Metagenomic Analysis of Respiratory Tract DNA Viral Communities in Cystic Fibrosis and Non-Cystic Fibrosis Individuals

The human respiratory tract is constantly exposed to a wide variety of viruses, microbes and inorganic particulates from environmental air, water and food. Physical characteristics of inhaled particles and airway mucosal immunity determine which viruses and microbes will persist in the airways. Here we present the first metagenomic study of DNA viral communities in the airways of diseased and non-diseased individuals. We obtained sequences from sputum DNA viral communities in 5 individuals with cystic fibrosis (CF) and 5 individuals without the disease. Overall, diversity of viruses in the airways was low, with an average richness of 175 distinct viral genotypes. The majority of viral diversity was uncharacterized. CF phage communities were highly similar to each other, whereas Non-CF individuals had more distinct phage communities, which may reflect organisms in inhaled air. CF eukaryotic viral communities were dominated by a few viruses, including human herpesviruses and retroviruses. Functional metagenomics showed that all Non-CF viromes were similar, and that CF viromes were enriched in aromatic amino acid metabolism. The CF metagenomes occupied two different metabolic states, probably reflecting different disease states. There was one outlying CF virome which was characterized by an over-representation of Guanosine-5′-triphosphate,3′-diphosphate pyrophosphatase, an enzyme involved in the bacterial stringent response. Unique environments like the CF airway can drive functional adaptations, leading to shifts in metabolic profiles. These results have important clinical implications for CF, indicating that therapeutic measures may be more effective if used to change the respiratory environment, as opposed to shifting the taxonomic composition of resident microbiota

Energetic differences between bacterioplankton trophic groups and coral reef resistance

Coral reefs are among the most productive and diverse marine ecosystems on the Earth. They are also particularly sensitive to changing energetic requirements by different trophic levels. Microbialization specifically refers to the increase in the energetic metabolic demands of microbes relative to macrobes and is significantly correlated with increasing human influence on coral reefs. In this study, metabolic theory of ecology is used to quantify the relative contributions of two broad bacterioplankton groups, autotrophs and heterotrophs, to energy flux on 27 Pacific coral reef ecosystems experiencing human impact to varying degrees. The effective activation energy required for photosynthesis is lower than the average energy of activation for the biochemical reactions of the Krebs cycle, and changes in the proportional abundance of these two groups can greatly affect rates of energy and materials cycling. We show that reef-water communities with a higher proportional abundance of microbial autotrophs expend more metabolic energy per gram of microbial biomass. Increased energy and materials flux through fast energy channels (i.e. water-column associated microbial autotrophs) may dampen the detrimental effects of increased heterotrophic loads (e.g. coral disease) on coral reef systems experiencing anthropogenic disturbance

Assessing Coral Reefs on a Pacific-Wide Scale Using the Microbialization Score

The majority of the world’s coral reefs are in various stages of decline. While a suite of disturbances (overfishing, eutrophication, and global climate change) have been identified, the mechanism(s) of reef system decline remain elusive. Increased microbial and viral loading with higher percentages of opportunistic and specific microbial pathogens have been identified as potentially unifying features of coral reefs in decline. Due to their relative size and high per cell activity, a small change in microbial biomass may signal a large reallocation of available energy in an ecosystem; that is the microbialization of the coral reef. Our hypothesis was that human activities alter the energy budget of the reef system, specifically by altering the allocation of metabolic energy between microbes and macrobes. To determine if this is occurring on a regional scale, we calculated the basal metabolic rates for the fish and microbial communities at 99 sites on twenty-nine coral islands throughout the Pacific Ocean using previously established scaling relationships. From these metabolic rate predictions, we derived a new metric for assessing and comparing reef health called the microbialization score. The microbialization score represents the percentage of the combined fish and microbial predicted metabolic rate that is microbial. Our result

Confirmatory sequencing of full-genome amplicons generated by PCR.

<p>A. For each amplicons, the sequencing was carried out with primers other than those used for full-genome amplification. The linear sequencing template (black line), the PCR primers are shown at both ends of the amplicons. Bleu lines (amplicons 671F-892R), green lines (amplicons 6838F-6972R), red lines (amplicons 6838F-7076R) represent the sequenced regions attached to the corresponding sequencing primers. Numbers included in primer IDs represent the position relative to HPV_SD2 genome. B. Alignment of sequences generated by Sanger method along the HPV_SD2 generated by 454 sequencing. Bars in blue, green and red correspond to sequences shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058404#pone-0058404-g006" target="_blank">Figure 6A</a>. The newly generated sequences covered 4,622 nucleotides representing 63.3% of the total HPV_SD2 genome. C. Comparison of the sequences generated by Sanger method and HPV_SD2 contig generated by 454 sequencing. Size of each Sanger sequence and percentage identity are shown.</p

Taxonomical assignment of sequence reads convenient respiratory waste samples.

<p>A. Overall proportions of sequences with homolog in Genbank (the known, 7.8%) compared to sequences with no homolog in Genbank (Unknown, 92.2%). Unknown/Divergent indicates the proportion of highly divergent and/or novel sequences with no homology to NCBI. B. Proportion of sequences classified as eukaryotes, bacteria and viruses. Sequences were classified using BLASTn search against all non-redundant nucleotide sequences in the NCBI nt database with an E-value cutoff of 10⁻⁵.</p

Summary of known viruses detected in nasopharyngeal samples.

<p>Summary of known viruses detected in nasopharyngeal samples.</p

Composition of sample pools and methods for DNA and RNA extraction.

<p>Composition of sample pools and methods for DNA and RNA extraction.</p

Genomic organization of the HPV_SD2 virus.

<p>Open reading frames (L2, L1, E6, E7, E1, E2, E4) and a 530 bp non-coding long control region (LCR) are shown. B. Details of the LCR region showing the TATA box (TATAAA, positions 3735–3740), a polyadenylated site (AATAAA, positions 3336–3341), 3 palindrome sites (ACCG-N₄-CGGT; positions 3483–3494, 3650–3661, 3720–3731) and 1 degenerate palindrome (ACC-N₆-GGT, positions 3524–3535). C. Metal-binding domains in deduced E6 and E7 proteins.</p