Origen de la multicel·lularitat : una aproximació genòmica i funcional

En el present estudi s'analitza l'origen i evolució de 2 molècules claus pera entendre la multicel·lularitat dels animals: les molècules d'adhesió integrines i els factors de transcripció T-box. S’utilitzen els genomes recentment publicats de protists unicel•lulars parents propers dels animals. S’analitza l’origen i evolució d’aquests gens mitjançant anàlisi filogènic, determinació de motius funcionals i també tècniques de biologia molecular. A més, es documenta un cas de transferència gènica horitzontal des d'un eucariota cap a un procariota, fenomen poc habitual. Les principals conclusions són que tant l’adhesoma d'integrina com els gens T-box tenen un origen molt anterior als animals, en un context unicel•lular, i que després foren cooptats pel llinatge multicel•lular dels animals.

Mating system and avpr1a promoter variation in primates.

It has been suggested that primate mating and social behaviours may be influenced by variation in promoter region repetitive DNA of the vasopressin receptor 1a gene (avpr1a). We show that male mating behaviour does not covary in a simple way with promoter repetitive DNA in 12 Old World primates. We found that one microsatellite (-553 bp upstream) was present in all species, irrespective of their behaviour. By contrast, two microsatellites (-3956 and -3625 bp upstream) were present only in some species, yet this variation did not correlate with behaviour. These findings agree with a recent comparative analysis of voles and show that the variation in repetitive DNA in the avpr1a promoter region does not generally explain variation in male mating behaviour. Phylogenetic analysis revealed a GAGTA motif that has been independently deleted three times and involved in another larger deletion. Importantly, the presence/absence of this GAGTA motif leads to changes in predicted transcription factor-binding sites. Given the repeated loss of this motif, we speculate that it might be of functional relevance. We suggest that such non-repetitive variation, either in indels or in sequence variation, are likely to be important in explaining interspecific variation in avpr1a expression.

