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The microbiome of the female reproductive tract defies the convention that high biodiversity is a hallmark of an optimal ecosystem. Although not universally true, a homogeneous vaginal microbiome composed of species of Lactobacillus is generally associated with health, whereas vaginal microbiomes consisting of other taxa are generally associated with dysbiosis and a higher risk of disease.

Compared to taxonomic composition, the functional potential within a bacterial community is more relevant to establishing mechanistic understandings and causal relationships between the microbiome and health outcomes. Metagenome inference attempts to bridge the gap between 16S rRNA gene amplicon sequencing and whole-metagenome sequencing by predicting a microbiome’s gene content based on its taxonomic composition and annotated genome sequences of its members.

Single-step nonadaptive group testing approaches for reducing the number of tests required to detect a small subset of positive samples from a larger set require solving two algorithmic problems. First, how to design the samples-to-tests measurement matrix, and second, how to decode the results of the tests to uncover positive samples. In this study, we focus on the first challenge. We introduce real-valued group testing, which matches the characteristics of existing PCR testing pipelines more closely than combinatorial group testing or compressed sensing settings. We show a set of conditions that allow measurement matrices to guarantee unambiguous decoding of positives in this new setting. For small matrix sizes, we also propose an algorithm for constructing matrices that meet the proposed condition. On simulated data sets, we show that the matrices resulting from the algorithm can successfully recover positive samples at higher positivity rates than matrices designed for combinatorial group testing setting. We use wet laboratory experiments involving SARS-CoV-2 nasopharyngeal swab samples to further validate the approach.

Previous studies have investigated the associations between the vaginal microbiome and preterm birth, with the aim of determining whether differences in community patterns meaningfully alter risk and could therefore be the target of intervention. We report on vaginal microbial analysis of a nested case-control subset of the Pregnancy, Infection, and Nutrition (PIN) Study, including 464 White women (375 term birth and 89 spontaneous preterm birth, sPTB) and 360 Black women (276 term birth and 84 sPTB). We found that the microbiome of Black women has higher alpha-diversity, higher abundance of Lactobacillus iners, and lower abundance of Lactobacillus crispatus. However, among women who douche, there were no significant differences in microbiome by race. The sPTB-associated microbiome exhibited a lower abundance of L. crispatus, while alpha diversity and

Gardnerella vaginalis has recently been split into 13 distinct species. In this study, we tested the hypotheses that species-specific variations in the vaginolysin (VLY) amino acid sequence could influence the interaction between the toxin and vaginal epithelial cells and that VLY variation may be one factor that distinguishes less virulent or commensal strains from more virulent strains. This was assessed by bioinformatic analyses of publicly available Gardnerella spp. sequences and quantification of cytotoxicity and cytokine production from purified, recombinantly produced versions of VLY. After identifying conserved differences that could distinguish distinct VLY types, we analyzed metagenomic data from a cohort of female subjects from the Vaginal Human Microbiome Project to investigate whether these different VLY types exhibited any significant associations with symptoms or Gardnerella spp.-relative abundance in vaginal swab samples. While Type 1 VLY was most prevalent among the subjects and may be associated with increased reports of symptoms, subjects with Type 2 VLY dominant profiles exhibited increased relative Gardnerella spp. abundance. Our findings suggest that amino acid differences alter the interaction of VLY with vaginal keratinocytes, which may potentiate differences in bacterial vaginosis (BV) immunopathology in vivo.

We screened the genomes of a broad panel of kinetoplastid protists for genes encoding proteins associated with the RNA interference (RNAi) system using probes from the Argonaute (AGO1), Dicer1 (DCL1), and Dicer2 (DCL2) genes of Leishmania brasiliensis and Crithidia fasciculata. We identified homologs for all the three of these genes in the genomes of a subset of these organisms. However, several of these organisms lacked evidence for any of these genes, while others lacked only DCL2. The open reading frames encoding these putative proteins were structurally analyzed in silico. The alignments indicated that the genes are homologous with a high degree of confidence, and three-dimensional structural models strongly supported a functional relationship to previously characterized AGO1, DCL1, and DCL2 proteins. Phylogenetic analysis of these putative proteins showed that these genes, when present, evolved in parallel with other nuclear genes, arguing that the RNAi system genes share a common progenitor, likely across all Kinetoplastea. In addition, the genome segments bearing these genes are highly conserved and syntenic, even among those taxa in which they are absent. However, taxa in which these genes are apparently absent represent several widely divergent branches of kinetoplastids, arguing that these genes were independently lost at least six times in the evolutionary history of these organisms. The mechanisms responsible for the apparent coordinate loss of these RNAi system genes independently in several lineages of kinetoplastids, while being maintained in other related lineages, are currently unknown.

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.

