Programmed cell death by apoptosis of unnecessary or potentially harmful cells is clearly beneficial to multicellular organisms. Proper functioning of such a program demands that the removal of dying cells proceed without an inflammatory reaction. Phosphatidylserine (PS) is one of the ligands displayed by apoptotic cells that participates in their noninflammatory removal when recognized by neighboring phagocytes. PS ligation induces the release of transforming growth factor-beta (TGF-beta), an antiinflammatory cytokine that mediates the suppression of macrophage-mediated inflammation. In Hydra vulgaris, an organism that stands at the base of metazoan evolution, the selective advantage provided by apoptosis lies in the fact that Hydra can survive recycling apoptotic cells by phagocytosis. In unicellular organisms, it has been proposed that altruistic death benefits clonal populations of yeasts and trypanosomatids. Now we show that advantageous features of the apoptotic process can operate without death as the necessary outcome. Leishmania spp are able to evade the killing activity of phagocytes and establish themselves as obligate intracellular parasites. Amastigotes, responsible for disease propagation, similar to apoptotic cells, inhibit macrophage activity by exposing PS. Exposed PS participates in amastigote internalization. Recognition of this moiety by macrophages induces TGF-beta secretion and IL-10 synthesis, inhibits NO production, and increases susceptibility to intracellular leishmanial growth.
Mimicking mammalian apoptotic cells by exposing phosphatidylserine (PS) is a strategy used by virus and parasitic protozoa to escape host protective inflammatory responses. With Leishmania amazonensis (La), apoptotic mimicry is a prerogative of the intramacrophagic amastigote form of the parasite and is modulated by the host. Now we show that differently from what happens with amastigotes, promastigotes exposing PS are non-viable, non-infective cells, undergoing apoptotic death. As part of the normal metacyclogenic process occurring in axenic cultures and in the gut of sand fly vectors, a sub-population of metacyclic promastigotes exposes PS. Apoptotic death of the purified PS-positive (PSPOS) sub-population was confirmed by TUNEL staining and DNA laddering. Transmission electron microscopy revealed morphological alterations in PSPOS metacyclics such as DNA condensation, cytoplasm degradation and mitochondrion and kinetoplast destruction, both in in vitro cultures and in sand fly guts. TUNELPOS promastigotes were detected only in the anterior midgut to foregut boundary of infected sand flies. Interestingly, caspase inhibitors modulated parasite death and PS exposure, when added to parasite cultures in a specific time window. Efficient in vitro macrophage infections and in vivo lesions only occur when PSPOS and PS-negative (PSNEG) parasites were simultaneously added to the cell culture or inoculated in the mammalian host. The viable PSNEG promastigote was the infective form, as shown by following the fate of fluorescently labeled parasites, while the PSPOS apoptotic sub-population inhibited host macrophage inflammatory response. PS exposure and macrophage inhibition by a subpopulation of promastigotes is a different mechanism than the one previously described with amastigotes, where the entire population exposes PS. Both mechanisms co-exist and play a role in the transmission and development of the disease in case of infection by La. Since both processes confer selective advantages to the infective microorganism they justify the occurrence of apoptotic features in a unicellular pathogen.
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Background Rickettsia rickettsii is a tick-borne obligate intracellular bacterium that causes Rocky Mountain spotted fever, a life-threatening illness. To obtain an insight into the vector–pathogen interactions, we assessed the effects of infection with R. rickettsii on the proteome cells of the tick embryonic cell line BME26. Methods The proteome of BME26 cells was determined by label-free high-performance liquid chromatography coupled with tandem mass spectrometry analysis. Also evaluated were the effects of infection on the activity of caspase-3, assessed by the hydrolysis of a synthetic fluorogenic substrate in enzymatic assays, and on the exposition of phosphatidyserine, evaluated by live-cell fluorescence microscopy after labeling with annexin-V. Finally, the effects of activation or inhibition of caspase-3 activity on the growth of R. rickettsii in BME26 cells was determined. Results Tick proteins of different functional classes were modulated in a time-dependent manner by R. rickettsii infection. Regarding proteins involved in apoptosis, certain negative regulators were downregulated at the initial phase of the infection (6 h) but upregulated in the middle of the exponential phase of the bacterial growth (48 h). Microorganisms are known to be able to inhibit apoptosis of the host cell to ensure their survival and proliferation. We therefore evaluated the effects of infection on classic features of apoptotic cells and observed DNA fragmentation exclusively in noninfected cells. Moreover, both caspase-3 activity and phosphatidylserine exposition were lower in infected than in noninfected cells. Importantly, while the activation of caspase-3 exerted a detrimental effect on rickettsial proliferation, its inhibition increased bacterial growth. Conclusions Taken together, these results show that R. rickettsii modulates the proteome and exerts an inhibitory effect on apoptosis in tick cellsthat seems to be important to ensure cell colonization.
