Iron Overload Favors the Elimination of Leishmania

Iron plays a central role in host-parasite interactions, since both intervenients need iron for survival and growth, but are sensitive to iron-mediated toxicity. The host’s iron overload is often associated with susceptibility to infection. However, it has been previously reported that iron overload prevented the growth of Leishmania major, an agent of cutaneous leishmaniasis, in BALB/c mice. In order to further clarify the impact of iron modulation on the growth of Leishmania in vivo, we studied the effects of iron supplementation or deprivation on the growth of L. infantum, the causative agent of Mediterranean visceral leishmaniasis, in the mouse model. We found that dietary iron deficiency did not affect the protozoan growth, whereas iron overload decreased its replication in the liver and spleen of a susceptible mouse strain. The fact that the iron-induced inhibitory effect could not be seen in mice deficient in NADPH dependent oxidase or nitric oxide synthase 2 suggests that iron eliminates L. infantum in vivo through the interaction with reactive oxygen and nitrogen species. Iron overload did not significantly alter the mouse adaptive immune response against L. infantum. Furthermore, the inhibitory action of iron towards L. infantum was also observed, in a dose dependent manner, in axenic cultures of promastigotes and amastigotes. Importantly, high iron concentrations were needed to achieve such effects. In conclusion, externally added iron synergizes with the host’s oxidative mechanisms of defense in eliminating L. infantum from mouse tissues. Additionally, the direct toxicity of iron against Leishmania suggests a potential use of this metal as a therapeutic tool or the further exploration of iron anti-parasitic mechanisms for the design of new drugs.

Natural malaria infection reduces starvation resistance of nutritionally stressed mosquitoes.

In disease ecology, there is growing evidence that environmental quality interacts with parasite and host to determine host susceptibility to an infection. Most studies of malaria parasites have focused on the infection costs incurred by the hosts, and few have investigated the costs on mosquito vectors. The interplay between the environment, the vector and the parasite has therefore mostly been ignored and often relied on unnatural or allopatric Plasmodium/vector associations. Here, we investigated the effects of natural avian malaria infection on both fecundity and survival of field-caught female Culex pipiens mosquitoes, individually maintained in laboratory conditions. We manipulated environmental quality by providing mosquitoes with different concentrations of glucose-feeding solution prior to submitting them to a starvation challenge. We used molecular-based methods to assess mosquitoes' infection status. We found that mosquitoes infected with Plasmodium had lower starvation resistance than uninfected ones only under low nutritional conditions. The effect of nutritional stress varied with time, with the difference of starvation resistance between optimally and suboptimally fed mosquitoes increasing from spring to summer, as shown by a significant interaction between diet treatment and months of capture. Infected and uninfected mosquitoes had similar clutch size, indicating no effect of infection on fecundity. Overall, this study suggests that avian malaria vectors may suffer Plasmodium infection costs in their natural habitat, under certain environmental conditions. This may have major implications for disease transmission in the wild.

Aspergillus flavus infections in Galleria mellonella : a pathogen-host model system for the study of emerging diseases

To study emerging diseases, I employed a model pathogen-host system involving infections of insect larvae with the opportunistic fungus Aspergillus flavus, providing insight into three mechanisms ofpathogen evolution namely de novo mutation, genome decay, and virulence factoracquisition In Chapter 2 as a foundational experiment, A. flavus was serially propagated through insects to study the evolution of an opportunistic pathogen during repeated exposure to a single host. While A. flavus displayed de novo phenotypic alterations, namely decreased saprobic capacity, analysis of genotypic variation in Chapter 3 signified a host-imposed bottleneck on the pathogen population, emphasizing the host's role in shaping pathogen population structure. Described in Chapter 4, the serial passage scheme enabled the isolation of an A. flavus cysteine/methionine auxotroph with characteristics reminiscent of an obligate insect pathogen, suggesting that lost biosynthetic capacity may restrict host range based on nutrient availability and provide selection pressure for further evolution. As outlined in Chapter 6, cysteine/methionine auxotrophy had the pleiotrophic effect of increasing virulence factor production, affording the slow-growing auxotroph with a modified pathogenic strategy such that virulence was not reduced. Moreover in Chapter 7, transformation with a virulence factor from a facultative insect pathogen failed to increase virulence, demonstrating the necessity of an appropriate genetic background for virulence factor acquisition to instigate pathogen evolution.

