Case of Wound Myiasis in a Squamous Cell Carcinoma Lesion of the Scalp.

Myiasis refers to the infestation of living humans and vertebrate animals by dipterous larvae. Many organs can be infested by fly larvae, but cutaneous and wound myiases are the most frequently encountered clinical forms. Persistent ulcer or non-healing wound is one of the symptoms of squamous cell carcinoma which is the second most common skin cancer in the world. Here we report a case of an elderly man with a severe wound myiasis in a squamous cell carcinoma lesion of the scalp. The maggots were confirmed to be Lucilia sericata which are widespread flies in Japan. Human myiasis is rarely reported in Japan, but patients with necrotic, hemorrhaging, or pus-filled wounds are susceptible to infestation. It is necessary for doctors and nurses to ensure that their patients change their dressings daily and keep their wounds clean.

Corrigendum: ArfX2 GTPase Regulates Trafficking From the Trans-Golgi to Lysosomes and Is Necessary for Liver Abscess Formation in the Protozoan Parasite Entamoeba histolytica.

[This corrects the article DOI: 10.3389/fcimb.2021.794152.].

Immunoglobulin G responses to variant forms of Plasmodium vivax merozoite surface protein 9 upon natural infection in Thailand.

Merozoite surface protein 9 (MSP9) constitutes a ligand complex involved in erythrocyte invasion by malarial merozoites and is a promising vaccine target. Plasmodium vivax MSP9 (PvMSP9) is immunogenic upon natural malaria exposure. To address whether sequence diversity in PvMSP9 among field isolates could affect natural antibody responses, the recombinant proteins representing two variants each for the N- and the C-terminal domains of PvMSP-9 were used as antigens to assess antibody reactivity among 246 P. vivax-infected patients' sera from Tak and Ubon Ratchathani Provinces in Thailand. Results revealed that the seropositivity rates of IgG antibodies to the N-terminal antigens were higher than those to the C-terminal antigens (87.80% vs. 67.48%). Most seropositive sera were reactive to both variants, suggesting the presence of common epitopes. Variant-specific antibodies to the N- and the C-terminal antigens were detected in 15.85% and 16.70% of serum samples, respectively. These seropositivity rates were not significant difference between provinces. The seropositivity rates, levels and avidity of anti-PvMSP9 antibodies exhibited positive trends towards increasing malaria episodes. The IgG isotype responses to the N- and the C-terminal antigens were mainly IgG1 and IgG3. The profile of IgG responses may have implications for development of PvMSP9-based vaccine.

ArfX2 GTPase Regulates Trafficking From the Trans-Golgi to Lysosomes and Is Necessary for Liver Abscess Formation in the Protozoan Parasite Entamoeba histolytica.

Entamoeba histolytica is the causative agent of amoebic dysentery and liver abscess in humans. The parasitic lifestyle and the virulence of the protist require elaborate biological processes, including vesicular traffic and stress management against a variety of reactive oxygen and nitrogen species produced by the host immune response. Although the mechanisms for intracellular traffic of representative virulence factors have been investigated at molecular levels, it remains poorly understood whether and how intracellular traffic is involved in the defense against reactive oxygen and nitrogen species. Here, we demonstrate that EhArfX2, one of the Arf family of GTPases known to be involved in the regulation of vesicular traffic, was identified by comparative transcriptomic analysis of two isogenic strains: an animal-passaged highly virulent HM-1:IMSS Cl6 and in vitro maintained attenuated avirulent strain. EhArfX2 was identified as one of the most highly upregulated genes in the highly virulent strain. EhArfX2 was localized to small vesicle-like structures and largely colocalized with the marker for the trans-Golgi network SNARE, EhYkt6, but neither with the endoplasmic reticulum (ER)-resident chaperon, EhBip, nor the cis-Golgi SNARE, EhSed5, and Golgi-luminal galactosyl transferase, EhGalT. Expression of the dominant-active mutant form of EhArfX2 caused an increase in the number of lysosomes, while expression of the dominant-negative mutant led to a defect in lysosome formation and cysteine protease transport to lysosomes. Expression of the dominant-negative mutant in the virulent E. histolytica strain caused a reduction of the size of liver abscesses in a hamster model. This defect in liver abscess formation was likely at least partially attributed to reduced resistance to nitrosative, but not oxidative stress in vitro. These results showed that the EhArfX2-mediated traffic is necessary for the nitrosative stress response and virulence in the host.

