Glória Regina Franco

Swine and Poultry Pathogens: the Complete Genome Sequences of Two Strains of Mycoplasma hyopneumoniae and a Strain of Mycoplasma synoviae

This work reports the results of analyses of three complete mycoplasma genomes, a pathogenic (7448) and a nonpathogenic (J) strain of the swine pathogen Mycoplasma hyopneumoniae and a strain of the avian pathogen Mycoplasma synoviae; the genome sizes of the three strains were 920,079 bp, 897,405 bp, and 799,476 bp, respectively. These genomes were compared with other sequenced mycoplasma genomes reported in the literature to examine several aspects of mycoplasma evolution. Strain-specific regions, including integrative and conjugal elements, and genome rearrangements and alterations in adhesin sequences were observed in the M. hyopneumoniae strains, and all of these were potentially related to pathogenicity. Genomic comparisons revealed that reduction in genome size implied loss of redundant metabolic pathways, with maintenance of alternative routes in different species. Horizontal gene transfer was consistently observed between M. synoviae and Mycoplasma gallisepticum. Our analyses indicated a likely transfer event of hemagglutinin-coding DNA sequences from M. gallisepticum to M. synoviae.

The complete genome sequence of Chromobacterium violaceum reveals remarkable and exploitable bacterial adaptability

Chromobacterium violaceum is one of millions of species of free-living microorganisms that populate the soil and water in the extant areas of tropical biodiversity around the world. Its complete genome sequence reveals (i) extensive alternative pathways for energy generation, (ii) ≈500 ORFs for transport-related proteins, (iii) complex and extensive systems for stress adaptation and motility, and (iv) widespread utilization of quorum sensing for control of inducible systems, all of which underpin the versatility and adaptability of the organism. The genome also contains extensive but incomplete arrays of ORFs coding for proteins associated with mammalian pathogenicity, possibly involved in the occasional but often fatal cases of human C. violaceum infection. There is, in addition, a series of previously unknown but important enzymes and secondary metabolites including paraquat-inducible proteins, drug and heavy-metal-resistance proteins, multiple chitinases, and proteins for the detoxification of xenobiotics that may have biotechnological applications.

Virus-host coevolution: common patterns of nucleotide motif usage in Flaviviridae and their hosts.

Virus-host biological interaction is a continuous coevolutionary process involving both host immune system and viral escape mechanisms. Flaviviridae family is composed of fast evolving RNA viruses that infects vertebrate (mammals and birds) and/or invertebrate (ticks and mosquitoes) organisms. These host groups are very distinct life forms separated by a long evolutionary time, so lineage-specific anti-viral mechanisms are likely to have evolved. Flaviviridae viruses which infect a single host lineage would be subjected to specific host-induced pressures and, therefore, selected by them. In this work we compare the genomic evolutionary patterns of Flaviviridae viruses and their hosts in an attempt to uncover coevolutionary processes inducing common features in such disparate groups. Especially, we have analyzed dinucleotide and codon usage patterns in the coding regions of vertebrate and invertebrate organisms as well as in Flaviviridae viruses which specifically infect one or both host types. The two host groups possess very distinctive dinucleotide and codon usage patterns. A pronounced CpG under-representation was found in the vertebrate group, possibly induced by the methylation-deamination process, as well as a prominent TpA decrease. The invertebrate group displayed only a TpA frequency reduction bias. Flaviviridae viruses mimicked host nucleotide motif usage in a host-specific manner. Vertebrate-infecting viruses possessed under-representation of CpG and TpA, and insect-only viruses displayed only a TpA under-representation bias. Single-host Flaviviridae members which persistently infect mammals or insect hosts (Hepacivirus and insect-only Flavivirus, respectively) were found to posses a codon usage profile more similar to that of their hosts than to related Flaviviridae. We demonstrated that vertebrates and mosquitoes genomes are under very distinct lineage-specific constraints, and Flaviviridae viruses which specifically infect these lineages appear to be subject to the same evolutionary pressures that shaped their host coding regions, evidencing the lineage-specific coevolutionary processes between the viral and host groups.

