The importance of individuals and scale: moving towards single cell microbiology.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
David A Relman
Publications and source records attributed to David A Relman.
The role of microbes in Crohn's disease.
Despite decades of research, the etiology of Crohn's disease (CD) remains unknown. Its pathogenesis may involve a complex interplay between host genetics, immune dysfunction, and microbial or environmental factors. Microorganisms, including pathogens and members of the indigenous microbiota, may initiate or propagate the inflammatory process in CD. The pathogenesis of CD has been difficult to study, owing to the broad spectrum of typically nonspecific clinical manifestations, the complexity of environmental and genetic factors, the lack of an accurate model of disease, and the limitations of microbiological methods. A more useful and relevant paradigm for the etiology of CD might be based on the idea of a pathogenic microbial community profile and might emphasize the role of interactive sets of microbes, rather than the role of individual organisms. We review how microbes may participate in the pathogenesis of CD and how they may inappropriately activate the mucosal immune system in genetically predisposed individuals.
Crohn Disease↗
Assembly of the human intestinal microbiota.
Complex microbial ecosystems occupy the skin, mucosa and alimentary tract of all mammals, including humans. Recent advances have highlighted the tremendous diversity of these microbial communities and their importance to host physiology, but questions remain about the ecological processes that establish and maintain the microbiota throughout life. The prevailing view, that the gastrointestinal microbiota of adult humans is a climax community comprised of the superior competitors for a stable set of niches, does not account for all of the experimental data. We argue here that the unique history of each community and intrinsic temporal dynamics also influence the structure of human intestinal communities.
Humans↗
Metagenomic analysis of the human distal gut microbiome.
The human intestinal microbiota is composed of 10(13) to 10(14) microorganisms whose collective genome ("microbiome") contains at least 100 times as many genes as our own genome. We analyzed approximately 78 million base pairs of unique DNA sequence and 2062 polymerase chain reaction-amplified 16S ribosomal DNA sequences obtained from the fecal DNAs of two healthy adults. Using metabolic function analyses of identified genes, we compared our human genome with the average content of previously sequenced microbial genomes. Our microbiome has significantly enriched metabolism of glycans, amino acids, and xenobiotics; methanogenesis; and 2-methyl-d-erythritol 4-phosphate pathway-mediated biosynthesis of vitamins and isoprenoids. Thus, humans are superorganisms whose metabolism represents an amalgamation of microbial and human attributes.
Adult↗
Early days: genomics and human responses to infection.
DNA microarray-based gene transcript-profiling of the responses of primates to infection has begun to yield new insights into host-pathogen interactions; this approach, however, remains plagued by challenges and complexities that have yet to be adequately addressed. The rapidly changing nature over time of acute infectious diseases in a host, and the genetic diversity of microbial pathogens present unique problems for the design and interpretation of functional-genomic studies in this field. In addition, there are the more common problems related to heterogeneity within clinical samples, the complex, non-standardized confounding variables associated with human subjects and the complexities posed by the analysis and validation of highly parallel data. Whereas various approaches have been developed to address each of these issues, there are significant limitations that remain to be overcome. The resolution of these problems should lead to a better understanding of the dialogue between the host and pathogen.
Animals↗
In search of biosecurity.
Explore the source record for details and available documents.
Biomedical Research↗
Bioterrorism--preparing to fight the next war.
Explore the source record for details and available documents.
Biological Warfare↗
Rapid quantitative profiling of complex microbial populations.
Diverse and complex microbial ecosystems are found in virtually every environment on earth, yet we know very little about their composition and ecology. Comprehensive identification and quantification of the constituents of these microbial communities--a 'census'--is an essential foundation for understanding their biology. To address this problem, we developed, tested and optimized a DNA oligonucleotide microarray composed of 10,462 small subunit (SSU) ribosomal DNA (rDNA) probes (7167 unique sequences) selected to provide quantitative information on the taxonomic composition of diverse microbial populations. Using our optimized experimental approach, this microarray enabled detection and quantification of individual bacterial species present at fractional abundances of <0.1% in complex synthetic mixtures. The estimates of bacterial species abundance obtained using this microarray are similar to those obtained by phylogenetic analysis of SSU rDNA sequences from the same samples--the current 'gold standard' method for profiling microbial communities. Furthermore, probes designed to represent higher order taxonomic groups of bacterial species reliably detected microbes for which there were no species-specific probes. This simple, rapid microarray procedure can be used to explore and systematically characterize complex microbial communities, such as those found within the human body.
