RESUMO
Despite stringent procedures to secure the best HLA matching between donors and recipients, life-threatening complications continue to occur after hematopoietic stem cell transplantation (HSCT). Studying single nucleotide polymorphism (SNP) in genes encoding costimulatory molecules could help identify patients at risk for post-HSCT complications. In a stepwise approach we selected SNPs in key costimulatory molecules including CD274, CD40, CD154, CD28, and TNFSF4 and systematically analyzed their association with post-HSCT outcomes. Our discovery cohort analysis of 1157 HLA-A, -B, -C, -DRB1, and -DQB1 matched cases found that patients with donors homozygous for the C variant of rs10912564 in TNFSF4 (48%) had better disease-free survival (P = .029) and overall survival (P = .009) with less treatment-related mortality (P = .006). Our data demonstrate the TNFSF4C variant had a higher affinity for the nuclear transcription factor Myb and increased percentage of TNFSF4-positive B cells after stimulation compared with CT or TT genotypes. However, these associations were not validated in a more recent cohort, potentially because of changes in standard of practice or absence of a true association. Given the discovery cohort, functional data, and importance of TNFSF4 in infection clearance, TNFSF4C may associate with outcomes and warrants future studies.
Assuntos
Neoplasias Hematológicas/genética , Transplante de Células-Tronco Hematopoéticas , Homozigoto , Ligante OX40/genética , Adolescente , Adulto , Idoso , Antígenos CD , Linfócitos B , Estudos de Casos e Controles , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Antígenos HLA/genética , Neoplasias Hematológicas/mortalidade , Neoplasias Hematológicas/patologia , Neoplasias Hematológicas/terapia , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Proteínas Oncogênicas v-myb/genética , Polimorfismo de Nucleotídeo Único , Taxa de SobrevidaRESUMO
The recent emergence of artemisinin-resistant Plasmodium falciparum malaria in western Cambodia could threaten prospects for malaria elimination. Identification of the genetic basis of resistance would provide tools for molecular surveillance, aiding efforts to contain resistance. Clinical trials of artesunate efficacy were conducted in Bangladesh, in northwestern Thailand near the Myanmar border, and at two sites in western Cambodia. Parasites collected from trial participants were genotyped at 8,079 single nucleotide polymorphisms (SNPs) using a P. falciparum-specific SNP array. Parasite genotypes were examined for signatures of recent positive selection and association with parasite clearance phenotypes to identify regions of the genome associated with artemisinin resistance. Four SNPs on chromosomes 10 (one), 13 (two), and 14 (one) were significantly associated with delayed parasite clearance. The two SNPs on chromosome 13 are in a region of the genome that appears to be under strong recent positive selection in Cambodia. The SNPs on chromosomes 10 and 13 lie in or near genes involved in postreplication repair, a DNA damage-tolerance pathway. Replication and validation studies are needed to refine the location of loci responsible for artemisinin resistance and to understand the mechanism behind it; however, two SNPs on chromosomes 10 and 13 may be useful markers of delayed parasite clearance in surveillance for artemisinin resistance in Southeast Asia.
Assuntos
Artemisininas/farmacologia , Resistência a Medicamentos/genética , Loci Gênicos/genética , Plasmodium falciparum/genética , Seleção Genética , Sudeste Asiático , Marcadores Genéticos/genética , Genótipo , Funções Verossimilhança , Razão de Chances , Análise de Sequência com Séries de Oligonucleotídeos , Polimorfismo de Nucleotídeo Único/genética , Análise de Componente Principal , Análise de RegressãoRESUMO
Reverse genetics systems represent a key technique for studying replication and pathogenesis of viruses, including Ebola virus (EBOV). During the rescue of recombinant EBOV from Vero cells, a high frequency of mutations was observed throughout the genomes of rescued viruses, including at the RNA editing site of the glycoprotein gene. The influence that such genomic instability could have on downstream uses of rescued virus may be detrimental, and we therefore sought to improve the rescue system. Here we report an improved EBOV rescue system with higher efficiency and genome stability, using a modified full-length EBOV clone in Huh7 cells. Moreover, by evaluating a variety of cells lines, we revealed that EBOV genome instability is cell-type dependent, a fact that has significant implications for the preparation of standard virus stocks. Thus, our improved rescue system will have an impact on both basic and translational research in the filovirus field.
