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The diversity and dominant bacterial taxa in the vagina are reported to be influenced by multiple intrinsic and extrinsic factors, including but not limited to pregnancy, contraceptive use, pathogenic states, socioeconomic status, and ancestry. However, the extent to which host genetic factors influence variation in the vaginal microbiota is unclear. We used a biometrical genetic approach to determine whether host genetic factors contribute to inter-individual differences in taxa from a sample of 332 twins who self-identified as being of African (44 pairs) or European ancestry (122 pairs). Lactobacillus crispatus, a major determinant of vaginal health, was identified as heritable among European American women (narrow-sense heritability = 34.7%, P-value = 0.018). Heritability of L. crispatus is consistent with the reduced prevalence of adverse reproductive disorders, including bacterial vaginosis and preterm birth, among women of European ancestry., Wright et al. apply biometric genetic approach to identify the extent to which host genetic factors influence species-level variation in the vaginal microbiota. Their study suggests that Lactobacillus crispatus, a major determinant of vaginal health, is heritable among European American women, consistent with the reduced prevalence of adverse reproductive disorders in them.

Objective The aim of this study was to evaluate the differences between the vaginal microbiome of reproductive-aged women with overweight and obesity (Ow/Ob) compared with healthy weight (HW). Methods In this case-control study, a cohort of 367 nonpregnant women (18 to 40 years) with Ow/Ob (BMI ≥25 kg/m2 ) was case-matched with 367 women with HW (BMI 18.0 to 24.9 kg/m2 ). The study was a secondary analysis of 16S rRNA vaginal microbiome surveys through the Vaginal Human Microbiome Study (VaHMP). Groups were matched on age, race/ethnicity, income, and nulliparity status. Results Mean age and BMI of Ow/Ob and HW groups were 26.8 versus 26.7 years and 37.0 versus 22.1 kg/m2 , respectively. The overall vaginal microbiome composition differed between groups (PERMANOVA, p = 0.035). Women with Ow/Ob had higher alpha diversity compared with women with HW (Wilcoxon test, Shannon index p = 0.025; inverse Simpson index p = 0.026). Lactobacillus dominance (≥30% proportional abundance) was observed in a greater proportion of women with HW (48.7%) compared with Ow/Ob (40.1%; p = 0.026). Conclusions The vaginal microbiome differs in reproductive-aged women with Ow/Ob compared with women with HW, with increased alpha diversity and decreased predominance of Lactobacillus. Observed differences in the vaginal microbiome may partially explain differences in preterm birth and bacterial vaginosis risk between these populations.

The NIH Human Microbiome Project (HMP) has been carried out over ten years and two phases to provide resources, methods, and discoveries that link interactions between humans and their microbiomes to health-related outcomes. The recently completed second phase, the Integrative Human Microbiome Project, comprised studies of dynamic changes in the microbiome and host under three conditions: pregnancy and preterm birth; inflammatory bowel diseases; and stressors that affect individuals with prediabetes. The associated research begins to elucidate mechanisms of host–microbiome interactions under these conditions, provides unique data resources (at the HMP Data Coordination Center), and represents a paradigm for future multi-omic studies of the human microbiome., Over ten years, the Human Microbiome Project has provided resources for studying the microbiome and its relationship to disease; this Perspective summarizes the key achievements and findings of the project and its relationship to the broader field.

The particularly interdisciplinary nature of human microbiome research makes the organization and reporting of results spanning epidemiology, biology, bioinformatics, translational medicine and statistics a challenge. Commonly used reporting guidelines for observational or genetic epidemiology studies lack key features specific to microbiome studies. Therefore, a multidisciplinary group of microbiome epidemiology researchers adapted guidelines for observational and genetic studies to culture-independent human microbiome studies, and also developed new reporting elements for laboratory, bioinformatics and statistical analyses tailored to microbiome studies. The resulting tool, called ‘Strengthening The Organization and Reporting of Microbiome Studies’ (STORMS), is composed of a 17-item checklist organized into six sections that correspond to the typical sections of a scientific publication, presented as an editable table for inclusion in supplementary materials. The STORMS checklist provides guidance for concise and complete reporting of microbiome studies that will facilitate manuscript preparation, peer review, and reader comprehension of publications and comparative analysis of published results.