Import of Entamoeba histolytica Mitosomal ATP Sulfurylase Relies on Internal Targeting Sequences.

Mitochondrial matrix proteins synthesized in the cytosol often contain amino (N)-terminal targeting sequences (NTSs), or alternately internal targeting sequences (ITSs), which enable them to be properly translocated to the organelle. Such sequences are also required for proteins targeted to mitochondrion-related organelles (MROs) that are present in a few species of anaerobic eukaryotes. Similar to other MROs, the mitosomes of the human intestinal parasite Entamoeba histolytica are highly degenerate, because a majority of the components involved in various processes occurring in the canonical mitochondria are either missing or modified. As of yet, sulfate activation continues to be the only identified role of the relic mitochondria of Entamoeba. Mitosomes influence the parasitic nature of E. histolytica, as the downstream cytosolic products of sulfate activation have been reported to be essential in proliferation and encystation. Here, we investigated the position of the targeting sequence of one of the mitosomal matrix enzymes involved in the sulfate activation pathway, ATP sulfurylase (AS). We confirmed by immunofluorescence assay and subcellular fractionation that hemagluttinin (HA)-tagged EhAS was targeted to mitosomes. However, its ortholog in the δ-proteobacterium Desulfovibrio vulgaris, expressed as DvAS-HA in amoebic trophozoites, indicated cytosolic localization, suggesting a lack of recognizable mitosome targeting sequence in this protein. By expressing chimeric proteins containing swapped sequences between EhAS and DvAS in amoebic cells, we identified the ITSs responsible for mitosome targeting of EhAS. This observation is similar to other parasitic protozoans that harbor MROs, suggesting a convergent feature among various MROs in favoring ITS for the recognition and translocation of targeted proteins.

Rab5b-Associated Arf1 GTPase Regulates Export of N-Myristoylated Adenylate Kinase 2 From the Endoplasmic Reticulum in Plasmodium falciparum.

Plasmodium falciparum extensively remodels human erythrocytes by exporting hundreds of parasite proteins. This remodeling is closely linked to the Plasmodium virulence-related functions and immune evasion. The N-terminal export signal named PEXEL (Plasmodium export element) was identified to be important for the export of proteins beyond the PVM, however, the issue of how these PEXEL-positive proteins are transported and regulated by Rab GTPases from the endoplasmic reticulum (ER) to the cell surface has remained poorly understood. Previously, we identified new aspects of the trafficking of N-myristoylated adenylate kinase 2 (PfAK2), which lacks the PEXEL motif and is regulated by the PfRab5b GTPase. Overexpression of PfRab5b suppressed the transport of PfAK2 to the parasitophorous vacuole membrane and PfAK2 was accumulated in the punctate compartment within the parasite. Here, we report the identification of PfRab5b associated proteins and dissect the pathway regulated by PfRab5b. We isolated two membrane trafficking GTPases PfArf1 and PfRab1b by coimmunoprecipitation with PfRab5b and via mass analysis. PfArf1 and PfRab1b are both colocalized with PfRab5b adjacent to the ER in the early erythrocytic stage. A super-resolution microgram of the indirect immunofluorescence assay using PfArf1 or PfRab1b- expressing parasites revealed that PfArf1 and PfRab1b are localized to different ER subdomains. We used a genetic approach to expresses an active or inactive mutant of PfArf1 that specifically inhibited the trafficking of PfAK2 to the parasitophorous vacuole membrane. While expression of PfRab1b mutants did not affect in the PfAK2 transport. In contrast, the export of the PEXEL-positive protein Rifin was decreased by the expression of the inactive mutant of PfRab1b or PfArf1. These data indicate that the transport of PfAK2 and Rifin were recognized at the different ER subdomain by the two independent GTPases: PfAK2 is sorted by PfArf1 into the pathway for the PV, and the export of Rifin might be sequentially regulated by PfArf1 and PfRab1b.