Assuntos
Ebolavirus/genética , Genoma Viral/genética , Doença pelo Vírus Ebola/virologia , Animais , Células COS , Linhagem Celular , Linhagem Celular Tumoral , Chlorocebus aethiops , Instabilidade Genômica/genética , Glicoproteínas/genética , Células HEK293 , Humanos , Mutação/genética , Genética Reversa/métodos , Células Vero , Replicação Viral/genéticaRESUMO
UNLABELLED: Specific gene duplications can enable double-stranded DNA viruses to adapt rapidly to environmental pressures despite the low mutation rate of their high-fidelity DNA polymerases. We report on the rapid positive selection of a novel vaccinia virus genomic duplication mutant in the presence of the assembly inhibitor rifampin. Until now, all known rifampin-resistant vaccinia virus isolates have contained missense mutations in the D13L gene, which encodes a capsid-like scaffold protein required for stabilizing membrane curvature during the early stage of virion assembly. Here we describe a second pathway to rifampin resistance involving A17, a membrane protein that binds and anchors D13 to the immature virion. After one round of selection, a rifampin-resistant virus that contained a genomic duplication in the A17L-A21L region was recovered. The mutant had both C-terminally truncated and full-length A17L open reading frames. Expression of the truncated A17 protein was retained when the virus was passaged in the presence of rifampin but was lost in the absence of the drug, suggesting that the duplication decreased general fitness. Both forms of A17 were bound to the virion membrane and associated with D13. Moreover, insertion of an additional truncated or inducible full-length A17L open reading frame into the genome of the wild-type virus was sufficient to confer rifampin resistance. In summary, this report contains the first evidence of an alternate mechanism for resistance of poxviruses to rifampin, indicates a direct relationship between A17 levels and the resistance phenotype, and provides further evidence of the ability of double-stranded DNA viruses to acquire drug resistance through gene duplication. IMPORTANCE: The present study provides the first evidence of a new mechanism of resistance of a poxvirus to the antiviral drug rifampin. In addition, it affirms the importance of the interaction between the D13 scaffold protein and the A17 membrane protein for assembly of virus particles. Resistance to rifampin was linked to a partial duplication of the gene encoding the A17 protein, similar to the resistance to hydroxyurea enabled by duplication of the gene encoding the small subunit of ribonucleotide reductase and of the K3L gene to allow adaptation to the antiviral action of protein kinase R. Gene duplication may provide a way for poxviruses and other DNA viruses with high-fidelity DNA polymerases to adjust rapidly to changes in the environment.
Assuntos
Farmacorresistência Viral/genética , Duplicação Gênica , Genes Virais , Rifampina/farmacologia , Vaccinia virus/genética , Vírion/genética , Sequência de Aminoácidos , Animais , Linhagem Celular , Chlorocebus aethiops , Farmacorresistência Viral/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/virologia , Expressão Gênica , Loci Gênicos , Dados de Sequência Molecular , Mutagênese Insercional , Fases de Leitura Aberta , Alinhamento de Sequência , Vaccinia virus/efeitos dos fármacos , Vírion/efeitos dos fármacos , Montagem de VírusRESUMO
A diverse suite of effector immune responses provide protection against various pathogens. However, the array of effector responses must be immunologically regulated to limit pathogen- and immune-associated damage. CD4(+)Foxp3(+) regulatory T cells (Treg) calibrate immune responses; however, how Treg cells adapt to control different effector responses is unclear. To investigate the molecular mechanism of Treg diversity we used whole genome expression profiling and next generation small RNA sequencing of Treg cells isolated from type-1 or type-2 inflamed tissue following Leishmania major or Schistosoma mansoni infection, respectively. In-silico analyses identified two miRNA "regulatory hubs" miR-10a and miR-182 as critical miRNAs in Th1- or Th2-associated Treg cells, respectively. Functionally and mechanistically, in-vitro and in-vivo systems identified that an IL-12/IFNγ axis regulated miR-10a and its putative transcription factor, Creb. Importantly, reduced miR-10a in Th1-associated Treg cells was critical for Treg function and controlled a suite of genes preventing IFNγ production. In contrast, IL-4 regulated miR-182 and cMaf in Th2-associed Treg cells, which mitigated IL-2 secretion, in part through repression of IL2-promoting genes. Together, this study indicates that CD4(+)Foxp3(+) cells can be shaped by local environmental factors, which orchestrate distinct miRNA pathways preserving Treg stability and suppressor function.
