In SituImmunolocalization and Stage-Dependent Expression of a Secretory Serine Protease inLeishmania donovaniand Its Role as a Vaccine Candidate

Abstract

Proteases have been found to play essential roles in many biological processes, including the pathogenesis of leishmaniasis. Most parasites rely on their intracellular and extracellular protease repertoire to invade and multiply in mammalian host cells. However, few studies have addressed serine proteases in Leishmania and their role in host pathogenesis. Here we report the intracellular distribution of a novel L. donovani secretory serine protease in the flagellar pocket, as determined by immunogold labeling. Flow cytometry and confocal immunofluorescence analysis revealed that the expression of the protease diminishes sequentially from virulent to attenuated strains of this species and is also highly associated with the metacyclic stage of L. donovani promastigotes. The level of internalization of parasites treated with the anti-115-kDa antibody into host macrophages was significantly reduced from that of non-antibody-treated parasites, suggesting that this serine protease probably plays a role in the infection process. In vivo studies confirmed that this serine protease is a potential vaccine candidate. Altogether, the 115-kDa serine protease might play vital roles in L. donovani pathogenesis and hence could be recognized as a potential candidate for drug design.Leishmania species, belonging to the family Trypanosomatidae, are a group of protozoan pathogens that cause a spectrum of chronic diseases ranging from self-healing cutaneous lesions to a lethal visceral disorder. They represent a major health problem in tropical and subtropical areas around the world. Leishmania species have a relatively simple life cycle, with parasite stage differentiation regulated by environmental signals encountered in their different hosts. They are digenetic parasites: the flagellated promastigote forms, which can be derived in axenic culture, differentiate within the alimentary tracts of sandfly vectors from a replicating procyclic to a nonreplicating, infectious metacyclic stage, whereas obligately intracellular amastigotes live and replicate in mammalian mononuclear phagocytes, such as macrophages (1).Recent advances in genomic analysis of several of the major global parasites have revealed key factors involved in the pathogenesis of parasite diseases. Among the major virulence factors identified are parasite-derived proteases. Parasite proteases play significant roles in the pathogenesis of parasitic diseases; the proteases are involved in the invasion of the host via parasite migration through tissue barriers, degradation of host proteins for their nutrition, immune evasion, and activation of inflammation (21). The migration of the parasite in host tissues is mediated by the release of proteolytic enzymes that can degrade the tissue barriers. Potent proteolytic enzymes of cysteine, serine, and metalloprotease classes have been identified in secretory products of many of the parasites (8,19,26,27,30). Serine proteases have been reported to have strong hydrolytic activity against a wide range of extracellular matrix (ECM) c...

Enhancement of Experimental Cutaneous Leishmaniasis by Leishmania Molecules Is Dependent on Interleukin-4, Serine Protease/Esterase Activity, and Parasite and Host Genetic Backgrounds

“…This is in agreement with previously reported data showing that Leishmania mexicana parasites that had been genetically modified so as to lack a cysteine protease (which has been shown to promote Th2 immune responses) (26) are less virulent than the wild-type parasites and that the decreased virulence is associated with the development of a Th1 immune response (5). In addition to the cysteine protease, it has recently been reported that a serine protease of Leishmania donovani metacyclic promastigotes has been indirectly associated with parasite virulence (7). In the present work, the addition of specific serine protease inhibitors (27) to the LaE decreased its infection-enhancing activity, as measured both by lesion size and by lesion parasitism.…”

Immunocytochemistry of Proteases in the Study of Leishmania Physiology and Host-Parasite Interaction

“…Besides, this enzyme is particularly expressed in virulent strains and is also associated with the metacyclic stage of L. (L.) donovani promastigotes. It is postulated that 115 kDa serine protease could be a potential vaccine candidate since it plays important roles in the macrophage infection and is secreted to extracellular environments (Choudhury et al, 2010).…”

Serine Proteases and Vaccines against Leishmaniasis: A Dual Role

“…A similar serine protease was found in the supernatant culture of L. donovani with the same molecular weight of 115 KDa [27] and with the same localization, mainly in flagelar pockets by immunohistochemistry [28]. The expression of the protease was correlated to a high virulence for the parasite and to the metacyclic stage of L. donovani promastigotes [28].…”

“…The expression of the protease was correlated to a high virulence for the parasite and to the metacyclic stage of L. donovani promastigotes [28]. When pSP was evaluated as a vaccine against L. donovani infection, protection was only obtained when it was associated with IL-12 as an adjuvant, which led to an increase in IFN-γ and TNF-α with a decrease in IL-4 and IL-10 after vaccination [47].…”