Adult↗
Molecular analysis of the bacterial microbiota in the human stomach.
The microbiota of the human stomach and the influence of Helicobacter pylori colonization on its composition remain largely unknown. We characterized bacterial diversity within the human gastric mucosa by using a small subunit 16S rDNA clone library approach and analyzed 1,833 sequences generated by broad-range bacterial PCR from 23 gastric endoscopic biopsy samples. A diverse community of 128 phylotypes was identified, featuring diversity at this site greater than previously described. The majority of sequences were assigned to the Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Fusobacteria phyla. Ten percent of the phylotypes were previously uncharacterized, including a Deinococcus-related organism, relatives of which have been found in extreme environments but not reported before in humans. The gastric clone libraries from 19 subjects contained H. pylori rDNA; however, only 12 of these subjects tested positive for H. pylori by conventional laboratory methods. Statistical analysis revealed a large degree of intersubject variability of the gastric ecosystem. The presence of H. pylori did not affect the composition of the gastric community. This gastric bacterial rDNA data set was significantly different from sequence collections of the human mouth and esophagus described in other studies, indicating that the human stomach may be home to a distinct microbial eco-system. The gastric microbiota may play important, as-yet-undiscovered roles in human health and disease.
Adult↗
Growth phase- and nutrient limitation-associated transcript abundance regulation in Bordetella pertussis.
To survive in a host environment, microbial pathogens must sense local conditions, including nutrient availability, and adjust their growth state and virulence functions accordingly. No comprehensive investigation of growth phase-related gene regulation in Bordetella pertussis has been reported previously. We characterized changes in genome-wide transcript abundance of B. pertussis as a function of growth phase and availability of glutamate, a key nutrient for this organism. Using a Bordetella DNA microarray, we discovered significant changes in transcript abundance for 861 array elements during the transition from log phase to stationary phase, including declining transcript levels of many virulence factor genes. The responses to glutamate depletion exhibited similarities to the responses induced by exit from log phase, including decreased virulence factor transcript levels. However, only 23% of array elements that showed at least a fourfold growth phase-associated difference in transcript abundance also exhibited glutamate depletion-associated changes, suggesting that nutrient limitation may be one of several interacting factors affecting gene regulation during stationary phase. Transcript abundance patterns of a Bvg+ phase-locked mutant revealed that the BvgAS two-component regulatory system is a key determinant of growth phase- and nutrient limitation-related transcriptional control. Several adhesin genes exhibited lower transcript abundance during stationary phase and under glutamate restriction conditions. The predicted bacterial phenotype was confirmed: adherence to bronchoepithelial cells decreased 3.3- and 4.4-fold at stationary phase and with glutamate deprivation, respectively. Growth phase and nutrient availability may serve as cues by which B. pertussis regulates virulence according to the stage of infection or the location within the human airway.
Bacterial Proteins↗
Significant gene order and expression differences in Bordetella pertussis despite limited gene content variation.
Bordetella pertussis, an obligate human pathogen and the agent of whooping cough, is a clonal species, despite the dynamic selection pressures imposed by host immunity and vaccine usage. Because the generation of variation is critical for species evolution, we employed a variety of approaches to examine features of B. pertussis genetic variation. We found a high level of conservation of gene content among 137 B. pertussis strains with different geographical, temporal, and epidemiological associations, using comparative genomic hybridization. The limited number of regions of difference were frequently located adjacent to copies of the insertion element IS481, which is present in high numbers in the B. pertussis chromosome. This repeated sequence appears to provide targets for homologous recombination, resulting in deletion of intervening sequences. Using subtractive hybridization, we searched for previously undetected genes in diverse clinical isolates but did not detect any new genes, indicating that gene acquisition is rare in B. pertussis. In contrast, we found evidence of altered gene order in the several strains that were examined and again found an association of IS481 with sites of rearrangement. Finally, we compared whole-genome expression profiles of different strains and found significant changes in transcript abundance, even in the same strain after as few as 12 laboratory passages. This combination of approaches provides a detailed picture of a pathogenic species with little gene loss or gain but with the capacity to generate variation by rearranging its chromosome and altering gene expression. These findings have broad implications for host adaptation by microbial pathogens.