Assuntos
Leishmania major/imunologia , Leishmaniose Cutânea/imunologia , MicroRNAs/imunologia , Schistosoma mansoni/imunologia , Esquistossomose mansoni/imunologia , Linfócitos T Reguladores/imunologia , Animais , Citocinas/genética , Citocinas/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/parasitologia , Inflamação/patologia , Leishmaniose Cutânea/genética , Leishmaniose Cutânea/patologia , Camundongos , Camundongos Knockout , MicroRNAs/genética , Esquistossomose mansoni/genética , Esquistossomose mansoni/patologia , Linfócitos T Reguladores/patologia , Células Th1/imunologia , Células Th1/patologia , Células Th2/imunologia , Células Th2/patologiaRESUMO
BACKGROUND: Upon infection of a mammalian host, Bacillus anthracis responds to host cues, and particularly to elevated temperature (37°C) and bicarbonate/CO2 concentrations, with increased expression of virulence factors that include the anthrax toxins and extracellular capsular layer. This response requires the presence of the pXO1 virulence plasmid-encoded pleiotropic regulator AtxA. To better understand the genetic basis of this response, we utilized a controlled in vitro system and Next Generation sequencing to determine and compare RNA expression profiles of the parental strain and an isogenic AtxA-deficient strain in a 2 × 2 factorial design with growth environments containing or lacking carbon dioxide. RESULTS: We found 15 pXO1-encoded genes and 3 chromosomal genes that were strongly regulated by the separate or synergistic actions of AtxA and carbon dioxide. The majority of the regulated genes responded to both AtxA and carbon dioxide rather than to just one of these factors. Interestingly, we identified two previously unrecognized small RNAs that are highly expressed under physiological carbon dioxide concentrations in an AtxA-dependent manner. Expression levels of the two small RNAs were found to be higher than that of any other gene differentially expressed in response to these conditions. Secondary structure and small RNA-mRNA binding predictions for the two small RNAs suggest that they may perform important functions in regulating B. anthracis virulence. CONCLUSIONS: A majority of genes on the virulence plasmid pXO1 that are regulated by the presence of either CO2 or AtxA separately are also regulated synergistically in the presence of both. These results also elucidate novel pXO1-encoded small RNAs that are associated with virulence conditions.
Assuntos
Bacillus anthracis/genética , Proteínas de Bactérias/genética , Dióxido de Carbono/metabolismo , Transativadores/genética , Proteínas de Bactérias/metabolismo , Perfilação da Expressão Gênica , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Conformação de Ácido Nucleico , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Análise de Sequência de RNA , Transativadores/metabolismo , Regulação para Cima , Fatores de Virulência/genéticaRESUMO
Background: The taxonomic status of the relapsing fever spirochete Borrelia hermsii in western North America was established in 1942 and based solely on its specific association with the soft tick vector Ornithodoros hermsi. Multilocus sequence typing (MLST) of the 16S rRNA, flaB, gyrB, glpQ, and 16S-23S rRNA intergenic spacer of B. hermsii isolates collected over many years from various geographic locations and biological sources identified two distinct clades designated previously as B. hermsii Genomic Group I (GGI) and Genomic Group II (GGII). To better assess the taxonomic relationship of these two genomic groups to each other and other species of Borrelia, DNA sequences of the entire linear chromosome were determined. Materials and Methods: Genomic DNA samples were prepared from 11 spirochete isolates grown in Barbour-Stoenner-Kelly-H medium. From these preparations, DNA sequences of the entire linear chromosome of two isolates of B. hermsii belonging to each genomic group and seven additional species were determined. Results: Chromosomal sequences of four isolates of B. hermsii contained 919,212 to 922,307 base pairs. DNA sequence identities between the two genomic groups of B. hermsii were 95.86-95.99%, which were more divergent than chromosomal sequences comparing Borrelia parkeri and Borrelia turicatae (97.13%), Borrelia recurrentis and Borrelia duttonii (97.07%), and Borrelia crocidurae and B. duttonii (97.09%). The 3' end of the chromosome of the two GGII isolates also contained a unique intact oppA gene absent from all other species examined. Conclusion: Previous MLST and the chromosomal sequences presented herein support the division of the B. hermsii species complex into two species, B. hermsii sensu stricto ( = GGI) and Borrelia nietonii sp. nov. ( = GGII). We name this unique relapsing fever spirochete in honor of our late friend and colleague Dr. Nathan Nieto for his outstanding contributions to our understanding of tick-borne relapsing fever.