CBA mice macrophages (MØ) control infection by Leishmania major and are susceptive to Leishmania amazonensis, suggesting that both parasite species induce distinct responses that play important roles in infection outcome. To evaluate the MØ responses to infection arising from these two Leishmania species, a proteomic study using a Multidimensional Protein Identification Technology (MudPIT) approach with liquid chromatography tandem mass spectrometry (LC-MS/MS) was carried out on CBA mice bone-marrow MØ (BMMØ). Following SEQUEST analysis, which revealed 2,838 proteins detected in BMMØ, data mining approach found six proteins significantly associated with the tested conditions. To investigate their biological significance, enrichment analysis was performed using Ingenuity Pathway Analysis (IPA). A three steps IPA approach revealed 4 Canonical Pathways (CP) and 7 Upstream Transcriptional Factors (UTFs) strongly associated with the infection process. NRF2 signatures were present in both CPs and UTFs pathways. Proteins involved in iron metabolism, such as heme oxigenase 1 (HO-1) and ferritin besides sequestosome (SQSMT1 or p62) were found in the NRF2 CPs and the NRF2 UTFs. Differences in the involvement of iron metabolism pathway in Leishmania infection was revealed by the presence of HO-1 and ferritin. Noteworty, HO-1 was strongly associated with L. amazonensis infection, while ferritin was regulated by both species. As expected, higher HO-1 and p62 expressions were validated in L. amazonensis-infected BMMØ, in addition to decreased expression of ferritin and nitric oxide production. Moreover, BMMØ incubated with L. amazonensis LPG also expressed higher levels of HO-1 in comparison to those stimulated with L. major LPG. In addition, L. amazonensis-induced uptake of holoTf was higher than that induced by L. major in BMMØ, and holoTf was also detected at higher levels in vacuoles induced by L. amazonensis. Taken together, these findings indicate that NRF2 pathway activation and increased HO-1 production, together with higher levels of holoTf uptake, may promote permissiveness to L. amazonensis infection. In this context, differences in protein signatures triggered in the host by L. amazonensis and L. major infection could drive the outcomes in distinct clinical forms of leishmaniasis.

Trypanosomatids of the subfamily Strigomonadinae bear permanent intracellular bacterial symbionts acquired by the common ancestor of these flagellates. However, the cospeciation pattern inherent to such relationships was revealed to be broken upon the description of Angomonas ambiguus, which is sister to A. desouzai, but bears an endosymbiont genetically close to that of A. deanei. Based on phylogenetic inferences, it was proposed that the bacterium from A. deanei had been horizontally transferred to A. ambiguus. Here, we sequenced the bacterial genomes from two A. ambiguus isolates, including a new one from Papua New Guinea, and compared them with the published genome of the A. deanei endosymbiont, revealing differences below the interspecific level. Our phylogenetic analyses confirmed that the endosymbionts of A. ambiguus were obtained from A. deanei and, in addition, demonstrated that this occurred more than once. We propose that coinfection of the same blowfly host and the phylogenetic relatedness of the trypanosomatids facilitate such transitions, whereas the drastic difference in the occurrence of the two trypanosomatid species determines the observed direction of this process. This phenomenon is analogous to organelle (mitochondrion/plastid) capture described in multicellular organisms and, thereafter, we name it endosymbiont capture.

Background Recent advances in next-generation sequencing have revolutionized genomic research. 16S rRNA amplicon sequencing using paired-end sequencing on the MiSeq platform from Illumina, Inc., is being used to characterize the composition and dynamics of extremely complex/diverse microbial communities. For this analysis on the Illumina platform, merging and quality filtering of paired-end reads are essential first steps in data analysis to ensure the accuracy and reliability of downstream analysis. Results We have developed the Merging and Filtering Tool (MeFiT) to combine these pre-processing steps into one simple, intuitive pipeline. MeFiT invokes CASPER (context-aware scheme for paired-end reads) for merging paired-end reads and provides users the option to quality filter the reads using the traditional average Q-score metric or using a maximum expected error cut-off threshold. Conclusions MeFiT provides an open-source solution that permits users to merge and filter paired end illumina reads. The tool has been implemented in python and the source-code is freely available at https://github.com/nisheth/MeFiT. Electronic supplementary material The online version of this article (doi:10.1186/s12859-016-1358-1) contains supplementary material, which is available to authorized users.

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

In trypanosomatids, the regulation of gene expression occurs mainly at the post-transcriptional level. Previous studies have revealed nontranslated mRNA in the Trypanosoma cruzi cytoplasm. Previously, we have identified and cloned the TcDHH1 protein, a DEAD box RNA helicase. It has been reported that Dhh1 is involved in multiple RNA-related processes in various eukaryotes. It has also been reported to accumulate in stress granules and processing bodies of yeast, animal cells, Trypanosoma brucei and T. cruzi. TcDHH1 is localized to discrete cytoplasmic foci that vary depending on the life cycle status and nutritional conditions. To study the composition of mRNPs containing TcDHH1, we carried out immunoprecipitation assays with anti-TcDHH1 using epimastigote lysates. The protein content of mRNPs was determined by MS and pre-immune serum was used as control. We also carried out a ribonomic approach to identify the mRNAs present within the TcDHH1 immunoprecipitated complexes. For this purpose, competitive microarray hybridizations were performed against negative controls, the nonprecipitated fraction. Our results showed that mRNAs associated with TcDHH1 in the epimastigote stage are those mainly expressed in the other forms of the T. cruzi life cycle. These data suggest that mRNPs containing TcDHH1 are involved in mRNA metabolism, regulating the expression of at least epimastigote-specific genes.