Bordetella pertussis↗
Genomic features of Bordetella parapertussis clades with distinct host species specificity.
BACKGROUND: The respiratory pathogen Bordetella parapertussis is a valuable model in which to study the complex phenotype of host specificity because of its unique two-species host range. One subset of strains, including the sequenced representative, causes whooping cough in humans, while other strains infect only sheep. The disease process in sheep is not well understood, nor are the genetic and transcriptional differences that might provide the basis for host specificity among ovine and human strains. RESULTS: We found 40 previously unknown genomic regions in an ovine strain of B. parapertussis using subtractive hybridization, including unique lipopolysaccharide genes. A microarray survey of the gene contents of 71 human and ovine strains revealed further differences, with 47 regions of difference distinguishing the host-restricted subgroups. In addition, sheep and human strains displayed distinct whole-genome transcript abundance profiles. We developed an animal model in which sheep were inoculated with a sheep strain, human strain, or mixture of the two. We found that the ovine strain persisted in the nasal cavity for 12 to 14 days, while the human strain colonized at lower levels and was no longer detected by 7 days post-inoculation. The ovine strain induced less granulocyte infiltration of the nasal mucosa. CONCLUSION: Several factors may play a role in determining host range of B. parapertussis. Human- and ovine-associated strains have differences in content and sequence of genes encoding proteins that mediate host-pathogen contact, such as lipopolysaccharide and fimbriae, as well as variation in regulation of toxins, type III secretion genes, and other virulence-associated genes.
Animals↗
Bordetella pertussis, the causative agent of whooping cough, evolved from a distinct, human-associated lineage of B. bronchiseptica.
Bordetella pertussis, B. bronchiseptica, B. parapertussis(hu), and B. parapertussis(ov) are closely related respiratory pathogens that infect mammalian species. B. pertussis and B. parapertussis(hu) are exclusively human pathogens and cause whooping cough, or pertussis, a disease that has resurged despite vaccination. Although it most often infects animals, infrequently B. bronchiseptica is isolated from humans, and these infections are thought to be zoonotic. B. pertussis and B. parapertussis(hu) are assumed to have evolved from a B. bronchiseptica-like ancestor independently. To determine the phylogenetic relationships among these species, housekeeping and virulence genes were sequenced, comparative genomic hybridizations were performed using DNA microarrays, and the distribution of insertion sequence elements was determined, using a collection of 132 strains. This multifaceted approach distinguished four complexes, representing B. pertussis, B. parapertussis(hu), and two distinct B. bronchiseptica subpopulations, designated complexes I and IV. Of the two B. bronchiseptica complexes, complex IV was more closely related to B. pertussis. Of interest, while only 32% of the complex I strains were isolated from humans, 80% of the complex IV strains were human isolates. Comparative genomic hybridization analysis identified the absence of the pertussis toxin locus and dermonecrotic toxin gene, as well as a polymorphic lipopolysaccharide biosynthesis locus, as associated with adaptation of complex IV strains to the human host. Lipopolysaccharide structural diversity among these strains was confirmed by gel electrophoresis. Thus, complex IV strains may comprise a human-associated lineage of B. bronchiseptica from which B. pertussis evolved. These findings will facilitate the study of pathogen host-adaptation. Our results shed light on the origins of the disease pertussis and suggest that the association of B. pertussis with humans may be more ancient than previously assumed.
Journal Article↗
Genomewide analysis of the host response to malaria in Kenyan children.
Malaria is a global problem, and there is a critical need for further understanding of the disease process. When malarial parasites invade and develop within the bloodstream, they stimulate a profound host response whose main clinical sign is fever. To explore this response, we measured host gene expression in whole blood from Kenyan children hospitalized with either acute malaria or other febrile illnesses. Genomewide analysis of expression identified 2 principal gene-expression profiles related to neutrophil and erythroid activity. In addition to these general acute responses, a third gene-expression profile was associated with host parasitemia; mediators of erythrophagocytosis and cellular stress were notable components of this response. The delineation of subjects on the basis of patterns of gene expression provides a molecular perspective of the host response to malaria and further functional insight into the underlying processes of pathogenesis.
Child↗
Diversity of the human intestinal microbial flora.