Identification of new Schistosoma mansoni genes by the EST strategy using a directional cDNA library.

A directional size-selected cDNA library constructed from Schistosoma mansoni (Sm) adult worm RNA was used for the generation of expressed sequence tags (EST). From one or both ends of 429 distinct cDNA clones 607 EST were obtained. Of these, only 16% were previously known Sm genes. More than 22% of the clones had matches with entries for other organisms in the databases. These new Sm genes constituted a broad range of transcripts distributed among cytoplasmic structural and regulatory proteins, enzymes, membrane, nuclear and secretory proteins, and proteins with other functions. Almost 33% of the clones had no significant database matches and thus potentially represent Sm-specific genes. Among the latter, several clones, as judged by their redundancy in the library, appear to represent abundant transcripts. The data, taken as a whole, more than double the number of Sm genes identified by nucleotide sequencing and indicate the potential value of the adoption of genome sequencing strategies for the rapid increase in knowledge of complex disease-causing organisms.

Evidence for Reductive Genome Evolution and Lateral Acquisition of Virulence Functions in Two Corynebacterium pseudotuberculosis Strains

Background Corynebacterium pseudotuberculosis, a Gram-positive, facultative intracellular pathogen, is the etiologic agent of the disease known as caseous lymphadenitis (CL). CL mainly affects small ruminants, such as goats and sheep; it also causes infections in humans, though rarely. This species is distributed worldwide, but it has the most serious economic impact in Oceania, Africa and South America. Although C. pseudotuberculosis causes major health and productivity problems for livestock, little is known about the molecular basis of its pathogenicity. Methodology and Findings We characterized two C. pseudotuberculosis genomes (Cp1002, isolated from goats; and CpC231, isolated from sheep). Analysis of the predicted genomes showed high similarity in genomic architecture, gene content and genetic order. When C. pseudotuberculosis was compared with other Corynebacterium species, it became evident that this pathogenic species has lost numerous genes, resulting in one of the smallest genomes in the genus. Other differences that could be part of the adaptation to pathogenicity include a lower GC content, of about 52%, and a reduced gene repertoire. The C. pseudotuberculosis genome also includes seven putative pathogenicity islands, which contain several classical virulence factors, including genes for fimbrial subunits, adhesion factors, iron uptake and secreted toxins. Additionally, all of the virulence factors in the islands have characteristics that indicate horizontal transfer. Conclusions These particular genome characteristics of C. pseudotuberculosis, as well as its acquired virulence factors in pathogenicity islands, provide evidence of its lifestyle and of the pathogenicity pathways used by this pathogen in the infection process. All genomes cited in this study are available in the NCBI Genbank database (http://www.ncbi.nlm.nih.gov/genbank/) under accession numbers CP001809 and CP001829.

Phenotypic screen of early-developing larvae of the blood fluke, schistosoma mansoni, using RNA interference.