Assuntos
Borrelia , Ornithodoros , Filogenia , Febre Recorrente , Borrelia/genética , Borrelia/isolamento & purificação , Borrelia/classificação , Ornithodoros/microbiologia , Animais , Febre Recorrente/microbiologia , DNA Bacteriano/genética , Tipagem de Sequências Multilocus , RNA Ribossômico 16S/genética , Genoma BacterianoRESUMO
Relatively little is understood about the dynamics of global host-pathogen transcriptome changes that occur during bacterial infection of mucosal surfaces. To test the hypothesis that group A Streptococcus (GAS) infection of the oropharynx provokes a distinct host transcriptome response, we performed genome-wide transcriptome analysis using a nonhuman primate model of experimental pharyngitis. We also identified host and pathogen biological processes and individual host and pathogen gene pairs with correlated patterns of expression, suggesting interaction. For this study, 509 host genes and seven biological pathways were differentially expressed throughout the entire 32-day infection cycle. GAS infection produced an initial widespread significant decrease in expression of many host genes, including those involved in cytokine production, vesicle formation, metabolism, and signal transduction. This repression lasted until day 4, at which time a large increase in expression of host genes was observed, including those involved in protein translation, antigen presentation, and GTP-mediated signaling. The interactome analysis identified 73 host and pathogen gene pairs with correlated expression levels. We discovered significant correlations between transcripts of GAS genes involved in hyaluronic capsule production and host endocytic vesicle formation, GAS GTPases and host fibrinolytic genes, and GAS response to interaction with neutrophils. We also identified a strong signal, suggesting interaction between host gammadelta T cells and genes in the GAS mevalonic acid synthesis pathway responsible for production of isopentenyl-pyrophosphate, a short-chain phospholipid that stimulates these T cells. Taken together, our results are unique in providing a comprehensive understanding of the host-pathogen interactome during mucosal infection by a bacterial pathogen.
Assuntos
Perfilação da Expressão Gênica , Macaca fascicularis/genética , Faringe/metabolismo , Streptococcus pyogenes/genética , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Citocinas/metabolismo , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Interações Hospedeiro-Patógeno , Ácido Hialurônico/metabolismo , Macaca fascicularis/metabolismo , Macaca fascicularis/microbiologia , Neutrófilos/metabolismo , Neutrófilos/microbiologia , Neutrófilos/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Faringite/genética , Faringite/microbiologia , Faringe/microbiologia , Faringe/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Streptococcus pyogenes/metabolismo , Streptococcus pyogenes/fisiologiaRESUMO
BACKGROUND: Human immunodeficiency virus (HIV) superinfection has been documented in high-risk individuals; however, the rate of superinfection among HIV-infected individuals within a general population remains unknown. METHODS: A novel next-generation ultra-deep sequencing technique was utilized to determine the rate of HIV superinfection in a heterosexual population by examining two regions of the viral genome in longitudinal samples from recent HIV seroconverters (n=149) in Rakai District, Uganda. RESULTS: The rate of superinfection was 1.44 per 100 person years (PYs) (95% confidence interval [CI], .4-2.5) and consisted of both inter- and intrasubtype superinfections. This was compared to primary HIV incidence in 20 220 initially HIV-negative individuals in the general population in Rakai (1.15 per 100 PYs; 95% CI, 1.1-1.2; P= .26). Propensity score matching (PS) was used to control for differences in sociodemographic and behavioral characteristics between the HIV-positive individuals at risk for superinfection and the HIV-negative population at baseline and follow-up. After PS matching, the estimated rate of primary incidence was 3.28 per 100 PYs (95% CI, 2.0-5.3; P = .07) controlling for baseline differences and 2.51 per 100 PYs (95% CI, 1.5-4.3; P = .24) controlling for follow-up differences. CONCLUSIONS: This suggests that the rate of HIV superinfection in a general population is substantial, which could have a significant impact on future public health and HIV vaccine strategies.
Assuntos
Infecções por HIV/epidemiologia , Superinfecção/epidemiologia , Superinfecção/virologia , Adolescente , Adulto , Estudos de Coortes , Feminino , Infecções por HIV/patologia , Infecções por HIV/virologia , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Fatores de Risco , Uganda/epidemiologia , Adulto JovemRESUMO
Since emerging in French Polynesia and Brazil in the 2010s, Zika virus (ZIKV) has been associated with fetal congenital disease. Previous studies have compared ancestral and epidemic ZIKV strains to identify strain differences that may contribute to vertical transmission and fetal disease. However, within-host diversity in ZIKV populations during vertical transmission has not been well studied. Here, we used the established anti-interferon treated Rag1-/- mouse model of ZIKV vertical transmission to compare genomic variation within ZIKV populations in matched placentas, fetal bodies, and fetal brains via RNASeq. At early stages of vertical transmission, the ZIKV populations in the matched placentas and fetal bodies were similar. Most ZIKV single nucleotide variants were present in both tissues, indicating little to no restriction in transmission of ZIKV variants from placenta to fetus. In contrast, at later stages of fetal infection there was a sharp reduction in ZIKV diversity in fetal bodies and fetal brains. All fetal brain ZIKV populations were comprised of one of two haplotypes, containing either a single variant or three variants together, as largely homogenous populations. In most cases, the dominant haplotype present in the fetal brain was also the dominant haplotype present in the matched fetal body. However, in two of ten fetal brains the dominant ZIKV haplotype was undetectable or present at low frequencies in the matched placenta and fetal body ZIKV populations, suggesting evidence of a strict selective bottleneck and possible selection for certain variants during neuroinvasion of ZIKV into fetal brains.