A 3-Year Overall Survival Update From a Phase 2 Study of Chemoselection With DCF and Subsequent Conversion Surgery for Locally Advanced Unresectable Esophageal Cancer.

BACKGROUND: A multicenter phase 2 trial analysed chemoselection with docetaxel plus 5-fluorouracil and cisplatin (DCF) induction chemotherapy (ICT) and subsequent conversion surgery (CS) for locally advanced unresectable esophageal cancer. This study presents updated 3-year analyses to further characterize the impact of DCF-ICT followed by CS. METHODS: Esophageal cancer patients with clinical T4 disease, unresectable supraclavicular lymph node metastasis, or both were eligible for this study. The treatment starts with DCF-ICT, followed by CS if the cancer is resectable, or by concurrent chemoradiation if it is not resectable. This updated analysis presents 3-year overall survival (OS), 3-year progression-free survival (PFS), and pattern of relapse. RESULTS: The median follow-up period for the patients surviving without death was 39.3 months. The estimated 1-year OS was 66.7%, and the lower limit of the 80% confidence interval (CI) was 54.6%. The estimated 3-year OS was 46.6% (95% CI 34.2-63.5%). The OS for the patients who underwent R0 resection (n = 19) was significantly longer than for those who did not (3-year OS: 71.4% vs. 30.1%). The estimated 1-year PFS was 50.6%, and the 3-year PFS was 39.6%. The PFS for R0 was significantly longer than for non-R0 (3-year PFS: 61.3% vs 25.0%). Recurrence or progression at the primary site was observed in 31% of the non-R0 group. The rate of distant metastasis did not differ significantly between the non-R0 and R0 groups (21% vs 16%). CONCLUSIONS: Long-term follow-up evaluation confirmed that DCF chemoselection aimed at CS is feasible and promising in terms of survival for patients with locally advanced esophageal cancer.

Whole genome sequencing of Entamoeba nuttalli reveals mammalian host-related molecular signatures and a novel octapeptide-repeat surface protein.

The enteric protozoa Entamoeba histolytica is the causative agent of amebiasis, which is one of the most common parasitic diseases in developed and developing countries. Entamoeba nuttalli is the genetically closest species to E. histolytica in current phylogenetic analyses of Entamoeba species, and is prevalent in wild macaques. Therefore, E. nuttalli may be a key organism in which to investigate molecules required for infection of human or non-human primates. To explore the molecular signatures of host-parasite interactions, we conducted de novo assembly of the E. nuttalli genome, utilizing self-correction of PacBio long reads and polishing corrected reads using Illumina short reads, followed by comparative genomic analysis with two other mammalian and a reptilian Entamoeba species. The final draft assembly of E. nuttalli included 395 contigs with a total length of approximately 23 Mb, and 9,647 predicted genes, of which 6,940 were conserved with E. histolytica. In addition, we found an E. histolytica-specific repeat known as ERE2 in the E. nuttalli genome. GO-term enrichment analysis of mammalian host-related molecules indicated diversification of transmembrane proteins, including AIG1 family and BspA-like proteins that may be involved in the host-parasite interaction. Furthermore, we identified an E. nuttalli-specific protein that contained 42 repeats of an octapeptide ([G,E]KPTDTPS). This protein was shown to be localized on the cell surface using immunofluorescence. Since many repeat-containing proteins in parasites play important roles in interactions with host cells, this unique octapeptide repeat-containing protein may be involved in colonization of E. nuttalli in the intestine of macaques. Overall, our draft assembly provides a valuable resource for studying Entamoeba evolution and host-parasite selection.

