(1) Immunotyping the response to metastatic Leishmania and their endosymbiotic viral antigens—Diagnosis, treatment and prevention of complicated cutaneous leishmaniasis.
As veterans of infection, Leishmania guyanensis parasites have been plaguing humankind for centuries, provoking a deleterious hyper-inflammatory immune response, destroying host tissue and forming the ulcerating lesions, which typify most forms of the disease. About 15% of patients develop secondary lesions in the mouth and nose, where parasites metastasise to mucocutaneous tissues creating debilitating and exceptionally disfiguring inflammation.
Our lab has recently shown that a virus within metastatic Leishmania parasites (Leishmania RNA virus: LRV) can act as an independently immunogenic entity, where its RNA-based nucleic acid acted as a potent innate immunogen, triggering a destructive hyper-inflammatory cascade through Toll-Like-Receptor 3 recognition.
Because viral replication depends on the vigor of its host, many viruses have evolved incentives of fitness to pay their keep. When the viral host is a human pathogen, these fitness factors can surface as virulence: creating a Russian doll of pathogenesis where parasites within parasites complicate the disease process.
We dub this process “hyperpathogenism” and have exposed its importance as major clinical consideration in metastatic leishmaniasis.
Interestingly, our preliminary data show this LRV-specific immune response can be rendered protective if primed before the infection as a vaccine, raising the importance of cross talk between the innate responses to the various components of this nested infection.
By appreciating the microbial-virus as a backseat driver of human disease, we may be able to better formulate appropriate clinical intervention or even exploit its presence for clinical benefit. Further, its presence could act as a biomarker, guiding diagnostics, treatment and phylogenetics as well as a having potential as novel molecular target for therapeutic and prophylactic intervention.
Leishmania RNA virus (Red) in Leishmania guyanensis
infected macrophages (Nuclei of macrophages stained
(2) Characterizing and inducing cell death in Leishmania—Metacaspase as a drug target.
Dubbed as the ‘executioner’ enzymes, capases have a well-described role in eukaryotic cell death. Although some caspase-like peptidase activity has been reported in dying Leishmania parasites, a caspase gene is not present in its genome. A single copy of the analogous metacaspase gene is, however, present in Leishmania major. Structurally, L. major metacaspase (LmjMCA) comprises a potential N-terminal mitochondrial localization sequence (MLS), a central domain with the catalytic dyad histidine/cysteine described for caspases and a C-terminal proline-rich sequence. The S. cerevisiae metacaspase YCA1 has been implicated in the death of aging cells, cells defective in some biological functions, and cells exposed to different environmental stresses. In our study of LmjMCA, we demonstrated the functional heterologous complementation of a S. cerevisiae yca1 null mutant with the L. major metacaspase (LmjMCA). Here, cell death was successfully induced in yeast by LmjMCA after oxidative stress. We completed the previous study by investigating LmjMCA activation and cellular localization following its processing. Although the LmjMCA polypeptide precursor form harbors a functional MLS, we demonstrated that LmjMCA is mainly localized in the cytoplasm due to an amino acid sequence in the catalytic domain blocking its transport into the mitochondrion. Finally, in stress conditions, overexpression of the LmjMCA catalytic domain enhanced sensitivity of parasites to ROS. This was the first report describing the activation, release of the catalytic domain (via auto-processing) and cytoplasmic localization of LmjMCA. Further, we demonstrated that LmjMCA increased susceptibility to ROS during stress, which, we predict is through perturbing mitochondrial membrane integrity (Zalila et al., 2011). These results suggest that metacaspases are members of a family of peptidases with an evolutionarily conserved role in cell death function. The identification of an enzyme sensitizing this parasitic pathogen to cell death holds much clinical potential for leishmaniasis: A neglected disease with an aged and problematic therapeutic arsenal, in need of new drug targets.