Expression of cysteine proteinases and cystatins in parasites and use of cysteine proteinase inhibitors in parasitic diseases. Part III: Protozoa (1)

Out of five classes of proteases (cysteine, serine, threonine, aspartate and glutamate), cysteine proteases (CPs) are responsible for hydrolysis of peptide bonds essential in several biological activities. In protozoa, as with helminths, not only do CPs play the major role in nutrients digestion, but they also have several functions for parasite survival such as differentiation of life cycle stages, immunomodulation of host immune response, and autophagy. Most wellcharacterized CPs in protozoa that were investigated in the last two decades belong to papain-family enzymes (clan CA, family C1). The present review highlights, in general, several aspects of CPs functions in protozoal survival and different strategies utilized in development of potent CPIs. The review also includes detailed data regarding T. gondii CPs, and their inhibitors wether exogenous (CPIs) or endogenous cystatins (CYSs). Abbreviations CALP: calpain; CATH: Cathepsin; CP: Cysteine proteinase; CPB: Cathepsin B; CPC: Cathepsin C; CPI: Cysteine proteinase inhibitor; CPL: Cathepsin L; CYS: Cystatin; MCA: Metacaspase; MIC: Microneme; PCD: Programmed cell death; PV: Parasitophorous vacuole; ROP: Rhoptry; VAC: Vacuolar compartment. CPs, CYSs, CPIs and T. gondii Abaza 9 succeeded to define 27, 24 and 18 genes, respectively. Amino acid sequences of the defined genes revealed high modular structure, suggesting the feasibility to utilize specific primers as diagnostic markers[5]. Recently, Siqueira-Neto et al.,[6] reviewed the proposed functions of the most characterized 29 CPs only in seven protozoa; E. histolytica (six), Leishmania spp. (six), Plasmodium spp. (five), T. gondii (five), T. cruzi (three), T. brucei (two), and Cryptosporidium spp. (two). It is evident that the most common proposed character of these CPs is a virulence factor to facilitate parasite survival and invasion. For each CP, the reviewers presented the mechanism(s) to achieve parasite invasion including induction of macrophage pro-inflammatory response, degradation of extracellular matrix, differentiation of life cycle stages, modulation of parasite metabolism, and autophagy. Mechanisms involved in immunoevasion and immunomodulation of host immune response are also proposed in all reviewed protozoa. There are other proposed mechanisms specified for some protozoa such as encystation-excystation transformation, and degradation of host IgA and IgG (E. histolytica), crossing blood brain barrier (T. brucei), hemoglobin degradation, enhancement of oocysts production, sporozoites invasion of hepatocytes, and apicoplast development and homeostasis (Plasmodium spp.), and high expression in tachyzoites for digestion of cytosolic proteins (T. gondii). Beside their role in parasite invasion, CPs of apicomplexan protozoa are required for pathogen exit from the infected cells to invade other cells and continue the infection. In Plasmodium spp. and T. gondii, being obligate intracellular pathogens, schizogony or endodyogeny, involve replication within a specialized parasitophorous vacuole (PV) to yield multiple merozoites or tachyzoites, respectively. Both host calcium and CALP-1 are implicated in rupture of the infected cells, while apicomplexan CALPs are implicated in escape of merozoites and tachyzoites from the PV by their proteolysis-dependent mechanism[7]. In another report published in 2009, the investigators discussed the role of CALP of apicomplexans Plasmodium spp. and T. gondii in parasite egress. They showed that in vitro addition of DCG04 (a derivative of nonspecific papain family protease inhibitor E64) to Plasmodium-infected erythrocytes revealed blocking in schizont-stage and trapping of merozoites in PV within intact red blood cell membranes. Infected RBCs were treated with saponin to dissolve PV membranes, then centrifuged to remove parasite cells, and pelleted to produce purified soluble fraction. Mass spectrometry identified only host CALP-1, confirming its involvement in RBCs rupture. When CALP-1 depleted erythrocytes were treated with DCG04, parasite kinetics was improved to some extent, suggesting the importance of apicomplexan CALPs in parasite egress. The investigators concluded that both CALPs of Plasmodium spp. and T. gondii exploit host cell CALPs to facilitate escape from PV or host plasma membrane, but they failed to explore the precise mechanism[8]. Apoptosis Apoptosis is an essential host pathway contributing both innate and acquired immune responses. It can be induced via either intrinsic or extrinsic pathways. The first is stimulated by cellular stress signals such as DNA damage, lack of essential growth factors, or infection. The extrinsic pathways are activated via death receptor ligation mechanism used by cytotoxic cells (T, natural killer, and non-lymphoid cells) to induce cell death[9]. It was reported that some cytotoxic cells can induce cell death via the perforin-dependent