Assuntos
Doenças Fetais , Complicações Infecciosas na Gravidez , Infecção por Zika virus , Zika virus , Gravidez , Humanos , Feminino , Animais , Camundongos , Zika virus/genética , Placenta , Transmissão Vertical de Doenças Infecciosas , Feto , EncéfaloRESUMO
Despite continual improvement, morbidity and mortality after hematopoietic stem cell transplantation (HSCT) remain high. The importance of chemokines in HSCT lies in their regulation of immune responses that determine transplantation outcomes. We investigated the role of recipient and donor chemokine system gene polymorphisms by using a candidate gene approach on the incidence of graft-versus-host disease and posttransplantation outcomes in 1370 extensively human leukocyte antigen-matched, unrelated donor-recipient pairs by using multivariate Cox regression models. Our analysis identified that recipients homozygous for a common CCR5 haplotype (H1/H1) had better disease-free survival (DFS; P = .005) and overall survival (P = .021). When the same genotype of both the donor and recipient were considered in the models, a highly significant association with DFS and overall survival was noted (P < .001 and P = .007, respectively) with absolute differences in survival of up to 20% seen between the groups at 3 years after transplantation (50% DFS for pairs with recipient CCR5 H1/H1 vs 30% for pairs with donor CCR5 H1/H1). This finding suggests that donor and/or recipient CCR5 genotypes may be associated with HSCT outcome and suggests new diagnostic and therapeutic strategies for optimizing therapy.
Assuntos
Transplante de Medula Óssea/mortalidade , Haplótipos/genética , Receptores CCR5/genética , Doadores de Tecidos , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Intervalo Livre de Doença , Feminino , Humanos , Lactente , Masculino , Pessoa de Meia-Idade , Análise Multivariada , Condicionamento Pré-Transplante , Adulto JovemRESUMO
BACKGROUND: The HIV Prevention Trials Network (HPTN) 052 trial demonstrated that early initiation of antiretroviral therapy (ART) reduces human immunodeficiency virus (HIV) transmission from HIV-infected adults (index participants) to their HIV-uninfected sexual partners. We analyzed HIV from 38 index-partner pairs and 80 unrelated index participants (controls) to assess the linkage of seroconversion events. METHODS: Linkage was assessed using phylogenetic analysis of HIV pol sequences and Bayesian analysis of genetic distances between pol sequences from index-partner pairs and controls. Selected samples were also analyzed using next-generation sequencing (env region). RESULTS: In 29 of the 38 (76.3%) cases analyzed, the index was the likely source of the partner's HIV infection (linked). In 7 cases (18.4%), the partner was most likely infected from a source other than the index participant (unlinked). In 2 cases (5.3%), linkage status could not be definitively established. CONCLUSIONS: Nearly one-fifth of the seroconversion events in HPTN 052 were unlinked. The association of early ART and reduced HIV transmission was stronger when the analysis included only linked events. This underscores the importance of assessing the genetic linkage of HIV seroconversion events in HIV prevention studies involving serodiscordant couples.
Assuntos
Ligação Genética , Soropositividade para HIV/genética , Soropositividade para HIV/virologia , HIV-1/genética , HIV-1/imunologia , Parceiros Sexuais , Adulto , Teorema de Bayes , Feminino , Soropositividade para HIV/transmissão , Humanos , Masculino , Filogenia , Análise de Sequência de DNA , Produtos do Gene pol do Vírus da Imunodeficiência Humana/genéticaRESUMO
Cationic amino acid transporters (CAT) are important regulators of NOS2 and ARG1 activity because they regulate L-arginine availability. However, their role in the development of Th1/Th2 effector functions following infection has not been investigated. Here we dissect the function of CAT2 by studying two infectious disease models characterized by the development of polarized Th1 or Th2-type responses. We show that CAT2(-/-) mice are significantly more susceptible to the Th1-inducing pathogen Toxoplasma gondii. Although T. gondii infected CAT2(-/-) mice developed stronger IFN-gamma responses, nitric oxide (NO) production was significantly impaired, which contributed to their enhanced susceptibility. In contrast, CAT2(-/-) mice infected with the Th2-inducing pathogen Schistosoma mansoni displayed no change in susceptibility to infection, although they succumbed to schistosomiasis at an accelerated rate. Granuloma formation and fibrosis, pathological features regulated by Th2 cytokines, were also exacerbated even though their Th2 response was reduced. Finally, while IL-13 blockade was highly efficacious in wild-type mice, the development of fibrosis in CAT2(-/-) mice was largely IL-13-independent. Instead, the exacerbated pathology was associated with increased arginase activity in fibroblasts and alternatively activated macrophages, both in vitro and in vivo. Thus, by controlling NOS2 and arginase activity, CAT2 functions as a potent regulator of immunity.
