the identified proteins such as apolipoprotein A and E, serum amyloid A and P, haptoglobin, ceruloplasmin, and hemopexin are interesting from a diagnostic point of view and could further be studied as potential serum biomarkers. The differentially expressed serum proteins in vivax malaria identified in this study were subjected to functional pathway analysis using multiple software, including Ingenuity Pathway Analysis (IPA), Protein ANalysis THrough Evolutionary Relationships (PANTHER) and Database for Annotation, Visualization and Integrated Discovery (DAVID) functional annotation tool for better understanding of the biological context of the identified proteins, their involvement in various physiological pathways and association with disease pathogenesis. Functional pathway analysis of the differentially expressed proteins suggested the modulation of multiple vital physiological pathways, including acute phase response signaling, complement and coagulation cascades, hemostasis and vitamin D metabolism pathway due to this parasitic infection. This article is part of a Special Issue entitled: Proteomics: The clinical link.     

Molecular phylogenetic analysis of Commiphora (Burseraceae) yields insight on the evolution and historical biogeography of an "impossible" genus

Expansion of the arid zone of sub-Saharan tropical Africa during the Miocene is posited as a significant contributing factor in the evolution of contemporary African flora. Nevertheless, few molecular phylogenetic studies have tested this hypothesis using reconstructed historical biogeographies of plants within this zone. Here, we present a molecular phylogeny of Commiphora, a predominantly tropical African, arid-adapted tree genus, in order to test the monophyly of its taxonomic sections and identify clades that will help direct future study of this species-rich and geographically widespread taxon. We then use multiple fossil calibrations of Commiphora phylogeny to determine the timing of well-supported diversification events within the genus and interpret these age estimates to determine the relative contribution of vicariance and dispersal in the expansion of Commiphora's geographic range. We find that Commiphora is sister to Vietnamese Bursera tonkinensis and that its crown group radiation corresponds with the onset of the Miocene.     

Comparative mapping of human Chromosome 19 with the chicken shows conserved synteny and gives an insight into chromosomal evolution

Human Chromosome 19 (HSA19) is virtually completely sequenced. A complete physical contig map made up of BACs and cosmids is also available for this chromosome. It is, therefore, a rich source of information that we have used as the basis for a comparative mapping study with the chicken. Various orthologs of genes known to map to HSA19 have been mapped in the chicken. Five chicken microchromosomes (two of which were previously undefined) are seen to show conserved synteny with this chromosome, along with individual gene homologs on Chr 1 and another tiny microchromosome. Compared with the mouse, which has 12 chromosomal regions homologous to HSA19, the chicken genotype displays fewer evolutionary rearrangements. The ancestral nature of the chicken karyotype is demonstrated and may prove to be an excellent tool for studying genome evolution.     

New insight into the role of dendritic cells in malaria immune pathogenesis

The mechanism by which the host develops protective immunity to malaria remains poorly understood. Dendritic cells (DCs) are central to the initiation and regulation of the adaptive immune response. Modulation of DC function might enable Plasmodium to evade the immune system. Millington et al. propose one mechanism by which malaria inhibits DC-T-cell interactions without interfering directly with T-cell receptor engagement. The consequence is a decrease in the co-stimulation required to develop an effective immune response.     