Background Trypanosoma conorhini and Trypanosoma rangeli, like Trypanosoma cruzi, are kinetoplastid protist parasites of mammals displaying divergent hosts, geographic ranges and lifestyles. Largely nonpathogenic T. rangeli and T. conorhini represent clades that are phylogenetically closely related to the T. cruzi and T. cruzi-like taxa and provide insights into the evolution of pathogenicity in those parasites. T. rangeli, like T. cruzi is endemic in many Latin American countries, whereas T. conorhini is tropicopolitan. T. rangeli and T. conorhini are exclusively extracellular, while T. cruzi has an intracellular stage in the mammalian host. Results Here we provide the first comprehensive sequence analysis of T. rangeli AM80 and T. conorhini 025E, and provide a comparison of their genomes to those of T. cruzi G and T. cruzi CL, respectively members of T. cruzi lineages TcI and TcVI. We report de novo assembled genome sequences of the low-virulent T. cruzi G, T. rangeli AM80, and T. conorhini 025E ranging from ~ 21–25 Mbp, with ~ 10,000 to 13,000 genes, and for the highly virulent and hybrid T. cruzi CL we present a ~ 65 Mbp in-house assembled haplotyped genome with ~ 12,500 genes per haplotype. Single copy orthologs of the two T. cruzi strains exhibited ~ 97% amino acid identity, and ~ 78% identity to proteins of T. rangeli or T. conorhini. Proteins of the latter two organisms exhibited ~ 84% identity. T. cruzi CL exhibited the highest heterozygosity. T. rangeli and T. conorhini displayed greater metabolic capabilities for utilization of complex carbohydrates, and contained fewer retrotransposons and multigene family copies, i.e. trans-sialidases, mucins, DGF-1, and MASP, compared to T. cruzi. Conclusions Our analyses of the T. rangeli and T. conorhini genomes closely reflected their phylogenetic proximity to the T. cruzi clade, and were largely consistent with their divergent life cycles. Our results provide a greater context for understanding the life cycles, host range expansion, immunity evasion, and pathogenesis of these trypanosomatids. Electronic supplementary material The online version of this article (10.1186/s12864-018-5112-0) contains supplementary material, which is available to authorized users.

A mechanistic link between trimethylamine N-oxide (TMAO) and atherogenesis has been reported. TMAO is generated enzymatically in the liver by the oxidation of trimethylamine (TMA), which is produced from dietary choline, carnitine and betaine by gut bacteria. It is known that certain members of methanogenic archaea (MA) could use methylated amines such as trimethylamine as growth substrates in culture. Therefore, we investigated the efficacy of gut colonization with MA on lowering plasma TMAO concentrations. Initially, we screened for the colonization potential and TMAO lowering efficacy of five MA species in C57BL/6 mice fed with high choline/TMA supplemented diet, and found out that all five species could colonize and lover plasma TMAO levels, although with different efficacies. The top performing MA, Methanobrevibacter smithii, Methanosarcina mazei, and Methanomicrococcus blatticola, were transplanted into Apoe−/− mice fed with high choline/TMA supplemented diet. Similar to C57BL/6 mice, following initial provision of the MA, there was progressive attrition of MA within fecal microbial communities post-transplantation during the initial 3 weeks of the study. In general, plasma TMAO concentrations decreased significantly in proportion to the level of MA colonization. In a subsequent experiment, use of antibiotics and repeated transplantation of Apoe−/− mice with M. smithii, led to high engraftment levels during the 9 weeks of the study, resulting in a sustained and significantly lower average plasma TMAO concentrations (18.2 ± 19.6 μM) compared to that in mock-transplanted control mice (120.8 ± 13.0 μM, p Apoe−/− mice, M. smithii-colonized mice also had a 44% decrease in aortic plaque area (8,570 μm [95% CI 19587–151821] vs. 15,369 μm [95% CI [70058–237321], p = 0.34), and 52% reduction in the fat content in the atherosclerotic plaques (14,283 μm [95% CI 4,957–23,608] vs. 29,870 μm [95% CI 18,074–41,666], p = 0.10), although these differences did not reach significance. Gut colonization with M. smithii leads to a significant reduction in plasma TMAO levels, with a tendency for attenuation of atherosclerosis burden in Apoe−/− mice. The anti-atherogenic potential of MA should be further tested in adequately powered experiments.

Vibrio vulnificus is a Gram-negative bacterium and an opportunistic pathogen that can cause septicemia or necrotizing fasciitis. Here, we report the draft genome sequences of 42 environmental V. vulnificus strains collected from the northern Gulf of Mexico. These data will allow for more robust comparisons between clinical and environmental strains.

The microbiome of the female reproductive tract has implications for women’s reproductive health. We examined the vaginal microbiome in two cohorts of women who experienced normal term births: a cross-sectionally sampled cohort of 613 pregnant and 1,969 non-pregnant women, focusing on 300 pregnant and 300 non-pregnant women of African, Hispanic or European ancestry case-matched for race, gestational age and household income; and a longitudinally sampled cohort of 90 pregnant women of African or non-African ancestry. In these women, the vaginal microbiome shifted during pregnancy toward Lactobacillus-dominated profiles at the expense of taxa often associated with vaginal dysbiosis. The shifts occurred early in pregnancy, followed predictable patterns, were associated with simplification of the metabolic capacity of the microbiome and were significant only in women of African or Hispanic ancestry. Both genomic and environmental factors are likely contributors to these trends, with socioeconomic status as a likely environmental influence. Ancestry and socioeconomic factors influence predictable changes in the vaginal microbiome that occur early in pregnancy in women who experience normal term birth.