Assuntos
Arginase/metabolismo , Transportador 2 de Aminoácidos Catiônicos/fisiologia , Macrófagos/enzimologia , Animais , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fibroblastos/citologia , Fibroblastos/enzimologia , Fibrose/parasitologia , Fibrose/patologia , Expressão Gênica , Inativação Gênica , Granuloma/parasitologia , Granuloma/patologia , Imunidade , Fígado/metabolismo , Fígado/parasitologia , Fígado/patologia , Pulmão/metabolismo , Pulmão/parasitologia , Pulmão/patologia , Pneumopatias Parasitárias/metabolismo , Pneumopatias Parasitárias/parasitologia , Pneumopatias Parasitárias/patologia , Linfonodos/parasitologia , Linfonodos/patologia , Ativação de Macrófagos , Macrófagos/imunologia , Masculino , Camundongos , Camundongos Knockout , Óxido Nítrico/metabolismo , Schistosoma mansoni/isolamento & purificação , Schistosoma mansoni/patogenicidade , Schistosoma mansoni/fisiologia , Esquistossomose mansoni/enzimologia , Esquistossomose mansoni/genética , Esquistossomose mansoni/imunologia , Células Th1/enzimologia , Células Th1/imunologia , Células Th2/enzimologia , Células Th2/imunologiaRESUMO
RATIONALE: Pulmonary nontuberculous mycobacterial (PNTM) disease is increasing, but predisposing features have been elusive. OBJECTIVES: To prospectively determine the morphotype, immunophenotype, and cystic fibrosis transmembrane conductance regulator genotype in a large cohort with PNTM. METHODS: We prospectively enrolled 63 patients with PNTM infection, each of whom had computerized tomography, echocardiogram, pulmonary function, and flow cytometry of peripheral blood. In vitro cytokine production in response to mitogen, LPS, and cytokines was performed. Anthropometric measurements were compared with National Health and Nutrition Examination Survey (NHANES) age- and ethnicity-matched female control subjects extracted from the NHANES 2001-2002 dataset. MEASUREMENTS AND MAIN RESULTS: Patients were 59.9 (+/-9.8 yr [SD]) old, and 5.4 (+/-7.9 yr) from diagnosis to enrollment. Patients were 95% female, 91% white, and 68% lifetime nonsmokers. A total of 46 were infected with Mycobacterium avium complex, M. xenopi, or M. kansasii; 17 were infected with rapidly growing mycobacteria. Female patients were significantly taller (164.7 vs. 161.0 cm; P < 0.001) and thinner (body mass index, 21.1 vs. 28.2; P < 0.001) than matched NHANES control subjects, and thinner (body mass index, 21.1 vs. 26.8; P = 0.002) than patients with disseminated nontuberculous mycobacterial infection. A total of 51% of patients had scoliosis, 11% pectus excavatum, and 9% mitral valve prolapse, all significantly more than reference populations. Stimulated cytokine production was similar to that of healthy control subjects, including the IFN-gamma/IL-12 pathway. CD4(+), CD8(+), B, and natural killer cell numbers were normal. A total of 36% of patients had mutations in the cystic fibrosis transmembrane conductance regulator gene. CONCLUSIONS: Patients with PNTM infection are taller and leaner than control subjects, with high rates of scoliosis, pectus excavatum, mitral valve prolapse, and cystic fibrosis transmembrane conductance regulator mutations, but without recognized immune defects.
Assuntos
Infecções por Mycobacterium não Tuberculosas/etiologia , Pneumonia Bacteriana/etiologia , Idoso , Estatura , Estudos de Casos e Controles , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Feminino , Tórax em Funil/complicações , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Infecções por Mycobacterium não Tuberculosas/genética , Infecções por Mycobacterium não Tuberculosas/imunologia , Fenótipo , Estudos Prospectivos , Fatores de Risco , Escoliose/complicações , Fatores Sexuais , Fumar/efeitos adversos , Síndrome , Magreza/complicaçõesRESUMO
Reston virus (RESTV) was discovered in 1989-1990 during three connected epizootics of highly lethal viral hemorrhagic fever among captive macaques in primate housing facilities in the United States and Philippines. Currently, only one RESTV isolate from that outbreak (named Pennsylvania) has been sequenced. Here, we report the sequence of a second isolate, Reston virus/M.fascicularis-tc/USA/1990/Philippines89-AZ1435.
