Functional genomics and immunological approaches toward a comprehensive view of protozoan parasite virulence factors

The protozoan parasites, which cause important diseases in humans and a number of other mammalian species, have complex life cycles including different developmental stages. Most of these parasites are transmitted by insect vectors and invade a range of different tissues in their mammalian hosts. These series of transitions involve major modifications in morphology, gene expression and cell cycle in order to survive environmental extremes, such as toxic metabolites and acidic pH, of the host cell phagolysosomes. Therefore, cells at different periods of the life cycle may differ significantly. A number of regulatory components or processes have been identified that control or regulate metabolism, growth and parasite development. Gene expression determines the state of differentiation and has a role in the regulation of activity of a given parasite form under a physiological state.

The breakthrough in methods exploiting the high frequency of homologous recombination and antisense RNA has made possible not only the identification of parasite proteins crucial for infectivity but also those involved in persistence and disease development. The last 10 years has witnessed the appearance of a substantial number of studies, in which genetic engineering of target molecules in transgenic parasites has been employed to unravel their biological functions. Moreover, despite the fact that active sites of enzymes are often highly conserved across species and that certain biochemical pathways could be shared between the parasite and the mammalian host, there are usually some functional differences. For instance, while complete disruption of the mannose pathway and the resulting absence of mannose-containing glycoconjugates is incompatible with eukaryotic life, this appears not to be the case for the pathogenic kinetoplastids Leishmania spp., which are among the most ancient eukaryotes, causing a diverse group of diseases collectively known as leishmaniasis. Indeed, although Leishmania spp. produce large amounts of unusual mannose-rich cell surface-associated and secreted glycoconjugates, whose synthesis depends directly or indirectly on the availability of GDP-mannose, transgenic inactivation of GDP-mannose biosynthesis through targeted replacement of the GDP-mannose pyrophosphorylase gene did not affect the parasite viability [1].

Another example concerns the Leishmania cytosolic SIR2 homolog-encoding gene (LSIR2RP1). In fact, proteins of the SIR2 family share a common conserved core domain of approximately 250 residues responsible for a deacetylase activity capable of removing the acetyl moiety from the ε-amino group of lysine residues in protein substrate. All these enzymes couple deacetylation to the hydrolysis of NAD⁺, transferring the acetyl group from substrate to ADP-ribose, thereby generating a novel compound, O-acetyl-ADP-ribose [2,3]. Functional analyses of such proteins have been carried out in a number of prokaryotic and eukaryotic organisms but, until now, none have described an essential function for any SIR2 gene. In a recent report using a genetic approach, it has been shown for the first time that a cytosolic SIR2 homolog in Leishmania is determinant to parasite survival [4].

In the case of the African trypanosome Trypanosoma brucei, a kinetoplastid parasite that grows in the mammalian vasculature and causes sleeping sickness in humans and Nagana in cattle, the SIR2-related protein termed TbSIR2RP1, homologous to LSIR2RP1, appears to have distinct features since it expresses NAD-dependent histone ADP-ribosyltransferase, as well as deacetylase activities. Downregulation of TbSIR2RP1 resulted in decreased resistance to DNA damage [5]. However, in a more recent study, three genes encoding SIR2-related proteins (SIR2RP1–3) have been characterized [6]. SIR2RP1 localizes to the nucleus, whereas SIR2RP2 and SIR2RP3 are both mitochondrial proteins. The nuclear protein SIR2RP1 controls DNA repair and repression of RNA polymerase I-mediated expression immediately adjacent to telomeres. However, antigenic variation appears to be independent of SIR2RP1 [6]. Therefore, although the parasites belonging to the same family may share closely related gene sequences, the products may differ in their localization pattern and functional properties. Thus, given the degree of homology between members of this large family of enzymes and the putative redundancy of their functions, pharmacological inhibitors may have limited selectivity or specificity. However, studies of enzymatic activities of members of the same family have revealed some differences that could reflect biochemical peculiarities (i.e., substrate specificities and cofactor requirement;) features that perhaps may be exploitable for drug design.

Therefore, investigation of parasite metabolic pathways may not only allow us to uncover new distinctive features, which will become the focus of intensive research interest in these model organisms, but will also shed light on potential structural and functional differences in target gene products to be exploited for the design of selective means (chemotherapeutic/immunological) to interfere with their biological function [7].