RNA interference (RNAi) represents the only method currently available for manipulating gene-specific expression in Schistosoma spp., although application of this technology as a functional genomic profiling tool has yet to be explored. In the present study 32 genes, including antioxidants, transcription factors, cell signaling molecules and metabolic enzymes, were selected to determine if gene knockdown by RNAi was associated with morphologically definable phenotypic changes in early intramolluscan larval development. Transcript selection was based on their high expression in in vitro cultured S. mansoni primary sporocysts and/or their potential involvement in developmental processes. Miracidia were allowed to transform to sporocysts in the presence of synthesized double-stranded RNAs (dsRNAs) and cultivated for 7 days, during which time developing larvae were closely observed for phenotypic changes including failure/delay in transformation, loss of motility, altered growth and death. Of the phenotypes evaluated, only one was consistently detected; namely a reduction in sporocyst size based on length measurements. The size-reducing phenotype was observed in 11 of the 33 (33%) dsRNA treatment groups, and of these 11 phenotype-associated genes (superoxide dismutase, Smad1, RHO2, Smad2, Cav2A, ring box, GST26, calcineurin B, Smad4, lactate dehydrogenase and EF1α), only 6 demonstrated a significant and consistent knockdown of specific transcript expression. Unexpectedly one phenotype-linked gene, superoxide dismutase (SOD), was highly induced (∼1600-fold) upon dsRNA exposure. Variation in dsRNA-mediated silencing effects also was evident in the group of sporocysts that lacked any definable phenotype. Out of 22 nonphenotype-expressing dsRNA treatments (myosin, PKCB, HEXBP, calcium channel, Sma2, RHO1, PKC receptor, DHHC, PepcK, calreticulin, calpain, Smeg, 14.3.3, K5, SPO1, SmZF1, fibrillarin, GST28, GPx, TPx1, TPx2 and TPx2/TPx1), 12 were assessed for the transcript levels. Of those, 6 genes exhibited consistent reductions in steady-state transcript levels, while expression level for the rest remained unchanged. Results demonstrate that the efficacy of dsRNA-treatment in producing consistent phenotypic changes and/or altered gene expression levels in S. mansoni sporocysts is highly dependent on the selected gene (or the specific dsRNA sequence used) and the timing of evaluation after treatment. Although RNAi holds great promise as a functional genomics tool for larval schistosomes, our finding of potential off-target or nonspecific effects of some dsRNA treatments and variable efficiencies in specific gene knockdown indicate a critical need for gene-specific testing and optimization as an essential part of experimental design, execution and data interpretation.

The Schistosoma gene discovery program: state of the art

Schistosoma are dioecious digenetic trematodes carrying a large (270 Mb) genome. Gaining knowledge about the genome of these parasites is of importance for the understanding of their biology, mechanisms of drug resistance and antigenic variation that determine escape from the hosts immune system. This review will provide an update on the Schistosoma Gene Discovery Program, which is part of the Schistosoma Genome Project created in 1992. One of the main objectives of this program is the discovery and characterisation of new genes of Schistosoma mansoni and Schistosoma japonicum in an attempt to search for new targets for drugs and vaccine development. The success of the Schistosoma Gene Discovery Program is demonstrated by the number of catalogued genes, that now reaches 15 to 20% of the full gene complement of its genome.

Helminth genome analysis: The current status of the filarial and schistosome genome projects

Genome projects for the parasitic helminths Brugia malayi (a representative filarial nematode) and Schistosoma were initiated in 1995 by the World Health Organization with the ultimate objectives of identifying new vaccine candidates and drug targets and of developing low resolution genome maps. Because no genetic maps are available, and very few genes have been characterized from either parasite group, the first goal of both Initiatives has been to catalogue new genes for future placement on chromosome and physical maps. These genes have been identified by the expressed sequence tag (EST) approach, utilising cDNA libraries constructed from diverse life cycle stages. To date, the Initiatives have deposited over 16,000 Brugia ESTs and nearly 8000 Schistosoma ESTs in Genbanks dbEST database, corresponding to 6000 and over 3600 genes respectively (33% of Brugias estimated gene compliment, 18-24% of that of Schistosoma). Large fragment, genomic libraries have been constructed in BAC and YAC vectors for studies of genomic organization and for physical and chromosome mapping, and public, hypertext genomic databases have been established to facilitate data access. We present a summary of progress within the helminth genome initiatives and give several examples of important gene discoveries and future applications of these data.