Background The human placenta has been traditionally viewed as sterile, and microbial invasion of this organ has been associated with adverse pregnancy outcomes. Yet, recent studies that utilized sequencing techniques reported that the human placenta at term contains a unique microbiota. These conclusions are largely based on the results derived from the sequencing of placental samples. However, such an approach carries the risk of capturing background-contaminating DNA (from DNA extraction kits, polymerase chain reaction reagents, and laboratory environments) when low microbial biomass samples are studied. Objective To determine whether the human placenta delivered at term in patients without labor who undergo cesarean delivery harbors a resident microbiota (“the assemblage of microorganisms present in a defined niche or environment”). Study Design This cross-sectional study included placentas from 29 women who had a cesarean delivery without labor at term. The study also included technical controls to account for potential background-contaminating DNA, inclusive in DNA extraction kits, polymerase chain reaction reagents, and laboratory environments. Bacterial profiles of placental tissues and background technical controls were characterized and compared with the use of bacterial culture, quantitative real-time polymerase chain reaction, 16S ribosomal RNA gene sequencing, and metagenomic surveys. Results (1) Twenty-eight of 29 placental tissues had a negative culture for microorganisms. The microorganisms retrieved by culture from the remaining sample were likely contaminants because corresponding 16S ribosomal RNA genes were not detected in the same sample. (2) Quantitative real-time polymerase chain reaction did not indicate greater abundances of bacterial 16S ribosomal RNA genes in placental tissues than in technical controls. Therefore, there was no evidence of the presence of microorganisms above background contamination from reagents in the placentas. (3) 16S ribosomal RNA gene sequencing did not reveal consistent differences in the composition or structure of bacterial profiles between placental samples and background technical controls. (4) Most of the bacterial sequences obtained from metagenomic surveys of placental tissues were from cyanobacteria, aquatic bacteria, or plant pathogens, which are microbes unlikely to populate the human placenta. Coprobacillus, which constituted 30.5% of the bacterial sequences obtained through metagenomic sequencing of placental samples, was not identified in any of the 16S ribosomal RNA gene surveys of these samples. These observations cast doubt as to whether this organism is really present in the placenta of patients at term not in labor. Conclusion With the use of multiple modes of microbiologic inquiry, a resident microbiota could not be identified in human placentas delivered at term from women without labor. A consistently significant difference in the abundance and/or presence of a microbiota between placental tissue and background technical controls could not be found. All cultures of placental tissue, except 1, did not yield bacteria. Incorporating technical controls for potential sources of background-contaminating DNA for studies of low microbial biomass samples, such as the placenta, is necessary to derive reliable conclusions.

Small Heat-Shock Proteins (sHSPs) and other proteins bearing alpha-crystallin domains (ACD) participate in defense against heat and oxidative stress and play important roles in cell cycle, cytoskeleton dynamics, and immunological and pathological mechanisms in eukaryotes. However, little is known about these proteins in early-diverging lineages of protists such as the kinetoplastids. Here, ACD-like proteins (ACDp) were investigated in genomes of 61 species of 12 kinetoplastid genera, including Trypanosoma spp. (23 species of mammals, reptiles and frogs), Leishmania spp. (mammals and lizards), trypanosomatids of insects, Phytomonas spp. of plants, and bodonids. Comparison of ACDps based on domain architecture, predicted tertiary structure, phylogeny and genome organization reveals a kinetoplastid evolutionarily conserved repertoire, which diversified prior to trypanosomatid adaptation to parasitic life. We identified 9 ACDp orthologs classified in 8 families of TryACD: four previously recognized (HSP20, Tryp23A, Tryp23B and ATOM69), and four characterized for the first time in kinetoplastids (TryACDP, TrySGT1, TryDYX1C1 and TryNudC). A single copy of each ortholog was identified in each genome alongside TryNudC1/TrypNudC2 homologs and, overall, ACDPs were under strong selection pressures at main phylogenetic lineages. Transcripts of all ACDPs were identified across the life stages of T. cruzi, T. brucei and Leishmania spp., but proteomic profiles suggested that most ACDPs may be species- and stage-regulated. Our findings establish the basis for functional studies, and provided evolutionary and structural support for an underestimated repertoire of ACDps in the kinetoplastids.

Background Bacterial vaginosis (BV) is the leading dysbiosis of the vaginal microbiome. The pathways leading towards the development of BV are not well understood. Gardnerella vaginalis is frequently associated with BV. G. vaginalis produces the cholesterol-dependent cytolysin (CDC), vaginolysin, which can lyse a variety of human cells and is thought to play a role in pathogenesis. Because membrane cholesterol is required for vaginolysin to function, and because HMG-CoA reductase inhibitors (statins) affect not only serum levels of cholesterol but membrane levels as well, we hypothesized that statins might affect the vaginal microbiome. Methods To investigate the relationship between use of the statins and the vaginal microbiome, we analyzed 16S rRNA gene taxonomic surveys performed on vaginal samples from 133 women who participated in the Vaginal Human Microbiome Project and who were taking statins at the time of sampling, 152 women who reported high cholesterol levels but were not taking statins, and 316 women who did not report high cholesterol. To examine the effect of statins on the cytolytic effect of vaginolysin, the cholesterol-dependent cytolysin (CDC) produced by Gardnerella vaginalis, we assessed the effect of simvastatin pretreatment of VK2E6/E7 vaginal epithelial cells on vaginolysin-mediated cytotoxicity. Results The mean proportion of G. vaginalis among women taking statins was significantly lower relative to women not using statins. Women using statins had higher mean proportions of Lactobacillus crispatus relative to women with normal cholesterol levels, and higher levels of Lactobacillus jensenii relative to women with high cholesterol but not taking statins. In vitro, vaginal epithelial cells pretreated with simvastatin were relatively resistant to vaginolysin and this effect was inhibited by cholesterol. Conclusions In this cross-sectional study, statin use was associated with reduced proportions of G. vaginalis and greater proportions of beneficial lactobacilli within the vaginal microbiome. The negative association between statin use and G. vaginalis may be related to inhibition of vaginolysin function.

