Diversity of Culturable Bacteria Isolated From the Feces of Wild Anopheles darlingi (Diptera: Culicidae) Mosquitoes From the Brazilian Amazon.

Microorganisms living in the midgut of Anopheles mosquitoes have been studied to fight vector-borne diseases, such as malaria. Studies on the microbiota of the Neotropical Anopheles darlingi, the most important Brazilian vector for malaria, have been reported for the same purpose. Our aims were to isolate and identify culturable bacteria from An. darlingi mosquito guts through their feces and to estimate the species richness and the frequency distribution of the sampled bacteria. Sixty wild females of An. darlingi mosquitoes were captured at two rural locations, near Porto Velho, Rondônia, Brazil. Bacteria were isolated from mosquito feces, which were collected using cages which permit the collection of feces on LB nutrient agar plates. Sixty bacterial colonies were isolated and stored in glycerol at -80°C. Bacteria were identified by sequencing their 16S rRNA gene obtained using PCR and Sanger sequencing. To aid in species identification, MALDI-TOF, VITEK2, and BBL Crystal were used as complementary protocols. The sequences obtained from the 60 bacterial isolates were compared to sequences deposited in GenBank (NCBI) using BLAST. Homology greater than 97% between the query and the subject was used as the criteria for assigning the identity of each isolate. Fourteen species from eight different genera were identified among the 60 isolates. The most frequent species were Serratia liquefaciens (20%) and Serratia marcescens (15%). Due to their established apathogenicity and according to previous studies, we suggest Serratia and Pantoea species as suitable for paratransgenesis development to fight malaria in Brazilian Amazon.

Determination of subtypes of Blastocystis sp. in Chilean patients with and without inflammatory bowel syndrome, A preliminary report.

BACKGROUND & METHODS: Blastocystis sp. is one of the most prevalent unicellular eukaryote of the human large intestine in Chile and worldwide. It is classified in subtypes (STs), where using the polymorphic sequences of its 18S rRNA genes currently recognizes 22. STs 1-9 and ST12 have been reported in humans. It has been hypothesized that different STs of Blastocystis sp. differentially affect the clinical severity of the digestive disease in Irritable Bowel Syndrome (IBS) patients, but more studies ar4e needed to establish this statement. To contribute in the elucidation of the potential relationship between Blastocystis sp. subtypes and IBS severity, 37 IBS patient fecal samples were collected at hospitals in Santiago (Chile) and were screened for the presence of vacuolated forms of Blastocystis sp. by using conventional microscopy. Positive samples were submitted to PCR and sequencing for determining STs. The same procedure was performed in fecal samples from five non-IBS Blastocystis sp. carriers for preliminary comparative purpose. RESULTS AND DISCUSSION: Four out of the 37 samples from the IBS patients were found positive for Blastocystis sp. (10.81%) by using microscopy. The presence of this microorganism in these four samples were confirmed by PCR and sequencing. Subtypes and their respective closest match alleles were searched and the ST1, ST2 and ST4 subtypes were found in these patients. ST4 subtype is scarcely detected in South America countries, being reported previously only in Colombia and Brazil. In this ST4 subtype we determined the allele 42 which is the most frequent allele observed in human Blastocystis isolates. In the non-IBS individuals' carriers, three subtypes were found: ST1, ST2 and ST3, even belonging to the same family group. Closest match alleles: 2, 12 and 34 here detected were also commonly reported globally. Instead of the small number of IBS patients studied here, the frequency of blastocystosis detected (10.81%) was lower than the prevalence of Blastocystis sp. infections described for the Chilean general population (30.4%). In Chile, clear correlation of Blastocystis sp. subtypes and IBS severity is still lacking with this study but it may lead and contribute to a better understanding of its pathogenicity and worldwide epidemiology.

Molecular cloning and structural modelling of gamma-phospholipase A2 inhibitors from Bothrops atrox and Micrurus lemniscatus snakes.

Phospholipases A2 inhibitors (PLIs) produced by venomous and non-venomous snakes play essential role in this resistance. These endogenous inhibitors may be classified by their fold in PLIα, PLIß and PLIγ. Phospholipases A2 (PLA2s) develop myonecrosis in snake envenomation, a consequence that is not efficiently neutralized by antivenom treatment. This work aimed to identify and characterize two PLIs from Amazonian snake species, Bothrops atrox and Micrurus lemniscatus. Liver tissues RNA of specimens from each species were isolated and amplified by RT-PCR using PCR primers based on known PLIγ gene sequences, followed by cloning and sequencing of amplified fragments. Sequence similarity studies showed elevated identity with inhibitor PLIγ gene sequences from other snake species. Molecular models of translated inhibitors' gene sequences resemble canonical three finger fold from PLIγ and support the hypothesis that the decapeptide (residues 107-116) may be responsible for PLA2 inhibition. Structural studies and action mechanism of these PLIs may provide necessary information to evaluate their potential as antivenom or as complement of the current ophidian accident treatment.

