Patricio Manque

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.

Proteomic and network analysis characterize stage-specific metabolism in Trypanosoma cruzi.

BackgroundTrypanosoma cruzi is a Kinetoplastid parasite of humans and is the cause of Chagas disease, a potentially lethal condition affecting the cardiovascular, gastrointestinal, and nervous systems of the human host. Constraint-based modeling has emerged in the last decade as a useful approach to integrating genomic and other high-throughput data sets with more traditional, experimental data acquired through decades of research and published in the literature.ResultsWe present a validated, constraint-based model of the core metabolism of Trypanosoma cruzi strain CL Brener. The model includes four compartments (extracellular space, cytosol, mitochondrion, glycosome), 51 transport reactions, and 93 metabolic reactions covering carbohydrate, amino acid, and energy metabolism. In addition, we make use of several replicate high-throughput proteomic data sets to specifically examine metabolism of the morphological form of T. cruzi in the insect gut (epimastigote stage).ConclusionThis work demonstrates the utility of constraint-based models for integrating various sources of data (e.g., genomics, primary biochemical literature, proteomics) to generate testable hypotheses. This model represents an approach for the systematic study of T. cruzi metabolism under a wide range of conditions and perturbations, and should eventually aid in the identification of urgently needed novel chemotherapeutic targets.

Protein and mRNA content of TcDHH1‐containing mRNPs in Trypanosoma cruzi

In trypanosomatids, the regulation of gene expression occurs mainly at the post‐transcriptional level. Previous studies have revealed nontranslated mRNA in the Trypanosoma cruzi cytoplasm. Previously, we have identified and cloned the TcDHH1 protein, a DEAD box RNA helicase. It has been reported that Dhh1 is involved in multiple RNA‐related processes in various eukaryotes. It has also been reported to accumulate in stress granules and processing bodies of yeast, animal cells, Trypanosoma brucei and T. cruzi. TcDHH1 is localized to discrete cytoplasmic foci that vary depending on the life cycle status and nutritional conditions. To study the composition of mRNPs containing TcDHH1, we carried out immunoprecipitation assays with anti‐TcDHH1 using epimastigote lysates. The protein content of mRNPs was determined by MS and pre‐immune serum was used as control. We also carried out a ribonomic approach to identify the mRNAs present within the TcDHH1 immunoprecipitated complexes. For this purpose, competitive microarray hybridizations were performed against negative controls, the nonprecipitated fraction. Our results showed that mRNAs associated with TcDHH1 in the epimastigote stage are those mainly expressed in the other forms of the T. cruzi life cycle. These data suggest that mRNPs containing TcDHH1 are involved in mRNA metabolism, regulating the expression of at least epimastigote‐specific genes.

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.

Proteomic analysis reveals the dynamic association of proteins with translated mRNAs in Trypanosoma cruzi.

Gene regulation is mainly post-transcriptional in trypanosomatids. The stability of mRNA and access to polysomes are thought to be tightly regulated, allowing Trypanosoma cruzi to adapt to the different environmental conditions during its life cycle. Post-transcriptional regulation requires the association between mRNAs and certain proteins to form mRNP complexes. We investigated the dynamic association between proteins and mRNAs, using poly(T) beads to isolate and characterize proteins and protein complexes bound to poly-A+ mRNAs. The protein content of these fractions was analyzed by mass spectrometry (LC-MS/MS). We identified 542 protein component of the mRNP complexes associated with mRNAs. Twenty-four of the proteins obtained were present in all fractions, whereas some other proteins were exclusive to a particular fraction: epimastigote polysomal (0.37%) and post-polysomal (2.95%) fractions; stress polysomal (13.8%) and post-polysomal (40.78%) fractions. Several proteins known to be involved in mRNA metabolism were identified, and this was considered important as it made it possible to confirm the reliability of our mRNP isolation approach. This procedure allowed us to have a first insight into the composition and dynamics of mRNPs in T. cruzi.