Background: Recent studies of various human microbiome habitats have revealed thousands of bacterial species and the existence of large variation in communities of microorganisms in the same habitats across individual human subjects. Previous efforts to summarize this diversity, notably in the human gut and vagina, have categorized microbiome profiles by clustering them into community state types (CSTs). The functional relevance of specific CSTs has not been established. Objective: We investigate whether CSTs can be used to assess dynamics in the microbiome. Design: We conduct a re-analysis of five sequencing-based microbiome surveys derived from vaginal samples with repeated measures. Results: We observe that detection of a CST transition is largely insensitive to choices in methods for normalization or clustering. We find that healthy subjects persist in a CST for two to three weeks or more on average, while those with evidence of dysbiosis tend to change more often. Changes in CST can be gradual or occur over less than one day. Upcoming CST changes and switches to high-risk CSTs can be predicted with high accuracy in certain scenarios. Finally, we observe that presence of Gardnerella vaginalis is a strong predictor of an upcoming CST change. Conclusion: Overall, our results show that the CST concept is useful for studying microbiome dynamics.

Quantitative relationship between the magnitude of variation in minor histocompatibility antigens (mHA) and graft versus host disease (GVHD) pathophysiology in stem cell transplant (SCT) donor-recipient pairs (DRP) is not established. In order to elucidate this relationship, whole exome sequencing (WES) was performed on 27 HLA matched related (MRD), & 50 unrelated donors (URD), to identify nonsynonymous single nucleotide polymorphisms (SNPs). An average 2,463 SNPs were identified in MRD, and 4,287 in URD DRP (p

The association between human cytomegalovirus (hCMV) reactivation and the development of graft-versus-host-disease (GVHD) has been observed in stem cell transplantation (SCT). Seventy seven SCT donor-recipient pairs (DRP) (HLA matched unrelated donor (MUD), n=50; matched related donor (MRD), n=27) underwent whole exome sequencing to identify single nucleotide polymorphisms (SNPs) generating alloreactive peptide libraries for each DRP (9-mer peptide-HLA complexes); Human CMV CROSS (Cross-Reactive Open Source Sequence) Database was compiled from NCBI; HLA class I binding affinity for each DRPs HLA was calculated by NetMHCpan 2.8 and hCMV- derived 9-mers algorithmically compared to the alloreactive peptide-HLA complex libraries. Short consecutive (6 or greater) amino acid (AA) sequence homology matching hCMV to recipient peptides was considered for HLA-bound-peptide (IC50, Pre-submission manuscript, 4 tables, 5 figures, 2 supplements & 2 Appendices-available upon request from first author

Cryptosporidium is a major cause of severe diarrhea, especially in malnourished children. Using a murine model of C. parvum oocyst challenge that recapitulates clinical features of severe cryptosporidiosis during malnutrition, we interrogated the effect of protein malnutrition (PM) on primary and secondary responses to C. parvum challenge, and tested the differential ability of mucosal priming strategies to overcome the PM-induced susceptibility. We determined that while PM fundamentally alters systemic and mucosal primary immune responses to Cryptosporidium, priming with C. parvum (106 oocysts) provides robust protective immunity against re-challenge despite ongoing PM. C. parvum priming restores mucosal Th1-type effectors (CD3+CD8+CD103+ T-cells) and cytokines (IFNγ, and IL12p40) that otherwise decrease with ongoing PM. Vaccination strategies with Cryptosporidium antigens expressed in the S. Typhi vector 908htr, however, do not enhance Th1-type responses to C. parvum challenge during PM, even though vaccination strongly boosts immunity in challenged fully nourished hosts. Remote non-specific exposures to the attenuated S. Typhi vector alone or the TLR9 agonist CpG ODN-1668 can partially attenuate C. parvum severity during PM, but neither as effectively as viable C. parvum priming. We conclude that although PM interferes with basal and vaccine-boosted immune responses to C. parvum, sustained reductions in disease severity are possible through mucosal activators of host defenses, and specifically C. parvum priming can elicit impressively robust Th1-type protective immunity despite ongoing protein malnutrition. These findings add insight into potential correlates of Cryptosporidium immunity and future vaccine strategies in malnourished children., Author Summary Cryptosporidium attributable morbidities in malnourished children are increasingly recognized. Exactly how malnutrition interferes with host mucosal immunity to diarrheal pathogens and mucosal vaccine responses remains unclear. Dissecting these interactions in an experimental model of cryptosporidiosis can uncover new insights into novel therapeutic approaches against a pathogen for which effective therapies and vaccines are currently unavailable. We demonstrate that although malnutrition diminishes baseline (primary) Th1-type mucosal immunity these deficits can be partially overcome via non-specific mucosal strategies (S. Typhi and CpG) and completely restored after a sub-clinical (low-dose) exposure to viable C. parvum. These results add insight into preventive strategies to help alleviate Cryptosporidium-specific diarrhea in children in low-resource settings and abrogate prolonged post-infection sequelae.