The human endogenous intestinal microflora is an essential "organ" in providing nourishment, regulating epithelial development, and instructing innate immunity; yet, surprisingly, basic features remain poorly described. We examined 13,355 prokaryotic ribosomal RNA gene sequences from multiple colonic mucosal sites and feces of healthy subjects to improve our understanding of gut microbial diversity. A majority of the bacterial sequences corresponded to uncultivated species and novel microorganisms. We discovered significant intersubject variability and differences between stool and mucosa community composition. Characterization of this immensely diverse ecosystem is the first step in elucidating its role in health and disease.
Adult↗
VCAM-1 expression on CD8+ cells correlates with enhanced anti-HIV suppressing activity.
CD8(+) cells from HIV-infected individuals showing the CD8(+) cell noncytotoxic antiviral response unexpectedly revealed mRNA for VCAM-1, a cell surface molecule found on endothelial cells. Uninfected subjects had undetectable levels of VCAM-1 mRNA in their CD8(+) cells. Flow cytometry analysis showed that up to 12% of the CD8(+) cells from HIV-positive individuals expressed VCAM-1 compared with 0.8% of the CD8(+) cells of HIV-negative individuals. Enrichment of the CD8(+)VCAM-1(+) cell population and subsequent coculture with CD4(+) cells acutely infected with HIV-1 showed that the VCAM-1(+)CD8(+) cells were able to suppress viral replication with 50% less input cells than the unseparated CD8(+) cell population. This study demonstrates, for the first time, the expression of VCAM-1 on CD8(+) cells. Moreover, the CD8(+)VCAM-1(+) cells show enhanced CD8(+) cell noncytotoxic antiviral response activity that could have clinical importance in HIV infection.
Acute Disease↗
The host response to smallpox: analysis of the gene expression program in peripheral blood cells in a nonhuman primate model.
Smallpox has played an unparalleled role in human history and remains a significant potential threat to public health. Despite the historical significance of this disease, we know little about the underlying pathophysiology or the virulence mechanisms of the causative agent, variola virus. To improve our understanding of variola pathogenesis and variola-host interactions, we examined the molecular and cellular features of hemorrhagic smallpox in cynomolgus macaques. We used cDNA microarrays to analyze host gene expression patterns in sequential blood samples from each of 22 infected animals. Variola infection elicited striking and temporally coordinated patterns of gene expression in peripheral blood. Of particular interest were features that appear to represent an IFN response, cell proliferation, immunoglobulin gene expression, viral dose-dependent gene expression patterns, and viral modulation of the host immune response. The virtual absence of a tumor necrosis factor alpha/NF-kappaB-activated transcriptional program in the face of an overwhelming systemic infection suggests that variola gene products may ablate this response. These results provide a detailed picture of the host transcriptional response during smallpox infection, and may help guide the development of diagnostic, therapeutic, and prophylactic strategies.
Animals↗
Exploring the potential of variola virus infection of cynomolgus macaques as a model for human smallpox.
Smallpox virus (variola) poses a significant threat as an agent of bioterrorism. To mitigate this risk, antiviral drugs and an improved vaccine are urgently needed. Satisfactory demonstration of protective efficacy against authentic variola will require development of an animal model in which variola produces a disease course with features consistent with human smallpox. Toward this end, cynomolgus macaques were exposed to several variola strains through aerosol and/or i.v. routes. Two strains, Harper and India 7124, produced uniform acute lethality when inoculated i.v. in high doses (10(9) plaque-forming units). Lower doses resulted in less fulminant, systemic disease and lower mortality. Animals that died had profound leukocytosis, thrombocytopenia, and elevated serum creatinine levels. After inoculation, variola was disseminated by means of a monocytic cell-associated viremia. Distribution of viral antigens by immunohistochemistry correlated with the presence of replicating viral particles demonstrated by electron microscopy and pathology in the lymphoid tissues, skin, oral mucosa, gastrointestinal tract, reproductive system, and liver. These particles resembled those seen in human smallpox. High viral burdens in target tissues were associated with organ dysfunction and multisystem failure. Evidence of coagulation cascade activation (D dimers) corroborated histologic evidence of hemorrhagic diathesis. Depletion of T cell-dependent areas of lymphoid tissues occurred, probably as a consequence of bystander apoptotic mechanisms initiated by infected macrophages. Elaboration of cytokines, including IL-6 and IFN-gamma, contribute to a cytokine storm formerly known as "toxemia." A more precise understanding of disease pathogenesis should provide targets for therapeutic intervention, to be used alone or in combination with inhibitors of variola virus replication.
Animals↗