João M. P. Alves

The genome of Cryptosporidium hominis

Cryptosporidium species cause acute gastroenteritis and diarrhoea worldwide. They are members of the Apicomplexa—protozoan pathogens that invade host cells by using a specialized apical complex and are usually transmitted by an invertebrate vector or intermediate host. In contrast to other Apicomplexans, Cryptosporidium is transmitted by ingestion of oocysts and completes its life cycle in a single host. No therapy is available, and control focuses on eliminating oocysts in water supplies. Two species, C. hominis and C. parvum, which differ in host range, genotype and pathogenicity, are most relevant to humans. C. hominis is restricted to humans, whereas C. parvum also infects other mammals. Here we describe the eight-chromosome ∼9.2-million-base genome of C. hominis. The complement of C. hominis protein-coding genes shows a striking concordance with the requirements imposed by the environmental niches the parasite inhabits. Energy metabolism is largely from glycolysis. Both aerobic and anaerobic metabolisms are available, the former requiring an alternative electron transport system in a simplified mitochondrion. Biosynthesis capabilities are limited, explaining an extensive array of transporters. Evidence of an apicoplast is absent, but genes associated with apical complex organelles are present. C. hominis and C. parvum exhibit very similar gene complements, and phenotypic differences between these parasites must be due to subtle sequence divergence.

Genome of the Opportunistic Pathogen Streptococcus sanguinis

The genome of Streptococcus sanguinis is a circular DNA molecule consisting of 2,388,435 bp and is 177 to 590 kb larger than the other 21 streptococcal genomes that have been sequenced. The G+C content of the S. sanguinis genome is 43.4%, which is considerably higher than the G+C contents of other streptococci. The genome encodes 2,274 predicted proteins, 61 tRNAs, and four rRNA operons. A 70-kb region encoding pathways for vitamin B(12) biosynthesis and degradation of ethanolamine and propanediol was apparently acquired by horizontal gene transfer. The gene complement suggests new hypotheses for the pathogenesis and virulence of S. sanguinis and differs from the gene complements of other pathogenic and nonpathogenic streptococci. In particular, S. sanguinis possesses a remarkable abundance of putative surface proteins, which may permit it to be a primary colonizer of the oral cavity and agent of streptococcal endocarditis and infection in neutropenic patients.

Cirrhosis, bile acids and gut microbiota: Unraveling a complex relationship

A picture is now starting to emerge regarding the liver-bile acid-microbiome axis. Increasing levels of the primary bile acid cholic acid (CA) causes a dramatic shift toward the Firmicutes, particularly Clostridium cluster XIVa and increasing production of the harmful secondary bile acid deoxycholic acid (DCA). During progression of cirrhosis, the microbiome, both through their metabolism, cell wall components (LPS) and translocation lead to inflammation. Inflammation suppresses synthesis of bile acids in the liver leading to a positive-feedback mechanism. Decrease in bile acids entering the intestines appears to favor overgrowth of pathogenic and pro-inflammatory members of the microbiome including Porphyromonadaceae and Enterobacteriaceae. Decreasing bile acid concentration in the colon in cirrhosis is also associated with decreases in Clostridium cluster XIVa, which includes bile acid 7α-dehydroxylating bacteria which produce DCA. Rifaximin treatment appears to act by suppressing DCA production, reducing endotoxemia and harmful metabolites without significantly altering microbiome structure. Taken together, the bile acid pool size and composition appear to be a major regulator of microbiome structure, which in turn appears to be an important regulator of bile acid pool size and composition. The balance between this equilibrium is critical for human health and disease.

Endosymbiosis in trypanosomatids: the genomic cooperation between bacterium and host in the synthesis of essential amino acids is heavily influenced by multiple horizontal gene transfers

BackgroundTrypanosomatids of the genera Angomonas and Strigomonas live in a mutualistic association characterized by extensive metabolic cooperation with obligate endosymbiotic Betaproteobacteria. However, the role played by the symbiont has been more guessed by indirect means than evidenced. Symbiont-harboring trypanosomatids, in contrast to their counterparts lacking symbionts, exhibit lower nutritional requirements and are autotrophic for essential amino acids. To evidence the symbiont’s contributions to this autotrophy, entire genomes of symbionts and trypanosomatids with and without symbionts were sequenced here.ResultsAnalyses of the essential amino acid pathways revealed that most biosynthetic routes are in the symbiont genome. By contrast, the host trypanosomatid genome contains fewer genes, about half of which originated from different bacterial groups, perhaps only one of which (ornithine cyclodeaminase, EC:4.3.1.12) derived from the symbiont. Nutritional, enzymatic, and genomic data were jointly analyzed to construct an integrated view of essential amino acid metabolism in symbiont-harboring trypanosomatids. This comprehensive analysis showed perfect concordance among all these data, and revealed that the symbiont contains genes for enzymes that complete essential biosynthetic routes for the host amino acid production, thus explaining the low requirement for these elements in symbiont-harboring trypanosomatids. Phylogenetic analyses show that the cooperation between symbionts and their hosts is complemented by multiple horizontal gene transfers, from bacterial lineages to trypanosomatids, that occurred several times in the course of their evolution. Transfers occur preferentially in parts of the pathways that are missing from other eukaryotes.ConclusionWe have herein uncovered the genetic and evolutionary bases of essential amino acid biosynthesis in several trypanosomatids with and without endosymbionts, explaining and complementing decades of experimental results. We uncovered the remarkable plasticity in essential amino acid biosynthesis pathway evolution in these protozoans, demonstrating heavy influence of horizontal gene transfer events, from Bacteria to trypanosomatid nuclei, in the evolution of these pathways.