The apicomplexan Cryptosporidium is a protozoan parasite of humans and other mammals. Cryptosporidium species cause acute gastroenteritis and diarrheal disease in healthy humans and animals, and cause life-threatening infection in immunocompromised individuals such as people with AIDS. The parasite has a one-host life cycle and commonly invades intestinal epithelial cells. The current genome annotation of C. hominis, the most serious human pathogen, predicts 3884 genes of which ca. 1581 have predicted functional annotations. Using a combination of bioinformatics analysis, biochemical evidence, and high-throughput data, we have constructed a genome-scale metabolic model of C. hominis. The model is comprised of 213 gene-associated enzymes involved in 540 reactions among the major metabolic pathways and provides a link between the genotype and the phenotype of the organism, making it possible to study and predict behavior based upon genome content. This model was also used to analyze the two life stages of the parasite by integrating the stage-specific proteomic data for oocyst and sporozoite stages. Overall, this model provides a computational framework to systematically study and analyze various functional behaviors of C. hominis with respect to its life cycle and pathogenicity.

Motivation: Although metabolic reactions are unquestionably shaped by evolutionary processes, the degree to which the overall structure and complexity of their interconnections are linked to the phylogeny of species has not been evaluated in depth. Here, we apply an original metabolome representation, termed Network of Interacting Pathways or NIP, with a combination of graph theoretical and machine learning strategies, to address this question. NIPs compress the information of the metabolic network exhibited by a species into much smaller networks of overlapping metabolic pathways, where nodes are pathways and links are the metabolites they exchange. Results: Our analysis shows that a small set of descriptors of the structure and complexity of the NIPs combined into regression models reproduce very accurately reference phylogenetic distances derived from 16S rRNA sequences (10-fold cross-validation correlation coefficient higher than 0.9). Our method also showed better scores than previous work on metabolism-based phylogenetic reconstructions, as assessed by branch distances score, topological similarity and second cousins score. Thus, our metabolome representation as network of overlapping metabolic pathways captures sufficient information about the underlying evolutionary events leading to the formation of metabolic networks and species phylogeny. It is important to note that precise knowledge of all of the reactions in these pathways is not required for these reconstructions. These observations underscore the potential for the use of abstract, modular representations of metabolic reactions as tools in studying the evolution of species. Contact: aurelien.mazurie@pasteur.fr Supplementary information: Supplementary data are available at Bioinformatics online.

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 potential of the cellular-automata (CA) method for modeling biological networks is demonstrated for the mitogen-activated protein kinase (MAPK) signaling cascade. The models derived reproduced the high signal amplification through the cascade and the deviation of the cascade enzymes from the Michaelis-Menten kinetics, evidencing cooperativity effects. The patterns of pathway change upon varying substrate concentrations and enzyme efficiencies were identified and used to show the ways for controlling pathway processes. Guidance in the selection of enzyme inhibition targets with minimum side effects is one outcome of the study.

In trypanosomatids, the spliced leader RNA, or SL RNA, donates its 5′ 39 nucleotides to mature nuclear mRNAs in a process termed trans-splicing. We have previously characterized the SL RNA gene from Trypanosoma cruzi and identified its transcription promoter, including a 14 nt proximal sequence element, or PSE, that binds a putative transcription factor and activates transcription of the gene. Herein, we describe establishment of a yeast one-hybrid system using the 14 nt PSE as bait, and use this system to select T. cruzi cDNAs encoding a putative transcription factor that activates transcription of the SL RNA gene. The cDNA was selected from a normalized library and encodes an ∼45 kDa putative PSE promoter-binding protein, PPB1. PPB1 in vitro translated or overexpressed in and isolated from transformed E. coli , showed PSE-specific binding activity by electrophoretic mobility shift assays. Finally, overexpression of PPB1 in T. cruzi led to increased expression of the SL RNA gene as well as reporter genes in episomal constructs under the control of the SL RNA gene promoter. These observations suggest that PPB1 is a transcription factor that plays an important role in SL RNA gene expression.

Human cryptosporidiosis is attributed to two major Cryptosporidium parvum genotypes of which type 1 appears to be the predominant. Most laboratory investigations however are performed using genotype 2 isolates, the only type which readily infects laboratory animals. So far type 1 has only been identified in humans and primates. A type 1 isolate, obtained from an individual with HIV and cryptosporidiosis, was successfully adapted to propagate in gnotobiotic piglets. Genotypic characterization of oocyst DNA from this isolate using multiple restriction fragment length polymorphisms, a genotype-specific PCR marker, and direct sequence analysis of two polymorphic loci confirmed that this isolate, designated NEMC1, is indeed type 1. No changes in the genetic profile were identified during multiple passages in piglets. In contrast, the time period between infection and onset of fecal oocyst shedding, an indicator of adaptation, decreased with increasing number of passages. Consistent with other type 1 isolates, NEMC1 failed to infect mice. A preliminary survey of the NEMC1 genome covering approximately 2% of the genome and encompassing 200 kb of unique sequence showed an average similarity of approximately 95% between type 1 and 2 sequences. Twenty-four percent of the NEMC1 sequences were homologous to previously determined genotype 2 C. parvum sequences. To our knowledge, this is the first successful serial propagation of genotype 1 in animals, which should facilitate characterization of the unique features of this human pathogen.

Tools for the genetic manipulation of Trypanosoma cruzi are largely unavailable, although several vectors for transfection of epimastigotes and expression of foreign or recombinant genes have been developed. We have previously constructed several plasmid vectors in which recombinant genes are expressed in T. cruzi using the rRNA promoter. In this report, we demonstrate that one of these vectors can simultaneously mediate expression of neomycin phosphotransferase and green fluorescent protein when used to stably transfect cultured epimastigotes. These stably transfected epimastigotes can be selected and cloned as unique colonies on solid medium. We describe a simple colony PCR approach to the screening of these T. cruzi colonies for relevant genes. Thus, the methodologies outlined herein provide important new tools for the genetic dissection of this important parasite.