Isolation and partial characterization of host cell wall surface glycoproteins: their involvement in agglutination, attachment and appressorium formation by piptocephalis virginiana

Cell surface proteins obtained by alkaline extraction from isolated cell walls of Mortierella pusilla and M. candelabrum, host and nonhost, respectively, to the mycoparasite, Piptocephalis virginiana, were tested for their ability to agglutinate mycoparasite spores. The host cell wall protein extract had a high agglutinating activity (788 a.u. mg- t ) as compared with the nonhost extract (21 a.li. mg- t ). SDS-polyacrylamide gel electrophoresis of the cell wall proteins revealed four protein bands, a, b, c, and d (Mr 117, 100, 85 and 64 kd, respectively) at the host surface, but not at the nonhost surface, except for the faint band c. Deletion of proteins b or c from the host cell wall protein extract significantly reduced its agglutinating activity. Proteins band c, obtained as purified preparations by a series of procedures, were shown to be two glycoproteins. Carbohydrate analysis by gas chromatography demonstrated that glucose and Nacetylglucosamine were the major carbohydrate components of the glycoproteins. It was further shown that the agglutinating activity of the pure preparation containing both band c was 500-850 times that of the single glycoproteins, suggesting the involvement of both glycoproteins in agglutination. The results suggest that the glycoproteins band c are the two subunits of agglutinin present at the host cell surface. The two glycoproteins band c purified from the host cell wall protein extract were further examined after various treatments for their possible role in agglutination, attachment and appressorium formation by the mycoparasite. Results obtained by agglutination and attachment tests showed: (1) the two glycoprotein-s are not only an agglutinin responsible for the mycoparasite spore agglutination, but may also serve as a receptor for the specific recognition, attachment and appressorium formation by the mycoparasite; (2) treatment of the rnycoparasite spores with various sugars revealed that arabinose, glucose and N-acetylglucosamine inhibited the agglutination and attachment activity of the glycoproteins, however, the relative percentage of appressorium formation was not affected by the above sugars; (3) the two glycoproteins are relatively stable with respect to their agglutinin and receptor functions. The present results suggest that the agglutination and attachment may be mediated directly by certain sugars present at the host and mycoparasite cell surfaces while the appressorlum formation may be the response of complementary combinations of both sugar and protein, the two parts of the glycoproteins at the interacting surfaces of two fungi.

Isolation and partial characterization of a complementary protein from the mycoparasite, Piptocephalis virginiana, which specifically binds to two glycoproteins b and c of the host cell surface

Presence of surface glycoprotein in Piptocephalis virginiana that recognizes the host glycoproteins band c, reported earlier from our laboratory, was detected by immunofluorescence microscopy. Germinated spores of P. virginiana treated with Mortierella pusilla cell wall protein extract, primary antibodies prepared against glycoproteins band c and FITC-goat anti-rabbit IgG conjugate showed fluorescence. This indicated that on the surfaces of the biotrophic mycoparasite P. virginiana , there might be a complementary molecule which recognizes the glycoproteins band c from M. pusilla. Immunobinding analysis identified a glycoprotein of Mr 100 kDa from the mycoparasite which binds with the host glycoproteins band c, separately as well as collectively. Purification of this glycoprotein was achieved by (i) 60% ammonium sulfate precipitation, (ii) followed by heat treatment, and (iii) Sephadex G-IOO gel filtration. The glycoprotein was isolated by preparative polyacrylamide gel electrophoresis by cutting and elution. The purity of the protein ·was ascertained by SDS-PAGE and Western blot analysis. Positive reaction to periodic acid-Schiff reagent revealed the glycoprotein nature of this 100 kDa protein. Mannose was identified as a major sugar component of this glycoprotein by using a BoehringerMannheim Glycan Differentiation Kit. Electrophoretically purified glycoprotein was used to raIse polyclonal antibody in rabbit. The specificity of the antibody was determined by dot-immunobinding test and western-blot analysis. Immunofluorescence mIcroscopy revealed surface localization of the protein on the germ tube of Piptocephalis virginiana. Fluorescence was also observed at the surfaceJ of the germinated spores and hyphae of the host, M. pusilla after treatment with complementary protein from P. virginiana, primary antibody prepared against the complementary protein and FITC-goat anti-rabbit IgG conjugate.

