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Development of genomic resources for a thraustochytrid pathogen and investigation of temperature influences on gene expression.

ABSTRACT: Understanding how environmental changes influence the pathogenicity and virulence of infectious agents is critical for predicting epidemiological patterns of disease. Thraustochytrids, part of the larger taxonomic class Labyrinthulomycetes, contain several highly pathogenic species, including the hard clam pathogen quahog parasite unknown (QPX). QPX has been associated with large-scale mortality events along the northeastern coast of North America. Growth and physiology of QPX is temperature-dependent, and changes in local temperature profiles influence pathogenicity. In this study we characterize the partial genome of QPX and examine the influence of temperature on gene expression. Genes involved in several biological processes are differentially expressed upon temperature change, including those associated with altered growth and metabolism and virulence. The genomic and transcriptomic resources developed in this study provide a foundation for better understanding virulence, pathogenicity and life history of thraustochytrid pathogens.

SUBMITTER: Garcia-Vedrenne AE

PROVIDER: S-EPMC3775781 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Development of genomic resources for a thraustochytrid pathogen and investigation of temperature influences on gene expression.

Garcia-Vedrenne Ana Elisa AE Groner Maya M Page-Karjian Annie A Siegmund Gregor-Fausto GF Singhal Sonia S Sziklay Jamie J Roberts Steven S

PloS one 20130917 9

Understanding how environmental changes influence the pathogenicity and virulence of infectious agents is critical for predicting epidemiological patterns of disease. Thraustochytrids, part of the larger taxonomic class Labyrinthulomycetes, contain several highly pathogenic species, including the hard clam pathogen quahog parasite unknown (QPX). QPX has been associated with large-scale mortality events along the northeastern coast of North America. Growth and physiology of QPX is temperature-dep ...[more]

PMID: 24069279

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Project description:BackgroundThe hard clam or northern quahog, Mercenaria mercenaria, is one of the most valuable seafood products in the United States representing the first marine resource in some Northeastern states. Severe episodes of hard clam mortality have been consistently associated with infections caused by a thraustochytrid parasite called Quahog Parasite Unknown (QPX). QPX is considered as a cold/temperate water organism since the disease occurs only in the coastal waters of the northwestern Atlantic Ocean from Maritime Canada to Virginia. High disease development at cold temperatures was also confirmed in laboratory studies and is thought to be caused predominantly by immunosuppression of the clam host even though the effect of temperature on QPX virulence has not been fully investigated. In this study, the QPX transcriptome was sequenced using Roche 454 technology to better characterize this microbe and initiate research on the molecular basis of QPX virulence towards hard clams.ResultsClose to 18,000 transcriptomic sequences were generated and functionally annotated. Results revealed a wide array of QPX putative virulence factors including a variety of peptidases, antioxidant enzymes, and proteins involved in extracellular mucus production and other secretory proteins potentially involved in interactions with the clam host. Furthermore, a 15 K oligonucleotide array was constructed and used to investigate the effect of temperature on QPX fitness and virulence factors. Results identified a set of QPX molecular chaperones that could explain its adaptation to cold temperatures. Finally, several virulence-related factors were up-regulated at low temperature providing molecular targets for further investigations of increased QPX pathogenicity in cold water conditions.ConclusionsThis is one of the first studies to characterize the transcriptome of a parasitic labyrinthulid, offering new insights into the molecular bases of the pathogenicity of members of this group. Results from the oligoarray study demonstrated the ability of QPX to cope with a wide range of environmental temperatures, including those considered to be suboptimal for clam immunity (low temperature) providing a mechanistic scenario for disease distribution in the field and for high disease prevalence and intensity at low temperature. These results will serve as basis for studies aimed at a better characterization of specific putative virulence factors.