Genomics of "chlamydia"-related bacteria

AbstractThe Chlamydiales order is an important bacterial phylum that comprises some of the most successful human pathogens such as Chlamydia trachomatis, the leading infectious cause of blindness worldwide. Since some years, several new bacteria related to Chlamydia have been discovered in clinical or environmental samples and might represent emerging pathogens. The genome sequencing of classical Chlamydia has brought invaluable information on these obligate intracellular bacteria otherwise difficult to study due to the lack of tools to perform basic genetic manipulation. The recent emergence of high-throughput sequencing technologies yielding millions of reads in a short time lowered the costs of genome sequencing and thus represented a unique opportunity to study Chlamydia-re\ated bacteria. Based on the sequencing and the analysis of Chlamydiales genomes, this thesis provides significant insights into the genetic determinants of the intracellular lifestyle, the pathogenicity, the metabolism and the evolution of Chlamydia-related bacteria. A first approach showed the efficacy of rapid sequencing coupled to proteomics to identify immunogenic proteins. This method, particularly useful for an emerging pathogen such as Parachlamydia acanthamoebae, enabled us to discover good candidates for the development of diagnostic tools that would permit to evaluate at larger scale the role of this bacterium in disease. Second, the complete genome of Waddlia chondrophila, a potential agent of miscarriage, encodes numerous virulence factors to manipulate its host cell and resist to environmental stresses. The reconstruction of metabolic pathways showed that the bacterium possesses extensive capabilities compared to related organisms. However, it is still incapable of synthesizing some essential components and thus has to import them from its host. Third, the genome comparison of Protochlamydia naegleriophila to its closest known relative Protochlamydia amoebophila revealed a particular evolutionary dynamic with the occurrence of an unexpected genome rearrangement. Fourth, a phylogenetic analysis of P. acanthamoebae and Legionella drancourtii identified several genes probably exchanged by horizontal gene transfer with other intracellular bacteria that might occur within their amoebal host. These genes often encode mechanisms for resistance to metal or toxic compounds. As a whole, the analysis of the different genomes enabled us to highlight a large diversity in size, GC percentage, repeat content as well as plasmid organization. The abundant genomic data obtained during this thesis have a wide impact since they provide the necessary bases for detailed investigations on countless aspects of the biology and the evolution of Chlamydia-related bacteria, whether in wet lab or by bioinformatical analyses.RésuméL'ordre des Chlamydiales est un important phylum bactérien qui comprend de nombreuses espèces pathogènes pour l'homme et les animaux, dont Chlamydia trachomatis, responsable du trachome, la cause majeure de cécité d'origine infectieuse à travers le monde. Durant ces dernières décennies, de nombreuses bactéries apparentées aux Chlamydia ont été découvertes dans des échantillons environnementaux ou cliniques mais leur éventuel rôle pathogène dans le développement de maladies reste peu connu. Ces bactéries sont des intracellulaires obligatoires car elles ont besoin d'une cellule hôte pour se multiplier, ce qui rend leur étude particulièrement difficile. Le développement de nouvelles technologies permettant de séquencer le génome d'un organisme rapidement et à moindre coût ainsi que l'essor des méthodes d'analyse s'y rapportant représentent une opportunité exceptionnelle d'étudier ces organismes. Dans ce contexte, cette thèse démontre l'utilité de la génomique pour développer de nouveaux outils diagnostiques ainsi que pour étudier le métabolisme de ces bactéries, leurs facteurs de virulence et leur évolution.Ainsi, une première approche a illustré l'utilité d'un séquençage rapide pour obtenir les informations nécessaires à l'identification de protéines qui sont reconnues par des anticorps humains ou animaux. Cette méthode, particulièrement utile pour un pathogène émergent tel que Parachlamydia acanthamoebae, a permis de découvrir de bons candidats pour le développement d'un outil diagnostique qui permettrait d'évaluer à plus large échelle le rôle de cette bactérie notamment dans la pneumonie. L'analyse du contenu génique de Waddlia chondrophila, un autre germe qui pourrait être impliqué dans les avortements et tes fausses-couches, a en outre mis en évidence la présence de nombreux facteurs connus qui lui permettent de manipuler son hôte. Cette bactérie possède de plus grandes capacités métaboliques que les autres Chlamydia, mais elle est incapable de synthétiser certains composants et doit donc les importer de son hôte pour subvenir à ses besoins. La comparaison du génome de Protochlamydia naegleriophila à son plus proche parent, Protochlamydia amoebophila, a dévoilé une évolution dynamique particulière avec l'occurrence d'un réarrangement majeur inattendu après la séparation de ces deux espèces. En outre, ces études ont montré l'occurrence de plusieurs transferts de gène avec d'autres organismes plus éloignés, notamment d'autres intracellulaires d'amibes, souvent pour l'acquisition de mécanismes de résistances à des composés toxiques. Les données génomiques acquises durant ce travail posent les fondements nécessaires a de nombreuses analyses qui permettront progressivement de mieux comprendre de nombreux aspects de ces bactéries fascinantes.

HIV type 1 drug resistance among naive patients from Venezuela.

In this study, we characterize proviral DNA of 20 HIV-1 asymptomatic antiretroviral-naive patients from Venezuela in env, gag, and pol genes regions. Results from both env/gag HMA subtyping and phylogenetic analysis of pol partial sequences led to the description of clade B in all cases. Nevertheless, the high prevalence of polymorphisms was particularly evident among the protease sequences. A 10% prevalence of major resistance mutations to RTIs was found. Our data also suggested that the protease polymorphisms I62T and V77T could be considered as molecular markers of the subtype B local epidemic. In addition, we show how proviral DNA can be used as a reliable tool to follow trends of resistance mutation transmission.

The Evolution of Trypanosomes Infecting Humans and Primates

Based on phylogenetic analysis of 18S rRNA sequences and clade taxon composition, this paper adopts a biogeographical approach to understanding the evolutionary relationships of the human and primate infective trypanosomes, Trypanosoma cruzi, T. brucei, T. rangeli and T. cyclops. Results indicate that these parasites have divergent origins and fundamentally different patterns of evolution. T. cruzi is placed in a clade with T. rangeli and trypanosomes specific to bats and a kangaroo. The predominantly South American and Australian origins of parasites within this clade suggest an ancient southern super-continent origin for ancestral T. cruzi, possibly in marsupials. T. brucei clusters exclusively with mammalian, salivarian trypanosomes of African origin, suggesting an evolutionary history confined to Africa, while T. cyclops, from an Asian primate appears to have evolved separately and is placed in a clade with T. (Megatrypanum) species. Relating clade taxon composition to palaeogeographic evidence, the divergence of T. brucei and T. cruzi can be dated to the mid-Cretaceous, around 100 million years before present, following the separation of Africa, South America and Euramerica. Such an estimate of divergence time is considerably more recent than those of most previous studies based on molecular clock methods. Perhaps significantly, Salivarian trypanosomes appear, from these data, to be evolving several times faster than Schizotrypanum species, a factor which may have contributed to previous anomalous estimates of divergence times.

