Gregory Gimenez

Microbial culturomics: paradigm shift in the human gut microbiome study

Comprehensive determination of the microbial composition of the gut microbiota and the relationships with health and disease are major challenges in the 21st century. Metagenomic analysis of the human gut microbiota detects mostly uncultured bacteria. We studied stools from two lean Africans and one obese European, using 212 different culture conditions (microbial culturomics), and tested the colonies by using mass spectrometry and 16S rRNA amplification and sequencing. In parallel, we analysed the same three samples by pyrosequencing 16S rRNA amplicons targeting the V6 region. The 32 500 colonies obtained by culturomics have yielded 340 species of bacteria from seven phyla and 117 genera, including two species from rare phyla (Deinococcus-Thermus and Synergistetes, five fungi, and a giant virus (Senegalvirus). The microbiome identified by culturomics included 174 species never described previously in the human gut, including 31 new species and genera for which the genomes were sequenced, generating c. 10 000 new unknown genes (ORFans), which will help in future molecular studies. Among these, the new species Microvirga massiliensis has the largest bacterial genome so far obtained from a human, and Senegalvirus is the largest virus reported in the human gut. Concurrent metagenomic analysis of the same samples produced 698 phylotypes, including 282 known species, 51 of which overlapped with the microbiome identified by culturomics. Thus, culturomics complements metagenomics by overcoming the depth bias inherent in metagenomic approaches.

Phylogenetic and Phyletic Studies of Informational Genes in Genomes Highlight Existence of a 4th Domain of Life Including Giant Viruses

The discovery of Mimivirus, with its very large genome content, made it possible to identify genes common to the three domains of life (Eukarya, Bacteria and Archaea) and to generate controversial phylogenomic trees congruent with that of ribosomal genes, branching Mimivirus at its root. Here we used sequences from metagenomic databases, Marseillevirus and three new viruses extending the Mimiviridae family to generate the phylogenetic trees of eight proteins involved in different steps of DNA processing. Compared to the three ribosomal defined domains, we report a single common origin for Nucleocytoplasmic Large DNA Viruses (NCLDV), DNA processing genes rooted between Archaea and Eukarya, with a topology congruent with that of the ribosomal tree. As for translation, we found in our new viruses, together with Mimivirus, five proteins rooted deeply in the eukaryotic clade. In addition, comparison of informational genes repertoire based on phyletic pattern analysis supports existence of a clade containing NCLDVs clearly distinct from that of Eukarya, Bacteria and Archaea. We hypothesize that the core genome of NCLDV is as ancient as the three currently accepted domains of life.

Minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE)

One purpose of the biomedical literature is to report results in sufficient detail that the methods of data collection and analysis can be independently replicated and verified. Here we present reporting guidelines for gene expression localization experiments: the minimum information specification for in situ hybridization and immunohistochemistry experiments (MISFISHIE). MISFISHIE is modeled after the Minimum Information About a Microarray Experiment (MIAME) specification for microarray experiments. Both guidelines define what information should be reported without dictating a format for encoding that information. MISFISHIE describes six types of information to be provided for each experiment: experimental design, biomaterials and treatments, reporters, staining, imaging data and image characterizations. This specification has benefited the consortium within which it was developed and is expected to benefit the wider research community. We welcome feedback from the scientific community to help improve our proposal.