Biotinidase cleaves biotin from biocytin, thereby recycling the vitamin. We have determined the structure of the human biotinidase gene. A genomic clone, containing three exons that code for the mature enzyme, was obtained by screening a human genomic bacteriophage library with the biotinidase cDNA by plaque hybridization. To obtain a clone containing the most 5' exon of the biotinidase cDNA, a human PAC library by PCR was screened. The human biotinidase gene is organized into four exons and spans at least 23 kb. The 5'-flanking region of exon 1 contains a CCAAT element, three initiator sequences, an octamer sequence, three methylation consensus sites, two GC boxes, and one HNF-5 site, but has no TATA element. The region from nt -600 to +400 has features of a CpG island and resembles a housekeeping gene promoter. The structure and sequence of this gene are useful for identifying and characterizing mutations that cause biotinidase deficiency.

We have previously identified a major proximal sequence element (PSE) responsible for transcription of the spliced leader (SL) gene from Trypanosoma cruzi strain CL, and showed that the sequence encompassing this PSE exhibits approximately 30% divergence between two major groups of T. cruzi isolates, but strong conservation within the groups. In this report, we show that the SL RNA gene promoter from the CL strain (group I) is efficiently expressed only in T. cruzi isolates from group I. Similarly, the sequence of the approximately 643 bp promoter region of the T. cruzi rRNA is strongly conserved within, but diverged approximately 20% between, the two groups. Reporter constructs driven by the rRNA promoter sequences from group I strains are strongly expressed after electroporation into other group I strains, but not expressed in group II strains. In contrast, constructs bearing rRNA promoter sequences from group II strains are active in strains from both groups. Phylogenetic analyses performed with both the rRNA and the SL RNA gene promoter sequences yielded similar trees, and these trees strongly reinforce the partitioning of known T. cruzi into two major groups that parallel the observed functional specificity of the promoters. Given the well-documented species specific pattern of both rRNA promoters and PSEs in higher eukaryotes, these results suggest an ancient evolutionary divergence among organisms currently classified as T. cruzi.

Biotinidase recycles the vitamin biotin from biocytin upon the degradation of the biotin-dependent carboxylases. We have identified a novel point mutation within the biotinidase gene that encodes the signal peptide in two unrelated individuals with profound biotinidase deficiency. Sequence analysis of genomic DNA from these individuals revealed a G to A transition (G100-->A) located 57 bases downstream of the authentic splice acceptor site in exon B. Although this mutation predicts a G34S substitution, it also generates a 3' splice acceptor site. Sequence of the PCR-amplified cDNA from the homozygous child revealed that all the product was shorter than that of normal individuals and was the result of aberrant splicing. The aberrantly spliced transcript lacked 57 bases, including a second in-frame ATG, that encode most of the putative signal peptide and results in an in-frame deletion of 19 amino acids. The mutation results in failure to secrete the aberrant protein into the blood. This is the first reported example in which a point mutation creates a cryptic 3' splice acceptor site motif that is used preferentially over the upstream authentic splice site. The preferential usage of the downstream splice site is not consistent with the 5'-3' scanning model, but is consistent with the exon definition model of RNA splicing.

A putative promoter element responsible for transcription of the spliced leader (SL) gene of Trypanosoma cruzi was identified by overlapping deletion and linker scanning analyses of the upstream flanking sequences using the bacterial chloramphenicol acetyltransferase (CAT) gene as a reporter in transient transfections of cultured epimastigotes. Deletion or substitution of a proximal sequence element (PSE) between positions −53 and −40 relative to the transcription start point eliminated CAT gene expression. Comparison of SL genes from several strains of T. cruzi revealed two alternative sequence patterns for the putative SL PSE, both composed of a short run of purines followed by a run of pyrimidines. Moreover, an examination of these sequences supports the subdivision of T. cruzi isolates into two divergent groups. Double-stranded oligonucleotides containing the sequence of the PSE exhibited specific gel mobility shifts after incubation with T. cruzi nuclear extracts, suggesting that a transcription factor binds this site. Finally, experiments designed to increase the level of CAT expression from the SL promoter suggest that it is not a strong promoter in cultured T. cruzi epimastigotes. © 1997 Elsevier Science B.V. All rights reserved.

Biotinidase deficiency is an autosomal recessively inherited disorder in the recycling of the vitamin biotin. The most common mutation that causes profound biotinidase deficiency in symptomatic individuals is a deletion/insertion (G98:d7i3) that occurs in exon B of the biotinidase gene. We now report the second most common mutation, a C-to-T substitution (position 1612) in a CpG dinucleotide in exon D of the biotinidase gene. This mutation results in the substitution of a cysteine for arginine538 (designated R538C) and was found in 10 of 30 symptomatic children with profound biotinidase deficiency, 5 of whom also have the G98:d7i3 mutation. This mutation was not found in DNA samples from 32 individuals with normal biotinidase activity, but was found in one individual with enzyme activity in the heterozygous range. This mutation was not detected in 371 randomly selected, normal individuals using allele-specific oligonucleotide hybridization analysis. Aberrant biotinidase protein was not detectable in extracts of fibroblasts from a child who is homozygous for the R538C mutation, but was present in less than normal concentration in identical extracts treated with beta-mercaptoethanol. Because there is no detectable biotinidase protein in sera of children who are homozygous for the R538C mutation and in combination with the deletion/insertion mutation, the R538C mutation likely results in inappropriate intra- or intermolecular disulfide bond formation, more rapid degradation of the aberrant enzyme, and failure to secrete the residual aberrant enzyme from the cells into blood.