Meloidogne incognita (nematode) parasitism of Lycopersicon esculentum (tomato) plants : Ethylene action in susceptible and resistant host responses

Involvement of ethylene in the etiology of tomato plants (Lycopersicon esculentum) infected with the root-knot nematode (Meloidogyne incognita) was investigated. Endogenous root concentrations of ethylene were not significantly different in uninfected resistant var. Anahu and susceptible var. Vendor plants. Exposure of resistant plants to high doses of infectious nematode larvae did not affect root ethylene concentrations during the subsequent 30 day period. The possibility that ethylene may be involved in the mechanism of resistance is therefore not supported by these experiments. In no experiments did ethylene concentrations in roots of susceptible plants increase significantly subsequent to ~ incognita infestation. This result is not consistent with the hypothesis in the literature which suggests that increased ethylene production accompanies gall formation. Growth of susceptible tomato plants was affected by ~ incognita infestation such that root weights increased (due to galling), stem heights decreased and top weights increased. The possibility that alterations in stem growth resulted from increased production of 'stress' ethylene is discussed. Growth of resistant plants was unaffected by exposure to high doses of ~ incognita and galls were never detected on the roots of these plants. Root ethane concentrations generally varied in parallel with root ethylene concentrations although ethane concentrations were without exception greater. In 4 of 6 experiments conducted ethane/ethylene ratios increased significantly with time. These results are discussed in the light of published data on the relationship between ethane and ethylene synthesis. The term infested is used throughout this thesis in reference to plants whose root systems had been exposed to nematodes and does not distinguish between the susceptible and resistant response.

Interaction entre le virus SRRP et Actinobacillus pleuropneumoniae dans un modèle d'infection en culture cellulaire

Le virus du syndrome reproducteur et respiratoire porcin (VSRRP) est un pathogène d’importance dans l’industrie porcine et est responsable d’importantes pertes économiques. Il n’existe pas d’antiviral efficace contre celui-ci. Il a récemment été mis en évidence que le surnageant de culture d’Actinobacillus pleuropneumoniae, l’agent étiologique de la pleuropneumonie porcine, possédait une activité antivirale in vitro contre le VSRRP dans la lignée cellulaire SJPL. Les objectifs de mon projet sont (i) d’étudier les mécanismes cellulaires menant à l’activité antivirale causée par le surnageant de culture d’A. pleuropneumoniae, et (ii) de caractériser les molécules actives présentes dans le surnageant de culture d’A. pleuropneumoniae. Dans un premier temps, des analyses de protéome ont été effectuées et ont permis d’observer que le surnageant de culture modulait la régulation du cycle cellulaire. Dans le but d’analyser le cycle cellulaire des cellules SJPL, la cytométrie en flux a été utilisée et a permis de démontrer que le surnageant de culture induisait un arrêt du cycle cellulaire en phase G2/M. Deux inhibiteurs de la phase G2/M ont alors été utilisé. Il s'est avéré que ces inhibiteurs avaient la capacité d’inhiber le VSRRP dans les cellules SJPL. Enfin, la spectrométrie de masse a été utilisée dans le but de caractériser les molécules actives présentes dans le surnageant de culture d’A. pleuropneumoniae et d’identifier deux molécules. Ce projet a permis de démontrer pour la première fois qu’A. pleuropneumoniae est capable de perturber le cycle cellulaire et que ce dernier était un élément important dans l’effet antiviral contre le VSRRP.

Host-symbiont interactions of the primary endosymbiont of human head and body lice

The first mycetome was discovered more than 340 yr ago in the human louse. Despite the remarkable biology and medical and social importance of human lice, its primary endosymbiont has eluded identification and characterization. Here, we report the host-symbiont interaction of the mycetomic bacterium of the head louse Pediculus humanus capitis and the body louse P. h. humanus. The endosymbiont represents a new bacterial lineage in the -Proteobacteria. Its closest sequenced relative is Arsenophonus nasoniae, from which it differs by more than 10%. A. nasoniae is a male-killing endosymbiont of jewel wasps. Using microdissection and multiphoton confocal microscopy, we show the remarkable interaction of this bacterium with its host. This endosymbiont is unique because it occupies sequentially four different mycetomes during the development of its host, undergoes three cycles of proliferation, changes in length from 2–4 µm to more than 100 µm, and has two extracellular migrations, during one of which the endosymbionts have to outrun its host’s immune cells. The host and its symbiont have evolved one of the most complex interactions: two provisional or transitory mycetomes, a main mycetome and a paired filial mycetome. Despite the close relatedness of body and head lice, differences are present in the mycetomic provisioning and the immunological response.—Perotti, M. A., Allen, J. M., Reed, D. L., Braig, H. R. Host-symbiont interactions of the primary endosymbiont of human head and body lice.