Analysis of the gene expression profile of Schistosoma mansoni cercariae using the expressed sequence tag approach

ESTs constitute rapid and informative tools with which to study gene-expression profiles of the diverse stages of the schistosome life cycle. Following a comprehensive EST study of adult worms, analysis has now targeted the cercaria, the parasite larval form responsible for infection of the vertebrate host. Two Schistosoma mansoni cercarial cDNA libraries were examined and partial sequence obtained from 957 randomly selected clones. On the basis of database searches, 551 (57.6%) ESTs generated had no homologs in the public databases whilst 308 (32.2%) were putatively identified, totaling 859 informative ESTs. The remaining 98 (10.2%) were uninformative ESTs (ribosomal RNA and non-coding mitochondrial sequences). By clustering analysis we have identified 453 different genes. The most common sequences in both libraries represented Sm8 calcium binding protein (8% of ESTs), fructose-1,6-bisphosphate aldolase, glyceraldehyde-3-phosphate dehydrogenase, cytochrome oxidase subunit 1, ATP guanidine kinase and triose phosphate isomerase. One hundred and nineteen identified genes were sorted into 11 functional categories, with genes associated with energy metabolism being the most abundant (13%) and diverse. The diversity and abundance of genes associated with the transcription/translation machinery and with regulatory/signaling functions were also marked. A paramyosin transcript was identified, indicating that this gene is not exclusively expressed in adult worms and sporocysts (as had been suggested previously). The possible physiological relevance to cercariae of the presence of transcripts with homology to calcium binding proteins of the EF-hand superfamily, Gq-coupled rhodopsin photoreceptor, rod phosphodiesterase 8 subunit and peripheral-type benzodiazepine receptor is discussed.

Role of the Endogenous Antioxidant System in the Protection of Schistosoma mansoni Primary Sporocysts against Exogenous Oxidative Stress

Antioxidants produced by the parasite Schistosoma mansoni are believed to be involved in the maintenance of cellular redox balance, thus contributing to larval survival in their intermediate snail host, Biomphalaria glabrata. Here, we focused on specific antioxidant enzymes, including glutathione-S-transferases 26 and 28 (GST26 and 28), glutathione peroxidase (GPx), peroxiredoxin 1 and 2 (Prx1 and 2) and Cu/Zn superoxide dismutase (SOD), known to be involved in cellular redox reactions, in an attempt to evaluate their endogenous antioxidant function in the early-developing primary sporocyst stage of S. mansoni. Previously we demonstrated a specific and consistent RNA interference (RNAi)-mediated knockdown of GST26 and 28, Prx1 and 2, and GPx transcripts, and an unexpected elevation of SOD transcripts in sporocysts treated with gene-specific double-stranded (ds)RNA. In the present followup study, in vitro transforming sporocysts were exposed to dsRNAs for GST26 and 28, combined Prx1/2, GPx, SOD or green-fluorescent protein (GFP, control) for 7 days in culture, followed by assessment of the effects of specific dsRNA treatments on protein levels using semi-quantitative Western blot analysis (GST26, Prx1/2 only), and larval susceptibility to exogenous oxidative stress in in vitro killing assays. Significant decreases (80% and 50%) in immunoreactive GST26 and Prx1/2, respectively, were observed in sporocysts treated with specific dsRNA, compared to control larvae treated with GFP dsRNA. Sporocysts cultured with dsRNAs for GST26, GST28, Prx1/2 and GPx, but not SOD dsRNA, were significantly increased in their susceptibility to H2O2 oxidative stress (60–80% mortalities at 48 hr) compared to GFP dsRNA controls (∼18% mortality). H2O2-mediated killing was abrogated by bovine catalase, further supporting a protective role for endogenous sporocyst antioxidants. Finally, in vitro killing of S. mansoni sporocysts by hemocytes of susceptible NMRI B. glabrata snails was increased in larvae treated with Prx1/2, GST26 and GST28 dsRNA, compared to those treated with GFP or SOD dsRNAs. Results of these experiments strongly support the hypothesis that endogenous expression and regulation of larval antioxidant enzymes serve a direct role in protection against external oxidative stress, including immune-mediated cytotoxic reactions. Moreover, these findings illustrate the efficacy of a RNAi-type approach in investigating gene function in larval schistosomes.