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.

We have isolated, characterized, and examined the expression of the genes encoding BiP endoplasmic reticulum (ER) resident chaperonins responsible for transport, maturation, and proper folding of membrane and secreted proteins from two divergent strains of Pneumocystis carinii. The BiP genes, Pcbip and Prbip, from the P. c. carinii (prototype) strain and the P. c. rattus (variant) strain, respectively, are single-copy genes that reside on chromosomes of approximately 330 and approximately 350 kbp. Both genes encode approximately 72.5-kDa proteins that are most homologous to BiP genes from other organisms and exhibit the amino-terminal signal peptides and carboxyl-terminal ER retention sequences that are hallmarks of BiP proteins. We established short-term P. carinii cultures to examine expression and induction of Pcbip in response to heat shock, glucose starvation, inhibition of protein transport or N-linked glycosylation, and other conditions known to affect proper transport, glycosylation, and maturation of membrane and secreted proteins. These studies indicated that Pcbip mRNA is constitutively expressed but induced under conditions known to induce BiP expression in other organisms. In contrast to mammalian BiP genes but like other fungal BiP genes, P. carinii BiP mRNA levels are induced by heat shock. Finally, the Prbip and Pcbip coding sequences surprisingly exhibit only approximately 83% DNA and approximately 90% amino acid sequence identity and show only limited conservation in noncoding flanking and intron sequences. Analyses of the P. carinii BiP gene sequences support inclusion of P. carinii among the fungi but suggest a large divergence and possible speciation among P. carinii strains infecting a given host.

Plasmid constructs containing a putative Trypanosoma cruzi rRNA promoter and transcription start point upstream from the bacterial chloramphenicol acetyltransferase (CAT) reporter gene were transfected into cultured T. cruzi epimastigotes to verify the presence of a promoter activity. Constructs bearing the putative promoter and a 3′ trans -splicing acceptor site in the proper orientation yielded approx. two orders of magnitude greater CAT expression than that previously observed with the T. cruzi spliced leader (SL) gene promoter. In contrast, similar constructs lacking the known 3′ splice site yielded reduced but readily measurable expression suggesting that sequences near the promoter may function as cryptic 3′ splice sites. A repeated sequence upstream from the putative basal rRNA promoter in a position analogous to rRNA gene enhancer elements in other eukaryotes did not enhance expression from the T. cruzi rRNA promoter. Finally, these constructs were functional in some but not all T. cruzi isolates, and were inactive in other kinetoplastid species, suggesting that the T. cruzi rRNA promoter may have a limited host range.

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.

Biotinidase (EC 3.5.1.12) catalyzes the hydrolysis of biocytin, the product of biotin-dependent carboxylase degradation, to biotin and lysine. Biotinidase deficiency is an inherited metabolic disorder of biotin recycling that is characterized by neurological and cutaneous abnormalities, and can be successfully treated with biotin supplementation. Sequences of tryptic peptides of the purified human serum enzyme were used to design oligonucleotide primers for polymerase chain reaction amplification from human hepatic total RNA to generate putative biotinidase cDNA fragments. Sequence analysis of a cDNA isolated from a human liver library by plaque hybridization with the largest cDNA probe revealed an open reading frame of 1629 bases encoding a protein of 543 amino acid residues, including 41 amino acids of a potential signal peptide. Comparison of the open reading frame with the known biotinidase tryptic peptides and recognition of the expressed protein encoded by this cDNA by monoclonal antibodies prepared against purified biotinidase demonstrated the identity of this cDNA. Southern analyses suggested that biotinidase is a single copy gene and revealed that human cDNA probes hybridized to genomic DNA from mammals, but not from chicken or yeast. Northern analysis indicated the presence of biotinidase mRNA in human heart, brain, placenta, liver, lung, skeletal muscle, kidney, and pancreas.

Although Pneumocystis carinii is the most common opportunistic pathogen infecting individuals with AIDS, very little is known of the basic biology of the organism. We have examined the ribosomal RNA (rRNA) and the DNA encoding it (rDNA) in P. carinii in an attempt to clarify its taxonomic position and to begin to study its genetic processes. Electrophoretic analysis showed that the sizes of the P. carinii rRNAs are quite similar to the sizes of the corresponding rRNAs from Saccharomyces cerevisiae. Direct sequence analysis of approx. 60% of the 18S small subunit-rRNA (Ss-rRNA) confirmed that its sequence is similar to that of yeast-like fungi and that a putative group-I intron previously observed in the 18S rDNA is, in fact, excised from the mature rRNA. PCR analysis of the intron in P. carinii genomic DNA showed that each of the multiple rDNA genes bears the group-I intron and in vitro transcripts of the intron autocatalytically excise from the rRNA primary transcript in the presence of GTP. Finally, analogues of GTP inhibit the self-splicing reaction, indicating that the guano sine-binding site of the intron closely resembles that of other well-characterized group-I introns. Since no group-I introns have been found in higher eukaryotes, this self-splicing process represents a viable target for chemotherapy of P. carinii pneumonia (PCP).

