Calpains of Leishmania braziliensis: genome analysis, differential expression, and functional analysis

Abstract

BACKGROUND Calpains are proteins belonging to the multi-gene family of calcium-dependent cysteine peptidases that undergo tight on/off regulation, and uncontrolled proteolysis of calpains is associated with severe human pathologies. Calpain orthologues are expanded and diversified in the trypanosomatids genome.OBJECTIVES Here, we characterised calpains in Leishmania braziliensis, the main causative agent of cutaneous leishmaniasis in Brazil.METHODS/FINDINGS In total, 34 predicted calpain-like genes were identified. After domain structure evaluation, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) during in vitro metacyclogenesis revealed (i) five genes with enhanced expression in the procyclic stage, (ii) one augmented gene in the metacyclic stage, and (iii) one procyclic-exclusive transcript. Western blot analysis revealed that an antibody against a consensus-conserved peptide reacted with multiple calpain-like proteins, which is consistent with the multi-gene family characteristic. Flow cytometry and immunocytochemistry analyses revealed the presence of calpain-like molecules mainly in the cytoplasm, to a lesser extent in the plasma membrane, and negligible levels in the nucleus, which are all consistent with calpain localisation. Eventually, the calpain inhibitor MDL28170 was used for functional studies revealing (i) a leishmaniostatic effect, (ii) a reduction in the association index in mouse macrophages, (iii) ultra-structural alterations conceivable with autophagy, and (iv) an enhanced expression of the virulence factor GP63.CONCLUSION This report adds novel insights into the domain structure, expression, and localisation of L. braziliensis calpain-like molecules.

Expression and cellular localisation of Trypanosoma cruzi calpains

“…(16) Here, small sequences were not considered for further investigations, although two proteomic studies reported the protein expression of SKCRPs in epimastigotes, metacyclic and bloodstream trypomastigotes. (17,18) As previously observed in a calpain screening in L. braziliensis, (14) T. cruzi also presents in chromosome 17 an ortholog sequence that possesses a kinesin motor domain associated to three armadillo/beta-catenin-like repeats (ARM) positioned before the CBSW domain (Tc-CLB.511307.10). In T. brucei, this orphan kinesin (KIN-E) enrichment at the flagellar tip depends on the CBSW domain, and the KIN-E depletion in the trypomastigote form causes major morphology changes generating epimastigote-like parasites.…”

“…These data are in agreement with our previous work with Leishmania braziliensis, whereas the presence of a large diversity of domain arrangement was revealed in 34 calpain-related sequences distributed in 13 different chromosomes. (14) However, microtubule interacting and trafficking domain (MIT) was not found associated with T. cruzi calpains.…”

“…(20) In this sense, it has been shown a link between differential expression levels of calpains' transcripts and protein expression during trypanosomatids differentiation. (14,21,22,23) Here, we evaluated by qPCR the relative gene expression of the calpains with the most conserved domain architecture, such as the presence of the CysPc, and removed from the analysis the repeat-rich and small sequences. From the 26 selected sequences, 20 were identical, which precluded the design of specific primers, resulting in the exclusion of 10 sequences from the subsequent analysis.…”

“…In L. braziliensis, the anti-tritryp-calpain antibody detected polypeptides migrating at approximately 70, 45 and 40 kDa that were strongly recognised, along with faint bands either in high (150 and 225 kDa) or in low (47 and 31 kDa) molecular masses. (14) A slight reactivity with molecules at approximately 127, 63, 43 and 35 kDa were observed in Vero cells extracts incubated with anti-tritryp-calpain antibody (Fig. 1A), which could be justified by the conservation of the peptide sequence in calpain 3 and 12 from the African green monkey (Chlorocebus aethiops) genome from which this cell line is derived from.…”

“…Reviewed in (14) *: indicates calpain domain presence; 1 : repeat rich regions do not correspond to traditionally domains, but they are important to conservate the protein structure. (30) CLB.508999.200), which was 2.5 times higher in epimastigotes submitted to nutritional stress preceding metacyclogenesis in relation to parasites in nutrient-rich medium.…”