Clostridium scindens: a human gut microbe with a high potential to convert glucocorticoids into androgens

Clostridium scindens American Type Culture Collection 35704 is capable of converting primary bile acids to toxic secondary bile acids, as well as converting glucocorticoids to androgens by side-chain cleavage. The molecular structure of the side-chain cleavage product of cortisol produced by C. scindens was determined to be 11β-hydroxyandrost-4-ene-3,17-dione (11β-OHA) by high-resolution mass spectrometry, 1H and 13C NMR spectroscopy, and X-ray crystallography. Using RNA-Seq technology, we identified a cortisol-inducible (∼1,000-fold) operon (desABCD) encoding at least one enzyme involved in anaerobic side-chain cleavage. The desC gene was cloned, overexpressed, purified, and found to encode a 20α-hydroxysteroid dehydrogenase (HSDH). This operon also encodes a putative “transketolase” (desAB) hypothesized to have steroid-17,20-desmolase/oxidase activity, and a possible corticosteroid transporter (desD). RNA-Seq data suggests that the two-carbon side chain of glucocorticords may feed into the pentose-phosphate pathway and are used as a carbon source. The 20α-HSDH is hypothesized to function as a metabolic “rheostat” controlling rates of side-chain cleavage. Phylogenetic analysis suggests this operon is rare in nature and the desC gene evolved from a gene encoding threonine dehydrogenase. The physiological effect of 11β-OHAD on the host or other gut microbes is currently unknown.

Identification and Phylogenetic Analysis of Heme Synthesis Genes in Trypanosomatids and Their Bacterial Endosymbionts

It has been known for decades that some insect-infecting trypanosomatids can survive in culture without heme supplementation while others cannot, and that this capability is associated with the presence of a betaproteobacterial endosymbiont in the flagellates cytoplasm. However, the specific mechanisms involved in this process remained obscure. In this work, we sequence and phylogenetically analyze the heme pathway genes from the symbionts and from their hosts, as well as from a number of heme synthesis-deficient Kinetoplastida. Our results show that the enzymes responsible for synthesis of heme are encoded on the symbiont genomes and produced in close cooperation with the flagellate host. Our evidence suggests that this synergistic relationship is the end result of a history of extensive gene loss and multiple lateral gene transfer events in different branches of the phylogeny of the Trypanosomatidae.

Biosynthesis of Vitamins and Cofactors in Bacterium-Harbouring Trypanosomatids Depends on the Symbiotic Association as Revealed by Genomic Analyses

Some non-pathogenic trypanosomatids maintain a mutualistic relationship with a betaproteobacterium of the Alcaligenaceae family. Intensive nutritional exchanges have been reported between the two partners, indicating that these protozoa are excellent biological models to study metabolic co-evolution. We previously sequenced and herein investigate the entire genomes of five trypanosomatids which harbor a symbiotic bacterium (SHTs for Symbiont-Haboring Trypanosomatids) and the respective bacteria (TPEs for Trypanosomatid Proteobacterial Endosymbiont), as well as two trypanosomatids without symbionts (RTs for Regular Trypanosomatids), for the presence of genes of the classical pathways for vitamin biosynthesis. Our data show that genes for the biosynthetic pathways of thiamine, biotin, and nicotinic acid are absent from all trypanosomatid genomes. This is in agreement with the absolute growth requirement for these vitamins in all protozoa of the family. Also absent from the genomes of RTs are the genes for the synthesis of pantothenic acid, folic acid, riboflavin, and vitamin B6. This is also in agreement with the available data showing that RTs are auxotrophic for these essential vitamins. On the other hand, SHTs are autotrophic for such vitamins. Indeed, all the genes of the corresponding biosynthetic pathways were identified, most of them in the symbiont genomes, while a few genes, mostly of eukaryotic origin, were found in the host genomes. The only exceptions to the latter are: the gene coding for the enzyme ketopantoate reductase (EC:1.1.1.169) which is related instead to the Firmicutes bacteria; and two other genes, one involved in the salvage pathway of pantothenic acid and the other in the synthesis of ubiquinone, that are related to Gammaproteobacteria. Their presence in trypanosomatids may result from lateral gene transfer. Taken together, our results reinforce the idea that the low nutritional requirement of SHTs is associated with the presence of the symbiotic bacterium, which contains most genes for vitamin production.