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.

Background Comparison of metabolic networks across species is a key to understanding how evolutionary pressures shape these networks. By selecting taxa representative of different lineages or lifestyles and using a comprehensive set of descriptors of the structure and complexity of their metabolic networks, one can highlight both qualitative and quantitative differences in the metabolic organization of species subject to distinct evolutionary paths or environmental constraints. Results We used a novel representation of metabolic networks, termed network of interacting pathways or NIP, to focus on the modular, high-level organization of the metabolic capabilities of the cell. Using machine learning techniques we identified the most relevant aspects of cellular organization that change under evolutionary pressures. We considered the transitions from prokarya to eukarya (with a focus on the transitions among the archaea, bacteria and eukarya), from unicellular to multicellular eukarya, from free living to host-associated bacteria, from anaerobic to aerobic, as well as the acquisition of cell motility or growth in an environment of various levels of salinity or temperature. Intuitively, we expect organisms with more complex lifestyles to have more complex and robust metabolic networks. Here we demonstrate for the first time that such organisms are not only characterized by larger, denser networks of metabolic pathways but also have more efficiently organized cross communications, as revealed by subtle changes in network topology. These changes are unevenly distributed among metabolic pathways, with specific categories of pathways being promoted to more central locations as an answer to environmental constraints. Conclusions Combining methods from graph theory and machine learning, we have shown here that evolutionary pressures not only affects gene and protein sequences, but also specific details of the complex wiring of functional modules in the cell. This approach allows the identification and quantification of those changes, and provides an overview of the evolution of intracellular systems.

Background Trypanosoma cruzi is a Kinetoplastid parasite of humans and is the cause of Chagas disease, a potentially lethal condition affecting the cardiovascular, gastrointestinal, and nervous systems of the human host. Constraint-based modeling has emerged in the last decade as a useful approach to integrating genomic and other high-throughput data sets with more traditional, experimental data acquired through decades of research and published in the literature. Results We present a validated, constraint-based model of the core metabolism of Trypanosoma cruzi strain CL Brener. The model includes four compartments (extracellular space, cytosol, mitochondrion, glycosome), 51 transport reactions, and 93 metabolic reactions covering carbohydrate, amino acid, and energy metabolism. In addition, we make use of several replicate high-throughput proteomic data sets to specifically examine metabolism of the morphological form of T. cruzi in the insect gut (epimastigote stage). Conclusion This work demonstrates the utility of constraint-based models for integrating various sources of data (e.g., genomics, primary biochemical literature, proteomics) to generate testable hypotheses. This model represents an approach for the systematic study of T. cruzi metabolism under a wide range of conditions and perturbations, and should eventually aid in the identification of urgently needed novel chemotherapeutic targets.

We have constructed a plasmid vector system into which a segment of the Trypanosoma cruzi gene encoding the spliced leader (SL) has been inserted to drive expression of a downstream gene encoding bacterial chloramphenicol acetyltransferase (CAT). We used this construct to establish conditions that permit reproducible transfection of cultured T. cruzi epimastigotes where transfection was mediated by electroporation and measured by assaying expression of CAT. CAT activity was detected only in T. cruzi lysates from cells transfected with constructs containing a properly oriented SL gene; constructs in which the gene was inserted in reverse orientation did not express CAT. The optimal electric field strength, cell density and DNA concentration for efficient transfection were established. This and similar systems will permit genetic dissection of this parasite.

Trypanosoma cruzi is the protozoan parasite that causes Chagas disease or American trypanosomiasis. Kinetoplastid parasites could be considered as model organisms for studying factors involved in posttranscriptional regulation because they control gene expression almost exclusively at this level. the PUF(Pumilio/FBF1) protein family regulates mRNA stability and translation in eukaryotes, and several members have been identified in trypano-somatids. We used a ribonomic approach to identify the putative target mRNAs associated with TcPUF6, a member of the T. cruzi PUF family. TcPUF6 is expressed in discrete sites in the cytoplasm at various stages of the parasite life cycle and is not associated with the translation machinery. the overexpression of a tandem affinity purification-tagged TcPUF6 protein allowed the identification of associated mRNAs by affinity purification assays and microarray hybridization yielding nine putative target mRNAs. Whole expression analysis of transfected parasites showed that the mRNAs associated with TcPUF6 were down-regulated in populations overexpressing TcPUF6. the association of TcPUF6 with the TcDhh1 helicase in vivo and the cellular co-localization of these proteins in epimastigote forms suggest that TcPUF6 promotes degradation of its associated mRNAs through interaction with RNA degradation complexes. Analysis of the mRNA levels of the putative TcPUF6-regulated genes during the parasite life cycle showed that their transcripts were up-regulated in metacyclic trypomastigotes. in these infective forms no co-localization between TcPUF6 and TcDhh1 was observed. Our results suggest that TcPUF6 regulates the half-lives of its associated transcripts via differential association with mRNA degradation complexes throughout its life cycle. Inst Biol Mol Parana, BR-81350010 Curitiba, Parana, Brazil Fundacao Oswaldo Cruz, BR-21040900 Rio de Janeiro, Brazil Fac Ciencias, Lab Interacc Mol, Montevideo 11400, Uruguay Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, Brazil Virginia Commonwealth Univ, Ctr Study Biol Complex, Richmond, VA 23284 USA Universidade Federal de São Paulo, Dept Microbiol Imunol & Parasitol, BR-04023062 São Paulo, Brazil Web of Science

