RESUMEN
Gut microbial communities can respond to antibiotic perturbations by rapidly altering their taxonomic and functional composition. However, little is known about the strain-level processes that drive this collective response. Here, we characterize the gut microbiome of a single individual at high temporal and genetic resolution through a period of health, disease, antibiotic treatment, and recovery. We used deep, linked-read metagenomic sequencing to track the longitudinal trajectories of thousands of single nucleotide variants within 36 species, which allowed us to contrast these genetic dynamics with the ecological fluctuations at the species level. We found that antibiotics can drive rapid shifts in the genetic composition of individual species, often involving incomplete genome-wide sweeps of pre-existing variants. These genetic changes were frequently observed in species without obvious changes in species abundance, emphasizing the importance of monitoring diversity below the species level. We also found that many sweeping variants quickly reverted to their baseline levels once antibiotic treatment had concluded, demonstrating that the ecological resilience of the microbiota can sometimes extend all the way down to the genetic level. Our results provide new insights into the population genetic forces that shape individual microbiomes on therapeutically relevant timescales, with potential implications for personalized health and disease.
Asunto(s)
Microbioma Gastrointestinal , Microbiota , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Microbioma Gastrointestinal/genética , Humanos , Metagenoma , Metagenómica/métodos , Microbiota/genéticaRESUMEN
The expression of ten tuberculosis candidate genes in lung and lymph nodes of cynomolgus macaques vaccinated and experimentally infected with Mycobacterium tuberculosis (Mtb) was quantified. The expression of TNFα, IL10, IL1ß, TLR4, IL17, IL6, IL12, and iNOS in the lungs of vaccinated animals was higher than that of non-vaccinated animals.
Asunto(s)
Vacuna BCG/inmunología , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Macaca fascicularis , Tuberculosis Pulmonar/genética , Tuberculosis Pulmonar/inmunología , Animales , Citocinas/genética , Citocinas/metabolismo , ADN Complementario/genética , ADN Complementario/metabolismo , Pulmón/inmunología , Pulmón/microbiología , Ganglios Linfáticos/inmunología , Mycobacterium tuberculosis/fisiología , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Polimorfismo de Nucleótido Simple , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Tuberculosis Pulmonar/microbiologíaRESUMEN
As our closest living relatives, non-human primates uniquely enable explorations of human health, disease, development, and evolution. Considerable effort has thus been devoted to generating induced pluripotent stem cells (iPSCs) from multiple non-human primate species. Here, we establish improved culture methods for chimpanzee (Pan troglodytes) and pig-tailed macaque (Macaca nemestrina) iPSCs. Such iPSCs spontaneously differentiate in conventional culture conditions, but can be readily propagated by inhibiting endogenous WNT signaling. As a unique functional test of these iPSCs, we injected them into the pre-implantation embryos of another non-human species, rhesus macaques (Macaca mulatta). Ectopic expression of gene BCL2 enhances the survival and proliferation of chimpanzee and pig-tailed macaque iPSCs within the pre-implantation embryo, although the identity and long-term contribution of the transplanted cells warrants further investigation. In summary, we disclose transcriptomic and proteomic data, cell lines, and cell culture resources that may be broadly enabling for non-human primate iPSCs research.
Asunto(s)
Células Madre Pluripotentes Inducidas , Pan troglodytes , Animales , Macaca mulatta , Macaca nemestrina/genética , ProteómicaRESUMEN
BACKGROUND: Macaque species share >93% genome homology with humans and develop many disease phenotypes similar to those of humans, making them valuable animal models for the study of human diseases (e.g., HIV and neurodegenerative diseases). However, the quality of genome assembly and annotation for several macaque species lags behind the human genome effort. RESULTS: To close this gap and enhance functional genomics approaches, we used a combination of de novo linked-read assembly and scaffolding using proximity ligation assay (HiC) to assemble the pig-tailed macaque (Macaca nemestrina) genome. This combinatorial method yielded large scaffolds at chromosome level with a scaffold N50 of 127.5 Mb; the 23 largest scaffolds covered 90% of the entire genome. This assembly revealed large-scale rearrangements between pig-tailed macaque chromosomes 7, 12, and 13 and human chromosomes 2, 14, and 15. We subsequently annotated the genome using transcriptome and proteomics data from personalized induced pluripotent stem cells derived from the same animal. Reconstruction of the evolutionary tree using whole-genome annotation and orthologous comparisons among 3 macaque species, human, and mouse genomes revealed extensive homology between human and pig-tailed macaques with regards to both pluripotent stem cell genes and innate immune gene pathways. Our results confirm that rhesus and cynomolgus macaques exhibit a closer evolutionary distance to each other than either species exhibits to humans or pig-tailed macaques. CONCLUSIONS: These findings demonstrate that pig-tailed macaques can serve as an excellent animal model for the study of many human diseases particularly with regards to pluripotency and innate immune pathways.
