Myrna G. Serrano

The genome of Cryptosporidium hominis

Cryptosporidium species cause acute gastroenteritis and diarrhoea worldwide. They are members of the Apicomplexa—protozoan pathogens that invade host cells by using a specialized apical complex and are usually transmitted by an invertebrate vector or intermediate host. In contrast to other Apicomplexans, Cryptosporidium is transmitted by ingestion of oocysts and completes its life cycle in a single host. No therapy is available, and control focuses on eliminating oocysts in water supplies. Two species, C. hominis and C. parvum, which differ in host range, genotype and pathogenicity, are most relevant to humans. C. hominis is restricted to humans, whereas C. parvum also infects other mammals. Here we describe the eight-chromosome ∼9.2-million-base genome of C. hominis. The complement of C. hominis protein-coding genes shows a striking concordance with the requirements imposed by the environmental niches the parasite inhabits. Energy metabolism is largely from glycolysis. Both aerobic and anaerobic metabolisms are available, the former requiring an alternative electron transport system in a simplified mitochondrion. Biosynthesis capabilities are limited, explaining an extensive array of transporters. Evidence of an apicoplast is absent, but genes associated with apical complex organelles are present. C. hominis and C. parvum exhibit very similar gene complements, and phenotypic differences between these parasites must be due to subtle sequence divergence.

Genome of the Opportunistic Pathogen Streptococcus sanguinis

The genome of Streptococcus sanguinis is a circular DNA molecule consisting of 2,388,435 bp and is 177 to 590 kb larger than the other 21 streptococcal genomes that have been sequenced. The G+C content of the S. sanguinis genome is 43.4%, which is considerably higher than the G+C contents of other streptococci. The genome encodes 2,274 predicted proteins, 61 tRNAs, and four rRNA operons. A 70-kb region encoding pathways for vitamin B(12) biosynthesis and degradation of ethanolamine and propanediol was apparently acquired by horizontal gene transfer. The gene complement suggests new hypotheses for the pathogenesis and virulence of S. sanguinis and differs from the gene complements of other pathogenic and nonpathogenic streptococci. In particular, S. sanguinis possesses a remarkable abundance of putative surface proteins, which may permit it to be a primary colonizer of the oral cavity and agent of streptococcal endocarditis and infection in neutropenic patients.

The truth about metagenomics: quantifying and counteracting bias in 16S rRNA studies

BackgroundCharacterizing microbial communities via next-generation sequencing is subject to a number of pitfalls involving sample processing. The observed community composition can be a severe distortion of the quantities of bacteria actually present in the microbiome, hampering analysis and threatening the validity of conclusions from metagenomic studies. We introduce an experimental protocol using mock communities for quantifying and characterizing bias introduced in the sample processing pipeline. We used 80 bacterial mock communities comprised of prescribed proportions of cells from seven vaginally-relevant bacterial strains to assess the bias introduced in the sample processing pipeline. We created two additional sets of 80 mock communities by mixing prescribed quantities of DNA and PCR product to quantify the relative contribution to bias of (1) DNA extraction, (2) PCR amplification, and (3) sequencing and taxonomic classification for particular choices of protocols for each step. We developed models to predict the “true” composition of environmental samples based on the observed proportions, and applied them to a set of clinical vaginal samples from a single subject during four visits.ResultsWe observed that using different DNA extraction kits can produce dramatically different results but bias is introduced regardless of the choice of kit. We observed error rates from bias of over 85% in some samples, while technical variation was very low at less than 5% for most bacteria. The effects of DNA extraction and PCR amplification for our protocols were much larger than those due to sequencing and classification. The processing steps affected different bacteria in different ways, resulting in amplified and suppressed observed proportions of a community. When predictive models were applied to clinical samples from a subject, the predicted microbiome profiles were better reflections of the physiology and diagnosis of the subject at the visits than the observed community compositions.ConclusionsBias in 16S studies due to DNA extraction and PCR amplification will continue to require attention despite further advances in sequencing technology. Analysis of mock communities can help assess bias and facilitate the interpretation of results from environmental samples.

Differences in vaginal microbiome in African American women versus women of European ancestry.

Women of European ancestry are more likely to harbour a Lactobacillus-dominated microbiome, whereas African American women are more likely to exhibit a diverse microbial profile. African American women are also twice as likely to be diagnosed with bacterial vaginosis and are twice as likely to experience preterm birth. The objective of this study was to further characterize and contrast the vaginal microbial profiles in African American versus European ancestry women. Through the Vaginal Human Microbiome Project at Virginia Commonwealth University, 16S rRNA gene sequence analysis was used to compare the microbiomes of vaginal samples from 1268 African American women and 416 women of European ancestry. The results confirmed significant differences in the vaginal microbiomes of the two groups and identified several taxa relevant to these differences. Major community types were dominated by Gardnerella vaginalis and the uncultivated bacterial vaginosis-associated bacterium-1 (BVAB1) that were common among African Americans. Moreover, the prevalence of multiple bacterial taxa that are associated with microbial invasion of the amniotic cavity and preterm birth, including Mycoplasma, Gardnerella, Prevotella and Sneathia, differed between the two ethnic groups. We investigated the contributions of intrinsic and extrinsic factors, including pregnancy, body mass index, diet, smoking and alcohol use, number of sexual partners, and household income, to vaginal community composition. Ethnicity, pregnancy and alcohol use correlated significantly with the relative abundance of bacterial vaginosis-associated species. Trends between microbial profiles and smoking and number of sexual partners were observed; however, these associations were not statistically significant. These results support and extend previous findings that there are significant differences in the vaginal microbiome related to ethnicity and demonstrate that these differences are pronounced even in healthy women.