A variety of microbial communities and their genes (the microbiome) exist throughout the human body, with fundamental roles in human health and disease. The National Institutes of Health (NIH)-funded Human Microbiome Project Consortium has established a population-scale framework to develop metagenomic protocols, resulting in a broad range of quality-controlled resources and data including standardized methods for creating, processing and interpreting distinct types of high-throughput metagenomic data available to the scientific community. Here we present resources from a population of 242 healthy adults sampled at 15 or 18 body sites up to three times, which have generated 5,177 microbial taxonomic profiles from 16S ribosomal RNA genes and over 3.5 terabases of metagenomic sequence so far. In parallel, approximately 800 reference strains isolated from the human body have been sequenced. Collectively, these data represent the largest resource describing the abundance and variety of the human microbiome, while providing a framework for current and future studies.

Vaginal lactobacilli can inhibit colonization by and growth of other bacteria, thereby preventing development of bacterial vaginosis (BV). Amongst the lactobacilli, Lactobacillus crispatus appears to be particularly effective at inhibiting growth of BV-associated bacteria. Nonetheless, some women who are colonized with this species can still develop clinical BV. Therefore, we sought to determine whether strains of L. crispatus that colonize women with lactobacilli-dominated vaginal microbiomes are distinct from strains that colonize women who develop BV. The genomes of L. crispatus isolates from four women with lactobacilli-dominated vaginal microbiomes ( 12 % 16S rRNA reads from bacterial taxa associated with BV) were sequenced and compared. Lactic acid production by the different strains was quantified. Phage induction in the strains was also analysed. There was considerable genetic diversity between strains, and several genes were exclusive to either the strains from Lactobacillus-dominated microbiomes or those containing BV-associated bacteria. Overall, strains from microbiomes dominated by lactobacilli did not differ from strains from microbiomes containing BV-associated bacteria with respect to lactic acid production. All of the strains contained multiple phage, but there was no clear distinction between the presence or absence of BV-associated bacteria with respect to phage-induced lysis. Genes found to be exclusive to the Lactobacillus-dominated versus BV-associated bacteria-containing microbiomes could play a role in the maintenance of vaginal health and the development of BV, respectively.

The oral cavity represents an initial entry way for oral and gut indigenous colonization. Skin-to-skin (STS) care, in which the mother holds the diaper clad naked preterm (PT) infant between her breasts, is associated with improved digestive function, decreased stress, and improved survival. This study evaluated the development of oral microbial colonization repertoires and health characteristics in PT infants with or without STS exposure.Saliva from 42 PT infants (32 weeks of gestation at birth) was collected prospectively at 1 month and/or at discharge. High-throughput 16S rRNA sequencing identified microbial diversity and prevalence of bacterial signatures correlated with clinical STS or non-STS care.Corrected for gestational age (CGA) at sampling, bacterial taxa demonstrated increased Streptococcus as a signature of oral repertoire maturation. STS was associated with increased Streptococcus (p 0.024), while non-STS was associated with greater Corynebacterium (p 0.023) and Pseudomonas (p 0.019) in infants ≤ 32 weeks CGA. In infants 32 weeks CGA, Neisseria and Acinetobacter were more prevalent, 50 vs. 16.7% and 40 vs. 0%, respectively. STS care was associated with shorter hospitalization (p 0.039).STS care during earlier gestation was associated with a distinct microbial pattern and an accelerated pace of oral microbial repertoire maturity.

Objective Microbial invasion of the amniotic cavity is associated with spontaneous preterm labor and adverse pregnancy outcome, and Mycoplasma hominis often is present. However, the pathogenic process by which M hominis invades the amniotic cavity and gestational tissues, often resulting in chorioamnionitis and preterm birth, remains unknown. We hypothesized that strains of M hominis vary genetically with regards to their potential to invade and colonize the amniotic cavity and placenta. Study Design We sequenced the entire genomes of 2 amniotic fluid isolates and a placental isolate of M hominis from pregnancies that resulted in preterm births and compared them with the previously sequenced genome of the type strain PG21. We identified genes that were specific to the amniotic fluid/placental isolates. We then determined the microbial burden and the presence of these genes in another set of subjects from whom samples of amniotic fluid had been collected and were positive for M hominis. Results We identified 2 genes that encode surface-located membrane proteins (Lmp1 and Lmp-like) in the sequenced amniotic fluid/placental isolates that were truncated severely in PG21. We also identified, for the first time, a microbial gene of unknown function that is referred to in this study as gene of interest C that was associated significantly with bacterial burden in amniotic fluid and the risk of preterm delivery in patients with preterm labor. Conclusion A gene in M hominis was identified that is associated significantly with colonization and/or infection of the upper reproductive tract during pregnancy and with preterm birth.

Humans are colonized by thousands of bacterial species, but it is difficult to assess the metabolic and pathogenic potential of the majority of these because they have yet to be cultured. Here, we characterize an uncultivated vaginal mycoplasma tightly associated with trichomoniasis that was previously known by its 16S rRNA sequence as “Mnola.” In this study, the mycoplasma was found almost exclusively in women infected with the sexually transmitted pathogen Trichomonas vaginalis, but rarely observed in women with no diagnosed disease. The genomes of four strains of this species were reconstructed using metagenome sequencing and assembly of DNA from four discrete mid-vaginal samples, one of which was obtained from a pregnant woman with trichomoniasis who delivered prematurely. These bacteria harbor several putative virulence factors and display unique metabolic strategies. Genes encoding proteins with high similarity to potential virulence factors include two collagenases, a hemolysin, an O-sialoglycoprotein endopeptidase and a feoB-type ferrous iron transport system. We propose the name “Candidatus Mycoplasma girerdii” for this potential new pathogen.