RESUMO
The virulence of many bacterial pathogens, including the important human pathogen Staphylococcus aureus, depends on the secretion of frequently large amounts of toxins. Toxin production involves the need for the bacteria to make physiological adjustments for energy conservation. While toxins are primarily targets of gene regulation, such changes may be accomplished by regulatory functions of the toxins themselves. However, mechanisms by which toxins regulate gene expression have remained poorly understood. We show here that the staphylococcal phenol-soluble modulin (PSM) toxins have gene regulatory functions that, in particular, include inducing expression of their own transport system by direct interference with a GntR-type repressor protein. This capacity was most pronounced in PSMs with low cytolytic capacity, demonstrating functional specification among closely related members of that toxin family during evolution. Our study presents a molecular mechanism of gene regulation by a bacterial toxin that adapts bacterial physiology to enhanced toxin production. IMPORTANCE: Toxins play a major role in many bacterial diseases. When toxins are produced during infection, the bacteria need to balance this energy-consuming task with other physiological processes. However, it has remained poorly understood how toxins can impact gene expression to trigger such adaptations. We found that specific members of a toxin family in the major human pathogen Staphylococcus aureus have evolved for gene regulatory purposes. These specific toxins interact with a DNA-binding regulator protein to enable production of the toxin export machinery and ascertain that the machinery is not expressed when toxins are not made and it is not needed. Our study gives mechanistic insight into how toxins may directly adjust bacterial physiology to times of toxin production during infection.
Assuntos
Toxinas Bacterianas/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Mapeamento de Interação de Proteínas , Transporte ProteicoRESUMO
Several human pathogens (e.g., Bacillus anthracis, Yersinia pestis, Bordetella pertussis, Plasmodium falciparum, and Mycobacterium tuberculosis) have very restricted unselected allelic variation in structural genes, which hinders study of the genetic relationships among strains and strain-trait correlations. To address this problem in a representative pathogen, 432 M. tuberculosis complex strains from global sources were genotyped on the basis of 230 synonymous (silent) single nucleotide polymorphisms (sSNPs) identified by comparison of four genome sequences. Eight major clusters of related genotypes were identified in M. tuberculosis sensu stricto, including a single cluster representing organisms responsible for several large outbreaks in the United States and Asia. All M. tuberculosis sensu stricto isolates of previously unknown phylogenetic position could be rapidly and unambiguously assigned to one of the eight major clusters, thus providing a facile strategy for identifying organisms that are clonally related by descent. Common clones of M. tuberculosis sensu stricto and M. bovis are distinct, deeply branching genotypic complexes whose extant members did not emerge directly from one another in the recent past. sSNP genotyping rapidly delineates relationships among closely related strains of pathogenic microbes and allows construction of genetic frameworks for examining the distribution of biomedically relevant traits such as virulence, transmissibility, and host range.
Assuntos
Mycobacterium tuberculosis/genética , Alelos , Animais , Variação Genética , Genoma Bacteriano , Genótipo , Humanos , Epidemiologia Molecular , Mycobacterium bovis/genética , Mycobacterium bovis/isolamento & purificação , Mycobacterium tuberculosis/classificação , Mycobacterium tuberculosis/isolamento & purificação , Mycobacterium tuberculosis/patogenicidade , Filogenia , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo Único , Especificidade da Espécie , Virulência/genéticaRESUMO
UNLABELLED: Sarcocystis neurona is a member of the coccidia, a clade of single-celled parasites of medical and veterinary importance including Eimeria, Sarcocystis, Neospora, and Toxoplasma. Unlike Eimeria, a single-host enteric pathogen, Sarcocystis, Neospora, and Toxoplasma are two-host parasites that infect and produce infectious tissue cysts in a wide range of intermediate hosts. As a genus, Sarcocystis is one of the most successful protozoan parasites; all vertebrates, including birds, reptiles, fish, and mammals are hosts to at least one Sarcocystis species. Here we sequenced Sarcocystis neurona, the causal agent of fatal equine protozoal myeloencephalitis. The S. neurona genome is 127 Mbp, more than twice the size of other sequenced coccidian genomes. Comparative analyses identified conservation of the invasion machinery among the coccidia. However, many dense-granule and rhoptry kinase genes, responsible for altering host effector pathways in Toxoplasma and Neospora, are absent from S. neurona. Further, S. neurona has a divergent repertoire of SRS proteins, previously implicated in tissue cyst formation in Toxoplasma. Systems-based analyses identified a series of metabolic innovations, including the ability to exploit alternative sources of energy. Finally, we present an S. neurona model detailing conserved molecular innovations that promote the transition from a purely enteric lifestyle (Eimeria) to a heteroxenous parasite capable of infecting a wide range of intermediate hosts. IMPORTANCE: Sarcocystis neurona is a member of the coccidia, a clade of single-celled apicomplexan parasites responsible for major economic and health care burdens worldwide. A cousin of Plasmodium, Cryptosporidium, Theileria, and Eimeria, Sarcocystis is one of the most successful parasite genera; it is capable of infecting all vertebrates (fish, reptiles, birds, and mammals-including humans). The past decade has witnessed an increasing number of human outbreaks of clinical significance associated with acute sarcocystosis. Among Sarcocystis species, S. neurona has a wide host range and causes fatal encephalitis in horses, marine mammals, and several other mammals. To provide insights into the transition from a purely enteric parasite (e.g., Eimeria) to one that forms tissue cysts (Toxoplasma), we present the first genome sequence of S. neurona. Comparisons with other coccidian genomes highlight the molecular innovations that drive its distinct life cycle strategies.