granule exocytosis pathway[10]. In intracellular pathogens such as viruses, bacteria and protozoa, host cytotoxicity plays an important role to establish efficient immune defense mechanism(s). On the other hand, intracellular pathogens must interfere with cell apoptosis to protect their host cells, and themselves, from cell death[11]. In a review published in 2011[12], the British scientists claimed that apoptosis is an essential phenomenon for normal development and survival in multicellular parasites, whereas its occurrence in unicellular protozoa seems strange since they have to evolve strategies to increase their replication, not death; i.e., self-regulate the intensity of infection in the host or vector. The first question in their review was “Do protozoa commit suicide to survive?" First, they drew a diagram showing that cell death is either passive, due to extrinsic factors, leading to rapid irreversible necrosis with membrane disruption and damage of organelles; or active, due to intrinsic factors, leading to programmed cell death (PCD), involved in a regulated step-manner which can be reversible before the final stage is reached. PCD is either slow leading to autophagy or fast resulting in apoptosis. In autophagy, there is downregulation of metabolic processes with digestion of organisms, while in apoptosis, there is controlled cascade with morphological events and functional cell breakdown and eventually cell death. Markers of cell apoptosis include DNA fragmentation, chromatin condensation, membrane’ blebs, cell shrinking, proteins cleavage by proteolysis, and release of proteins from mitochondria. The second question was "how cell apoptosis with parasite number reduction can assist the survivors?" The answer was that it depends on density of parasites, and that logically, no gained benefits will be obtained for low parasite density survivors. In contrast, if the parasite number is high enough to cause host or vector survival at risk, the best strategy for unicellular protozoa is to undergo apoptosis to maintain a sub-lethal density, i.e. higher apoptotic parasite number leads to bigger benefits to survivors. The third question was "how parasites get information about low or high density? Or is it the time to commit suicide or to proliferate?" The answer is it doesn’t matter to have this information, as natural PARASITOLOGISTS UNITED JOURNAL 10 selection shapes parasite strategy usually in line with the parasite density. The reviewers pointed out that “one-size-fits-all” strategy is the least outcome when variation in parasite density is an achieved experience during infections in different hosts. However, more sophisticated strategies become possible in case of parasites ability to get that information; as with other parasite strategies, e.g. gene mutations attempted in drug resistance. Previous studies reported that P. falciparum[13] and T. brucei[14] can determine the genetic diversity of their infections suggesting its ability to estimate the density or proliferation rate of their clonemates. Because caspases are limited to metazoans, the first description of caspase orthologues was proposed as paracaspases from animals and metacaspases (MCAs) from unicellular pathogens such as fungi and protozoa[15]. All caspases and orthologues are clan CD, family C14, however, they show difference only in substrate specificity. MCAs, with their highly acidic S1 pocket, have high basic specificity for arginine and lysine at the P1 position, rather than aspartic acid specificity for caspases[16]. Two types of MCAs are known, however, only type I was detected in protozoa. It is characterized by having a N-terminal prodomain, while type II MCAs have a linkage between the p20 and p10 domains instead[17]. It is worth mentioning that some, not all, of apoptotic markers were observed in unicellular protozoa such as T. cruzi, P. berghei, Leishmania spp., Blastocystis spp., and T. vaginalis[18-22]. On the other hand, MCAs role in parasite apoptosis was investigated in T. cruzi, L. major, and T. gondii[23-25]. Cysteine proteinase inhibitors (CPIs) Several strategies are employed to identify or synthesize safe and potent CPs inhibitory compounds. Virtual screening of 241 thousand compounds in ChemBridge database identified 24 CP inhibitors (CPIs), among them four compounds showed efficient CPs inhibition of P. falciparum and L. donovani[26]. Screening a library including synthesized thio-semicarbazones identified several promising leading compounds that showed high activity against falcipain-2, rhodesain and cruzain; the major CPs in P. falciparum, T. brucei, and T. cruzi, respectively. In addition, their toxicity was tested in mice and only one compound showed observable toxicity after 62 h. The investigators recommended further studies to validate use of thio-semicarbazones as CPIs[27]. Several gold compounds were investigated against CATH L-like and CATH B-like; the major CPs of T. brucei rhodesiense and L. mexicana, respectively. According to the promising results, some gold compounds showed