Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology.

Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for α(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form

Murine model for Fusarium oxysporum invasive fusariosis reveals organ-specific structures for dissemination and long-term persistence

Peer reviewedPublisher PD

Structural Changes of the Paraflagellar Rod during Flagellar Beating in Trypanosoma cruzi

, the agent of Chagas disease, is a protozoan member of the Kinetoplastidae family characterized for the presence of specific and unique structures that are involved in different cell activities. One of them is the paraflagellar rod (PFR), a complex array of filaments connected to the flagellar axoneme. Although the function played by the PFR is not well established, it has been shown that silencing of the synthesis of its major proteins by either knockout of RNAi impairs and/or modifies the flagellar motility.Here, we present results obtained by atomic force microscopy (AFM) and transmission electron microscopy (TEM) of replicas of quick-frozen, freeze-fractured, deep-etched and rotary-replicated cells to obtain detailed information of the PFR structures in regions of the flagellum in straight and in bent state. The images obtained show that the PFR is not a fixed and static structure. The pattern of organization of the PFR filament network differs between regions of the flagellum in a straight state and those in a bent state. Measurements of the distances between the PFR filaments and the filaments that connect the PFR to the axoneme as well as of the angles between the intercrossed filaments supported this idea.Graphic computation based on the information obtained allowed the proposal of an animated model for the PFR structure during flagellar beating and provided a new way of observing PFR filaments during flagellar beating

Cell cycle perturbations and apoptosis induced by isohomohalichondrin B (IHB), a natural marine compound

Isohomohalichondrin B (IHB), a novel marine compound with anti-tumoral activity, extracted from the Lissodendorix sponge, inhibits GTP binding to tubulin, preventing microtubule assembly. Cell cycle perturbations and apoptosis induced by IHB were investigated on selected human cancer cell lines by using flow cytometric and biochemical techniques. Monoparameter flow cytometric analysis showed that 1 h IHB exposure caused a delayed progression through S-phase, a dramatic block in G2M phase of the cell cycle and the appearance of tetraploid cell population in LoVo, LoVo/DX, MOLT-4 and K562 cells. At 24 h after IHB exposure, the majority of cells blocked in G2M were in prophase as assessed by morphological analysis and by the fact that they expressed high levels of cyclin A/cdc2 and cyclin B1/cdc2. At 48 h, all cells were tetraploid as assessed by biparameter cyclin A/DNA and cyclin B1/DNA content analysis. Apoptotic death was detected in both leukaemic MOLT-4 and K562 cells, which express wild-type and mutated p53 respectively, when the cells were blocked in mitotic prophase. In conclusion, IHB is a novel potent anti-tumour drug that causes delayed S-phase progression, mitotic block, tetraploidy and apoptosis in cancer cell lines. © 1999 Cancer Research Campaig

Estudo da formação de aderências e da cicatrização de anastomoses colônicas em ratos com sepse peritoneal induzida