Evidence of the megavirome in humans

BACKGROUND Megavirales is a proposed new virus order composed of Mimivirus, Marseillevirus and closely related viruses, as well as members of the families Poxviridae, Iridoviridae, Ascoviridae, Phycodnaviridae and Asfarviridae. The Megavirales virome, which we refer to as the megavirome, has been largely neglected until now because of the use of technical procedures that have jeopardized the discovery of giant viruses, particularly the use of filters with pore sizes in the 0.2-0.45-μm range. Concurrently, there has been accumulating evidence supporting the role of Mimivirus, discovered while investigating a pneumonia outbreak using amoebal coculture, as a causative agent in pneumonia. OBJECTIVES In this paper, we describe the detection of sequences related to Mimivirus and Marseillevirus in the gut microbiota from a young Senegalese man. We also searched for sequences related to Megavirales in human metagenomes publicly available in sequence databases. RESULTS We serendipitously detected Mimivirus- and Marseillevirus-like sequences while using a new metagenomic approach targeting bacterial DNA that subsequently led to the isolation of a new member of the family Marseilleviridae, named Senegalvirus, from human stools. This discovery demonstrates the possibility of the presence of giant viruses of amoebae in humans. In addition, we detected sequences related to Megavirales members in several human metagenomes, which adds to previous findings by several groups. CONCLUSIONS Overall, we present convergent evidence of the presence of mimiviruses and marseilleviruses in humans. Our findings suggest that we should re-evaluate the human megavirome and investigate the prevalence, diversity and potential pathogenicity of giant viruses in humans.

Non-contiguous finished genome sequence and description of Herbaspirillum massiliense sp. nov.

Herbaspirillum massiliense strain JC206T sp. nov. is the type strain of H. massiliense sp. nov., a new species within the genus Herbaspirillum. This strain, whose genome is described here, was isolated from the fecal flora of a healthy Senegalese patient. H. massiliense is an aerobic rod. Here we describe the features of this organism, together with the complete genome sequence and annotation. The 4,186,486 bp long genome (one chromosome but no plasmid) contains 3,847 protein-coding and 54 RNA genes, including 3 rRNA genes.

Lineage-specific enhancers activate self-renewal genes in macrophages and embryonic stem cells

Genetic programming for self-renewal Instead of repopulating themselves from tissue-resident stem cell pools like most types of differentiated cells, tissue macrophages maintain themselves by self-renewing. The underlying genetic programs that allow for this, however, are unknown. Soucie et al. now report that in macrophages at homeostasis, a pair of transcription factors (MafB and c-Maf) bind to and repress the enhancers of genes regulating self-renewal. When macrophages need to replenish their stocks, for example in response to injury, they transiently decrease MafB and c-Maf expression so they can self-renew. A parallel pathway also operates to control the self-renewal of embryonic stem cells. Science, this issue p. 10.1126/science.aad5510 Tissue macrophages and embryonic stem cells use similar genetic programs to self-renew. INTRODUCTION In many organs of the body, differentiated cells are frequently lost and need to be replaced as part of normal homeostatic tissue maintenance or in response to injury. In most cases, this regeneration is assured by differentiation from tissue-specific stem cells. Together with a few other cell types, tissue macrophages represent a rare exception to this pathway, as they can be maintained independently of blood stem cells by local proliferation. Under certain conditions, mature macrophages can also be expanded and maintained long term in culture without transformation or loss of differentiation status. The gene regulatory mechanisms that allow such differentiated cells to self-renew while maintaining cell type–specific identity have so far remained unknown. Self-renewing macrophages provide a rare opportunity to study this question. RATIONALE Molecularly, cell identity can be defined by the genomic positions of gene regulatory enhancer elements. The cell type–specific signatures and activity status of such elements have been characterized by the analysis of specific histone modifications and the binding of regulatory proteins. To identify the regulatory mechanisms that enable macrophage self-renewal capacity to be integrated into the overall program of epigenetic macrophage identity, we have compared the enhancer repertoires of quiescent and self-renewing macrophages. Based on our previous observations that deletion of MafB and c-Maf transcription factors results in an extended self-renewal capacity of macrophages, we further investigated how the absence of Maf transcription factors affects the enhancers of specific self-renewal genes and how these mechanisms activate macrophage self-renewal under homeostatic and challenge conditions in vivo. RESULTS Compared to quiescent macrophages, self-renewing macrophages showed no appreciable difference with respect to genome-wide enhancer positions but displayed an increase in the activation status of many enhancers that were also bound by the lineage-specifying transcription factor PU.1 in both cell types. This finding suggests that these poised macrophage-specific enhancers became active in self-renewing macrophages. We found activated enhancers to be associated with a network of genes, centered on Myc and Klf2, that were up-regulated and functionally important for self-renewal in these cells. The same genes were also required for embryonic stem (ES) cell self-renewal but were associated with a distinct, ES cell–specific set of enhancers. We observed that activated self-renewal–associated macrophage enhancers were directly repressed by MafB binding. The loss of MafB and c-Maf expression relieved this repression and led to activation of the self-renewal gene network in MafB and cMaf knockout macrophages, as well as in alveolar macrophages that express constitutively low levels of these transcription factors. In vivo single-cell analysis further revealed that, both in the steady state and in response to immune stimulation, proliferating resident macrophages could access this network by transient down-regulation of Maf transcription factors. CONCLUSION Our results demonstrate that self-renewal in macrophages involves down-regulation of MafB and cMaf, as well as concomitant activation of a self-renewal gene network shared with ES cells but controlled from cell type–specific enhancers. Macrophage enhancers associated with self-renewal genes are already present in quiescent cells and can become activated when direct repression by Maf transcription factors is relieved. Our findings provide a general molecular rationale for the compatibility of self-renewal and differentiated cell functions and may also be more generally relevant for the direct activation of self-renewal activity in other differentiated cell types with therapeutic potential. The self-renewal potential of both ES cells and differentiated macrophages is dependent on a shared network of self-renewal genes (left) that are controlled by distinct lineage-specific enhancers (right). In quiescent macrophages, the transcription factor MafB binds and represses these enhancers. The loss of MafB expression results in enhancer activation and enables macrophage self-renewal. At bottom left, red arrows indicate activation; blue bars represent inhibition. Circle size is a function of the number of times the target is affected by other regulators. MΦ, macrophage; E, enhancer; P, promoter. ILLUSTRATION: SERENA BIELLI Differentiated macrophages can self-renew in tissues and expand long term in culture, but the gene regulatory mechanisms that accomplish self-renewal in the differentiated state have remained unknown. Here we show that in mice, the transcription factors MafB and c-Maf repress a macrophage-specific enhancer repertoire associated with a gene network that controls self-renewal. Single-cell analysis revealed that, in vivo, proliferating resident macrophages can access this network by transient down-regulation of Maf transcription factors. The network also controls embryonic stem cell self-renewal but is associated with distinct embryonic stem cell–specific enhancers. This indicates that distinct lineage-specific enhancer platforms regulate a shared network of genes that control self-renewal potential in both stem and mature cells.