Asunto(s)
Cromosomas , Genoma , Genómica , Macaca nemestrina/genética , Animales , Biología Computacional/métodos , Genómica/métodos , Humanos , Cariotipificación/métodos , Masculino , Anotación de Secuencia Molecular , Proteómica/métodos , Secuencias Repetitivas de Ácidos NucleicosRESUMEN
Mycobacterial infections are of primary health concern in NHP colonies in biomedical research. NHP are constantly monitored and screened for Mycobacterium spp. We report 6 Chinese-origin rhesus macaques infected with Mycobacterium kansasii that exhibited positive tuberculin skin tests in the absence of disease. Two of these macaques were being used for research purposes; the remaining 4 macaques were residing at the contract quarantine company. Histopathology and acid-fast staining of fixed tissues from all macaques showed that all were free of disease. Thoracic radiographs were negative for any signs of disease or infection. Samples from bronchial lavage and tissues including lung, spleen, hilar and mesenteric lymph nodes tested negative by PCR assay for Mycobacterium spp. One of the research macaques tested culture-positive for M. kansasii and a poorly characterized M. avium complex organism. One macaque from the contract quarantine facility tested culture positive for M. kansasii. Genomic testing and target gene RNA expression analysis of the 2 M. kansasii isolates were performed to evaluate possible kinship and affected genes that might contribute to susceptibility to mycobacterial infection. Genotyping of the 2 isolates revealed 2 genetically distinct strains (strains 1 and 4). The presence of positive tuberculin skin tests in the absence of disease raises serious concerns regarding diagnostic methods used for infected NHP.
Asunto(s)
Enfermedades de los Monos/microbiología , Infecciones por Mycobacterium no Tuberculosas/veterinaria , Mycobacterium kansasii/aislamiento & purificación , Prueba de Tuberculina/veterinaria , Animales , Técnicas Bacteriológicas/veterinaria , Células Cultivadas , Reacciones Falso Positivas , Genotipo , Interacciones Huésped-Patógeno , Macaca mulatta , Enfermedades de los Monos/diagnóstico , Enfermedades de los Monos/inmunología , Reacción en Cadena de la Polimerasa Multiplex/veterinaria , Infecciones por Mycobacterium no Tuberculosas/diagnóstico , Infecciones por Mycobacterium no Tuberculosas/inmunología , Infecciones por Mycobacterium no Tuberculosas/microbiología , Mycobacterium kansasii/genética , Mycobacterium kansasii/inmunología , Mycobacterium kansasii/patogenicidad , Valor Predictivo de las Pruebas , Reproducibilidad de los ResultadosRESUMEN
We use the Reciprocal Smallest Distance (RSD) algorithm to identify amino acid sequence orthologs in the Chinese and Indian rhesus macaque draft sequences and estimate the evolutionary distance between such orthologs. We then use GOanna to map gene function annotations and human gene identifiers to the rhesus macaque amino acid sequences. We conclude methodologically by cross-tabulating a list of amino acid orthologs with large divergence scores with a list of genes known to be involved in SIV or HIV pathogenesis. We find that many of the amino acid sequences with large evolutionary divergence scores, as calculated by the RSD algorithm, have been shown to be related to HIV pathogenesis in previous laboratory studies. Four of the strongest candidate genes for SIVmac resistance in Chinese rhesus macaques identified in this study are CDK9, CXCL12, TRIM21, and TRIM32. Additionally, ANKRD30A, CTSZ, GORASP2, GTF2H1, IL13RA1, MUC16, NMDAR1, Notch1, NT5M, PDCD5, RAD50, and TM9SF2 were identified as possible candidates, among others. We failed to find many laboratory experiments contrasting the effects of Indian and Chinese orthologs at these sites on SIVmac pathogenesis, but future comparative studies might hold fertile ground for research into the biological mechanisms underlying innate resistance to SIVmac in Chinese rhesus macaques.
