An actin‐like protein is involved in regulation of mitochondrial and flagellar functions as well as in intramacrophage survival of Leishmania donovani

Abstract

SummaryActin-related proteins are ubiquitous actin-like proteins that show high similarity with actin in terms of their amino acid sequence and three-dimensional structure. However, in lower eukaryotes, such as trypanosomatids, their functions have not yet been explored. Here, we show that a novel actin-related protein (ORF LmjF.13.0950) is localized mainly in the Leishmania mitochondrion. We further reveal that depletion of the intracellular levels of this protein leads to an appreciable decrease in the mitochondrial membrane potential as well as in the ATP production, which appears to be accompanied with impairment in the flagellum assembly and motility. Additionally, we report that the mutants so generated fail to survive inside the mouse peritoneal macrophages. These abnormalities are, however, reversed by the episomal gene complementation. Our results, for the first time indicate that apart from their classical roles in the cytoplasm and nucleus, actin-related proteins may also regulate the mitochondrial function, and in case of Leishmania donovani they may also serve as the essential factor for their survival in the host cells.

Emerging Functions of Actins and Actin Binding Proteins in Trypanosomatids

“…Following the reverse genetic approach, depletion of the intracellular levels of LdALP3 resulted in decreased mitochondrial membrane potential and the ATP content together with shortening of the flagellum length ( Singh et al, 2014 ). Similar shortening of the flagellum has also been observed earlier in case of LdCof and LdMyo13 heterozygous mutants ( Tammana et al, 2008 ; Katta et al, 2010 ).…”

“…Although morphologies of the LdCof and LdMyo13 mutants closely resembled the nonmotile clones of LdALP3 mutants, there were two distinct features that made them different from each other – (1) the flexible and fast wriggling flagellum of the LdALP3 mutants, which in case of LdCof and LdMyo13 mutants was completely immotile ( Tammana et al, 2008 ; Katta et al, 2010 ), and (2) the assembly of PFR (though poor) in LdALP3 mutants, which was absent in the LdCof and LdMyo13 mutants. Based on these facts, it has been concluded that LdALP3 perhaps operates through a mechanism, that appears to be different from the mechanisms through which actin-based LdMyo13 motor functions in assembly of the Leishmania flagellum ( Singh et al, 2014 ). However, a cross-talk between the actin-based LdMyo13 motor and LdALP3 operated mechanisms cannot be ruled out.…”

“…Similar distribution has also been observed earlier by overexpressing fluorescently tagged version of ALP3 in T. brucei ( Ersfeld and Gull, 2001 ). However, in L. donovani , ALP3 (earlier classified as Arp1) was predominantly localized to the mitochondrion, besides localizing to the cytoplasm and the flagellum ( Singh et al, 2014 ). And depletion of its intracellular levels resulted in decreased mitochondrion membrane potential and the ATP content, and also in shortening of the flagella length.…”

“…And depletion of its intracellular levels resulted in decreased mitochondrion membrane potential and the ATP content, and also in shortening of the flagella length. These effects were, however, reversed by episomal complementation of LdALP3 gene ( Singh et al, 2014 ), suggesting that ALP3 regulates mitochondrion potential, ATP synthesis and flagellum length in Leishmania promastigotes. The difference observed between intracellular distributions of TbALP3 and LdALP3 may perhaps be attributed to the larger size of LdALP3 (483 amino acids), compared with TbALP3 (433 amino acids), which is perhaps caused by insertions that confer distinctive properties to this protein.…”

Evaluation of Modulators of cAMP-Response in Terms of Their Impact on Cell Cycle and Mitochondrial Activity of Leishmania donovani

“…Live Leishmania cells were visualized under bright field of Olympus IX 81 microscope with FV1000 confocal system on grooved slides which provides the freedom of movement to the parasites. Flagellar movements of both treated and control cells were analyzed (Singh et al, 2014). Numerous fields were used, and the distribution of counting areas was chosen in a nonbiased manner along the total surface area of the cover-slip for graphical representation of the motile parasites.…”