Melatonin is a ubiquitous molecule and exists across kingdoms including plant species. Studies on melatonin in plants have mainly focused on its physiological influence on growth and development, and on its biosynthesis. Much less attention has been drawn to its affect on genome-wide gene expression. To comprehensively investigate the role(s) of melatonin at the genomics level, we utilized mRNA-seq technology to analyze Arabidopsis plants subjected to a 16-hour 100 pM (low) and 1 mM (high) melatonin treatment. The expression profiles were analyzed to identify differentially expressed genes. 100 pM melatonin treatment significantly affected the expression of only 81 genes with 51 down-regulated and 30 up-regulated. However, 1 mM melatonin significantly altered 1308 genes with 566 up-regulated and 742 down-regulated. Not all genes altered by low melatonin were affected by high melatonin, indicating different roles of melatonin in regulation of plant growth and development under low and high concentrations. Furthermore, a large number of genes altered by melatonin were involved in plant stress defense. Transcript levels for many stress receptors, kinases, and stress-associated calcium signals were up-regulated. The majority of transcription factors identified were also involved in plant stress defense. Additionally, most identified genes in ABA, ET, SA and JA pathways were up-regulated, while genes pertaining to auxin responses and signaling, peroxidases, and those associated with cell wall synthesis and modifications were mostly down-regulated. Our results indicate critical roles of melatonin in plant defense against various environmental stresses, and provide a framework for functional analysis of genes in melatonin-mediated signaling pathways.

Until recently, the apicomplexan parasites,Cryptosporidium hominisandC. parvum, were considered the same species. However, the two parasites, now considered distinct species, exhibit significant differences in host range, infectivity, and pathogenicity, and their sequenced genomes exhibit only 95–97% identity. The availability of the complete genome sequences of these organisms provides the potential to identify the genetic variations that are responsible for the phenotypic differences between the two parasites. We compared the genome organization and structure, gene composition, the metabolic and other pathways, and the local sequence identity between the genes of these twoCryptosporidiumspecies. Our observations show that the phenotypic differences betweenC. hominisandC. parvumare not due to gross genome rearrangements, structural alterations, gene deletions or insertions, metabolic capabilities, or other obvious genomic alterations. Rather, the results indicate that these genomes exhibit a remarkable structural and compositional conservation and suggest that the phenotypic differences observed are due to subtle variations in the sequences of proteins that act at the interface between the parasite and its host.

International audience; 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.

Texto completo. Acesso restrito. p. 579–591 Submitted by Santiago Fabio (fabio.ssantiago@hotmail.com) on 2013-10-16T20:33:46Z No. of bitstreams: 1 5555555555.pdf: 1459187 bytes, checksum: 8d7186a26071a6aedf0690a3c922fa6a (MD5) Made available in DSpace on 2013-10-16T20:33:47Z (GMT). No. of bitstreams: 1 5555555555.pdf: 1459187 bytes, checksum: 8d7186a26071a6aedf0690a3c922fa6a (MD5) Previous issue date: 2013 CBA macrophages effectively control Leishmania major infection, yet are permissive to Leishmania amazonensis. Employing a transcriptomic approach, we previously showed the up-regulation of the genes involved in the classical pathway of macrophage activation in resistant mice. However, microarray analyses do not evaluate changes in gene expression that occur after translation. To circumvent this analytical limitation, we employed a proteomics approach to increase our understanding of the modulations that occur during infection and identify novel targets for the control of Leishmania infection. To identify proteins whose expression changes in CBA macrophages infected with L. major or L. amazonensis, protein extracts were obtained and digested and the peptides were characterized using multi-dimensional liquid chromatography coupled with tandem mass spectrometry analyses. A total of 162 proteins were selected as potentially modulated. Using biological network analyses, these proteins were classified as primarily involved in cellular metabolism and grouped into cellular development biological networks. This study is the first to use a proteomics approach to describe the protein modulations involved in cellular metabolism during the initial events of Leishmania–macrophage interaction. Based on these findings, we hypothesize that these differentially expressed proteins likely play a pivotal role in determining the course of infection. Salvador