Identification and Immunological Characterization of Three Potential Vaccinogens against Cryptosporidium Species

ABSTRACT Cryptosporidiosis is a ubiquitous infectious disease, caused by the protozoan parasites Cryptosporidium hominis and Cryptosporidium parvum, leading to acute, persistent, and chronic diarrhea with life-threatening consequences in immunocompromised individuals. In developing countries, cryptosporidiosis in early childhood has been associated with subsequent significant impairment in growth, physical fitness, and intellectual abilities. Currently, vaccines are unavailable and chemotherapeutics are toxic and impractical, and agents for immunoprophylaxis or treatment of cryptosporidiosis are a high priority. Availability of the genome sequences for C. hominis and C. parvum provides new opportunities to procure and examine novel vaccine candidates. Using the novel approach of “reverse vaccinology,” we identified several new potential vaccine candidates. Three of these antigens—Cp15, profilin, and a Cryptosporidium apyrase—were delivered in heterologous prime-boost regimens as fusions with cytolysin A (ClyA) in a Salmonella live vaccine vector and as purified recombinant antigens, and they were found to induce specific and potent humoral and cellular immune responses, suggesting their potential as new vaccinogens against Cryptosporidium infection.

Genome Evolution and Phylogenomic Analysis of Candidatus Kinetoplastibacterium, the Betaproteobacterial Endosymbionts of Strigomonas and Angomonas

It has been long known that insect-infecting trypanosomatid flagellates from the genera Angomonas and Strigomonas harbor bacterial endosymbionts (Candidatus Kinetoplastibacterium or TPE [trypanosomatid proteobacterial endosymbiont]) that supplement the host metabolism. Based on previous analyses of other bacterial endosymbiont genomes from other lineages, a stereotypical path of genome evolution in such bacteria over the duration of their association with the eukaryotic host has been characterized. In this work, we sequence and analyze the genomes of five TPEs, perform their metabolic reconstruction, do an extensive phylogenomic analyses with all available Betaproteobacteria, and compare the TPEs with their nearest betaproteobacterial relatives. We also identify a number of housekeeping and central metabolism genes that seem to have undergone positive selection. Our genome structure analyses show total synteny among the five TPEs despite millions of years of divergence, and that this lineage follows the common path of genome evolution observed in other endosymbionts of diverse ancestries. As previously suggested by cell biology and biochemistry experiments, Ca. Kinetoplastibacterium spp. preferentially maintain those genes necessary for the biosynthesis of compounds needed by their hosts. We have also shown that metabolic and informational genes related to the cooperation with the host are overrepresented amongst genes shown to be under positive selection. Finally, our phylogenomic analysis shows that, while being in the Alcaligenaceae family of Betaproteobacteria, the closest relatives of these endosymbionts are not in the genus Bordetella as previously reported, but more likely in the Taylorella genus.

Repertoire, Genealogy and Genomic Organization of Cruzipain and Homologous Genes in Trypanosoma cruzi, T. cruzi-Like and Other Trypanosome Species

Trypanosoma cruzi, the agent of Chagas disease, is a complex of genetically diverse isolates highly phylogenetically related to T. cruzi-like species, Trypanosoma cruzi marinkellei and Trypanosoma dionisii, all sharing morphology of blood and culture forms and development within cells. However, they differ in hosts, vectors and pathogenicity: T. cruzi is a human pathogen infective to virtually all mammals whilst the other two species are non-pathogenic and bat restricted. Previous studies suggest that variations in expression levels and genetic diversity of cruzipain, the major isoform of cathepsin L-like (CATL) enzymes of T. cruzi, correlate with levels of cellular invasion, differentiation, virulence and pathogenicity of distinct strains. In this study, we compared 80 sequences of genes encoding cruzipain from 25 T. cruzi isolates representative of all discrete typing units (DTUs TcI-TcVI) and the new genotype Tcbat and 10 sequences of homologous genes from other species. The catalytic domain repertoires diverged according to DTUs and trypanosome species. Relatively homogeneous sequences are found within and among isolates of the same DTU except TcV and TcVI, which displayed sequences unique or identical to those of TcII and TcIII, supporting their origin from the hybridization between these two DTUs. In network genealogies, sequences from T. cruzi clustered tightly together and closer to T. c. marinkellei than to T. dionisii and largely differed from homologues of T. rangeli and T. b. brucei. Here, analysis of isolates representative of the overall biological and genetic diversity of T. cruzi and closest T. cruzi-like species evidenced DTU- and species-specific polymorphisms corroborating phylogenetic relationships inferred with other genes. Comparison of both phylogenetically close and distant trypanosomes is valuable to understand host-parasite interactions, virulence and pathogenicity. Our findings corroborate cruzipain as valuable target for drugs, vaccine, diagnostic and genotyping approaches.