Sepsis is a life-threatening disorder characterized by organ dysfunction and a major cause of mortality worldwide. The major challenge in studying sepsis is its diversity in such factors as age, source of infection and etiology. Recently, genomic and proteomic approaches have improved our understanding of its complex pathogenesis. In the present study, we use quantitative proteomics to evaluate the host proteome response in septic patients secondary to community-acquired pneumonia (CAP). Samples obtained at admission and after 7 days of follow-up were analyzed according to the outcomes of septic patients. The patients’ proteome profiles were compared with age- and gender-matched healthy volunteers. Bioinformatic analyses of differentially expressed proteins showed alteration in the cytoskeleton, cellular assembly, movement, lipid metabolism and immune responses in septic patients. Actin and gelsolin changes were assessed in mononuclear cells using immunofluorescence, and a higher expression of gelsolin and depletion of actin were observed in survivor patients. Regarding lipid metabolism, changes in cholesterol, HDL and apolipoproteins were confirmed using enzymatic colorimetric methods in plasma. Transcriptomic studies revealed a massive change in gene expression in sepsis. Our proteomic results stressed important changes in cellular structure and metabolism, which are possible targets for future interventions of sepsis.
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.
Trypanosoma cruzi is the etiologic agent of Chagas’ disease, which affects 6–7 million people worldwide. Different strains of T. cruzi present specific genotypic and phenotypic characteristics that affect the host–pathogen interactions, and thus, the parasite has been classified into six groups (TcI to TcVI). T. cruzi infection presents two clinical phases, acute and chronic, both with distinct characteristics and important participation by the immune system. However, the specific contributions of parasite and host factors in the disease phases are not yet fully understood. The murine model for Chagas’ disease is well-established and reproduces important features of the human infection, providing an experimental basis for the study of host lineages and parasite strains. Thus, we evaluated acute and chronic infection by the G (TcI) and CL (TcVI) strains of T. cruzi, which have distinct tropisms and infectivity, in two inbred mice lineages (C57BL/6 and BALB/c) that display variable degrees of susceptibility to different T. cruzi strains. Analysis of the parasite loads in host tissues by qPCR showed that CL strain established an infection faster than the G strain; at the same time, the response in BALB/c mice, although diverse in terms of cytokine secretion, was initiated earlier than that in C57BL/6 mice. At the parasitemia peak in the acute phase, we observed, either by confocal microscopy or by qPCR, that the infection was disseminated in all groups analyzed, with some differences concerning parasite tropism; at this point, all animals responded to infection by increasing the serum concentrations of cytokines. However, BALB/c mice seemed to better regulate the immune response than C57BL/6 mice. Indeed, in the chronic phase, C57BL/6 mice still presented exacerbated cytokine and chemokine responses. In summary, our results indicate that in these experimental models, the deregulation of immune response that is typical of chronic Chagas’ disease may be due to control loss over pro- and anti-inflammatory cytokines early in the acute phase of the disease, depending primarily on the host background rather than the parasite strain.
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.
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