Linking the coevolutionary and population dynamics of host-parasitoid interactions

The interplay between coevolutionary and population or community dynamics is currently the focus of much empirical and theoretical consideration. Here, we develop a simulation model to study the coevolutionary and population dynamics of a hypothetical host-parasitoid interaction. In the model, host resistance and parasitoid virulence are allowed to coevolve. We investigate how trade-offs associated with these traits modify the system's coevolutionary and population dynamics. The most important influence on these dynamics comes from the incorporation of density-dependent costs of resistance ability. We find three main outcomes. First, if the costs of resistance are high, then one or both of the players go extinct. Second, when the costs of resistance are intermediate to low, cycling population and coevolutionary dynamics are found, with slower evolutionary changes observed when the costs of virulence are also low. Third, when the costs associated with resistance and virulence are both high, the hosts trade-off resistance against fecundity and invest little in resistance. However, the parasitoids continue to invest in virulence, leading to stable host and parasitoid population sizes. These results support the hypothesis that costs associated with resistance and virulence will maintain the heritable variation in these traits found in natural populations and that the nature of these trade-offs will greatly influence the population dynamics of the interacting species.

Interactions of Paracoccidioides brasiliensis with host cells: recent advances

Host-fungal interactions are inherently complex and dynamic. In order to identify new microbial targets and develop more effective anti-fungal therapies, it is important to understand the cellular and molecular mechanisms of disease. Paracoccidioidomycosis provokes a variety of clinical symptoms, and Paracoccidioides brasiliensis can reach many tissues, but primarily attacks the lungs. The ability of the pathogen to interact with the host surface structures is essential to further colonization, invasion, and growth. Epithelial cells may represent the first host barrier or the preferential site of entry of the fungus. For this reason, interactions between P. brasiliensis and Vero/A549 epithelial cells were evaluated, with an emphasis on the adherence, induction of cytoskeletal alterations, and differential signaling activity of the various surface molecules. The adhesion to and invasion of epithelial cells by P. brasiliensis may represent strategies employed to thwart the initial host immune response, and may help in the subsequent dissemination of the pathogen throughout the body.

Surface-expressed enolase contributes to the adhesion of Paracoccidioides brasiliensis to host cells

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Distribuição espacial, formação de grupos, uso do hábitat e ecologia comportamental de jovens de tubarão limão, negaprion brevirostris (poey 1868), no arquipélago de fernando de noronha

Lemon sharks, Negaprion brevirostris, are common in the Fernando de Noronha Archipelago, but detailed information about the species in this site is lacking. The aim of this study was to describe the spatial distribution, grouping behavior, habitat use and behavioral ecology of juvenile lemon sharks in the archipelago, and their interaction with some environmental and ecological factors. During 2006 and 2007, the presence and spatial distribution of juvenile sharks were quantified through scuba diving and snorkeling at several sites of the archipelago. In 2008 the habitat use of juvenile sharks was quantified through visual census while snorkeling along 300 x 8 m strip transects. During these transects the grouping behavior of lemon sharks was quantified by ad libitum. Results indicate that Fernando de Noronha Archipelago is used as a nursery area for lemon sharks, and the parturition occurs from November to April. Juveniles preferred using shallower areas available by the tide variation and formed groups only in the presence of adult conspecifics. This preference for shallower habitats and the group behavior probably are anti-predatory tactics used by juvenile lemon sharks, in response to the low availability of shelter and high predation risk of the studied areas. Quantifications of prey availability and predation risk of juveniles showed that, in general, lemon sharks are trading-off food by security and investing in sites with higher possibility of energetic return. Behavioral observations enabled to record juvenile carangid fishes following juvenile lemon sharks, remora host-parasite and juvenile sharks foraging on schools of herrings and octopuses. We also recorded the behavior of juvenile sharks following conspecifics of similar size, circling with two or three individuals and smaller individuals giving way to larger juveniles. When adults are present, juvenile lemon sharks are more social than solitary, indicating that predation is one of the factors that contribute to social behaviors of the species. Results also suggest that when grouped the juveniles have a hierarchical organization according to body size. Furthermore, observation of large adult females with several fresh mating bites and scars in the same habitats used by juvenile lemon sharks, indicates that Fernando de Noronha Archipelago is used as nursery and mating grounds by this species

