Virulence factors

Parasites developed several strategies for their survival and host tissue invasion. Helminths express potent molecules mainly for immunomodulation, which is why they stay in their hosts for years. Helminths display several mechanisms not only to evade host immune response(s), but also to preserve the host for as long as they could live. In contrast, protozoa evolve several policies primarily for pathogenesis, and invasion. Therefore, variable clinical manifestations are reported in protozoal diseases. Both symptomatic and asymptomatic cases are commonly observed in amoebiasis, giardiasis, trichomoniasis, cryptosporidiosis and toxoplasmosis, while mild, moderate, and severe cases occur in malaria, leishmaniasis, African sleeping sickness and Chagas’ disease. This was primarily attributed to strains variability and to a lesser extent, to host immune response(s). With recent evolutionary technology in molecular parasitology and bioinformatics, several molecules are established as virulence factors. These factors encourage researchers and scientists to develop novel drug targets and/or vaccine candidates. The present review aims to highlight, and review virulence strategies adapted by parasites to invade host tissue, enhance its replication and spread, as well as other processes for immunomodulation or immunoevasion of host immune response(s). Abbreviations: CATH: Cathepsin; CP: Cysteine protease; CPI: Cysteine protease inhibitor; CYS: Cystatin; endogenous CPI; EMP1: Erythrocyte membrane protein 1; EVs: Extracellular vesicles; GP: Glycoprotein; HSP: Heat shock protein; MEROPS: Proteases database (www.ebi.ac.uk/merops/); MP: Metalloprotease; PV: Parasitophorous vacuole; SP: Serine protease; SUB: Subtilase, subtilisin-like proteases; VSPs: Variant surface proteins. Parasite virulence Abaza 77 communicate within their own populations for several functions including growth promotion, host immune system evasion, disease transmission, and manipulation of micro-environmental stress. Communication is also directed to the host through trafficking transfer of effector molecules to host cells to manipulate host gene expression, and consequently mediate parasite pathogenicity[7]. • Extracellular vesicles (EVs): These are nano-scale lipid bilayer membrane-bound structures. They contribute in the trafficking of virulence factors required for parasite nutrition, cytoadherence, host cell migration and invasion, cytotoxicity, and host immune system evasion[7]. Reviewing literature, EVs are classified into exosomes, microvesicles and apoptotic bodies. Exsomes and microvesicles are released with conserved biogenesis and functional roles. For example, exsomes in G. lamblia, T. vaginalis and pathogenic trypanosomatids are released at the flagellar pocket, whereas they are intracellularly released in apicomplexans as microvesicles[8]. It is worth mentioning that Plasmodium EVs include exonemes, micronemes, and mononemes. They are merozoite secretory apical organelles that express in the parasitophorous vacuole (PV) subtilases 1 and 2 (SUB1, SUB2) and rhomboid-1 (ROM1), respectively. Their role in egress and de novo invasion cascade will be discussed later[9]. • Egress cascade: A wide spectrum of pathogenic bacteria and protozoa adapt several strategies to enter and exit their host with optimum rates of survival, replication, progression through life cycle stages as well as transmission. Pathogen egress is of fundamental importance due to its close association with pathogen spread, transmission and inflammation processes. Accordingly, molecules involved in egress mechanism(s) are considered key steps in transmission and infection, i.e., they are considered indirect virulence factors. In their review Friedrich, and his colleagues[10] listed in a table several molecules involved in egress mechanism(s) in T. gondii, and species of Plasmodium, Trypanosoma and Leishmania. Egress strategies are designed to overcome host cellular membranes, cell cytoskeleton, and organelles. Pathogens utilize proteases, lipases, and pore-forming proteins as molecular effectors of active egress. Also, pathogens use molecular mimicry to simulate host cellular cytoskeleton dynamics. For instance, some pathogens such as T. cruzi escape PV to replicate in the host cell cytosol. However, the parasite has to control this first egress step to preserve host cell integrity. After replication, a second controlled egress event takes place to release replicates that infect new host cells[10]. In Plasmodium spp., egress and de novo invasion cascade involves the following steps: 1) while SUB1 is involved in merozoites egress, SUB2 is required for merozoites de novo RBCs invasion; 2) degradation of PV membrane; 3) breakdown of both RBC’ membrane and cytoskeleton is essentially done by SUB1, SUB2 and ROM1, and 4) serine repeat antigens (SERA5 and SERA6); merozoite surface proteins (MSP1, 6 and 7) released from SUB1, and SUB2 contribute with ROM1 to catalyze the