Function and localization of Leishmania mexicana MAP kinase 2

Leishmaniasis is a life threatening tropical disease caused by Leishmania parasites that are transmitted by blood feeding sand flies. This disease affects approximately 12 million people worldwide, leaving another 350 million more at risk in endemic areas. Roughly two million new cases are reported annually with nearly 60,000 mortality rate. Treatment improvements have been slow in the past century, resulting in ineffective single treatment approach patient care with toxic side effects to current drugs at an expensive cost. These flows accents the ugency for a safer, a 'Combination Regime' treatment approach at an affordable rate.;LmxMPK2, a mitogen-activated protein (MAP) kinase homologue of Leishmania mexicana, is expressed in the amastigote and promastigote life stages of Leishmania parasites. Previous research (PhD, Laura Munro 2013) suggested that LmxMPK2 is involved in the organisation of microtubules, influencing cell shape and cytokinesis. A homozygous gene knock out mutant (null mutant) for LmxMPK2 in the promastigote stage was generated with deletions that caused alterations in cell proliferation and morphology, resulting to variations in the cell shape with spiked posterior ends, as well as division channel ingression from the posterior end.;MAP kinases are known to be key regulatory elements in cell cycle progression, proliferation, differentiation, as well as in stress responses in Leishmania cells (Wiese et al., 2003). Therefore, understanding the function of MAP kinases in Leishmania is essential and therefore relevant to the pursuit of new treatment approaches for leishmaniasis. In this study two constructs were generated that allowed the expression of Green Fluorescence Protein-tagged MPK2 (MPK2GFP) in the ribosomal DNA locus and Red Fluorescence Protein-tagged Deflagellation inducible protein 13 (RFPDIP13) from a plasmid.;Live cell fluorescence microscopy of cloned cells confirmed the expression of MPK2GFP and RFPDIP13 in both the promastigote and amastigote life stages in Leishmania cells. GFP-tagged LmxMPK2 showed localisation primarily at the poles, with occasional localisation at the centre of the cells. L. mexicana wild type; two LmxMPK2 null mutant clones; two LmxMPK2 add back clones; and four LmxMPK2 add back clones co-expressing RFPDIP13, were differentiated from the promastigote to the amastigote life stage for further analysis.;Although all cells were differentiable into the amastigote form, only cells expressing LmxMPK2 showed proliferation after differentiation. This observation suggests that the null mutant Leishmania cells (knock out; no LmxMPK2) are unable to survive under amastigote conditions. Hence, targeting LmxMPK2 with a specific inhibitor in the amastigote stage could significantly impact the course of treatment for leishmaniasis.Leishmaniasis is a life threatening tropical disease caused by Leishmania parasites that are transmitted by blood feeding sand flies. This disease affects approximately 12 million people worldwide, leaving another 350 million more at risk in endemic areas. Roughly two million new cases are reported annually with nearly 60,000 mortality rate. Treatment improvements have been slow in the past century, resulting in ineffective single treatment approach patient care with toxic side effects to current drugs at an expensive cost. These flows accents the ugency for a safer, a 'Combination Regime' treatment approach at an affordable rate.;LmxMPK2, a mitogen-activated protein (MAP) kinase homologue of Leishmania mexicana, is expressed in the amastigote and promastigote life stages of Leishmania parasites. Previous research (PhD, Laura Munro 2013) suggested that LmxMPK2 is involved in the organisation of microtubules, influencing cell shape and cytokinesis. A homozygous gene knock out mutant (null mutant) for LmxMPK2 in the promastigote stage was generated with deletions that caused alterations in cell proliferation and morphology, resulting to variations in the cell shape with spiked posterior ends, as well as division channel ingression from the posterior end.;MAP kinases are known to be key regulatory elements in cell cycle progression, proliferation, differentiation, as well as in stress responses in Leishmania cells (Wiese et al., 2003). Therefore, understanding the function of MAP kinases in Leishmania is essential and therefore relevant to the pursuit of new treatment approaches for leishmaniasis. In this study two constructs were generated that allowed the expression of Green Fluorescence Protein-tagged MPK2 (MPK2GFP) in the ribosomal DNA locus and Red Fluorescence Protein-tagged Deflagellation inducible protein 13 (RFPDIP13) from a plasmid.;Live cell fluorescence microscopy of cloned cells confirmed the expression of MPK2GFP and RFPDIP13 in both the promastigote and amastigote life stages in Leishmania cells. GFP-tagged LmxMPK2 showed localisation primarily at the poles, with occasional localisation at the centre of the cells. L. mexicana wild type; two LmxMPK2 null mutant clones; two LmxMPK2 add back clones; and four LmxMPK2 add back clones co-expressing RFPDIP13, were differentiated from the promastigote to the amastigote life stage for further analysis.;Although all cells were differentiable into the amastigote form, only cells expressing LmxMPK2 showed proliferation after differentiation. This observation suggests that the null mutant Leishmania cells (knock out; no LmxMPK2) are unable to survive under amastigote conditions. Hence, targeting LmxMPK2 with a specific inhibitor in the amastigote stage could significantly impact the course of treatment for leishmaniasis