Putative cis-Regulatory Elements Associated with Heat Shock Genes Activated During Excystation of Cryptosporidium parvum

Abstract Background Cryptosporidiosis is a ubiquitous infectious disease, caused by the protozoan parasitesCryptosporidium 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 theCryptosporidium 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 ofCryptosporidium, these results provide important insight into the pathogenicity of the parasite

Comparative transcriptomic analysis of Gardnerella vaginalis biofilms vs. planktonic cultures using RNA-seq

Bacterial vaginosis is the most common gynecological disorder affecting women of reproductive age. Bacterial vaginosis is frequently associated with the development of a Gardnerella vaginalis biofilm. Recent data indicates that G. vaginalis biofilms are more tolerant to antibiotics and are able to incorporate other bacterial vaginosis -associated species, yielding a multi-species biofilm. However, despite its apparent role in bacterial vaginosis, little is known regarding the molecular determinants involved in biofilm formation by G. vaginalis. To gain insight into the role of G. vaginalis in the pathogenesis of bacterial vaginosis, we carried out comparative transcriptomic analysis between planktonic and biofilm phenotypes, using RNA-sequencing. Significant differences were found in the expression levels of 815 genes. A detailed analysis of the results obtained was performed based on direct and functional gene interactions. Similar to other bacterial species, expression of genes involved in antimicrobial resistance were elevated in biofilm cells. In addition, our data indicate that G. vaginalis biofilms assume a characteristic response to stress and starvation conditions. The abundance of transcripts encoding proteins involved in glucose and carbon metabolism was reduced in biofilms. Surprisingly, transcript levels of vaginolysin were reduced in biofilms relative to planktonic cultures. Overall, our data revealed that gene-regulated processes in G. vaginalis biofilms resulted in a protected form of bacterial growth, characterized by low metabolic activity. This phenotype may contribute towards the chronic and recurrent nature of bacterial vaginosis. This suggests that G. vaginalis is capable of drastically adjusting its phenotype through an extensive change of gene expressionThis work was presented at Biofilms7 meeting (26–28 June 2016, Porto, Portugal) and was awarded the Biofilms7—SPM Young Researcher Award. N.C. is an Investigador FCT. This work was funded by Fundação para a Ciência e a Tecnologia (FCT) by the strategic project of UID/BIO/04469/2013 unit and the project RECI/BBB-EBI/0179/2012 (FCOMP-01-0124-FEDER-027462), and by the National Institutes of Health; P60 MD002256 “VCU NIMHD Comprehensive Center of Excellence”. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The following authors had an individual FCT fellowship: J.C. (SFRH/BD/93963/2013) and A.F. (SFRH/BPD/99961/2014)

What happens to Gardnerella vaginalis when growing as a biofilm: a comparative transcriptomic analyses by RNA-seq

Bacteria assume distinct lifestyles during the planktonic and biofilm modes of growth. In biofilms, they are more tolerant to antibiotics and can evade the immune system response more effectively. However, little is known regarding the molecular determinants involved in biofilm formation by Gardnerella vaginalis, the predominant species found in the polymicrobial condition bacterial vaginosis (BV), the most common vaginal disorder of women in reproductive age.Hence, to gain insight into the pathogenesis of G. vaginalis, we carried out a comparative transcriptomic analysis between planktonic and biofilm phenotypes, using RNA-sequencing. Significant differences were found in the expression of 815 genes. A detailed analysis of the results obtained was performed based on direct and functional gene interactions. In biofilm bacteria, the cell envelope appeared to be very active since genes encoding binding proteins and proteins involved in the synthesis of murein were significantly up-regulated. In addition, our data showed that G. vaginalis reflects the typical adaptation to stress and starvation conditions. Interestingly, genes associated with glucose and carbon metabolism, as well as oxidoreductase activity were found down-regulated in biofilms.Furthermore, gene-regulated processes in G. vaginalis biofilms resulted in a protected form of bacterial growth, characterized by low metabolic activity, which is appropriate to guarantee long-term survival during BV recurrence. Therefore, our data suggested that G. vaginalis adjust its lifestyle during colonization and infection by means of an extensive change of gene expression

LA AMBIDIEXTRÍA ORGANIZACIONAL EN LAS SOCIEDADES COOPERATIVAS / ORGANIZATIONAL AMBIDIEXTRY IN COOPERATIVE SOCIETIES

Este trabajo explora la importancia de desarrollar y potencializar la asociatividad e intercooperación entre empresas cooperativas para incrementar las actividades de explotación y exploración de conocimientos asociativos o grupales que generen comportamientos innovadores, por lo que se propone la gestación de redes entre empresas cooperativas son necesarias para sembrar proyectos con recursos compartidos que generen valor, competitividad, innovación y desempeño sostenibles. A través de esta investigación se describe como se relaciona la ambidiextría organizacional, la innovación y la asociatividad cooperativa. Las actividades de ambidiextría como la explotación y la exploración son guías que se adaptan al entorno haciendo un cambio obligado y pasar de la obsolescencia a mejores prácticas, por lo que, si se aplican estas actividades, el mismo contexto empresarial nos va llevando de una mejora continua que con actividades de exploración de nuevos conocimientos se puede llegar a la innovación y poder aumentar el desempeño organizacional, con el fin de aprovechar el potencial de las empresas cooperativas, robustecerlas para que crezcan y contribuyan al desarrollo familiar, social, nacional y mundial. Identificar el impacto que la ambidiextría organizacional pudiera tener en las cooperativas conlleva a la actualización de la gerencia hacia la innovación adoptando actividades de explotación y exploración para la generación de conocimiento, para el éxito, la adaptación y la supervivencia organizacional