Novel lineage-specific transmembrane ß-barrel proteins in the endoplasmic reticulum of Entamoeba histolytica.

ß-barrel outer membrane proteins (BOMPs) are essential components of outer membranes of Gram-negative bacteria and endosymbiotic organelles, usually involved in the transport of proteins and substrates across the membrane. Based on the analysis of our in silico BOMP predictor data for the Entamoeba histolytica genome, we detected a new transmembrane ß-barrel domain-containing protein, EHI_192610. Sequence analysis revealed that this protein is unique to Entamoeba species, and it exclusively clusters with a homolog, EHI_099780, which is similarly lineage specific. Both proteins possess an N-terminal signal peptide sequence as well as multiple repeats that contain dyad hydrophobic periodicities. Data from immunofluorescence assay of trophozoites expressing the respective candidates showed the absence of colocalization with mitosomal marker, and interestingly demonstrated partial colocalization with endoplasmic reticulum (ER) proteins instead. Integration to organellar membrane was supported by carbonate fractionation assay and immunoelectron microscopy. CD analysis of reconstituted proteoliposomes containing EHI_192610 showed a spectrum demonstrating a predominant ß-sheet structure, suggesting that this protein is ß-strand rich. Furthermore, the presence of repeat regions with predicted transmembrane ß-strand pairs in both EHI_192610 and EHI_099780, is consistent with the hypothesis that BOMPs originated from the amplification of ßß-hairpin modules, suggesting that the two Entamoeba-specific proteins are novel ß-barrels, intriguingly localized partially to the ER membrane.

Influence of Heterologous Transplant of DNA-lacking Mitochondria from Entamoeba histolytica on Proliferation of Entamoeba invadens.

In mitochondria, compatibility of proteins encoded in mitochondrial DNA and nuclear DNA is essential for the normal functioning of the organelle. Incompatibility between mitochondrial and nuclear DNA can lead to dysfunctional respiration, mitochondrial diseases, and lethal problems, which suggests that the presence of heterologous mitochondria is unfavorable. In a previous study, we established a transplant method for DNA-lacking mitochondria (mitosomes) in the anaerobic protozoan Entamoeba histolytica. In this study, interspecies transplant of mitosomes from E. histolytica into Entamoeba invadens, which is a parasitic protozoon of reptiles, was performed using the microinjection method at various temperatures and injection volumes. When E. invadens was used as recipient, it showed higher tolerance to a lower temperature and larger injection volume, in comparison with E. histolytica. After microinjection, donor mitosomes expressing HA-tag conjugated protein were observed in recipient cells by immunofluorescent staining. The heterologous mitosomes-injected cells proliferated and growth rate of the microinjected-cells was similar to that of intact cells. Therefore, we conclude that interspecies transplant of DNA-lacking mitochondria does not result in incompatibility.

Reinventing an Organelle: The Reduced Mitochondrion in Parasitic Protists.

Mitochondria originated from the endosymbiotic event commencing from the engulfment of an ancestral α-proteobacterium by the first eukaryotic ancestor. Establishment of niches has led to various adaptations among eukaryotes. In anaerobic parasitic protists, the mitochondria have undergone modifications by combining features shared from the aerobic mitochondria with lineage-specific components and mechanisms; a diversified class of organelles emerged and are generally called mitochondrion-related organelles (MROs). In this review we summarize and discuss the recent advances in the knowledge of MROs from parasitic protists, particularly the themes such as metabolic functions, contribution to parasitism, dynamics, protein targeting, and novel lineage- specific proteins, with emphasis on the diversity among these organelles.

Naturally acquired IgG antibodies to thrombospondin-related anonymous protein of Plasmodium vivax (PvTRAP) in Thailand predominantly elicit immunological cross-reactivity.