Asunto(s)
Macaca mulatta/genética , Síndrome de Inmunodeficiencia Adquirida del Simio/genética , Virus de la Inmunodeficiencia de los Simios , Secuencia de Aminoácidos , Animales , Evolución Biológica , Evolución MolecularRESUMEN
Inducible nitric oxide synthase (iNOS) is an enzyme that plays a key role in intracellular immune response against respiratory infections. Since various species of nonhuman primates exhibit different levels of susceptibility to infectious respiratory diseases, and since variation in regulatory regions of genes is thought to play a key role in expression levels of genes, two candidate regulatory regions of iNOS were mapped, sequenced, and compared across five species of nonhuman primates: African green monkeys (Chlorocebus sabaeus), pig-tailed macaques (Macaca nemestrina), cynomolgus macaques (Macaca fascicularis), Indian rhesus macaques (Macaca mulatta), and Chinese rhesus macaques (M. mulatta). In addition, we conducted an in silico analysis of the transcription factor binding sites associated with genetic variation in these two candidate regulatory regions across species. We found that only one of the two candidate regions showed strong evidence of involvement in iNOS regulation. Specifically, we found evidence of 13 conserved binding site candidates linked to iNOS regulation: AP-1, C/EBPB, CREB, GATA-1, GATA-3, NF-AT, NF-AT5, NF-κB, KLF4, Oct-1, PEA3, SMAD3, and TCF11. Additionally, we found evidence of interspecies variation in binding sites for several regulatory elements linked to iNOS (GATA-3, GATA-4, KLF6, SRF, STAT-1, STAT-3, OLF-1 and HIF-1) across species, especially in African green monkeys relative to other species. Given the key role of iNOS in respiratory immune response, the findings of this study might help guide the direction of future studies aimed to uncover the molecular mechanisms underlying the increased susceptibility of African green monkeys to several viral and bacterial respiratory infections.
Asunto(s)
Variación Genética , Óxido Nítrico Sintasa de Tipo II/genética , Secuencias Reguladoras de Ácidos Nucleicos , Animales , Sitios de Unión , Análisis por Conglomerados , Biología Computacional/métodos , Factor 4 Similar a Kruppel , Macaca mulatta , Regiones Promotoras Genéticas , Alineación de Secuencia , Análisis de Secuencia de ADN , Factores de Transcripción/metabolismoRESUMEN
Tuberculosis (TB) pathologic lesions in rhesus macaques resemble those in humans. The expression levels of several host TB candidate genes in the peripheral blood mononuclear cells (PBMCs) of six rhesus macaques experimentally infected with Mycobacterium tuberculosis were quantified pre-infection and at several dates post-infection. Quantitative measures of TB histopathology in the lungs including: granuloma count, granuloma size, volume of granulomatous and non-granulomatous lesions, and direct bacterial load, were used as the outcomes of a multi-level Bayesian regression model in which expression levels of host genes at various dates were used as predictors. The results indicate that the expression levels of TR4, CD40, CD40L, FAS (CD95) and TNF in PBMC were associated with quantitative measures of the severity of TB histopathologic lesions in the lungs of the study animals. Moreover, no reliable association between the expression levels of IFNE in PBMCs and the severity of TB lesions in the lungs of the study animals was found. In conclusion, PBMC expression profiles derived from the above-listed host genes might be appropriate biomarkers for probabilistic diagnosis and/or prognosis of TB severity in rhesus macaques.