Natural Plasmodium infection in monkeys in the state of Rondônia (Brazilian Western Amazon).

BACKGROUND: Simian malaria is still an open question concerning the species of Plasmodium parasites and species of New World monkeys susceptible to the parasites. In addition, the lingering question as to whether these animals are reservoirs for human malaria might become important especially in a scenario of eradication of the disease. To aid in the answers to these questions, monkeys were surveyed for malaria parasite natural infection in the Amazonian state of Rondônia, Brazil, a state with intense environmental alterations due to human activities, which facilitated sampling of the animals. METHODS: Parasites were detected and identified in DNA from blood of monkeys, by PCR with primers for the 18S rRNA, CSP and MSP1 genes and sequencing of the amplified fragments. Multiplex PCR primers for the 18S rRNA genes were designed for the parasite species Plasmodium falciparum and Plasmodium vivax, Plasmodium malariae/Plasmodium brasilianum and Plasmodium simium. RESULTS: An overall infection rate of 10.9% was observed or 20 out 184 monkey specimens surveyed, mostly by P. brasilianum. However, four specimens of monkeys were found infected with P. falciparum, two of them doubly infected with P. brasilianum and P. falciparum. In addition, a species of monkey of the family Aotidae, Aotus nigriceps, is firstly reported here naturally infected with P. brasilianum. None of the monkeys surveyed was found infected with P. simium/P. vivax. CONCLUSION: The rate of natural Plasmodium infection in monkeys in the Brazilian state of Rondônia is in line with previous surveys of simian malaria in the Amazon region. The fact that a monkey species was found that had not previously been described to harbour malaria parasites indicates that the list of monkey species susceptible to Plasmodium infection is yet to be completed. Furthermore, finding monkeys in the region infected with P. falciparum clearly indicates parasite transfer from humans to the animals. Whether this parasite can be transferred back to humans and how persistent the parasite is in monkeys in the wild so to be efficient reservoirs of the disease, is yet to be evaluated. Finding different species of monkeys infected with this parasite species suggests indeed that these animals can act as reservoirs of human malaria.

Comparative genomics of cryptosporidium.

Until recently, the apicomplexan parasites, Cryptosporidium hominis and C. parvum, were considered the same species. However, the two parasites, now considered distinct species, exhibit significant differences in host range, infectivity, and pathogenicity, and their sequenced genomes exhibit only 95-97% identity. The availability of the complete genome sequences of these organisms provides the potential to identify the genetic variations that are responsible for the phenotypic differences between the two parasites. We compared the genome organization and structure, gene composition, the metabolic and other pathways, and the local sequence identity between the genes of these two Cryptosporidium species. Our observations show that the phenotypic differences between C. hominis and C. parvum are not due to gross genome rearrangements, structural alterations, gene deletions or insertions, metabolic capabilities, or other obvious genomic alterations. Rather, the results indicate that these genomes exhibit a remarkable structural and compositional conservation and suggest that the phenotypic differences observed are due to subtle variations in the sequences of proteins that act at the interface between the parasite and its host.

A comparison of two distinct murine macrophage gene expression profiles in response to Leishmania amazonensis infection.

BACKGROUND: The experimental murine model of leishmaniasis has been widely used to characterize the immune response against Leishmania. CBA mice develop severe lesions, while C57BL/6 present small chronic lesions under L. amazonensis infection. Employing a transcriptomic approach combined with biological network analysis, the gene expression profiles of C57BL/6 and CBA macrophages, before and after L. amazonensis infection in vitro, were compared. These strains were selected due to their different degrees of susceptibility to this parasite. RESULTS: The genes expressed by C57BL/6 and CBA macrophages, before and after infection, differ greatly, both with respect to absolute number as well as cell function. Uninfected C57BL/6 macrophages express genes involved in the deactivation pathway of macrophages at lower levels, while genes related to the activation of the host immune inflammatory response, including apoptosis and phagocytosis, have elevated expression levels. Several genes that participate in the apoptosis process were also observed to be up-regulated in C57BL/6 macrophages infected with L. amazonensis, which is very likely related to the capacity of these cells to control parasite infection. By contrast, genes involved in lipid metabolism were found to be up-regulated in CBA macrophages in response to infection, which supports the notion that L. amazonensis probably modulates parasitophorous vacuoles in order to survive and multiply in host cells. CONCLUSION: The transcriptomic profiles of C57BL/6 macrophages, before and after infection, were shown to be involved in the macrophage pathway of activation, which may aid in the control of L. amazonensis infection, in contrast to the profiles of CBA cells.