BACKGROUND: Thrombospondin-related anonymous protein (TRAP) is a prime candidate for a malaria vaccine. Antibodies to Plasmodium vivax TRAP (PvTRAP) occur upon natural infection while specific antigenic domains remain to be addressed. METHODS: The PvTRAP sequences were determined from 73 P. vivax isolates from Tak and Ubon Ratchathani provinces collected in 2013. The recombinant proteins representing four variants each for domain II (A domain) and domain IV (thrombospondin repeat region) of PvTRAP circulating in these areas were used as antigens in enzyme-linked immunosorbent assay against 246 serum samples from P. vivax-infected patients in both provinces collected during 2013 and 2014. RESULTS: The prevalence of total IgG antibodies to at least one variant antigen of domain II and domain IV was 63.8% and 71.5%, respectively. Differential IgG antibody responses to these variant antigens of each domain were observed. Total IgG antibody responses to the variant antigens of each domain upon pairwise comparisons were highly correlated, suggesting immunological cross-reactivity in the majority of serum samples. A smaller proportion of serum samples contained non-cross-reactive antibodies to variants of each domain; particularly domain II in which amino acid differences significantly influenced antibody recognition. Previous malaria exposure positively affected antibody responses to domain IV. Positive seroconversion and rising antibody titres occurred within a few weeks after resolution of infections. CONCLUSIONS: Both domains II and IV are targets of naturally acquired IgG antibodies. Despite sequence variation in these domains, most antibody responses were cross-reactive. A cross-sectional evaluation of antibodies to PvTRAP during acute infection could underestimate the seroprevalence.

Development of a sensitive immunochromatographic kit using fluorescent silica nanoparticles for rapid serodiagnosis of amebiasis.

We have previously shown that the C-terminal region of the intermediate subunit of Entamoeba histolytica galactose- and N-acetyl-D-galactosamine-inhibitable lectin (C-Igl) is a useful antigen for serodiagnosis of amebiasis. An immunochromatographic kit was developed using fluorescent silica nanoparticles coated with C-Igl prepared in Escherichia coli. Samples for examination were added to the freeze-dried particles and then applied to the immunochromatographic device, in which a test line on the membrane was also coated with C-Igl. Fluorescent intensity was measured using a hand-held reader. In an evaluation of the kit using a human monoclonal antibody, the minimum amount of C-Igl specific antibody showing positive results was 100 pg. In the evaluation of serum samples with different antibody titers in indirect immunofluorescent antibody tests in the kit, 20 µL of serum was sufficient to obtain positive results at 30 min. Serum samples from symptomatic patients with amebic colitis and amebic liver abscess and those from asymptomatic E. histolytica-cyst carriers showed positive results in the kit. Based on evaluation using sera from healthy controls and patients with other infectious diseases, the sensitivity and specificity of the kit were 100 and 97.6%, respectively. Therefore, we conclude that the newly developed kit is useful for rapid serodiagnosis of amebiasis.

Hetero-oligomer of dynamin-related proteins participates in the fission of highly divergent mitochondria from Entamoeba histolytica.

Entamoeba histolytica is an anaerobic parasitic protist and possesses mitosomes, one of the most highly divergent mitochondrion-related organelles (MROs). Although unique metabolism and protein/metabolite transport machinery have been demonstrated in Entamoeba mitosomes, the mechanism of mitosomal fusion and fission remains to be elucidated. In this study, we demonstrate that two dynamin-related proteins (DRPs) are cooperatively involved in the fission of Entamoeba mitosomes. Expression of a dominant negative form of EhDrpA and EhDrpB, and alternatively, repression of gene expression of EhDrpA and EhDrpB genes, caused elongation of mitosomes, reflecting inhibition of mitosomal fission. Moreover, EhDrpA and EhDrpB formed an unprecedented hetero-oligomeric complex with an approximate 1:2 to 1:3 ratio, suggesting that the observed elongation of mitosomes is likely caused by the disruption and instability of the complex caused by an imbalance in the two DRPs. Altogether, this is the first report of a hetero-oligomeric DRP complex which participates in the fission of mitochondria and MROs.

Comparison of hemolytic activity of the intermediate subunit of Entamoeba histolytica and Entamoeba dispar lectins.