The amphioxus genome provides unique insight into the evolution of immunity

Immune systems evolve as essential strategies to maintain homeostasis with the environment, prevent microbial assault and recycle damaged host tissues. The immune system is composed of two components, innate and adaptive immunity. The former is common to all animals while the latter consists of a vertebrate-specific system that relies on somatically derived lymphocytes and is associated with near limitless genetic diversity as well as long-term memory. Deuterostome invertebrates provide a view of immune repertoires in phyla that immediately predate the origins of vertebrates. Genomic studies in amphioxus, a cephalochordate, have revealed homologs of genes encoding most innate immune receptors found in vertebrates; however, many of the gene families have undergone dramatic expansions, greatly increasing the innate immune repertoire. In addition, domain-swapping accounts for the innovation of new predicted pathways of receptor function. In both amphioxus and Ciona, a urochordate, the VCBPs (variable region containing chitin-binding proteins), which consist of immunoglobulin V (variable) and chitin binding domains, mediate recognition through the V domains. The V domains of VCBPs in amphioxus exhibit high levels of allelic complexity that presumably relate to functional specificity. Various features of the amphioxus immune repertoire reflect novel selective pressures, which likely have resulted in innovative strategies. Functional genomic studies underscore the value of amphioxus as a model for studying innate immunity and may help reveal how unique relationships between innate immune receptors and both pathogens and symbionts factored in the evolution of adaptive immune systems.     

Behavior of dermal fibroblasts on microdot arrays yields insight into wound healing mechanisms

The behavior of fibroblasts on patterned substrates was examined in order to elucidate the role of dermal structure in wound healing. Dermal fibroblasts were cultured on micro-patterned silicone elastomer substrates designed to enforce cell adhesion only to fibronectin microdots. The morphology, expression of α-smooth muscle actin (α-SMA), proliferation, apoptotic cells, and soluble collagen production of cells were measured. Cells grown on patterned substrates showed some signs of a scar-fibroblast phenotype such as: elongated pseudopodia, enhanced expression of alpha smooth muscle actin (α-SMA), and increased collagen/pre-collagen, in comparison to unpatterned controls. Cells also showed low proliferation rates and high apoptotic index. The results showed that the microdot arrays, acting as a grid of limited focal adhesion sites, could force cells to adopt constrained morphologies and limited adhesion areas, which affect the cytoskeleton, ultimately leading to expression of a scar-tissue fibroblast phenotype. This study provides insight into the regulatory mechanisms of micro-topology on cell behavior in wound healing.     

Evolutionary breakpoint analysis on Y chromosomes of higher primates provides insight into human Y evolution

Comparative FISH mapping of PAC clones covering almost 3 Mb of the human AZFa region in Yq11.21 to metaphases of human and great apes unravels breakpoints that were involved in species-specific Y chromosome evolution. An astonishing clustering of evolutionary breakpoints was detected in the very proximal region on the long arm of the human Y chromosome in Yq11.21. These breakpoints were involved in deletions, one specific for the human and another for the orang-utan Y chromosome, in a duplicative translocation/transposition specific for bonobo and chimpanzee Y chromosomes and in a pericentric inversion specific for the gorilla Y chromosome. In addition, our comparative results allow the deduction of a model for the human Y chromosome evolution.     

Comparative analysis and insights into the evolution of gene clusters for glycopeptide antibiotic biosynthesis

The bal, cep, dbv, sta and tcp gene clusters specify the biosynthesis of the glycopeptide antibiotics balhimycin, chloroeremomycin, A40926, A47934 and teicoplanin, respectively. These structurally related compounds share a similar mechanism of action in their inhibition of bacterial cell wall formation. Comparative sequence analysis was performed on the five gene clusters. Extensive conserved synteny was observed between the bal and cep clusters, which direct the synthesis of very similar compounds but originate from two different species of the genus Amycolatopsis. All other cluster pairs show a limited degree of conserved synteny, involving biosynthetically functional gene cassettes: these include those involved in the synthesis of the carbon backbone of two non-proteinogenic amino acids; in the linkage of amino acids 1–3 and 4–7 in the heptapeptide; and in the formation of the aromatic cross-links. Furthermore, these segments of conserved synteny are often preceded by conserved intergenic regions. Phylogenetic analysis of protein families shows several instances in which relatedness in the chemical structure of the glycopeptides is not reflected in the extent of the relationship of the corresponding polypeptides. Coherent branchings are observed for all polypeptides encoded by the syntenous gene cassettes. These results suggest that the acquisition of distinct, functional genetic elements has played a significant role in the evolution of glycopeptide gene clusters, giving them a mosaic structure. In addition, the synthesis of the structurally similar compounds A40926 and teicoplanin appears as the result of convergent evolution.     

