Optimization of a medium composition for the heterologous production of Alcaligenes faecalis penicillin G acylase in Bacillus megaterium.

Penicillin G acylase (PGA) is a strategic enzyme in the production processes of beta-lactam antibiotics. High demand for ß-lactam semisynthetic antibiotics explain the genetic and biochemical engineering strategies devoted towards novel ways for PGA production and application. This work presents a fermentation process for the heterologous production of PGA from Alcaligenes faecalis in Bacillus megaterium with optimization. The thermal stability from A. faecalis PGA is considerably higher than other described PGA and the recombinant enzyme is secreted to the culture medium by B. megaterium, which facilitates the separation and purification steps. Media optimization using fractional factorial design experiments was used to identify factors related to PGA activity detection in supernatant and cell lysates. The optimized medium resulted in almost 6-fold increased activity in the supernatant samples when compared with the basal medium. Maximum enzyme activity in optimized medium composition achieves values between 135 and 140 IU/ml. The results suggest a promising model for recombinant production of PGA in B. megaterium with possible extracellular expression of the active enzyme.

CD4+ T Cell-Dependent Macrophage Activation Modulates Sustained PS Exposure on Intracellular Amastigotes of Leishmania amazonensis.

Leishmania amazonensis amastigotes can make use of surface-exposed phosphatidylserine (PS) molecules to promote infection and non-classical activation of macrophages (MΦ), leading to uncontrolled intracellular proliferation of the parasites. This mechanism was quoted as apoptotic mimicry. Moreover, the amount of PS molecules exposed on the surface of amastigotes correlates with the susceptibility of the host. In this study, we tested whether host cellular responses influence PS expression on intracellular amastigotes. We found that the level of PS exposure on intracellular amastigotes was modulated by CD4+ T cell and MΦ activation status in vitro and in vivo. L. amazonensis infection generated a Th1/Th2-mixed cytokine profile, providing the optimal MΦ stimulation that favored PS exposure on intracellular amastigotes. Maintenance of PS exposed on the parasite was dependent on low, but sustained, levels of nitric oxide and polyamine production. Amastigotes obtained from lymphopenic nude mice did not expose PS on their surface, and adoptive transfer of CD4+ T cells reversed this phenotype. In addition, histopathological analysis of mice treated with anti-PS antibodies showed increased inflammation and similarities to nude mouse lesions. Collectively, our data confirm the role of pathogenic CD4+ T cells for disease progression and point to PS as a critical parasite strategy to subvert host immune responses.

Microvesicles and exosomes as vehicles between protozoan and host cell communication.

Cells release extracellular vesicles in response to external factors or in a physiological way. Microvesicles and exosomes originate in cells in different ways and, depending on their contents, may have multiple biological effects on other cells and the environment. The host cell-parasite relationship could be changed dramatically by the plasticity of a new type of communication through extracellular vesicles. In the present paper, we discuss how protozoans use this new resource to evade the immune system and establish infection.

Exposure of phosphatidylserine on Leishmania amazonensis isolates is associated with diffuse cutaneous leishmaniasis and parasite infectivity.

Diffuse cutaneous leishmaniasis (DCL) is a rare clinical manifestation of leishmaniasis, characterized by an inefficient parasite-specific cellular response and heavily parasitized macrophages. In Brazil, Leishmania (Leishmania) amazonensis is the main species involved in DCL cases. In the experimental model, recognition of phosphatidylserine (PS) molecules exposed on the surface of amastigotes forms of L. amazonensis inhibits the inflammatory response of infected macrophages as a strategy to evade the host immune surveillance. In this study, we examined whether PS exposure on L. amazonensis isolates from DCL patients operated as a parasite pathogenic factor and as a putative suppression mechanism of immune response during the infection. Peritoneal macrophages from F1 mice (BALB/c×C57BL/6) were infected with different L. amazonensis isolates from patients with localized cutaneous leishmaniasis (LCL) or DCL. DCL isolates showed higher PS exposure than their counterparts from LCL patients. In addition, PS exposure was positively correlated with clinical parameters of the human infection (number of lesions and time of disease) and with characteristics of the experimental infection (macrophage infection and anti-inflammatory cytokine induction). Furthermore, parasites isolated from DCL patients displayed an increased area in parasitophorous vacuoles (PV) when compared to those isolated from LCL patients. Thus, this study shows for the first time that a parasite factor (exposed PS) might be associated with parasite survival/persistence in macrophages and lesion exacerbation during the course of DCL, providing new insights regarding pathogenic mechanism in this rare chronic disease.