In Silico Derivation of HLA-Specific Alloreactivity Potential from Whole Exome Sequencing of Stem Cell Transplant Donors and Recipients: Understanding the Quantitative Immuno-biology of Allogeneic Transplantation

Donor T cell mediated graft vs. host effects may result from the aggregate alloreactivity to minor histocompatibility antigens (mHA) presented by the HLA in each donor-recipient pair (DRP) undergoing stem cell transplantation (SCT). Whole exome sequencing has demonstrated extensive nucleotide sequence variation in HLA-matched DRP. Non-synonymous single nucleotide polymorphisms (nsSNPs) in the GVH direction (polymorphisms present in recipient and absent in donor) were identified in 4 HLA-matched related and 5 unrelated DRP. The nucleotide sequence flanking each SNP was obtained utilizing the ANNOVAR software package. All possible nonameric-peptides encoded by the non-synonymous SNP were then interrogated in-silico for their likelihood to be presented by the HLA class I molecules in individual DRP, using the Immune-Epitope Database (IEDB) SMM algorithm. The IEDB-SMM algorithm predicted a median 18,396 peptides/DRP which bound HLA with an IC50 of <500nM, and 2254 peptides/DRP with an IC50 of <50nM. Unrelated donors generally had higher numbers of peptides presented by the HLA. A similarly large library of presented peptides was identified when the data was interrogated using the Net MHCPan algorithm. These peptides were uniformly distributed in the various organ systems. The bioinformatic algorithm presented here demonstrates that there may be a high level of minor histocompatibility antigen variation in HLA-matched individuals, constituting an HLA-specific alloreactivity potential. These data provide a possible explanation for how relatively minor adjustments in GVHD prophylaxis yield relatively similar outcomes in HLA matched and mismatched SCT recipients.Comment: Abstract: 235, Words: 6422, Figures: 7, Tables: 3, Supplementary figures: 2, Supplementary tables:

Determining the Quantitative Principles of T Cell Response to Antigenic Disparity in Stem Cell Transplantation

Alloreactivity compromising clinical outcomes in stem cell transplantation is observed despite HLA matching of donors and recipients. This has its origin in the variation between the exomes of the two, which provides the basis for minor histocompatibility antigens (mHA). The mHA presented on the HLA class I and II molecules and the ensuing T cell response to these antigens results in graft vs. host disease. In this paper, results of a whole exome sequencing study are presented, with resulting alloreactive polymorphic peptides and their HLA class I and HLA class II (DRB1) binding affinity quantified. Large libraries of potentially alloreactive recipient peptides binding both sets of molecules were identified, with HLA-DRB1 generally presenting a greater number of peptides. These results are used to develop a quantitative framework to understand the immunobiology of transplantation. A tensor-based approach is used to derive the equations needed to determine the alloreactive donor T cell response from the mHA-HLA binding affinity and protein expression data. This approach may be used in future studies to simulate the magnitude of expected donor T cell response and determine the risk for alloreactive complications in HLA matched or mismatched hematopoietic cell and solid organ transplantation

Genomic sequence analysis and characterization of Sneathia amnii sp. nov

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. BecauseSneathia 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

Stem Cell Transplantation As A Dynamical System: Are Clinical Outcomes Deterministic?

Outcomes in stem cell transplantation (SCT) are modeled using probability theory. However the clinical course following SCT appears to demonstrate many characteristics of dynamical systems, especially when outcomes are considered in the context of immune reconstitution. Dynamical systems tend to evolve over time according to mathematically determined rules. Characteristically, the future states of the system are predicated on the states preceding them, and there is sensitivity to initial conditions. In SCT, the interaction between donor T cells and the recipient may be considered as such a system in which, graft source, conditioning and early immunosuppression profoundly influence immune reconstitution over time. This eventually determines clinical outcomes, either the emergence of tolerance or the development of graft versus host disease. In this paper parallels between SCT and dynamical systems are explored and a conceptual framework for developing mathematical models to understand disparate transplant outcomes is proposed.Comment: 23 pages, 4 figures. Updated version with additional data, 2 new figures and editorial revisions. New authors adde

Species-level classification of the vaginal microbiome

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

Cytomegalovirus Antigenic Mimicry of Human Alloreactive Peptides: A Potential Trigger for Graft versus Host Disease

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<500 nM) cross reactivity. Of the 70,686 hCMV 9-mers contained within the hCMV CROSS database, 29,658.8 +/- 9038.5 were found to match MRD DRP alloreactive peptides and 52,910.2 +/- 16121.8 matched MUD DRP peptides (Student's T-test, p<0.001). In silico analysis revealed multiple high affinity, immunogenic CMV-Human peptide matches (IC50<500 nM) expressed in GVHD-affected tissue-specific manner (proteins expressed at 10 RPKM or greater). hCMV+GVHD was found in 18 patients, 13 developing hCMV viremia before GVHD onset with a subset analysis of 7 instances of hCMV viremia prior to acute GVHD onset (n=3), chronic GVHD (n=2) and acute + chronic GVHD (n=2) indicating cross reactive peptide expression within affected organs. We propose that based on our analysis and preliminary clinical correlations that hCMV immune cross-reactivity may cause antigenic mimicry of human alloreactive peptides triggering GVHD.Comment: Pre-submission manuscript, 4 tables, 5 figures, 2 supplements & 2 Appendices-available upon request from first autho