Experimental paracoccidioidomycosis in hamster: transmission electron microscopy of inoculation site lesion

Estudou-se sequencialmente, à microscopia eletrônica de transmissão, a interação entre Paracoccidioides brasiliensis (Pb) e células inflamatórias em hamsters inoculados por via intratesticular. Seis horas após inoculações havia predominância de neutrófilos, estando presentes algumas células mononucleares e eosinófilos. Os neutrófilos foram progressivamente substituídos por células mononucleares. Fungos viáveis apresentavam-se fagocitados ou circunscritos por células inflamatórias, geralmente com ampla interface hospedeiro-parasita. Fungos mortos ou degenerados eram acompanhados de interfase estreita. A camada externa da parede do Pb era às vezes quebrada quando em contacto com neutrófilos, em vários pontos, sendo os fragmentos dessa parede descamados e fagocitados. Células fúngicas pequenas com um único núcleo se relacionavam com ampla interfase enquanto as células maiores e multinucleadas apresentavam paredes irregulares, por vezes, contendo lomasoma e/ou estrutura semelhante à mielina. Diferentes padrões de interação do Pb com células do hospedeiro podem ser decorrentes do a fluxo de células inflamatórias funcionalmente diferentes ao local de inoculação ou à idade dos fungos ou ambos os fatores.

Poult intestinal organ culture for propagation of Turkey coronavirus and assessment of host-virus interactions

Infection of young poults with turkey coronavirus (TCoV) produces a syndrome characterized by acute enteritis, diarrhea, anorexia, ruffled feathers, decreased body weight gain and uneven flock growth. The objective of this study was to standardize an intestinal organ culture (IOC) in order to assess host-virus interaction related to apoptosis. For this purpose the Brazilian strain (TCoV/Brazil/2006 with GenBank accession number FJ188401), was used for infection. Infected IOC cells had mitochondrial dysfunction and initial nuclear activation with MTT value of 90.7 (± 2.4) and apoptotic factor 2.21 (± 2.1), considered statistically different from uninfected IOC cells (p > 0.05). The kinetics of TCoV antigens and viral RNA was directly correlated to annexin-V, caspases- 2 and -3, p53, BCl-2 antigens at 24, 72 and 96 h post-infection (p.i.). Morphological and biochemical features of apoptosis, such as in situ nuclear fragmentation (TUNEL and annexin-V) and DNA ladder formation were also detected in infected cells at all assayed p.i. intervals. Moreover, different from other coronaviruses, the expression of both effective caspase-2 and - 3 and p53 antigens were considered lower. However, at all p.i., the BCl-2 antigens were expressed quantitatively and qualitatively as viral antigen measured by immunofluorescence microscopy analysis. Because the diagnosis of TCoV infection is only performed by infecting embryonated poult eggs, the pathological characteri tics related to host-virus interaction remain unclear. This is the first report on apoptosis of TCoV infected IOC, and reveals that it may be useful immunological method to assess virus pathogenesis.

Biologic and genetics aspects of chagas disease at endemic areas

The etiologic agent of Chagas Disease is the Trypanosoma cruzi, transmitted through blood-sucking insect vectors of the Triatominae subfamily, representing one of the most serious public health concerns in Latin America. There are geographic variations in the prevalence of clinical forms and morbidity of Chagas disease, likely due to genetic variation of the T. cruzi and the host genetic and environmental features. Increasing evidence has supported that inflammatory cytokines and chemokines are responsible for the generation of the inflammatory infiltrate and tissue damage. Moreover, genetic polymorphisms, protein expression levels, and genomic imbalances are associated with disease progression. This paper discusses these key aspects. Large surveys were carried out in Brazil and served as baseline for definition of the control measures adopted. However, Chagas disease is still active, and aspects such as host-parasite interactions, genetic mechanisms of cellular interaction, genetic variability, and tropism need further investigations in the attempt to eradicate the disease. Copyright 2012 Marilanda Ferreira Bellini et al.