Species and Strain-specific Typing of Cryptosporidium Parasites in Clinical and Environmental Samples

Cryptosporidiosis has recently attracted attention as an emerging waterborne and foodborne disease as well as an opportunistic infection in HIV infected individuals. The lack of genetic information, however, has resulted in confusion in the taxonomy of Cryptosporidium parasites and in the development of molecular tools for the identification and typing of oocysts in environmental samples. Phylogenetic analysis of the small subunit ribosomal RNA (SSU rRNA) gene has shown that the genus Cryptosporidium is comprised of several distinct species. Our data show the presence of at least four species: C. parvum, C. muris, C. baileyi and C. serpentis (C. meleagridis, C. nasorum and C. felis were not studied). Within each species, there is some sequence variation. Thus, various genotypes (genotype 1, genotype 2, guinea pig genotype, monkey genotype and koala genotype, etc.) of C. parvum differ from each other in six regions of the SSU rRNA gene. Information on polymorphism in Cryptosporidium parasites has been used in the development of species and strain-specific diagnostic tools. Use of these tools in the characterization of oocysts various samples indicates that C. parvum genotype 1 is the strain responsible for most human Cryptosporidium infections. In contrast, genotype 2 is probably the major source for environmental contamination of environment, and has been found in most oysters examined from Chesapeake Bay that serve as biologic monitors of surface water. Parasites of Cryptosporidium species other than C. parvum have not been detected in HIV+ individuals, indicating that the disease in humans is caused only by C. parvum.

A Mitochondrial DNA Phylogeny Indicates Close Relationships between Populations of Lutzomyia whitmani (Diptera: Psychodidae, Phlebotominae) from the Rain-forest Regions of Amazônia and Northeast Brazil

Phylogenetic analysis of all 31 described mitochondrial (cytochrome b) haplotypes of Lutzomyia whitmani demonstrated that new material from the State of Rondônia, in southwest Amazônia, forms a clade within a lineage found only in the rain-forest regions of Brazil. This rain-forest lineage also contains two other clades of haplotypes, one from eastern Amazônia and one from the Atlantic forest zone of northeast Brazil (including the type locality of the species in Ilhéus, State of Bahia). These findings do not favour recognizing two allopatric cryptic species of L. whitmani, one associated with the silvatic transmission of Leishmania shawi in southeast Amazônia and the other with the peridomestic transmission of Le. braziliensis in northeast Brazil. Instead, they suggest that there is (or has been in the recent past) a continuum of inter-breeding populations of L. whitmani in the rain-forest regions of Brazil.

New respiratory enterovirus and recombinant rhinoviruses among circulating picornaviruses.

Rhinoviruses and enteroviruses are leading causes of respiratory infections. To evaluate genotypic diversity and identify forces shaping picornavirus evolution, we screened persons with respiratory illnesses by using rhinovirus-specific or generic real-time PCR assays. We then sequenced the 5 untranslated region, capsid protein VP1, and protease precursor 3CD regions of virus-positive samples. Subsequent phylogenetic analysis identified the large genotypic diversity of rhinoviruses circulating in humans. We identified and completed the genome sequence of a new enterovirus genotype associated with respiratory symptoms and acute otitis media, confirming the close relationship between rhinoviruses and enteroviruses and the need to detect both viruses in respiratory specimens. Finally, we identified recombinants among circulating rhinoviruses and mapped their recombination sites, thereby demonstrating that rhinoviruses can recombine in their natural host. This study clarifies the diversity and explains the reasons for evolution of these viruses.