Project description:Periodontitis is a microbial infection that destroys the structures that support the teeth. Although it is typically a chronic condition, rapidly progressing, aggressive forms are associated with the oral pathogen Aggregatibacter actinomycetemcomitans One of this bacterium's key virulence traits is its ability to attach to surfaces and form robust biofilms that resist killing by the host and antibiotics. Though much has been learned about A. actinomycetemcomitans since its initial discovery, we lack insight into a fundamental aspect of its basic biology, as we do not know the full set of genes that it requires for viability (the essential genome). Furthermore, research on A. actinomycetemcomitans is hampered by the field's lack of a mutant collection. To address these gaps, we used rapid transposon mutant sequencing (Tn-seq) to define the essential genomes of two strains of A. actinomycetemcomitans, revealing a core set of 319 genes. We then generated an arrayed mutant library comprising >1,500 unique insertions and used a sequencing-based approach to define each mutant's position (well and plate) in the library. To demonstrate its utility, we screened the library for mutants with weakened resistance to subinhibitory erythromycin, revealing the multidrug efflux pump AcrAB as a critical resistance factor. During the screen, we discovered that erythromycin induces A. actinomycetemcomitans to form biofilms. We therefore devised a novel Tn-seq-based screen to identify specific factors that mediate this phenotype and in follow-up experiments confirmed 4 mutants. Together, these studies present new insights and resources for investigating the basic biology and disease mechanisms of a human pathogen.IMPORTANCE Millions suffer from gum disease, which often is caused by Aggregatibacter actinomycetemcomitans, a bacterium that forms antibiotic-resistant biofilms. To fully understand any organism, we should be able to answer: what genes does it require for life? Here, we address this question for A. actinomycetemcomitans by determining the genes in its genome that cannot be mutated. As for the genes that can be mutated, we archived these mutants into a library, which we used to find genes that contribute to antibiotic resistance, leading us to discover that antibiotics cause A. actinomycetemcomitans to form biofilms. We then devised an approach to find genes that mediate this process and confirmed 4 genes. These results illuminate new fundamental traits of a human pathogen.

Project description:Chronic rhinosinusitis (CRS) is an inflammatory disease of the paranasal sinuses, and microbial dysbiosis associated with CRS is thought to be a key driver of host inflammation that contributes to disease progression. Staphylococcus aureus is a common upper respiratory tract (URT) pathobiont associated with higher carriage rates in CRS populations, where S. aureus-secreted toxins can be identified in CRS tissues. Although many genera of bacteria colonize the URT, few account for the majority of sequencing reads. These include S. aureus and several species belonging to the genus Corynebacterium, including Corynebacterium propinquum and Corynebacterium pseudodiphtheriticum, which are observed at high relative abundance in the healthy URT. Studies have examined bacterial interactions between major microbionts of the URT and S. aureus, but few have done so in the context of a healthy versus diseased URT environment. Here, we examine the role of temperature in commensal, pathogen, and epithelial dynamics using an air-liquid interface cell culture model mimicking the nasal epithelial environment. Healthy URT temperatures change from the nares to the nasopharynx and are increased during disease. Temperatures representative of the healthy URT increase persistence and aggregate formation of commensal C. propinquum and C. pseudodiphtheriticum, reduce S. aureus growth, and lower epithelial cytotoxicity compared to higher temperatures correlating with the diseased CRS sinus. Dual-species colonization revealed species-specific interactions between Corynebacterium species and S. aureus dependent on temperature. Our findings suggest URT mucosal temperature plays a significant role in mediating polymicrobial and host-bacterial interactions that may exacerbate microbial dysbiosis in chronic URT diseases.IMPORTANCEChronic rhinosinusitis is a complex inflammatory disease with a significant healthcare burden. Although presence of S. aureus and microbial dysbiosis are considered mediators of inflammation in CRS, no studies have examined the influence of temperature on S. aureus interactions with the nasal epithelium and the dominant genus of the healthy URT, Corynebacterium. Interactions between Corynebacterium species and S. aureus have been documented in several studies, but none to date have examined how environmental changes in the URT may alter their interactions with the epithelium or each other. This study utilizes a polarized epithelial cell culture model at air-liquid interface to study the colonization and spatial dynamics of S. aureus and clinical isolates of Corynebacterium from people with CRS to characterize the role temperature has in single- and dual-species dynamics on the nasal epithelium.

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Development of genomic resources for a thraustochytrid pathogen and investigation of temperature influences on gene expression.

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Development of genomic resources for a thraustochytrid pathogen and investigation of temperature influences on gene expression.

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