International audience; Some non-pathogenic trypanosomatids maintain a mutualistic relationship with a betaproteobacterium of the Alcaligenaceae family. Intensive nutritional exchanges have been reported between the two partners, indicating that these protozoa are excellent biological models to study metabolic co-evolution. We previously sequenced and herein investigate the entire genomes of five trypanosomatids which harbor a symbiotic bacterium (SHTs for Symbiont-Haboring Trypanosomatids) and the respective bacteria (TPEs for Trypanosomatid Proteobacterial Endosymbiont), as well as two trypanosomatids without symbionts (RTs for Regular Trypanosomatids), for the presence of genes of the classical pathways for vitamin biosynthesis. Our data show that genes for the biosynthetic pathways of thiamine, biotin, and nicotinic acid are absent from all trypanosomatid genomes. This is in agreement with the absolute growth requirement for these vitamins in all protozoa of the family. Also absent from the genomes of RTs are the genes for the synthesis of pantothenic acid, folic acid, riboflavin, and vitamin B6. This is also in agreement with the available data showing that RTs are auxotrophic for these essential vitamins. On the other hand, SHTs are autotrophic for such vitamins. Indeed, all the genes of the corresponding biosynthetic pathways were identified, most of them in the symbiont genomes, while a few genes, mostly of eukaryotic origin, were found in the host genomes. The only exceptions to the latter are: the gene coding for the enzyme ketopantoate reductase (EC:1.1.1.169) which is related instead to the Firmicutes bacteria; and two other genes, one involved in the salvage pathway of pantothenic acid and the other in the synthesis of ubiquinone, that are related to Gammaproteobacteria. Their presence in trypanosomatids may result from lateral gene transfer. Taken together, our results reinforce the idea that the low nutritional requirement of SHTs is associated with the presence of the symbiotic bacterium, which contains most genes for vitamin production.

Background Bacteria of the genus Sneathia are emerging as potential pathogens of the female reproductive tract. Species of Sneathia, which were formerly grouped with Leptotrichia, can be part of the normal microbiota of the genitourinary tracts of men and women, but they are also associated with a variety of clinical conditions including bacterial vaginosis, preeclampsia, preterm labor, spontaneous abortion, post-partum bacteremia and other invasive infections. Sneathia species also exhibit a significant correlation with sexually transmitted diseases and cervical cancer. Because Sneathia species are fastidious and rarely cultured successfully in vitro; and the genomes of members of the genus had until now not been characterized, very little is known about the physiology or the virulence of these organisms. Results Here, we describe a novel species, Sneathia amnii sp. nov, which closely resembles bacteria previously designated "Leptotrichia amnionii". As part of the Vaginal Human Microbiome Project at VCU, a vaginal isolate of S. amnii sp. nov. was identified, successfully cultured and bacteriologically cloned. The biochemical characteristics and virulence properties of the organism were examined in vitro, and the genome of the organism was sequenced, annotated and analyzed. The analysis revealed a reduced circular genome of ~1.34 Mbp, containing ~1,282 protein-coding genes. Metabolic reconstruction of the bacterium reflected its biochemical phenotype, and several genes potentially associated with pathogenicity were identified. Conclusions Bacteria with complex growth requirements frequently remain poorly characterized and, as a consequence, their roles in health and disease are unclear. Elucidation of the physiology and identification of genes putatively involved in the metabolism and virulence of S. amnii may lead to a better understanding of the role of this potential pathogen in bacterial vaginosis, preterm birth, and other issues associated with vaginal and reproductive health.

Trypanosoma cruzi, the agent of Chagas disease, is a complex of genetically diverse isolates highly phylogenetically related to T. cruzi-like species, Trypanosoma cruzi marinkellei and Trypanosoma dionisii, all sharing morphology of blood and culture forms and development within cells. However, they differ in hosts, vectors and pathogenicity: T. cruzi is a human pathogen infective to virtually all mammals whilst the other two species are non-pathogenic and bat restricted. Previous studies suggest that variations in expression levels and genetic diversity of cruzipain, the major isoform of cathepsin L-like (CATL) enzymes of T. cruzi, correlate with levels of cellular invasion, differentiation, virulence and pathogenicity of distinct strains. In this study, we compared 80 sequences of genes encoding cruzipain from 25 T. cruzi isolates representative of all discrete typing units (DTUs TcI-TcVI) and the new genotype Tcbat and 10 sequences of homologous genes from other species. The catalytic domain repertoires diverged according to DTUs and trypanosome species. Relatively homogeneous sequences are found within and among isolates of the same DTU except TcV and TcVI, which displayed sequences unique or identical to those of TcII and TcIII, supporting their origin from the hybridization between these two DTUs. In network genealogies, sequences from T. cruzi clustered tightly together and closer to T. c. marinkellei than to T. dionisii and largely differed from homologues of T. rangeli and T. b. brucei. Here, analysis of isolates representative of the overall biological and genetic diversity of T. cruzi and closest T. cruzi-like species evidenced DTU- and species-specific polymorphisms corroborating phylogenetic relationships inferred with other genes. Comparison of both phylogenetically close and distant trypanosomes is valuable to understand host-parasite interactions, virulence and pathogenicity. Our findings corroborate cruzipain as valuable target for drugs, vaccine, diagnostic and genotyping approaches.

Author Manuscript date: 2013 February 05., Studies of the human microbiome have revealed that even healthy individuals differ remarkably in the microbes that occupy habitats such as the gut, skin and vagina. Much of this diversity remains unexplained, although diet, environment, host genetics and early microbial exposure have all been implicated. Accordingly, to characterize the ecology of human-associated microbial communities, the Human Microbiome Project has analysed the largest cohort and set of distinct, clinically relevant body habitats so far. We found the diversity and abundance of each habitat’s signature microbes to vary widely even among healthy subjects, with strong niche specialization both within and among individuals. The project encountered an estimated 81–99% of the genera, enzyme families and community configurations occupied by the healthy Western microbiome. Metagenomic carriage of metabolic pathways was stable among individuals despite variation in community structure, and ethnic/racial background proved to be one of the strongest associations of both pathways and microbes with clinical metadata. These results thus delineate the range of structural and functional configurations normal in the microbial communities of a healthy population, enabling future characterization of the epidemiology, ecology and translational applications of the human microbiome.