A potential snail host of schistosomiasis in Bolivia: Biomphalaria amazonica paraense, 1966

Biomphalaria amazonica Paraense, 1996 was collected from a permanent pond in the outskirts of the Bolivian city of Santa Cruz. Identification of the collected specimens was made by comparison with the original description of the species and with topotypic material in the collection of Instituto Oswaldo Cruz. Phylogenetic analysis confirmed that these Bolivian specimens belong to B. amazonica.

Genomic characterization of a Brazilian TT Virus isolate closely related to SEN virus-F

SEN virus (SENV) is a circular, single stranded DNA virus that has been first characterized in the serum of a human immunodeficiency virus type 1 (HIV-1)-infected patient. Eight genotypes of SENV (A-H) have been identified and further recognized as variants of TT virus (TTV) in the family Circoviridae. Here we describe the first genomic characterization of a SENV isolate (5-A) from South America. Using 'universal' primers, able to amplify most, if not all, TTV/SENV genotypes, a segment of > 3 kb was amplified by polymerase chain reaction from the serum of an HIV-1 infected patient. The amplicon was cloned and a 3087-nucleotide sequence was determined, that showed a high (85%) homology with the sequence of the Italian isolate SENV-F. Proteins encoded by open reading frames (ORFs) 1 to 4 consisted of 758, 129, 276, and 267 amino acids, respectively. By phylogenetic analysis, isolate 5-A was classified into TTV genotype 19 (phylogenetic group 3), together with SENV-F and TTV isolate SAa-38.

Antiretroviral resistance and genetic diversity of human immunodeficiency virus type 1 isolates from the Federal District, Central Brazil

In the context of universal access to antiretroviral therapy, the surveillance of human immunodeficiency virus type 1 (HIV-1) genetic diversity and resistance becomes pivotal. In this work our purpose was to describe the genetic variability; prevalence of drug-resistance mutations; and genotypic resistance profiles in HIV-1 infected individuals under antiretroviral treatment, from the Federal District, Brasília, Central Brazil. The entire viral protease and codons 19 to 234 of the reverse transcriptase gene from 45 HIV-1 isolates were amplified and sequenced for subtyping and genotyping. By phylogenetic analysis, 96% of the samples clustered with subtype B and the remaining 4% with HIV-1 subtype F sequences. One major protease inhibitor resistance-associated mutation, I50V, was detected in 38% of the samples. Minor mutations were also found at the protease gene: L10I/V (7%), K20M (2%), M36I (11%), L63P (20%), A71T (2%), and V77I (7%). Many mutations associated with reduced susceptibility to nucleoside or non-nucleoside reverse transcriptase inhibitors were detected: M41L (11%), E44D (4%), D67N (11%), T69D (2%), K70R (11%), L74V (2%), L100I (4%), K103N (18%), V118I (9%), Y181C (11%), M184V (18%), G190A (4%), T215Y (4%), and K219E (4%). This study has shown that 84% of the studied population from the Federal District, showing evidences of therapy failure, presented viral genomic mutations associated with drug resistance. The main antiretrovirals to which this population showed resistance were the PI amprenavir (38%), the NNRTIs delavirdine, nevirapine (31%), and efavirenz (24%), and the NRTIs lamivudine (18%), abacavir, and zidovudine (13%).

Identification of human T-cell lymphotropic virus infection in a semi-isolated Afro-Brazilian quilombo located in the Marajó Island (Pará, Brazil)

Antibodies to human T-cell lymphotropic virus-1 and 2 (HTLV-1 and 2) were tested in 259 inhabitants (98 males and 161 females) of four villages of the Marajó Island (Pará, Brazil) using enzyme immunoassays (ELISA and Western blot). Types and subtypes of HTLV were determined by nested polymerase chain reaction (PCR) targeting the pX, env and 5´LTR regions. HTLV-1 infection was detected in Santana do Arari (2.06%) and Ponta de Pedras (1%). HTLV-2 was detected only in Santana do Arari (1.06%). Sequencing of the 5´LTR region of HTLV-1 and the phylogenetic analysis identified the virus as a member of the Cosmopolitan Group, subgroup Transcontinental. Santana do Arari is an Afro-Brazilian community and the current results represent the first report of HTLV-1 infection in a mocambo located in the Brazilian Amazon region.

Early infection and asymptomatic spread of hepatitis A virus in a public child care center in Rio de Janeiro, Brazil: should attending children under two years of age be vaccinated?