A Toxin-Antitoxin Module of Salmonella Promotes Virulence in Mice

Toxin-antitoxin (TA) modules are widely prevalent in both bacteria and archaea. Originally described as stabilizing elements of plasmids, TA modules are also widespread on bacterial chromosomes. These modules promote bacterial persistence in response to specific environmental stresses. So far, the possibility that TA modules could be involved in bacterial virulence has been largely neglected, but recent comparative genomic studies have shown that the presence of TA modules is significantly associated with the pathogenicity of bacteria. Using Salmonella as a model, we investigated whether TA modules help bacteria to overcome the stress conditions encountered during colonization, thereby supporting virulence in the host. By bioinformatics analyses, we found that the genome of the pathogenic bacterium Salmonella Typhimurium encodes at least 11 type II TA modules. Several of these are conserved in other pathogenic strains but absent from non-pathogenic species indicating that certain TA modules might play a role in Salmonella pathogenicity. We show that one TA module, hereafter referred to as sehAB, plays a transient role in virulence in perorally inoculated mice. The use of a transcriptional reporter demonstrated that bacteria in which sehAB is strongly activated are predominantly localized in the mesenteric lymph nodes. In addition, sehAB was shown to be important for the survival of Salmonella in these peripheral lymphoid organs. These data indicate that the transient activation of a type II TA module can bring a selective advantage favouring virulence and demonstrate that TA modules are engaged in Salmonella pathogenesis.