1. Introduction. –1.1.DefinitionofGeneEssentialityandCaveats.Geneessentialityreferstothephenotypiceffectsofdisruptingthefunctionofaspecificgene.Ageneissaidtobeessentialifsuchadisruptionresultsincelldeath,i.e.,inabilitytocompleteacertainnumberofcelldivisions.Incontrast,disruptionofnonessentialgenesmayslowgrowthorhaveotherphenotypiceffects,butthecellsremainviable.Thisseeminglystraightforwarddefinitionissubjecttoanumberofcaveats.First,essentialityisalwaysdefinedrelativetoaspecificsetofenvironmentalconditions.Forexample, the fruR gene of Escherichia coli is essential during growth on glycerol-supplemented minimal medium, but it is not essential during growth on glucose-supplemented minimal medium [1]. In fact, evidence suggests that a substantialfractionofE.coligenesmayberegardedaseitheressentialornonessential,dependingonthespecificsetofconditions[2].Becausetheabilitytopredictgeneessentialityisgenerallyevaluatedrelativetoexperimentalresultsusingrichculturemedium[3][4],weheredefineessentialgenesasthoserequiredforviabilityduringgrowthundertheseconditions.Second,asrecentlyhighlightedbyGerdesetal.[5],thereisadistinctionbetweengenes essential for survival and genes essential for fitness. When genes areexperimentally disrupted to assess their essentiality, the resulting mutant cells maybepropagatedasclones(nocompetitionduringoutgrowth)orasmixedpopulations(competition during outgrowth). One expects that the set of genes identified asessential in the absence of competition should be reduced compared to the setidentifiedincompetitiveconditions.Nocompetitionimpliesthatonlygenesperform-ing core living processes are required. This expectation has indeed been borne outexperimentally [4][6]. In the experimental findings reviewed below, essentialityprediction is almost always done for the yeast Saccharomyces cerevisiae. Again,becausethesepredictionsareevaluatedrelativetoavailableexperimentalresultsbasedonclonalpropagation[3][7],essentialinthisworkgenerallyreferstoessentialforsurvival.These criteria for essential genes are potentially problematic, but necessary ifessentiality predictions are to be verified using available experimental results. Oneconcernisthepossibilitythatgenesrequiredforviabilityonrichculturemediumarenot required in wild-type conditions. This would imply the existence of biological2618

Despite the growing number of genomes published or currently being sequenced, there is a relative paucity of software for functional classification of newly discovered genes and their assignment to metabolic pathways. Available software for such analyses has a very steep learning curve and requires the installation, configuration, and maintenance of large amounts of complex infrastructure, including complementary software and databases. Many such tools are restricted to one or a few data sources and classification schemes. In this work, we report an automated system for gene annotation and metabolic pathway reconstruction (ASGARD), which was designed to be powerful and generalizable, yet simple for the biologist to install and run on centralized, commonly available computers. It avoids the requirement for complex resources such as relational databases and web servers, as well as the need for administrator access to the operating system. Our methodology contributes to a more rapid investigation of the potential biochemical capabilities of genes and genomes by the biological researcher, and is useful in biochemical as well as comparative and evolutionary studies of pathways and networks.

A comparative analysis of the topological structure of molecules and molecular biology networks revealed both similarity and differences in the methods used, as well as in the essential features of the two types of systems. Molecular graphs are static and, due to the limitations in atomic valence, show neither power distribution of vertex degrees nor "small-world" properties, which are typical for dynamic evolutionary networks. Areas of mutual benefits from an exchange of methods and ideas are outlined for the two fields. More specifically, chemical graph theory might make use of some new descriptors of network structure. Of interest for quantitative structure-property relationship/quantitative structure-activity relationship and drug design might be the conclusion that descriptors based on distributions of vertex degrees, distances, and subgraphs seem to be more relevant to biological information than the single-number descriptors. The network concepts of centrality, clustering, and cliques provide a basis for similar studies in theoretical chemistry. The need of dynamic theory of molecular topology is advocated.

Trypanosoma cruziis thought to undergo predominant clonal evolution, as determined by population genetics studies. However, this model does not exclude occasional recombination, which existence is strongly suggested by several recent studies. We sequenced a portion of the maxicircle cytochrome b (CYb) gene and of the nuclear rRNA promoter region from representative strains of six T. cruzi genetic lineages isolated from anthroponotic environments and man (lineages IIb, IId and IIe), sylvatic environments (lineages IIa and IIc) or both (lineage I). Phylogenetic analyses based on the two genes were incongruent. Remarkably, in lineage IIe, CYb and rRNA sequences were very closely related to those of lineages IIc and IIb, respectively. One stock of lineage IId showed rRNA sequence heterogeneity, with both IIb-like and IIc-like copies. Analysis of the size variation of six distinct pairs of putative homologous chromosomes revealed a bimodal distribution of chromosomal sizes across T. cruzi. Notably, stocks of lineages IId and IIe had several chromosomal pairs distributed in distinct modes, with the corresponding modes individually found in lineages IIb and IIc. Together, these data indicate the origin of lineages IId and IIe by hybridization between representatives of lineages IIb and IIc. CYb and rRNA sequences clustered into three and four major lineages, respectively. Data were in agreement with the distinction of six genetic lineages, but not with their proposed grouping into two primary lineages, as lineage II was not monophyletic. Based on a CYb substitution rate of 1% per million years (Myr), the major lineages are estimated to have diverged around 10 million years ago. © 2002 Elsevier Science B.V. All rights reserved.