intermembrane cleavage leading to de novo RBCs invasion. Plasmepsins, and aspartyl proteases, also established their role in egress and de novo cascade, acting as a maturation factor for rhoptery proteins that control SUB1 maturation[9]. • Ubiquitin-proteasome system (UBS): The UPS has essential roles in several cellular pathways including those required for parasite biology and virulence, i.e. proliferation and cell differentiation, which are the key steps in protozoal colonization inside its host. Turnover of intracellular proteins is carried out by two proteolytic organelles: lysosomes and proteasomes, utilizing their molecules released by UPS[11]. The most commonly reported molecule is 20S, described as a barrel-shaped assembly of 28 protein subunits. For parasite proliferation and differentiation, 20S proteasome degrades its own proteins to oligopeptides (3-15 amino acids), followed by peptide hydrolysis. Therefore, hydrolyzed amino acids are used for biosynthesis of life cycle stages. Muñoz and her colleagues[12] from Chile reviewed roles and functions of 20S in E. histolytica, pathogenic trypanosomatids and T. gondii validating them as virulence factors and potential drug targets. They also claimed that UPS is not only a degrading machine, but it is also employed as regulatory factor involved in several pathways including cell growth, inflammatory response, and antigen processing[12]. Identification of virulence factors: No doubt that identification of virulence factors would help researchers to discover or develop new or synthetic inhibitors to be used as novel drugs and/or vaccine candidates, utilizing virtual or high-throughput screening. Reviewing literature, two approaches were utilized to identify virulence factors, either comparative transcriptomic analysis between virulent and avirulent isolates or gene knock-out, i.e. RNA interference to identify gene function(s). Mechanisms involved in parasite virulence: Parasites utilized several strategies to establish their persistence in the host, i.e. alive (survival) and active (virulent). Reported utilized mechanisms to achieve these tasks include proteolytic activity, antigenic variation, protein folding and mechanical mechanism (Table 1). Proteolytic activity, achieved by several proteases, is the main strategy reported in almost all parasites. Antigenic variation comes next and is most frequently observed in different species of Leishmania, Plasmodium, and T. cruzi. Protein folding achieved by genes encoding heat shock proteins (HSPs) is less frequently reported in few parasites. It is worth mentioning that the mechanical mechanism is only reported in G. lamblia. PARASITOLOGISTS UNITED JOURNAL 78 It should be considered that virulence may occur, in some instances, due to non-parasitic molecules such as missed diagnosis, ineffective treatment or drug resistance, immunosuppression and associated endosymbiosis. Prior to discussing parasitic virulence factors, two points are to be considered: endosymbiosis and trafficking of virulence factors through cellular membranes. Endosymbiosis: There is much controversy over the contribution of intestinal enteropathogenic Escherichia coli and E. histolytica virulence. Incubation of E. coli in E. histolytica cultures can decrease[22] or increase[23] its virulence. A recent study showed that either enteropathogenic E. coli or nonpathogenic Entamoeba coli modified E. histolytica virulence causing amoebiasis in cell line culture as well as in experimental models due to increased proteolytic activity of expressed EhCPs 1, 2, 4, and 5[24]. On the other hand, Burgess and her colleagues[25] focused in their review on the contribution of intestinal nonpathogenic microbiota not only on intestinal protozoa, but also on extra-intestinal protozoa as Plasmodium spp. and T. vaginalis. The reviewers attributed decreased virulence in intestinal protozoa to decreased parasite cytoadherence at the mucosal sites. However, intestinal microbiota may alter systemic immunity by alteration of granulopoiesis and/or adaptive immunity and by increasing virulence in non-intestinal protozoa. In addition, the reviewers tabulated commonly reported intestinal nonpathogenic microbiota associated with E. histolytica, G. lamblia, T. vaginalis. P. falciparum and species of Cryptosporidium and Blastocystis. Interestingly, it was concluded that treatment using microbiota may provide a costeffective prophylactic strategy for intestinal protozoal infections[25]. Treatment of filarial patients with tetracycline was suggested to cause worm sterility in symbioticaly associated filarial worms and Wolbachia. It was shown that recombinant Wolbachia surface protein predisposed to host immunoevasion, increasing disease pathogenicity and virulence[26]. Additionally, the role of T. vaginalis virus (TVV) in the parasite virulence was demonstrated in several studies; as induction of various phenotypic changes[27] and contribution in parasite cytoadherence[28,29]. Similar st