An insight into the origin and functional evolution of bacterial aromatic ring-hydroxylating oxygenases

Bacterial aromatic ring-hydroxylating oxygenases (RHOs) are multicomponent enzyme systems which have potential utility in bioremediation of aromatic compounds in the environment. To cope with the enormous diversity of aromatic compounds in the environment, this enzyme family has evolved remarkably exhibiting broad substrate specificity. RHOs are multicomponent enzymes comprising of a homo- or hetero-multimeric terminal oxygenase and one or more electron transport (ET) protein(s). The present study attempts in depicting the evolutionary scenarios that might have occurred during the evolution of RHOs, by analyzing a set of available sequences including those obtained from complete genomes. A modified classification scheme identifying four new RHO types has been suggested on the basis of their evolutionary and functional behaviours, in relation to structural configuration of substrates and preferred oxygenation site(s). The present scheme emphasizes on the fact that the phylogenetic affiliation of RHOs is distributed among four distinct 'Similarity classes', independent of the constituent ET components. Similar combination of RHO components that was previously considered to be equivalent and classified together [Kweon et al., BMC Biochemistry 9, 11 (2008)] were found here in distinct similarity classes indicating the role of substrate-binding terminal oxygenase in guiding the evolution of RHOs irrespective of the nature of constituent ET components. Finally, a model for evolution of the multicomponent RHO enzyme system has been proposed, beginning from genesis of the terminal oxygenase components followed by recruitment of constituent ET components, finally evolving into various 'extant' RHO types.     

Structure and evolution of neurexin genes: insight into the mechanism of alternative splicing

Neurexins are neuron-specific vertebrate proteins with hundreds of differentially spliced isoforms that may function in synapse organization. We now show that Drosophila melanogaster and Caenorhabditis elegans express a single gene encoding only an alpha-neurexin, whereas humans and mice express three genes, each of which encodes alpha- and beta-neurexins transcribed from separate promoters. The neurexin genes are very large (up to 1.62 Mb), with the neurexin-3 gene occupying almost 2% of human chromosome 14. Although invertebrate and vertebrate neurexins exhibit a high degree of evolutionary conservation, only vertebrate neurexins are subject to extensive alternative splicing that uses mechanisms ranging from strings of mini-exons to multiple alternative splice donor and acceptor sites. Consistent with their proposed role in synapse specification, neurexins thus have evolved from relatively simple genes in invertebrates to diversified genes in vertebrates with multiple promoters and extensive alternative splicing.     

Modeling bacterial evolution with comparative-genome-based marker systems: application to Mycobacterium tuberculosis evolution and pathogenesis

The comparative-genomic sequencing of two Mycobacterium tuberculosis strains enabled us to identify single nucleotide polymorphism (SNP) markers for studies of evolution, pathogenesis, and epidemiology in clinical M. tuberculosis. Phylogenetic analysis using these "comparative-genome markers" (CGMs) produced a highly unusual phylogeny with a complete absence of secondary branches. To investigate CGM-based phylogenies, we devised computer models to simulate sequence evolution and calculate new phylogenies based on an SNP format. We found that CGMs represent a distinct class of phylogenetic markers that depend critically on the genetic distances between compared "reference strains." Properly distanced reference strains generate CGMs that accurately depict evolutionary relationships, distorted only by branch collapse. Improperly distanced reference strains generate CGMs that distort and reroot outgroups. Applying this understanding to the CGM-based phylogeny of M. tuberculosis, we found evidence to suggest that this species is highly clonal without detectable lateral gene exchange. We noted indications of evolutionary bottlenecks, including one at the level of the PHRI "C" strain previously associated with particular virulence characteristics. Our evidence also suggests that loss of IS6110 to fewer than seven elements per genome is uncommon. Finally, we present population-based evidence that KasA, an important component of mycolic acid biosynthesis, develops G312S polymorphisms under selective pressure.