Species-level classification of the vaginal microbiome.

BackgroundThe application of next-generation sequencing to the study of the vaginal microbiome is revealing the spectrum of microbial communities that inhabit the human vagina. High-resolution identification of bacterial taxa, minimally to the species level, is necessary to fully understand the association of the vaginal microbiome with bacterial vaginosis, sexually transmitted infections, pregnancy complications, menopause, and other physiological and infectious conditions. However, most current taxonomic assignment strategies based on metagenomic 16S rDNA sequence analysis provide at best a genus-level resolution. While surveys of 16S rRNA gene sequences are common in microbiome studies, few well-curated, body-site-specific reference databases of 16S rRNA gene sequences are available, and no such resource is available for vaginal microbiome studies.ResultsWe constructed the Vaginal 16S rDNA Reference Database, a comprehensive and non-redundant database of 16S rDNA reference sequences for bacterial taxa likely to be associated with vaginal health, and we developed STIRRUPS, a new method that employs the USEARCH algorithm with a curated reference database for rapid species-level classification of 16S rDNA partial sequences. The method was applied to two datasets of V1-V3 16S rDNA reads: one generated from a mock community containing DNA from six bacterial strains associated with vaginal health, and a second generated from over 1,000 mid-vaginal samples collected as part of the Vaginal Human Microbiome Project at Virginia Commonwealth University. In both datasets, STIRRUPS, used in conjunction with the Vaginal 16S rDNA Reference Database, classified more than 95% of processed reads to a species-level taxon using a 97% global identity threshold for assignment.ConclusionsThis database and method provide accurate species-level classifications of metagenomic 16S rDNA sequence reads that will be useful for analysis and comparison of microbiome profiles from vaginal samples. STIRRUPS can be used to classify 16S rDNA sequence reads from other ecological niches if an appropriate reference database of 16S rDNA sequences is available.

Endosymbiosis in trypanosomatids: the genomic cooperation between bacterium and host in the synthesis of essential amino acids is heavily influenced by multiple horizontal gene transfers

BackgroundTrypanosomatids of the genera Angomonas and Strigomonas live in a mutualistic association characterized by extensive metabolic cooperation with obligate endosymbiotic Betaproteobacteria. However, the role played by the symbiont has been more guessed by indirect means than evidenced. Symbiont-harboring trypanosomatids, in contrast to their counterparts lacking symbionts, exhibit lower nutritional requirements and are autotrophic for essential amino acids. To evidence the symbiont’s contributions to this autotrophy, entire genomes of symbionts and trypanosomatids with and without symbionts were sequenced here.ResultsAnalyses of the essential amino acid pathways revealed that most biosynthetic routes are in the symbiont genome. By contrast, the host trypanosomatid genome contains fewer genes, about half of which originated from different bacterial groups, perhaps only one of which (ornithine cyclodeaminase, EC:4.3.1.12) derived from the symbiont. Nutritional, enzymatic, and genomic data were jointly analyzed to construct an integrated view of essential amino acid metabolism in symbiont-harboring trypanosomatids. This comprehensive analysis showed perfect concordance among all these data, and revealed that the symbiont contains genes for enzymes that complete essential biosynthetic routes for the host amino acid production, thus explaining the low requirement for these elements in symbiont-harboring trypanosomatids. Phylogenetic analyses show that the cooperation between symbionts and their hosts is complemented by multiple horizontal gene transfers, from bacterial lineages to trypanosomatids, that occurred several times in the course of their evolution. Transfers occur preferentially in parts of the pathways that are missing from other eukaryotes.ConclusionWe have herein uncovered the genetic and evolutionary bases of essential amino acid biosynthesis in several trypanosomatids with and without endosymbionts, explaining and complementing decades of experimental results. We uncovered the remarkable plasticity in essential amino acid biosynthesis pathway evolution in these protozoans, demonstrating heavy influence of horizontal gene transfer events, from Bacteria to trypanosomatid nuclei, in the evolution of these pathways.

