Expression of a Novel Leishmania Gene Encoding a Histone H1-Like Protein in Leishmania major Modulates Parasite Infectivity In Vitro

Abstract

We describe identification and characterization of a novel two-copy gene of the parasitic protozoan Leishmania that encodes a nuclear protein designated LNP18. This protein is highly conserved in the genus Leishmania, and it is developmentally regulated. It is an alanine-and lysine-rich protein with potential bipartite nuclear targeting sequence sites. LNP18 shows sequence similarity to H1 histones of trypanosomatids and of higher eukaryotes and in particular with histone H1 of Leishmania major. The nuclear localization of LNP18 was determined by indirect immunofluorescence and Western blot analysis of isolated nuclei by using antibodies raised against the recombinant protein as probes. The antibodies recognized predominantly a 18-kDa band or a 18-kDa-16-kDa doublet. Photochemical cross-linking of intact parasites followed by Western blot analysis provided evidence that LNP18 is indeed a DNA-binding protein. Generation of transfectants overexpressing LNP18 allowed us to determine the role of this protein in Leishmania infection of macrophages in vitro. These studies revealed that transfectants overexpressing LNP18 are significantly less infective than transfectants with the vector alone and suggested that the level of LNP18 expression modulates Leishmania infectivity, as assessed in vitro.Leishmania spp. are obligately intracellular protozoan parasites that are members of the family Trypanosomatidae, and they cause a wide spectrum of human diseases of major health importance, ranging from self-healing cutaneous lesions to severe visceral leishmaniasis or kala-azar with a high fatality rate (13). These parasites have two developmentally distinct stages; the amastigotes are nonmotile forms that survive and replicate within mammalian host macrophages, and the motile flagellated promastigotes survive and replicate inside the insect vector (13

Overexpression of histone H2A modulates drug susceptibility in Leishmania parasites

“…Leishmania parasites possess core histones H2A, H2B, H3, H4 [28,29] and linker histone H1 [30][31][32] that facilitate the formation of higher-order chromatin structures. In eukaryotes, post-translational modifications of core histones act in diverse biological processes such as gene regulation, DNA repair and chromosome condensation (mitosis).…”

Canonical and variant histones of protozoan parasites

“…No difference in the rate of macrophage invasion in control and LeishH1overexpressing parasites was observed. However, mutants exhibited a delay in parasite cell cycle progression and differentiation (45,46). Additionally, in the presence of excess histone H1, parasites display resistance to MNase treatment and accumulate clusters of condensed chromatin (47).…”

Histones and histone modifications in protozoan parasites

“…Interestingly, with respect to virulence, H1 may prove helpful in the design of novel vaccine candidates. Transgenic parasites over‐expressing Leishmania H1 suffer delays in the cell cycle and differentiation, and are significantly less infective in vivo (Papageorgiou and Soteriadou, 2002; Smirlis et al ., 2006).…”

“…Protozoan histones may also be exploitable in novel therapeutic approaches as immunogens or by way of creating attenuated strains for vaccine applications. Transgenic parasites over‐expressing H1 have been examined as a vaccine candidate for leishmaniasis (Papageorgiou and Soteriadou, 2002). Further, the prophylactic value of a cocktail consisting of the four core Leishmania infantum histones was evaluated in the murine model of cutaneous leishmaniasis.…”