Genotyping of Human Lice Suggests Multiple Emergences of Body Lice from Local Head Louse Populations

Background Genetic analyses of human lice have shown that the current taxonomic classification of head lice (Pediculus humanus capitis) and body lice (Pediculus humanus humanus) does not reflect their phylogenetic organization. Three phylotypes of head lice A, B and C exist but body lice have been observed only in phylotype A. Head and body lice have different behaviours and only the latter have been involved in outbreaks of infectious diseases including epidemic typhus, trench fever and louse borne recurrent fever. Recent studies suggest that body lice arose several times from head louse populations. Methods and Findings By introducing a new genotyping technique, sequencing variable intergenic spacers which were selected from louse genomic sequence, we were able to evaluate the genotypic distribution of 207 human lice. Sequence variation of two intergenic spacers, S2 and S5, discriminated the 207 lice into 148 genotypes and sequence variation of another two intergenic spacers, PM1 and PM2, discriminated 174 lice into 77 genotypes. Concatenation of the four intergenic spacers discriminated a panel of 97 lice into 96 genotypes. These intergenic spacer sequence types were relatively specific geographically, and enabled us to identify two clusters in France, one cluster in Central Africa (where a large body louse outbreak has been observed) and one cluster in Russia. Interestingly, head and body lice were not genetically differentiated. Conclusions We propose a hypothesis for the emergence of body lice, and suggest that humans with both low hygiene and head louse infestations provide an opportunity for head louse variants, able to ingest a larger blood meal (a required characteristic of body lice), to colonize clothing. If this hypothesis is ultimately supported, it would help to explain why poor human hygiene often coincides with outbreaks of body lice. Additionally, if head lice act as a reservoir for body lice, and that any social degradation in human populations may allow the formation of new populations of body lice, then head louse populations are potentially a greater threat to humans than previously assumed.

The giant Cafeteria roenbergensis virus that infects a widespread marine phagocytic protist is a new member of the fourth domain of Life.

Background A recent work has provided strong arguments in favor of a fourth domain of Life composed of nucleo-cytoplasmic large DNA viruses (NCLDVs). This hypothesis was supported by phylogenetic and phyletic analyses based on a common set of proteins conserved in Eukarya, Archaea, Bacteria, and viruses, and implicated in the functions of information storage and processing. Recently, the genome of a new NCLDV, Cafeteria roenbergensis virus (CroV), was released. The present work aimed to determine if CroV supports the fourth domain of Life hypothesis. Methods A consensus phylogenetic tree of NCLDVs including CroV was generated from a concatenated alignment of four universal proteins of NCLDVs. Some features of the gene complement of CroV and its distribution along the genome were further analyzed. Phylogenetic and phyletic analyses were performed using the previously identified common set of informational genes present in Eukarya, Archaea, Bacteria, and NCLDVs, including CroV. Findings Phylogenetic reconstructions indicated that CroV is clearly related to the Mimiviridae family. The comparison between the gene repertoires of CroV and Mimivirus showed similarities regarding the gene contents and genome organization. In addition, the phyletic clustering based on the comparison of informational gene repertoire between Eukarya, Archaea, Bacteria, and NCLDVs unambiguously classified CroV with other NCLDVs and clearly included it in a fourth domain of Life. Taken together, these data suggest that Mimiviridae, including CroV, may have inherited a common gene content probably acquired from a common Mimiviridae ancestor. Conclusions This further analysis of the gene repertoire of CroV consolidated the fourth domain of Life hypothesis and contributed to outline a functional pan-genome for giant viruses infecting phagocytic protistan grazers.

Complete Genome Sequence of Methanomassiliicoccus luminyensis, the Largest Genome of a Human-Associated Archaea Species

The present study describes the complete and annotated genome sequence of Methanomassiliicoccus luminyensis strain B10 (DSM 24529(T), CSUR P135), which was isolated from human feces. The 2.6-Mb genome represents the largest genome of a methanogenic euryarchaeon isolated from humans. The genome data of M. luminyensis reveal unique features and horizontal gene transfer events, which might have occurred during its adaptation and/or evolution in the human ecosystem.