A New Era of the Vaginal Microbiome: Advances Using Next-Generation Sequencing

Until recently, bacterial species that inhabit the human vagina have been primarily studied using organism‐centric approaches. Understanding how these bacterial species interact with each other and the host vaginal epithelium is essential for a more complete understanding of vaginal health. Molecular approaches have already led to the identification of uncultivated bacterial taxa associated with bacterial vaginosis. Here, we review recent studies of the vaginal microbiome and discuss how culture‐independent approaches, such as applications of next‐generation sequencing, are advancing the field and shifting our understanding of how vaginal health is defined. This work may lead to improved diagnostic tools and treatments for women who suffer from, or are at risk for, vaginal imbalances, pregnancy complications, and sexually acquired infections. These approaches may also transform our understanding of how host genetic factors, physiological conditions (e.g., menopause), and environmental exposures (e.g., smoking, antibiotic usage) influence the vaginal microbiome.

Clostridium scindens: a human gut microbe with a high potential to convert glucocorticoids into androgens

Clostridium scindens American Type Culture Collection 35704 is capable of converting primary bile acids to toxic secondary bile acids, as well as converting glucocorticoids to androgens by side-chain cleavage. The molecular structure of the side-chain cleavage product of cortisol produced by C. scindens was determined to be 11β-hydroxyandrost-4-ene-3,17-dione (11β-OHA) by high-resolution mass spectrometry, 1H and 13C NMR spectroscopy, and X-ray crystallography. Using RNA-Seq technology, we identified a cortisol-inducible (∼1,000-fold) operon (desABCD) encoding at least one enzyme involved in anaerobic side-chain cleavage. The desC gene was cloned, overexpressed, purified, and found to encode a 20α-hydroxysteroid dehydrogenase (HSDH). This operon also encodes a putative “transketolase” (desAB) hypothesized to have steroid-17,20-desmolase/oxidase activity, and a possible corticosteroid transporter (desD). RNA-Seq data suggests that the two-carbon side chain of glucocorticords may feed into the pentose-phosphate pathway and are used as a carbon source. The 20α-HSDH is hypothesized to function as a metabolic “rheostat” controlling rates of side-chain cleavage. Phylogenetic analysis suggests this operon is rare in nature and the desC gene evolved from a gene encoding threonine dehydrogenase. The physiological effect of 11β-OHAD on the host or other gut microbes is currently unknown.

Genetic diversity of Trypanosoma cruzi in bats, and multilocus phylogenetic and phylogeographical analyses supporting Tcbat as an independent DTU (discrete typing unit).

Trypanosoma cruzi is a complex of phenotypically and genetically diverse isolates distributed in six discrete typing units (DTUs) designated as TcI-TcVI. Five years ago, T. cruzi isolates from Brazilian bats showing unique patterns of traditional ribosomal and spliced leader PCRs not clustering into any of the six DTUs were designated as the Tcbat genotype. In the present study, phylogenies inferred using SSU rRNA (small subunit of ribosomal rRNA), gGAPDH (glycosomal glyceraldehyde 3-phosphate dehydrogenase) and Cytb (cytochrome b) genes strongly supported Tcbat as a monophyletic lineage prevalent in Brazil, Panama and Colombia. Providing strong support for Tcbat, sequences from 37 of 47 nuclear and 12 mitochondrial genes (retrieved from a draft genome of Tcbat) and reference strains of all DTUs available in databanks corroborated Tcbat as an independent DTU. Consistent with previous studies, multilocus analysis of most nuclear genes corroborated the evolution of T. cruzi from bat trypanosomes its divergence into two main phylogenetic lineages: the basal TcII; and the lineage clustering TcIV, the clade comprising TcIII and the sister groups TcI-Tcbat. Most likely, the common ancestor of Tcbat and TcI was a bat trypanosome. However, the results of the present analysis did not support Tcbat as the ancestor of all DTUs. Despite the insights provided by reports of TcIII, TcIV and TcII in bats, including Amazonian bats harbouring TcII, further studies are necessary to understand the roles played by bats in the diversification of all DTUs. We also demonstrated that in addition to value as molecular markers for DTU assignment, Cytb, ITS rDNA and the spliced leader (SL) polymorphic sequences suggest spatially structured populations of Tcbat. Phylogenetic and phylogeographical analyses, multiple molecular markers specific to Tcbat, and the degrees of sequence divergence between Tcbat and the accepted DTUs strongly support the definitive classification of Tcbat as a new DTU.

Identification and Phylogenetic Analysis of Heme Synthesis Genes in Trypanosomatids and Their Bacterial Endosymbionts

It has been known for decades that some insect-infecting trypanosomatids can survive in culture without heme supplementation while others cannot, and that this capability is associated with the presence of a betaproteobacterial endosymbiont in the flagellates cytoplasm. However, the specific mechanisms involved in this process remained obscure. In this work, we sequence and phylogenetically analyze the heme pathway genes from the symbionts and from their hosts, as well as from a number of heme synthesis-deficient Kinetoplastida. Our results show that the enzymes responsible for synthesis of heme are encoded on the symbiont genomes and produced in close cooperation with the flagellate host. Our evidence suggests that this synergistic relationship is the end result of a history of extensive gene loss and multiple lateral gene transfer events in different branches of the phylogeny of the Trypanosomatidae.