Intranasal vaccination in mice with an attenuated Salmonella enterica Serovar 908htr A expressing Cp15 of Cryptosporidium: Impact of malnutrition with preservation of cytokine secretion

Cryptosporidium is a protozoan parasite associated with acute and persistent diarrhea that, even in asymptomatic persons, can impair normal growth and potentially cognitive and physical development in young children. The recent availability of the complete gene sequence for Cryptosporidium hominis antigen Cp15 allows examination of innovative vaccine regimens involving intra-nasal antigen priming with live bacterial vectors applicable to human populations. We used a recently described weaned mouse model of cryptosporidiosis, where nourished and malnourished vaccinated mice receive the Cp15 antigen recombinant with cytolysinA on a Salmonella serovar Typhi CVD 908-htr A vector, followed by parenteral exposure to antigen with adjuvant. After challenge with Cryptosporidium oocysts via gavage, parameters of infection and disease (stool shedding of parasites, growth rates) were quantified, and serum/lymphoid tissue harvested to elucidate the Cp15-specific adaptive immune response. In vaccinated nourished mice, the regimen was highly immunogenic, with strong antigen-specific IL-6 and IFN-γ secretion and robust Cp15-specific immunoglobulin titers. In vaccinated malnourished mice, secretion of cytokines, particularly IFN-γ, and antigen-specific humoral immunity were generally undiminished despite protein deprivation and stunted growth. In contrast, after natural (oral) challenge with an identical inoculum of Cryptosporidium oocysts, cytokine and humoral responses to Cp15 were less than one-fourth those in vaccinated mice. Nevertheless, vaccination resulted in only transient reduction in stool shedding of parasites and was not otherwise protective against disease. Overall, immunogenicity for a C. hominis antigen was documented in mice, even in the setting of prolonged malnutrition, using an innovative vaccine regimen involving intra-nasal antigen priming with a live enteric bacterial vector, that has potential applicability to vulnerable human populations irrespective of nutritional status.

A Genome-Scale Metabolic Model of Cryptosporidium hominis

The apicomplexan Cryptosporidium is a protozoan parasite of humans and other mammals. Cryptosporidium species cause acute gastroenteritis and diarrheal disease in healthy humans and animals, and cause life‐threatening infection in immunocompromised individuals such as people with AIDS. The parasite has a one‐host life cycle and commonly invades intestinal epithelial cells. The current genome annotation of C. hominis, the most serious human pathogen, predicts 3884 genes of which ca. 1581 have predicted functional annotations. Using a combination of bioinformatics analysis, biochemical evidence, and high‐throughput data, we have constructed a genome‐scale metabolic model of C. hominis. The model is comprised of 213 gene‐associated enzymes involved in 540 reactions among the major metabolic pathways and provides a link between the genotype and the phenotype of the organism, making it possible to study and predict behavior based upon genome content. This model was also used to analyze the two life stages of the parasite by integrating the stage‐specific proteomic data for oocyst and sporozoite stages. Overall, this model provides a computational framework to systematically study and analyze various functional behaviors of C. hominis with respect to its life cycle and pathogenicity.

Over-expression and localization of a host protein on the membrane of Cryptosporidium parvum infected epithelial cells.

The genus Cryptosporidium includes several species of intestinal protozoan parasites which multiply in intestinal epithelial cells. The impact of this infection on the transcriptome of cultured host cells was investigated using DNA microarray hybridizations. The expression of 14 genes found to be consistently up- or down-regulated in infected cell monolayers was validated with RT PCR. Using immunofluorescence we examined the expression of Protease Activated Receptor-2, which is encoded by one of the up-regulated genes. In infected cells this receptor localized to the host cell membrane which covers the intracellular trophozoites and meronts. This observation indicates that the composition of the host cell membrane is affected by the developing trophozoite, a phenomenon which has not been described previously.

Cryptosporidium hominis infection of the human respiratory tract.

Cryptosporidium oocysts, observed in a natural sputum sample of a patient with HIV, were further studied by using DNA markers to determine the species of the parasite. C. hominis was identified as the species infecting the patient’s respiratory tract, a finding that strengthens evidence regarding this pathogen’s role in human disease.