OBJETIVO: Avaliar os efeitos da sepse abdominal sobre a formação de aderências e a cicatrização de anastomoses colônicas em ratos. MÉTODOS: 40 ratos distribuídos em dois grupos contendo 20 animais, para anastomose do cólon esquerdo na presença (grupo S) ou ausência (grupo N) de indução de sepse por ligadura e punção do ceco (CLP). Cada grupo foi dividido em subgrupos para eutanásia no terceiro (N3 e S3) ou sétimo (N7 e S7) dia de pós-operatório (DPO). Foi avaliada a quantidade de aderências e removido um segmento colônico contendo a anastomose para análise histopatológica, força de ruptura, hidroxiprolina e conteúdo de colágeno tecidual. RESULTADOS: Os animais submetidos à CLP apresentaram maior quantidade de aderências intra-abdominais tanto no 3° DPO (p=0,00) quanto no 7° DPO (p=0,00). Tiveram menores valores de força de ruptura no 3° DPO (p=0,00), porém maiores valores no 7° DPO (p=0,00). Não houve diferença na variação da concentração de hidroxiprolina, conteúdo de colágeno e histopatologia. CONCLUSÕES: A infecção peritoneal desencadeada por CLP aumentou a quantidade de aderências intra-cavitárias. Houve diminuição da resistência de anastomoses cólicas no 3° DPO, com posterior aumento no 7° DPO, sem efeito sobre os outros parâmetros da cicatrização. ________________________________________________________________________________ ABSTRACTPURPOSE: To evaluate the effects of abdominal sepsis on adhesion formation and colon anastomosis healing in rats. METHODS: Forty rats were distributed in two groups containing 20 rats each for left colon anastomosis in the presence (Group S) or absence (Group N) of induced sepsis by cecal ligation and puncture. Each group was divided into subgroups for euthanasia on the third (N3 and S3) or seventh (N7 or S7) post-operative day. The amount of adhesions was evaluated and a segment of the colon was removed for histopathologic analysis, bursting strength assessment, hydroxyproline and the determination of tissue collagen. RESULTS: The subjects which underwent cecal ligation and puncture presented a higher amount of intra-abdominal adherences in both third (p=0,00) and seventh (p=0,00) post-operatory days. Smaller bursting strengths were found in the S3 subgroup, and greater bursting strengths were found in the S7 subgroup. There was no difference in the variations on the concentrations of hydroxyproline, tissue collagen and histopathology. CONCLUSIONS: The peritoneal infection which was developed by cecal ligation and puncture raised the amount of intra-cavitary adhesions. There was a decrease in the amount of colonic anastomosis on the third post-operatory day with a following raise on the seventh without any effects on other healing parameters

Microevolution of Helicobacter pylori during prolonged infection of single hosts and within families

Our understanding of basic evolutionary processes in bacteria is still very limited. For example, multiple recent dating estimates are based on a universal inter-species molecular clock rate, but that rate was calibrated using estimates of geological dates that are no longer accepted. We therefore estimated the short-term rates of mutation and recombination in Helicobacter pylori by sequencing an average of 39,300 bp in 78 gene fragments from 97 isolates. These isolates included 34 pairs of sequential samples, which were sampled at intervals of 0.25 to 10.2 years. They also included single isolates from 29 individuals (average age: 45 years) from 10 families. The accumulation of sequence diversity increased with time of separation in a clock-like manner in the sequential isolates. We used Approximate Bayesian Computation to estimate the rates of mutation, recombination, mean length of recombination tracts, and average diversity in those tracts. The estimates indicate that the short-term mutation rate is 1.4×10−6 (serial isolates) to 4.5×10−6 (family isolates) per nucleotide per year and that three times as many substitutions are introduced by recombination as by mutation. The long-term mutation rate over millennia is 5–17-fold lower, partly due to the removal of non-synonymous mutations due to purifying selection. Comparisons with the recent literature show that short-term mutation rates vary dramatically in different bacterial species and can span a range of several orders of magnitude

The molecular detection of different Leishmania species within sand flies from a cutaneous and visceral leishmaniasis sympatric area in Southeastern Brazil

Over the last 20 years, there has been an increase in the number of leishmaniasis cases in Brazil. Belo Horizonte (BH) is one of the most highly populated Brazilian cities that is affected by visceral leishmaniasis (VL). The health services in BH are coordinated by a central nucleus that is subdivided into nine sanitary districts. Historically, the highest level of human VL cases was found in the northeast sanitary district (NSD). The objective of our study was to detect Leishmania infection in the phlebotomine sand flies collected in the NSD by dissection and molecular approaches. Following the occurrence of human VL cases in 2005, entomological captures were performed from July 2006-June 2007. Out of the 245 sand flies dissected, only three Lutzomyia longipalpis spp contained flagellates. The female sand flies were grouped into 120 pools according to date, collection site and species, with approximately 10 individual sand flies in each pool. Subsquently, the DNA was extracted and Leishmania spp and other parasites were detected and identified by polymerase chain reaction (PCR) and PCR-restriction fragment length polymorfism. Leishmania infantum was present in at least 19% of the Lu. longipalpis collected, in 3.8% of the Nyssomiya whitmani collected, in 33.3% of the Evandromiya termitophila collected and in 14.3% of the Nyssomiya intermedia collected. When the females of the cortelezzii complex were compared with each other, 3.2% of the females were infected with Leishmania braziliensis, whereas 3.2% of the females were infected with trypanosomatids