Injection of Pseudomonas aeruginosa Exo Toxins into Host Cells Can Be Modulated by Host Factors at the Level of Translocon Assembly and/or Activity

Pseudomonas aeruginosa type III secretion apparatus exports and translocates four exotoxins into the cytoplasm of the host cell. The translocation requires two hydrophobic bacterial proteins, PopB and PopD, that are found associated with host cell membranes following infection. In this work we examined the influence of host cell elements on exotoxin translocation efficiency. We developed a quantitative flow cytometry based assay of translocation that used protein fusions between either ExoS or ExoY and the ß-lactamase reporter enzyme. In parallel, association of translocon proteins with host plasma membranes was evaluated by immunodetection of PopB/D following sucrose gradient fractionation of membranes. A pro-myelocytic cell line (HL-60) and a pro-monocytic cell line (U937) were found resistant to toxin injection even though PopB/D associated with host cell plasma membranes. Differentiation of these cells to either macrophage- or neutrophil-like cell lines resulted in injection-sensitive phenotype without significantly changing the level of membrane-inserted translocon proteins. As previous in vitro studies have indicated that the lysis of liposomes by PopB and PopD requires both cholesterol and phosphatidyl-serine, we first examined the role of cholesterol in translocation efficiency. Treatment of sensitive HL-60 cells with methyl-ß-cyclodextrine, a cholesterol-depleting agent, resulted in a diminished injection of ExoS-Bla. Moreover, the PopB translocator was found in the membrane fraction, obtained from sucrose-gradient purifications, containing the lipid-raft marker flotillin. Examination of components of signalling pathways influencing the toxin injection was further assayed through a pharmacological approach. A systematic detection of translocon proteins within host membranes showed that, in addition to membrane composition, some general signalling pathways involved in actin polymerization may be critical for the formation of a functional pore. In conclusion, we provide new insights in regulation of translocation process and suggest possible cross-talks between eukaryotic cell and the pathogen at the level of exotoxin translocation

Anurans of a riparian forest in Sao Carlos, state of Sao Paulo, Brazil

No presente estudo foi realizado um inventário das espécies de anfíbios anuros que ocorrem em um fragmento de mata ciliar do Córrego do Espraiado, localizado na Universidade Federal de São Carlos (UFSCar), Município de São Carlos, estado de São Paulo, sudeste do Brasil. Durante os meses de março de 2009 a fevereiro de 2010 foram realizadas saídas de campo semanais nas quais foram registradas 13 espécies de anuros. A atividade reprodutiva das espécies de anuros componentes da comunidade estudada mostrou-se sazonal, sendo que houve correlação positiva e significativa entre o número de espécies em atividade reprodutiva e o fotoperíodo. Verificamos que a comunidade estudada mostrou-se mais semelhante a comunidades de anuros de florestas de transição entre Mata Atlântica e Cerrado do que a fragmentos de Cerrado próximos da região.We surveyed anuran amphibians in a riparian forest fragment of the Córrego do Espraiado, located at the Universidade Federal de São Carlos (UFSCar), Municipality of São Carlos, state of São Paulo, southeastern Brazil. During March 2009 and February 2010 we did weekly surveys in which we recorded 13 anuran species. The species showed a seasonal reproductive activity. In addition, we found a positive correlation between the number of reproductively active species and photoperiod. We also found that the studied community was more similar to those of transitional areas between the Atlantic Forest and Cerrado than those of the nearby Cerrado fragments.Universidade Estadual Paulista Instituto de Biociências Departamento de ZoologiaUniversidade Estadual do Sudoeste da Bahia Departamento de Ciências BiológicasUniversidade Estadual Paulista Instituto de Biociências Departamento de Zoologi

Anurofauna da Floresta Estacional Semidecidual da Estação Ecológica dos Caetetus, Sudeste do Brasil

A Floresta Estacional Semidecidual é considerada uma das formações florestais mais ameaçadas e suprimidas pela expansão agrícola do início do século passado, com sua cobertura vegetal reduzida a menos de 8% da original. Estudos sobre taxocenoses de anfíbios anuros nessa formação florestal ainda são escassos, mesmo quando consideramos as poucas áreas de floresta primária que restaram. Apresentamos neste estudo os resultados de um levantamento de espécies de anfíbios anuros da Estação Ecológica dos Caetetus, uma unidade de conservação que abriga importante remanescente de Floresta Estacional Semidecidual na região Centro-oeste do estado de São Paulo, região considerada prioritária para conservação no estado. Foram registradas 34 espécies distribuídas em nove famílias, dentre as quais Hylidae e Leptodactylidae foram as que apresentaram as maiores riquezas, com 14 e oito espécies, respectivamente. A elevada riqueza de espécies da área, principalmente da família Hylidae, provavelmente está associada tanto à grande heterogeneidade ambiental dessa formação florestal como à sua localização em área de transição entre áreas de Cerrado e de Floresta Ombrófila. A alta riqueza de espécies, quando comparada à de outras áreas de mesma formação florestal, bem como a presença de representantes das famílias Centrolenidae, Craugastoridae e Hylodidae, que são dependentes de habitats florestais, atestam o bom estado de conservação e a importância regional desse remanescente florestal.Semi-deciduous Forest is one of the most threatened and deforested vegetation types of Brazil due to its land conversion to agriculture in the beginning of the 20th Century; its distribution is now reduced to less than 8% of its original extent. Studies about anuran taxocenosis associated to these forests are scarce, even when we consider the remaining areas of pristine forest. We present herein results from an amphibian survey at the Estação Ecológica dos Caetetus, a conservation unit with a singular preserved Semi-deciduous Forest from the Middle-West region of the State of São Paulo, located inside the priority areas for conservation on São Paulo State. We provided here data on species richness, composition, geographical distribution, and natural history notes for each taxa found in the area. We recorded 34 anuran species belonging to nine families, of which Hylidae and Leptodactylidae are the two most speciose families, with 14 and eight species respectively. The high species richness in the area, mainly in the family Hylidae, can be explained by both the high environmental heterogeneity of this remnant and its geographic location between Cerrado savannas and Atlantic rain forest areas. Its high species richness when compared with other localities of the same vegetation type, as well as the presence of forest dependent species in the families Centrolenidae, Hylodidae and Craugastoridae, indicate both the good conservation status and the regional importance of this forest remnant.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade de São Paulo Escola Superior de Agricultura Luiz de Queiroz Departamento de Ciências BiológicasUniv Estadual Paulista UNESP, Dept Zool & Bot, Inst Biociencias Letras & Ciencias Exatas, BR-15054000 Sao Jose do Rio Preto, SP, BrazilUniv Estadual Paulista UNESP, Dept Zool & Bot, Inst Biociencias Letras & Ciencias Exatas, BR-15054000 Sao Jose do Rio Preto, SP, BrazilFAPESP: 04/15938-5FAPESP: 99/09635-