Background Cryptosporidiosis is a ubiquitous infectious disease, caused by the protozoan parasites Cryptosporidium hominis and C. parvum, leading to acute, persistent and chronic diarrhea worldwide. Although the complications of this disease can be serious, even fatal, in immunocompromised patients of any age, they have also been found to lead to long term effects, including growth inhibition and impaired cognitive development, in infected immunocompetent children. The Cryptosporidium life cycle alternates between a dormant stage, the oocyst, and a highly replicative phase that includes both asexual vegetative stages as well as sexual stages, implying fine genetic regulatory mechanisms. The parasite is extremely difficult to study because it cannot be cultured in vitro and animal models are equally challenging. The recent publication of the genome sequence of C. hominis and C. parvum has, however, significantly advanced our understanding of the biology and pathogenesis of this parasite. Methodology/Principal Findings Herein, our goal was to identify cis-regulatory elements associated with heat shock response in Cryptosporidium using a combination of in silico and real time RT-PCR strategies. Analysis with Gibbs-Sampling algorithms of upstream non-translated regions of twelve genes annotated as heat shock proteins in the Cryptosporidium genome identified a highly conserved over-represented sequence motif in eleven of them. RT-PCR analyses, described herein and also by others, show that these eleven genes bearing the putative element are induced concurrent with excystation of parasite oocysts via heat shock. Conclusions/Significance Our analyses suggest that occurrences of a motif identified in the upstream regions of the Cryptosporidium heat shock genes represent parts of the transcriptional apparatus and function as stress response elements that activate expression of these genes during excystation, and possibly at other stages in the life cycle of the parasite. Since heat shock and excystation represent a critical step in the development of the infectious sporozoite form of Cryptosporidium, these results provide important insight into the pathogenicity of the parasite.

To study the transcriptional response of mammalian cells to infection with the intracellular apicomplexan parasite Cryptosporidium parvum, infected and uninfected cells were recovered from C. parvum-infected cell monolayers. This approach, which contrasts with a more conventional experimental design that compares infected to uninfected cell monolayers, enabled the identification of functional categories of genes that are differentially transcribed as a direct consequence of the presence of intracellular parasites. Among several categories of upregulated genes, glycoprotein metabolism was significantly overrepresented. To investigate whether these transcriptional changes affected the composition of the surface of infected cells, cells were probed with fluorescently labeled lectins. Among a panel of seven lectins, soybean agglutinin, which recognizes N-acetyl-D-galactosamine, generated the largest difference in fluorescence between infected and uninfected cells. The origin of the fluorescent signal emitted by infected cells was further investigated and attributed to the overexpression of glycoprotein on the surface of infected cells, as well as the presence of glycoprotein located in the proximity of intracellular parasites. Cryptosporidium parvum, an apicomplexan protozoan species, infects the intestine and sometimes the respiratory tract and bile duct of various mammalian hosts. In immunocompetent humans the infection causes self-limited diarrhea, but in immunocompromised individuals, primarily those with AIDS, the infection can become chronic and potentially cause lifethreatening syndromes (5). Infection with Cryptosporidium parasites occurs when food or water contaminated with oocysts is ingested, or possibly through the inhalation of oocysts. After entering the gastrointestinal tract of the host, four sporozoites released from the oocyst seek to invade the intestinal epithelial cells, where they multiply as meronts. Global gene expression profiles in cell monolayers infected with C. parvum have been studied using microarrays (3, 8, 20). These studies found that genes belonging to the cell proliferation, apoptosis, signal transduction, and transcription categories are overrepresented among significantly regulated genes (8, 20, 39). The overexpression of the osteoprotegerin gene in host cells after infection also was reported (3). Published microarray studies of C. parvum-infected cells are based on RNA extracted from cells in culture. The development of this parasite in culture is restricted in time, and the completion of the life cycle is only rarely observed (14, 32). High oocyst doses and long incubation times do not increase the proportion of infected cells (36) and may instead lead to the extensive perturbation of the monolayer caused primarily by apoptosis and mitosis (19, 21, 34, 38). Discerning which transcriptional changes occur directly in response to the infection, and which result from the perturbance of the monolayer (13, 36), is difficult. To eliminate the effect of partial infection and monolayer perturbation, we physically separated infected and uninfected cells to generate subpopulations that differed only with respect to the presence of intracellular parasites. We report on transcriptional and phenotypic changes identified by this approach, in particular the transcriptional upregulation and overexpression of cell surface glycoprotein in C. parvuminfected cells.

The genus Cryptosporidium includes several species of intestinal protozoan parasites which multiply in intestinal epithelial cells. The impact of this infection on the transcriptome of cultured host cells was investigated using DNA microarray hybridizations. The expression of 14 genes found to be consistently up- or down-regulated in infected cell monolayers was validated with RT PCR. Using immunofluorescence we examined the expression of Protease Activated Receptor-2, which is encoded by one of the up-regulated genes. In infected cells this receptor localized to the host cell membrane which covers the intracellular trophozoites and meronts. This observation indicates that the composition of the host cell membrane is affected by the developing trophozoite, a phenomenon which has not been described previously.