Secretome analysis of Trypanosoma cruzi by proteomics studies

International audienceBackgroundChagas disease is a debilitating often fatal disease resulting from infection by the protozoan parasite Trypanosoma cruzi. Chagas disease is endemic in 21 countries of the Americas, and it is an emerging disease in other countries as a result of migration. Given the chronic nature of the infection where intracellular parasites persist for years, the diagnosis of T. cruzi by direct detection is difficult, whereas serologic tests though sensitive may yield false-positive results. The development of new rapid test based on the identification of soluble parasitic antigens in serum would be a real innovation in the diagnosis of Chagas disease.MethodsTo identify new soluble biomarkers that may improve diagnostic tests, we investigated the proteins secreted by T. cruzi using mass spectrometric analyses of conditioned culture media devoid of serum collected during the emergence of trypomastigotes from infected Vero cells. In addition, we compared the secretomes of two T. cruzi strains from DTU Tc VI (VD and CL Brener).ResultsAnalysis of the secretome collected during the emergence of trypomastigotes from Vero cells led to the identification of 591 T. cruzi proteins. Three hundred sixty three proteins are common to both strains and most belong to different multigenic super families (i.e. TcS, GP63, MASP, and DGF1). Ultimately we have established a list of 94 secreted proteins, common to both DTU Tc VI strains that do not belong to members of multigene families.ConclusionsThis study provides the first comparative analysis of the secretomes from two distinct T. cruzi strains of DTU TcVI. This led us to identify a subset of common secreted proteins that could potentially serve as serum markers for T. cruzi infection. Their potential could now be evaluated, with specific antibodies using sera collected from patients and residents from endemic regions

Potent Antiplasmodial Derivatives of Dextromethorphan Reveal the Ent-Morphinan Pharmacophore of Tazopsine-Type Alkaloids

International audienceThe alkaloid tazopsine 1 was introduced in the late 2000s as a novel antiplasmodial hit compound active against Plasmodium falciparum hepatic stages, with the potential to develop prophylactic drugs based on this novel chemical scaffold. However, the structural determinants of tazopsine 1 bioactivity, together with the exact definition of the pharmacophore, remained elusive, impeding further development. We found that the antitussive drug dextromethorphan (DXM) 3, although lacking the complex pattern of stereospecific functionalization of the natural hit, was harboring significant antiplasmodial activity in vitro despite suboptimal prophylactic activity in a murine model of malaria, precluding its direct repurposing against the disease. The targeted N-alkylation of nor-DXM 15 produced a small library of analogues with greatly improved activity over DXM 3 against P. falciparum asexual stages. Amongst these, N-2’-pyrrolylmethyl-nor-DXM 16i showed a 2- to 36-fold superior inhibitory potency compared to tazopsine 1 and DXM 3 against P. falciparum liver and blood stages, with respectively 760 ± 130 nM and 2.1 ± 0.4 μM IC(50) values, as well as liver/blood phase selectivity of 2.8. Furthermore, cpd. 16i showed a 5- to 8-fold increase in activity relative to DXM 3 against P. falciparum stages I-II and V gametocytes, with 18.5 μM and 13.2 μM IC(50) values, respectively. Cpd. 16i can thus be considered a promising novel hit compound against malaria in the ent-morphinan series with putative pan cycle activity, paving the way for further therapeutic development (e.g., investigation of its prophylactic activity in vivo)

Analyse of trans-sialidase (TcS) proteins found in secretome of 2 strains.

<p>Classification of TcS proteins for each strain into 8 group previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185504#pone.0185504.ref022" target="_blank">22</a>]. Groups IV, V and VI are less than I, II, III groups VII and VIII for two strains. On the x-axis, the number of group is indicated. The Y-axis shows the percentage of each TcS identified in our analyses.</p

Overlap between secretomes of two different <i>T</i>. <i>cruzi</i> strains DTU Tc VI.

<p>A total of 591 proteins of <i>T</i>. <i>cruzi</i> were identified. We note that 78 proteins are specific to the CL Brener strain whereas 151 proteins are specific to VD strain. However, 363 proteins are common to both strains.</p

List of secreted proteins that are common in both <i>T</i>.<i>cruzi</i> strains without of multigene proteins.

<p>List of secreted proteins that are common in both <i>T</i>.<i>cruzi</i> strains without of multigene proteins.</p

Functional categories classification of <i>T</i>. <i>cruzi</i> proteins from CL Brener and VD strains.

<p>Proteins were classified into 15 functional categories using literature and UniprotKB annotation. Y-axis, categories are indicated. X-axis shows the percentage of each category for each strain.</p