Sílvia Regina Costa Dias

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.

Cytological and parasitological analysis of bronchoalveolar lavage fluid for the diagnosis of Angiostrongylus vasorum infection in dogs

Bronchoalveolar lavage (BAL) is a procedure that retrieves cells and other elements from the lungs for evaluation, which helps in the diagnosis of many pulmonary diseases. The aims of this work were to perform this procedure in dogs in the acute and chronic phases of an Angiostrongylus vasorum infection for cytological analysis and to evaluate the potential of this technique as a diagnostic method for this lung-heart worm. The BAL procedure was performed through the use of an endotracheal tube on seven A. vasorum infected dogs and on five non-infected dogs lined as a control group. Sixty days post-infection (dpi) active and live larvae were retrieved from the bronchoalveolar fluid (BALF) of all infected dogs. Furthermore, in one animal it was possible to retrieve larvae in its BALF before the pre-patent period. This work reports that the A. vasorum infection resulted in an increase of relative neutrophils and eosinophils counts. In contrast, there was a significant decrease in the alveolar macrophage relative count in infected animals from 60 to 330 dpi. This study shows that the BAL is an accurate technique for the diagnosis of canine angiostrongylosis. Moreover, the technique allows us to retrieve cells and other elements that line the lung surface for cytological evaluation, which provides information about inflammatory diseases, and the diagnosis and prognosis of pulmonary parasites such as A. vasorum.

Male-enriched transcription of genes encoding ASPs and Kunitz-type protease inhibitors in Ancylostoma species.

Various transcripts coding for proteins considered to be central to parasite-host interactions were identified previously as male-enriched in the hookworm Ancylostoma braziliense. Among these genes were an ASP-5-like homologue and a Kunitz-type protease inhibitor. The present study extends this previous work to investigate similar molecules in other hookworms (Ancylostomatidae). Specifically, partial cDNA sequences encoding three different ASP molecules and two different Kunitz-type protease inhibitors were isolated, and the differential transcription between adult male and female worms was compared by conventional and quantitative reverse transcription (RT)-PCR for three species, A. braziliense, Ancylostoma caninum and Ancylostoma ceylanicum. In accordance with previous findings, male-enriched transcription was observed for all molecules explored. Based on this information, it is hypothesized that adult males are responsible for producing proteins essential to the survival of hookworms inside the host and for supporting developmental and reproductive processes in female worms.

Evaluation of biochemical, hematological and parasitological parameters of protein-deficient hamsters infected with Ancylostoma ceylanicum.

Background Hookworms infect millions of people worldwide and can cause severe clinical symptoms in their hosts. Prospective cohort studies in Brazil show high rates of hookworm reinfection in malnourished children compared to well-nourished children, despite previous treatment. Additionally, soil-transmitted helminth (STH) infections can worsen the nutritional status of affected populations. Therefore, this study aims to clarify the effects of host malnutrition during Ancylostoma ceylanicum infection and how this infection affects host physiological parameters using a hamster model. Methodology/Principal Findings Hamsters were divided into four experimental groups: normal diet or low-protein diet (also referred to as “malnourished”) and A. ceylanicum infection or no infection. More severe pathogenesis was observed in the infected malnourished group, as demonstrated by significant decreases in the hemoglobin concentration, erythrocyte number and packed-cell volume compared to the non-infected malnourished group. Greater numbers of adult parasites and eggs were observed in the malnourished group compared to the control group; however, the oviposition rate was lower in the malnourished group. In general, greater values of total lipids were observed in malnourished animals compared to control animals, including lipids excreted in the stool. Conclusions In this work, we have demonstrated that animals fed an isocaloric low-protein diet presented more severe pathogenesis when infected with A. ceylanicum. The increased lipid concentration in the liver and blood is related to the conversion of the excess carbohydrate into fatty acids that increase the concentration of triglycerides in general. Triglycerides were excreted in the feces, indicating that infection associated with malnutrition caused a greater loss of these molecules for this group of animals and confirming the hypothesis that both nutrition and infection are responsible for the malabsorption syndrome. Taken together, the results found in this work confirm the hypothesis that the nutritional condition of the host greatly influences the course of the infection.