Another strategy that may eventually lead to new therapeutic options would be through immunological approaches, which might allow the definition of the precise protective responses to be induced in the host. The development of genetically modified animal models lacking genes encoding secreted cytokines and various surface molecules important in cellular signaling processes has provided new insights into the pathogenesis of parasitic infections. Moreover, these experimental systems represent one of the paradigms for genetic studies of disease susceptibility and are a topic of interest to link the immunological mechanisms with the responsible genes. Owing to the cross-reactivity between some parasite and host components, and toxicity of some parasite molecules, identification and isolation of molecules that might induce efficient protective immune mechanisms is required. Although significant progress has been made in our understanding of the immune response to parasites, no definitive step has yet been performed successfully in terms of operational vaccines against parasitic diseases.

Indeed, in a number of protozoan infections, different strategies were developed in order to identify parasite components that could be used as a vaccine candidate. The use of inactivated parasites, fractionated parasite material, irradiated noninfectious parasites, recombinant parasites lacking genes encoding virulence factors and parasite DNA has been shown to induce partial protection of mice against a lethal challenge infection [8–13]. However, evidence accumulated over the years has undoubtedly shed light on the importance of Treg cells in the development of an immunosuppressive environment that favors the parasite maintenance into the host during the chronic phase of the disease and this may limit the success of immunotherapeutic strategies based on vaccination [14].

In fact, numerous data pointed to the fact that, in contrast to the previous thought that Th2 cells were the only source of IL-10 [15], Th1 cells are also able to secrete IL-10 [16]. In addition, a number of other T-cell subsets, including CD8⁺ T cells, Treg cells (FOXP3⁺, CD4⁺ and CD25⁺) and antigen-driven regulatory CD4⁺ T cells, as well as other cell types (i.e., dendritic cells, macrophages, B cells and eosinophils), produce IL-10.

Studies conducted with a wide range of intracellular protozoan experimental infection models (i.e., Leishmania major, Trypanosoma cruzi and Toxoplasma gondii) have highlighted the central role of IL-10 in the outcome of parasitic infection. However, it is hard to conciliate all the studies attempting to delineate the nature of T cells involved in IL-10 secretion that play a role in different facets of disease progression or healing: clearance, persistence and development of immunopathological processes.

For instance, a fascinating case is that of L. major infection of genetically resistant C57BL/6 mice where the parasites persist in chronic sites after spontaneous healing of the dermal lesions. The release of IL-10 and IFN-γ by T cells appears to play a major role in this process [17]. Indeed, transient treatment of infected mice during the chronic phase with anti-IL-10 receptor antibodies achieved sterile cure. However, mice that were cleared of the parasite during the chronic stage of their primary infection failed to control a secondary parasite challenge. Thus, maintenance of a few parasites at the lesion site is necessary for the maintenance of acquired immunity to L. major. Taking into account the paradoxical function of Treg cells, suppressing effector T-cell functions at the site of infection and at the same time maintaining the recirculation of memory cells that confer powerful immunity to reinfection, it seems difficult to envisage the success of parasitic disease prevention by vaccination based on DNA and/or defined parasite-derived molecules, although studies have shown that a Leishmania-derived recombinant polyprotein, Leish-111f, could be a potential vaccine candidate [18].

It is interesting to remember some basic aspects mentioned previously that may explain the high complexity level of the host–parasite interplay at the immunological level: the parasites have complex life cycles (i.e., different developmental stages expressing various sets of antigens); different parasite strains coexist; a certain degree of variability at the genetic level (e.g., sequence polymorphism of genes encoding immunoregulatory factors); different tissue localization (i.e., spleen, liver and brain); and the genetic background of the host; among other factors.

The deficiency of key parasite metabolic enzymes, as achieved through targeted disruption of their genes, resulted in the production of parasite lines with low virulence in vitro and in vivo upon infection of animal models [4,12]. Although in certain cases the parasites may persist in vivo for a relatively long period of time, they did not cause disease [19]. Furthermore, using a Leishmania infantum lacking one SIR2RP1 gene copy as an immunizing agent in a murine model experimental infection, a strong correlation was found between the elimination of the parasites and an increased Leishmania-specific IFN-γ:IL-10 ratio, suggesting that the polarization to a high IFN-γ:low IL-10 ratio after challenge could be considered as a clear indicator of a vaccine success [20]. Taken together, these observations provide the hope that potential attenuated antiparasitic vaccine candidates could be developed in the future.

Financial disclosure

The author has no relevant financial interests related to this manuscript, including employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.