Assuntos
Genoma de Protozoário , Sarcocystis/crescimento & desenvolvimento , Sarcocystis/genética , Sarcocistose/parasitologia , Sarcocistose/veterinária , Animais , Humanos , Estágios do Ciclo de Vida , Filogenia , Proteínas de Protozoários/genética , Sarcocystis/classificação , Sarcocystis/metabolismoRESUMO
Giardia lamblia (syn G. intestinalis, G. duodenalis) is the most common pathogenic intestinal parasite of humans worldwide and is a frequent cause of endemic and epidemic diarrhea. G. lamblia is divided into eight genotypes (A-H) which infect a wide range of mammals and humans, but human infections are caused by Genotypes A and B. To unambiguously determine the relationship among genotypes, we sequenced GS and DH (Genotypes B and A2) to high depth coverage and compared the assemblies with the nearly completed WB genome and draft sequencing surveys of Genotypes E (P15; pig isolate) and B (GS; human isolate). Our results identified DH as the smallest Giardia genome sequenced to date, while GS is the largest. Our open reading frame analyses and phylogenetic analyses showed that GS was more distant from the other three genomes than any of the other three were from each other. Whole-genome comparisons of DH_A2 and GS_B with the optically mapped WB_A1 demonstrated substantial synteny across all five chromosomes but also included a number of rearrangements, inversions, and chromosomal translocations that were more common toward the chromosome ends. However, the WB_A1/GS_B alignment demonstrated only about 70% sequence identity across the syntenic regions. Our findings add to information presented in previous reports suggesting that GS is a different species of Giardia as supported by the degree of genomic diversity, coding capacity, heterozygosity, phylogenetic distance, and known biological differences from WB_A1 and other G. lamblia genotypes.
Assuntos
DNA de Protozoário/genética , Genoma de Protozoário , Giardia lamblia/classificação , Giardia lamblia/genética , Sequência de Bases , Bases de Dados de Ácidos Nucleicos , Evolução Molecular , Biblioteca Gênica , Genótipo , Giardia lamblia/isolamento & purificação , Giardíase/genética , Giardíase/parasitologia , Dados de Sequência Molecular , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Spirochetes are bacteria characterized in part by rotating periplasmic flagella that impart their helical or flat-wave morphology and motility. While most other bacteria rely on a transcriptional cascade to regulate the expression of motility genes, spirochetes employ post-transcriptional mechanism(s) that are only partially known. In the present study, we characterize a spontaneous non-motile mutant of the relapsing fever spirochete Borrelia hermsii that was straight, non-motile and deficient in periplasmic flagella. We used next generation DNA sequencing of the mutant's genome, which when compared to the wild-type genome identified a 142 bp deletion in the chromosomal gene encoding the flagellar export apparatus protein FliH. Immunoblot and transcription analyses showed that the mutant phenotype was linked to the posttranscriptional deficiency in the synthesis of the major periplasmic flagellar filament core protein FlaB. Despite the lack of FlaB, the amount of FlaA produced by the fliH mutant was similar to the wild-type level. The turnover of the residual pool of FlaB produced by the fliH mutant was comparable to the wild-type spirochete. The non-motile mutant was not infectious in mice and its inoculation did not induce an antibody response. Trans-complementation of the mutant with an intact fliH gene restored the synthesis of FlaB, a normal morphology, motility and infectivity in mice. Therefore, we propose that the flagellar export apparatus protein regulates motility of B. hermsii at the post-transcriptional level by influencing the synthesis of FlaB.