Wen, L.-M., Xu, P., Benegal, G., Carvaho, M. R. C., Butler, D. R., and Buck, G. A. 2001. Trypanosoma cruzi: Exogenously regulated gene expression. Experimental Parasitology97, 196–204. A regulated expression vector would provide a strong tool for the dissection of gene function in Trypanosoma cruzi. Herein, we establish a system in which genes in T. cruzi expression vectors can be exogenously regulated by tetracycline. We first generated strains of T. cruzi that stably express the repressor of the bacterial tetracycline resistance gene and T7 RNA polymerase. Based on these strains, we developed two T. cruzi expression systems regulated by tetracycline—the first by use of a regulated rRNA promoter and the second by use of a regulated T7 promoter. In the former, we constructed an expression vector in which tetracycline resistance gene operators flank the transcription start point of the T. cruzi rRNA gene promoter. Reporter gene activity from this modified promoter was regulated up to 20-fold in the presence of different concentrations of tetracycline. In the T7 system, tetracycline resistance gene operators flank the transcription start point of the T7 promoter. Reporter gene activity from this modified promoter was regulated up to 150-fold in the presence of different concentrations of tetracycline. Expression in these systems was repressed when tetracycline was removed even after full induction for extended periods in the presence of tetracycline. We are now using these two systems to test protein function in T. cruzi.

The ribosomal RNA (rRNA) gene promoter was used to construct plasmid vectors that simultaneously express multiple exogenous genes in Trypanosoma cruzi. Vector pBSPANEO expresses neomycin phosphotransferase, and pPAGFPAN expresses both green fluorescent protein and neomycin phosphotransferase from a single promoter. Both vectors require the presence of the rRNA promoter for stable transfection; epimastigotes transfected with pPAGFPAN strongly fluoresced due to green fluorescent protein expression. Intact plasmids were rescued from the T. cruzi-transfected population after8 mo of culture, indicating stable replication of these vectors. Vectors were integrated into the rRNA locus by homologous recombination and into other loci, presumably by illegitimate recombination. Parasites bearing tandem concatamers of plasmids were also found among the transfectants. Transfectants expressing green fluorescent protein showed a bright green fluorescence distributed throughout the cell. Fluorescence was also detected in amastigotes after infection of mammalian cells with transfected parasites, indicating that the rRNA promoter can drive efficient expression of these reporter genes in multiple life-cycle stages of the parasite. Expression of the heterologous genes was detected after passage in mice or in the insect vector. These vectors will be useful for the genetic dissection of T. cruzi biology and pathogenesis.

Nuclear mRNAs in trypanosomatids are generated by trans-splicing. Although trans-splicing resembles cis-splicing in many ways and most of the U RNA participants have been characterized, relatively few involved proteins have been identified. Herein, we employed a yeast three-hybrid system to identify a protein, XB1, which binds to the Trypanosoma cruzi SL RNA. XB1 is a 45 kDa protein which is homologous to the essential pre-mRNA-splicing factor PRP31p from Saccharomyces cere6isiae .G el shift assays and UV cross-linking experiments with recombinant XB1 confirmed that this T. cruzi protein binds the SL RNA in vitro. The binding site of XB1 on the SL RNA was mapped to stem-loop II by deletion of the SL RNA ‘bait’ in the three-hybrid system. Finally, UV cross-linking SL RNA with S100 extract indicated native XB1 protein and SL RNA interaction in T. cruzi extract. © 2001 Elsevier Science B.V. All rights reserved.

A PCR-based method was adapted for the amplification of DNA from methanol-fixed smears of insects and plants parasitized by trypanosomatids. The PCR target was the multicopy spliced leader (SL) gene. Amplicons were hybridized with an oligonucleotide probe (SL3') specific for Phytomonas. The method has the advantage of dispensing with the cultivation of parasites, many of which are very fastidious or non-cultivable. The technique was applied to archival glass slides and to newly collected material. It proved to specific for Phytomonas spp., enabling their detection in plants and insects. Sequence comparison of the amplicons obtained revealed the existence of different strains/species of Phytomonas circulating among diseased palsms and fruit.

We have dissected the topoisomerase II gene of members of the two recently characterized subgroups of Trypanosoma cruzi to obtain further evidence to support this dichotomy of isolates in this important parasite. Pulsed field gel electrophoresis showed a striking heterogeneity in the molecular karyotypes of the strains analyzed. Southern analysis of these chromosome gels also showed heterogeneity in the size and number of chromosomes containing the topoisomerase II gene. Analysis of DNA restriction fragment length polymorphisms of the topoisomerase II gene also showed two principal patterns consistent with the two previously characterized groups. Finally, the sequences of portions of the topoisomerase II genes from members of the T. cruzi groups showed two distinct patterns, again consistent with the previous grouping of this parasite. Thus, this work clearly supports previous observations suggesting an ancient divergence of known T. cruzi isolates into two main branches.