Também conhecida como leishmaniose mucocutânea, úlcera de Bauru, ferida brava, a LTA é dividida em leishmaniose cutânea (LC), com comprometimento exclusivamente da pele, e leishmaniose mucocutânea (LMC), acometendo as mucosas.A Organização Mundial da Saúde (OMS) estima que a leishmaniose tegumentar ocorra em 88 países e, desse total, 90% dos casos ocorrem em seis países (Brasil e Peru, no Novo Mundo, e Irã, Síria, Afeganistão e Arábia Saudita, no Velho Mundo) (Gontijo & Carvalho, 2003).A leishmaniose é uma zoonose disseminada por quase todo o continente americano. A incidência abrange o sul dos Estados Unidos da América, passando pelo Caribe e chegando até a América do Sul.A OMS considera a LTA como uma entre as seis principais doenças infecciosas, pela sua capacidade de produzir deformações e sua alta incidência.De 1980 a 2001, no Brasil, a LTA foi a sexta doença infecciosa com maior incidência confirmada, ficando atrás apenas das seguintes doenças: malária, dengue, tuberculose, sarampo e hanseníase (Fundação Nacional de Saúde -FUNASA, 1999).A LTA só afeta o homem por acidente quando ele entra nos ecótopos naturais dos reservatórios silvestres, apesar de hoje ela ter sofrido um processo de endemização em áreas de colonização antiga, como resultado da adaptação do parasito a cães, eqüinos e roedores, que hoje são importantes reservatórios das Leishmanias.O período de incubação da doença no homem pode variar entre duas semanas a dois anos. 2Atualmente, no Brasil, essa doença está em franco crescimento. Entre os fatores que contribuem para esse crescimento estão: casos oriundos dos focos antigos e/ou de áreas próximas a eles; aparecimento de surtos epidêmicos associados à aceleração do processo de expansão agrícola; garimpos; construção de estradas; expansão das periferias das cidades; novos pólos industriais e turísticos; entre outros (FUNASA, 1999).A LTA é encontrada em todas as regiões do Brasil, sendo mais freqüente nas regiões Norte e Nordeste e, em seguida, na região Centro- (Shaw & Lainson, 1975).A Leishmania (Leishmania) amazonensis, principal espécie utilizada em nossos estudos, encontra-se nas florestas primárias e secundárias da 3 Amazônia e também no Nordeste, principalmente na Bahia, no Sudeste (Minas Gerais e São Paulo) e no Centro-Oeste. Seus hospedeiros naturais são marsupiais e roedores, como oProechymis (rato-soiá) e o Oryzomys (FUNASA, 2000). Seus insetos vetores (Lutzomyia flaviscutellata, Lutzomyia reducta eLutzomyia olmeca) têm hábitos noturnos e vôo baixo e são pouco antropofílicos.Na doença humana, ocorre lesão ulcerada e geralmente única caracterizada por úlcera de borda elevada e com fundo granuloso e, em geral, indolor.Em alguns indivíduos, pode ocorrer o quadro clássico da leishmaniose cutânea difusa (LCD), em que se observa inicialmente mácula, pápula no local da inoculação e não se observa ulceração das lesões. Em poucos meses, ocorre disseminação hematogênica do parasita com aparecimento de lesões em outras partes do corpo, em que são abundantemente encontrados macrófagos vacuolizad...
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