A cross-sectional study was conducted in order to identify hepatitis A virus (HAV) serological markers in 418 individuals (mean age, 16.4 years; range, 1 month-80 years) at a public child care center in Rio de Janeiro, Brazil, as well as to analyze risk factors and determine circulating genotypes. Serum samples were tested using an enzyme immunoassay. Reverse transcription polymerase chain reaction (RT-PCR) was used to detect and characterize HAV RNA, and sequencing was performed. Anti-HAV antibodies and IgM anti-HAV antibodies were detected, respectively, in 89.5% (374/418) and 10.5% (44/418) of the individuals tested. Acute HAV infection in children was independently correlated with crawling (p < 0.05). In 56.8% (25/44) of the IgM anti-HAV-positive individuals and in 33.3% (5/15) of the IgM anti-HAV-negative individuals presenting clinical symptoms, HAV RNA was detected. Phylogenetic analysis revealed co-circulation of subgenotypes IA and IB in 93.3% (28/30) of the amplified samples. In present study, we verify that 79% (30/38) of children IgM anti-HAV-positive were asymptomatic. In child care centers, this asymptomatic spread is a more serious problem, promoting the infection of young children, who rarely show signs of infection. Therefore, vaccinating children below the age of two might prevent the asymptomatic spread of hepatitis A.

Characterization of the rice PHO1 gene family reveals a key role for OsPHO1;2 in phosphate homeostasis and the evolution of a distinct clade in dicotyledons.

Phosphate homeostasis was studied in a monocotyledonous model plant through the characterization of the PHO1 gene family in rice (Oryza sativa). Bioinformatics and phylogenetic analysis showed that the rice genome has three PHO1 homologs, which cluster with the Arabidopsis (Arabidopsis thaliana) AtPHO1 and AtPHO1;H1, the only two genes known to be involved in root-to-shoot transfer of phosphate. In contrast to the Arabidopsis PHO1 gene family, all three rice PHO1 genes have a cis-natural antisense transcript located at the 5 ' end of the genes. Strand-specific quantitative reverse transcription-PCR analyses revealed distinct patterns of expression for sense and antisense transcripts for all three genes, both at the level of tissue expression and in response to nutrient stress. The most abundantly expressed gene was OsPHO1;2 in the roots, for both sense and antisense transcripts. However, while the OsPHO1;2 sense transcript was relatively stable under various nutrient deficiencies, the antisense transcript was highly induced by inorganic phosphate (Pi) deficiency. Characterization of Ospho1;1 and Ospho1;2 insertion mutants revealed that only Ospho1;2 mutants had defects in Pi homeostasis, namely strong reduction in Pi transfer from root to shoot, which was accompanied by low-shoot and high-root Pi. Our data identify OsPHO1;2 as playing a key role in the transfer of Pi from roots to shoots in rice, and indicate that this gene could be regulated by its cis-natural antisense transcripts. Furthermore, phylogenetic analysis of PHO1 homologs in monocotyledons and dicotyledons revealed the emergence of a distinct clade of PHO1 genes in dicotyledons, which include members having roles other than long-distance Pi transport.

Molecular characterization of the NSP4 gene of human group A rotavirus samples from the West Central region of Brazil

Nonstructural protein 4 (NSP4), encoded by group A rotavirus genome segment 10, is a multifunctional protein and the first recognized virus-encoded enterotoxin. The NSP4 gene has been sequenced, and five distinct genetic groups have been described: genotypes A-E. NSP4 genotypes A, B, and C have been detected in humans. In this study, the NSP4-encoding gene of human rotavirus strains of different G and P genotypes collected from children between 1987 and 2003 in three cities of West Central region of Brazil was characterized. NSP4 gene of 153 rotavirus-positive fecal samples was amplified by reverse transcriptase-polymerase chain reaction and then sequenced. For phylogenetic analysis, NSP4 nucleotide sequences of these samples were compared to nucleotide sequences of reference strains available in GenBank. Two distinct NSP4 genotypes could be identified: 141 (92.2%) sequences clustered with NSP4 genotype B, and 12 sequences (7.8%) clustered with NSP4 genotype A. These results reinforce that further investigations are needed to assess the validity of NSP4 as a suitable target for epidemiologic surveillance of rotavirus infections and vaccine development.