Trypanosoma cruzi immune evasion mediated by host cell-derived microvesicles.

The innate immune system is the first mechanism of vertebrate defense against pathogen infection. In this study, we present evidence for a novel immune evasion mechanism of Trypanosoma cruzi, mediated by host cell plasma membrane-derived vesicles. We found that T. cruzi metacyclic trypomastigotes induced microvesicle release from blood cells early in infection. Upon their release, microvesicles formed a complex on the T. cruzi surface with the complement C3 convertase, leading to its stabilization and inhibition, and ultimately resulting in increased parasite survival. Furthermore, we found that TGF-ß-bearing microvesicles released from monocytes and lymphocytes promoted rapid cell invasion by T. cruzi, which also contributed to parasites escaping the complement attack. In addition, in vivo infection with T. cruzi showed a rapid increase of microvesicle levels in mouse plasma, and infection with exogenous microvesicles resulted in increased T. cruzi parasitemia. Altogether, these data support a role for microvesicles contributing to T. cruzi evasion of innate immunity.

L-amino acid oxidase activity present in fractions of Bothrops jararaca venom is responsible for the induction of programmed cell death in Trypanosoma cruzi.

Bothrops jararaca venom induces programmed cell death in epimastigotes of Trypanosoma cruzi. Here we fractionated the venom and observed that the anti-T. cruzi activity was associated with fractions that present L-amino acid oxidase (L-AAO) activity. L-AAO produces H(2)O(2), which is highly toxic. The addition of catalase to the medium, a H(2)O(2) scavenger, reverted the killing capacity of venom fractions. The anti-T. cruzi activity was also abolished when parasites were cultured in a medium without hydrophobic amino acids that are essential for L-AAO activity. These results were confirmed with a commercial purified L-AAO. Treatment for 24 h with fractions that present L-AAO activity induced parasites cytoplasmic retraction, mitochondrial swelling and DNA fragmentation, all morphological characteristics of programmed cell death. Similar changes were also observed when parasites were treated with H(2)O(2). These results indicate that H(2)O(2), the product of L-AAO reaction, induces programmed cell death explaining the anti-T. cruzi activity of B. jararaca venom.

Cooperation between apoptotic and viable metacyclics enhances the pathogenesis of Leishmaniasis.

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 (PS(POS)) sub-population was confirmed by TUNEL staining and DNA laddering. Transmission electron microscopy revealed morphological alterations in PS(POS) metacyclics such as DNA condensation, cytoplasm degradation and mitochondrion and kinetoplast destruction, both in in vitro cultures and in sand fly guts. TUNEL(POS) 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 PS(POS) and PS-negative (PS(NEG)) parasites were simultaneously added to the cell culture or inoculated in the mammalian host. The viable PS(NEG) promastigote was the infective form, as shown by following the fate of fluorescently labeled parasites, while the PS(POS) 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.

Exposição de fosfatidilserina em amastigotas intracelulares de Leishmania (L) amazonensis e seu papel na modulação da resposta macrofágica / Exposition of phosphatidylserine in amastigotes intracellular of Leishmania (L) amazonensis and its paper in the modulation of the macrophage reply