A high-throughput cloning system for reverse genetics in Trypanosoma cruzi.

BACKGROUND: The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches. RESULTS: We constructed a platform with a flexible structure allowing the exchange of various elements, such as promoters, fusion tags, intergenic regions or resistance markers. This platform is based on Gateway® technology, to ensure a fast and efficient cloning system. We obtained plasmid vectors carrying genes for fluorescent proteins (green, cyan or yellow), and sequences for the c-myc epitope, and tandem affinity purification or polyhistidine tags. The vectors were verified by successful subcellular localization of two previously characterized proteins (TcRab7 and PAR 2) and a putative centrin. For the tandem affinity purification tag, the purification of two protein complexes (ribosome and proteasome) was performed. CONCLUSIONS: We constructed plasmids with an efficient cloning system and suitable for use across various applications, such as protein localization and co-localization, protein partner identification and protein expression. This platform also allows vector customization, as the vectors were constructed to enable easy exchange of its elements. The development of this high-throughput platform is a step closer towards large-scale trypanosome applications and initiatives.

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.

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 approximately 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.

Role of mitochondrial carrier protein Mrs3/4 in iron acquisition and oxidative stress resistance of Cryptococcus neoformans.

Cryptococcus neoformans is a pathogenic basidiomycete that causes meningitis in immunocompromised patients. In this paper, we demonstrate that a previously described oxidant-sensitive mutant, oxy1, has constitutive ferric reductase and iron uptake, similar to a ferric reductase regulatory mutant, frr1. Through meiotic genetic analysis, we show that the two mutations are allelic. By complementation of frr1 with a genomic library, we isolated a gene, MRS3/4. The encoded protein is a putative solute transporter of the inner mitochondrial membrane. Disruption of this gene led to high ferric reductase, iron uptake and iron content, as well as increased sensitivity to hydrogen peroxide and slow growth in low iron medium. The disrupted gene is allelic with oxy1 and frr1. We sequenced the oxy1 and frr1 alleles of MRS3/4 and found that the frr1 mutation results in a premature stop codon, while the oxy1 mutation results in the substitution of a highly conserved glutamate residue with lysine. The Mrs3/4 protein appears to be involved in mitochondrial iron transport in eukaryotes. Resistance to strong oxidants requires stringent control of iron metabolism.

A study on the pathogenesis of human cerebral malaria and cerebral babesiosis

Cerebral complications are important, but poorly understood pathological features of infections caused by some species of Plasmodium and Babesia. Patients dying from P. falciparum were classified as cerebral or non-cerebral cases according to the cerebral malaria coma scale. Light microscopy revealed that cerebral microvessels of cerebral malaria patients were field with a mixture of parazited and unparazited erythrocytes, with 94% of the vessels showing parasitized red blood cell (PRBC) sequestration. Some degree of PRBC sequestration was also found in non-cerebral malaria patients, but the percentage of microvessls with sequestered PRBC was only 13% Electron microscopy demonstrated knobs on the membrane of PRBC that formed focal junctions with the capillary endothelium. A number of host cell molecules such as CD36, thrombospondim (TSP) and intracellular adhesion molecule I (ICAM-1) may function as endothelial cell surfacereports for P. falciparum-infected erythrocytes. Affinity labeling of CD36 and TSP to the PRBC surface showed these molecules specifically bind to the knobs. Babesia bovis infected erythrocytes procedure projections of the erythrocyte membrane that are similar to knobs. When brain tissue from B. bovis-infected cattle was examined, cerebral capillaries were packed with PRBC. Infected erythrocytes formed focal attachments with cerebral endothelial cells at the site of these knob-like projections. These findings indicate that cerebral pathology caused by B. bovis is similar to human cerebral malaria. A search for cytoadherence proteins in the endothelial cells may lead to a better understanding of the pathogenisis of cerebral babesiosis