Mevalonate kinase (MVK) is an essential enzyme acting in early steps of sterol isoprenoids biosynthesis, such as cholesterol in humans or ergosterol in trypanosomatids. MVK is conserved from bacteria to mammals, and localizes to glycosomes in trypanosomatids. During the course of T. cruzi MVK characterization, we found that, in addition to glycosomes, this enzyme may be secreted and modulate cell invasion. To evaluate the role of TcMVK in parasite-host cell interactions, TcMVK recombinant protein was produced and anti-TcMVK antibodies were raised in mice. TcMVK protein was detected in the supernatant of cultures of metacyclic trypomastigotes (MTs) and extracellular amastigotes (EAs) by Western blot analysis, confirming its secretion into extracellular medium. Recombinant TcMVK bound in a non-saturable dose-dependent manner to HeLa cells and positively modulated internalization of T. cruzi EAs but inhibited invasion by MTs. In HeLa cells, TcMVK induced phosphorylation of MAPK pathway components and proteins related to actin cytoskeleton modifications. We hypothesized that TcMVK is a bifunctional enzyme that in addition to playing a classical role in isoprenoid synthesis in glycosomes, it is secreted and may modulate host cell signaling required for T. cruzi invasion.

This study evaluated the participation of host cell Rho-family GTPases and their effector proteins in the actin-dependent invasion by Trypanosoma cruzi extracellular amastigotes (EAs). We observed that all proteins were recruited and colocalized with actin at EA invasion sites in live or fixed cells. EA internalization was inhibited in cells depleted in Rac1, N-WASP, and WAVE2. Time-lapse experiments with Rac1, N-WASP and WAVE2 depleted cells revealed that EA internalization kinetics is delayed even though no differences were observed in the proportion of EA-induced actin recruitment in these groups. Overexpression of constitutively active constructs of Rac1 and RhoA altered the morphology of actin recruitments to EA invasion sites. Additionally, EA internalization was increased in cells overexpressing CA-Rac1 but inhibited in cells overexpressing CA-RhoA. WT-Cdc42 expression increased EA internalization, but curiously, CA-Cdc42 inhibited it. Altogether, these results corroborate the hypothesis of EA internalization in non-phagocytic cells by a phagocytosis-like mechanism and present Rac1 as the key Rho-family GTPase in this process.

Species of Baccharis exhibit antibiotic, antiseptic, wound-healing, and anti-protozoal properties, and have been used in the traditional medicine of South America for the treatment of several diseases. In the present work, the fractionation of EtOH extract from aerial parts of Baccharis uncinella indicated that the isolated compounds caffeic acid and pectolinaringenin showed inhibitory activity against Leishmania (L.) amazonensis and Leishmania (V.) braziliensis promastigotes, respectively. Moreover, amastigote forms of both species were highly sensible to the fraction composed by oleanolic + ursolic acids and pectolinaringenin. Caffeic acid also inhibited amastigote forms of L. (L.) amazonensis, but this effect was weak in L. (V.) braziliensis amastigotes. The treatment of infected macrophages with these compounds did not alter the levels of nitrates, indicating a direct effect of the compounds on amastigote stages. The results presented herein suggest that the active components from B. uncinella can be important to the design of new drugs against American tegumentar leishmaniases.

Trypanosoma cruzi extracellular amastigotes (EAs) display unique mechanisms for cell invasion that are highly dependent on host actin filaments. Protein kinase D1 (PKD1) phosphorylates and modulates the activity of cortactin, a key regulator of actin dynamics. We evaluated the role of host cortactin and PKD1 in actin filament dynamics during HeLa cell invasion by EAs. Host cortactin, PKD1 and actin are recruited by EAs based on experiments in fixed and live cells by time lapse confocal microscopy. EAs trigger PKD1 and extracellular signal-regulated kinase 1/2 activation, but not Src family kinases, and selectively phosphorylate cortactin. Heat-killed EAs and non-infective epimastigotes both triggered distinct host responses and did not recruit the molecules studied herein. EA invasion was influenced by depletion or overexpression of host cortactin and PKD1, respectively, suggesting the involvement of both proteins in this event. Collectively, these results show new host cell mechanisms subverted during EA internalization into non-phagocytic cells.

Among the different infective stages that Trypanosoma cruzi employs to invade cells, extracellular amastigotes (EAs) have recently gained attention by our group. This is true primarily because these amastigotes are able to infect cultured cells and animals, establishing a sustainable infective cycle. EAs are thus an excellent means of adaptation and survival for T. cruzi, whose different infective stages each utilize unique mechanisms for attachment and penetration. Here we discuss some features of host cell invasion by EAs and the associated host cell signaling events that occur as part of the process.