Os macrófagos são as células preferenciais para a infecção e a proliferação de parasitas do gênero Leishmania. A sobrevivência do parasita e o desenvolvimento da infecção são resultado direto da evasão do parasita dos mecanismos microbicidas do hospedeiro. As formas amastigotas de Leishmania (L) amazonensis isoladas de lesãoes em camundongos espõem o fosfolipídio fosfatidilserina (PS) na face externa da sua membrana plasmática, um fenômeno descrito como Mimetismo Apoptótico. O reconhecimento deste fosfolipídio pelo macrófago induz a internalização do parasita por um mecanismo de macropinocitose, a produção de citocinas anti-inflamatórias IL-10 e TGF-Beta e inibe a produção de óxido nítrico (NO), contribuindo para o aumento da infectividade do parasita e sua proliferação na célula hospedeira. A exposição de PS na superfície das amastigotas pode ser modulada pelo hospedeiro: amastigotas isoladas de lesões em camundongos BALB/c expõem mais moléculas de PS na superfície que aquelas isoladas de camundongos C57BL/6. Neste trabalho demonstramos que amastigotas purificadas de macrófagos de camundongos BALB/c e C57BL/6 infectados in vitro não apresentam diferenças significativas na exposição de PS na superfície, sugerindo que as diferenças observadas no modelo in vivo são dependentes de uma ativação diferencial dos macrófagos no hospedeiro. Amastigotas isoladas de lesão em camundongos BALB/c(nu/nu) expõem menos PS na superfície que aquelas isoladas de lesões em camundongos BALB/c, indicando um papel das citocinas produzidas pelas células T na modulação da exposição de PS pelo parasita. Durante o processo de diferenciação de promastigota para amastigota no interior do vacúolo parasitóforo ocorre aumento na exposição de PS na superfície externa da membrana do parasita. Neste período, observamos indução de macropinocitose nos macrófagos infectados e fusão das vesículas macropinocíticas com o vacúolo contendo os parasitas, sugerindo que a exposição de PS pela...infecção.

Trypanosoma cruzi exposes phosphatidylserine as an evasion mechanism.

Chagas disease is caused by Trypanosoma cruzi and affects 18 million people in Central and South America. Here we analyzed the exposure of phosphatidylserine by the different forms of the parasite life cycle. Only the infective trypomastigotes, but not the epimastigotes or intracellular amastigotes, expose this phospholipid. This triggers a transforming growth factor beta signaling pathway, based on phosphorylated Smad 2 nuclear translocation, leading to iNOS disappearance in infected macrophages. This macrophage deactivation favors the survival of this intracellular parasite. Thus, phosphatidylserine exposure may be used by T. cruzi to evade innate immunity and be a common feature of obligate intracellular parasites that have to deal with activated macrophages.

Programmed cell death in Trypanosoma cruzi induced by Bothrops jararaca venom.

Cells die through a programmed process or accidental death, know as apoptosis or necrosis, respectively. Bothrops jararaca is a snake whose venom inhibits the growth of Trypanosoma cruzi epimastigote forms causing mitochondrion swelling and cell death. The aim of the present work was to determine the type of death induced in epimastigotes of T. cruzi by this venom. Parasite growth was inhibited after venom treatment, and 50% growth inhibition was obtained with 10 microg/ml. Ultrastructural observations confirmed mitochondrion swelling and kinetoplast disorganization. Furthermore, cytoplasmic condensation, loss of mitochondrion membrane potential, time-dependent increase in phosphatidylserine exposure at the outer leaflet plasma membrane followed by permeabilization, activation of caspase like protein and DNA fragmentation were observed in epimastigotes throughout a 24 h period of venom treatment. Taken together, these results indicate that the stress induced in epimastigote by this venom, triggers a programmed cell death process, similar to metazoan apoptosis, which leads to parasite death.

Programmed cell death in Trypanosoma cruzi induced by Bothrops jararaca venom

Cells die through a programmed process or accidental death, know as apoptosis or necrosis, respectively. Bothrops jararaca is a snake whose venom inhibits the growth of Trypanosoma cruzi epimastigote forms causing mitochondrion swelling and cell death. The aim of the present work was to determine the type of death induced in epimastigotes of T. cruzi by this venom. Parasite growth was inhibited after venom treatment, and 50 percent growth inhibition was obtained with 10 æg/ml. Ultrastructural observations confirmed mitochondrion swelling and kinetoplast disorganization. Furthermore, cytoplasmic condensation, loss of mitochondrion membrane potential, time-dependent increase in phosphatidylserine exposure at the outer leaflet plasma membrane followed by permeabilization, activation of caspase like protein and DNA fragmentation were observed in epimastigotes throughout a 24 h period of venom treatment. Taken together, these results indicate that the stress induced in epimastigote by this venom, triggers a programmed cell death process, similar to metazoan apoptosis, which leads to parasite death.