Prednisolone and cyclosporine A: Effects on an experimental model of ancylostomiasis

Corticosteroids and cyclosporine A (CsA) are important clinical immunosuppressive drugs used in the maintenance of organ transplants and in suppressing undesired autoimmune or allergic immune responses. To study the effect of CsA and prednisolone on the course of an Ancylostoma ceylanicum infection, hamsters were treated with commercially available prednisolone or CsA. For both drugs, half the recommended dose was sufficient to inhibit the proliferation of more than 70% of hamster lymph node cells. There was no difference in the recovery of adult worms; however, animals treated with prednisolone presented with low egg counts in the feces. Infection with A. ceylanicum resulted in an increase in specific antibodies against adult worm antigens, but hamsters treated with either drug presented with lower IgG titers. We observed that A. ceylanicum infection caused peripheral cellular immune suppression, which is characterized by a reduction in the total white cell count, neutropenia and lymphopenia. We also observed a lymphoplasmacytic pattern and few eosinophils in the mucosal inflammatory infiltrate for all the animals. The animals treated with prednisolone showed changes in the architecture of the intestine, including the loss of the mucosa, intense congestion and inflammation. In spleen, we observed hyperplasia of white pulp in all infected animals; in addition, there was a loss of tissue architecture in the animals treated with prednisolone. In conclusion, this work shows that an A. ceylanicum infection leads to acute peripheral cellular immune suppression in hamsters but not humoral immune suppression and that CsA treatment does not interfere with the process of infection. However, prednisolone treatment causes intestinal injury, what could hamper the parasite attachment to the intestinal wall, and as a result affects copulation and, consequently, decreases the number of eggs eliminated in the feces. Moreover, the possibility that the drug can also be exerting an effect on female fertility should be considered.

Identification of a new Schistosoma mansoni SMYB1 partner: Putative roles in RNA metabolism

SMYB1 is a Schistosoma mansoni protein highly similar to members of the Y-box binding protein family. Similar to other homologues, SMYB1 is able to bind double- and single-stranded DNA, as well as RNA molecules. The characterization of proteins involved in the regulation of gene expression in S. mansoni is of great importance for the understanding of molecular events that control morphological and physiological changes in this parasite. Here we demonstrate that SMYB1 is located in the cytoplasm of cells from different life-cycle stages of S. mansoni, suggesting that this protein is probably acting in mRNA metabolism in the cytoplasm and corroborating previous findings from our group that showed its ability to bind RNA. Protein-protein interactions are important events in all biological processes, since most proteins execute their functions through large supramolecular structures. Yeast two-hybrid screenings using SMYB1 as bait identified a partner in S. mansoni similar to the SmD3 protein of Drosophila melanogaster (SmRNP), which is important in the assembly of small nuclear ribonucleoprotein complexes. Also, pull-down assays were conducted using immobilized GST-SMYB1 proteins and confirmed the SMYB1-SmRNP interaction. The interaction of SMYB1 with a protein involved in mRNA processing suggests that it may act in processes such as turnover, transport and stabilization of RNA molecules.

Obesity induction in hamster that mimics the human clinical condition

Although obesity is well established in hamsters, studies using diets with high levels of simple carbohydrate associated with lipids are necessary to assess the impact of this type of food in the body. In this study a high sugar and butter diet (HSB) and high temperature were employed towards this end. Obesity was successfully induced at a temperature of 30.3°C to 30.9°C after 38 days feeding the animals an HSB diet. It was shown that although diet is important for the induction of obesity, temperature is also essential because at a temperature slightly below the one required, obesity was not induced, even when the animals were fed for a longer period (150 days).The obese clinical condition was accompanied by biochemical and hematological changes, as increased cholesterol and triglyceride levels and increased leukocyte numbers, similar to alterations observed in obese humans. Furthermore, it was demonstrated that increasing the intake of simple carbohydrates associated with lipids provided evidence of inflammation in obese animals.