Galactose and N-acetyl-D-galactosamine-inhibitable lectin of Entamoeba histolytica has roles in pathogenicity and induction of protective immunity in rodent models of amoebiasis. Recently, the intermediate subunit of the lectin, Igl1, of E. histolytica has been shown to have hemolytic activity. However, the corresponding lectin is also expressed in a non-virulent species, Entamoeba dispar, and another subunit, Igl2, is expressed in the protozoa. Therefore, in this study, we compared the activities of Igl1 and Igl2 subunits from E. histolytica and E. dispar using various regions of recombinant Igl proteins expressed in Escherichia coli. The recombinant E. dispar Igl proteins had comparable hemolytic activities with those of E. histolytica Igl proteins. Furthermore, Igl1 gene-silenced E. histolytica trophozoites showed less hemolytic activity compared with vector-transfected trophozoites, indicating that the expression level of Igl1 protein influences the activity. These results suggest that the lower hemolytic activity in E. dispar compared with E. histolytica reflects the lower expression level of Igl1 in the E. dispar parasite.

Behavior of DNA-lacking mitochondria in Entamoeba histolytica revealed by organelle transplant.

The anaerobic protozoan parasite Entamoeba histolytica has mitosomes that are mitochondria lacking some canonical functions and organelle DNA. Mitosomes play an important role in the life cycle of the parasite. The distribution of proteins in mitosomes is not uniform, and how mitosomes are maintained and retained is unknown. To answer these questions, we developed a transplant method for mitosomes with hemagglutinin-tagged protein into recipient cells containing mitosomes with Myc-tagged protein. Immunofluorescence staining showed that the two protein tags colocalized in single mitosomes in some recipient cells. These results suggest that our transplant method can be used in anaerobic protozoa and that donor mitosomes may obtain recipient proteins through fusion with other mitosomes or through de novo synthesis of proteins in recipient cells.

Heterogeneity of the serine synthetic pathway in Entamoeba species.

Phosphoserine phosphatase (PSP) catalyzes the third step of the phosphorylated serine biosynthetic pathway, and occurred multiple times in evolution, while enzymes catalyzing the first and second steps in the pathway have single respective origins. In the present study, we examined the existence of PSP among genus Entamoeba including a human enteric parasite, Entamoeba histolytica. E. histolytica as well as majority of Entamoeba species have the first and second enzymes, but lacks PSP. In contrast, a reptilian enteric parasite, Entamoeba invadens possesses canonical PSP. Thus, there are variations in the existence of the serine biosynthetic ability among Entamoeba species.

Differential remodelling of peroxisome function underpins the environmental and metabolic adaptability of diplonemids and kinetoplastids.

The remodelling of organelle function is increasingly appreciated as a central driver of eukaryotic biodiversity and evolution. Kinetoplastids including Trypanosoma and Leishmania have evolved specialized peroxisomes, called glycosomes. Glycosomes uniquely contain a glycolytic pathway as well as other enzymes, which underpin the physiological flexibility of these major human pathogens. The sister group of kinetoplastids are the diplonemids, which are among the most abundant eukaryotes in marine plankton. Here we demonstrate the compartmentalization of gluconeogenesis, or glycolysis in reverse, in the peroxisomes of the free-living marine diplonemid, Diplonema papillatum Our results suggest that peroxisome modification was already under way in the common ancestor of kinetoplastids and diplonemids, and raise the possibility that the central importance of gluconeogenesis to carbon metabolism in the heterotrophic free-living ancestor may have been an important selective driver. Our data indicate that peroxisome modification is not confined to the kinetoplastid lineage, but has also been a factor in the success of their free-living euglenozoan relatives.

A novel Mitosomal ß-barrel Outer Membrane Protein in Entamoeba.

Entamoeba possesses a highly divergent mitochondrion-related organelle known as the mitosome. Here, we report the discovery of a novel protein in Entamoeba, which we name Mitosomal ß-barrel Outer Membrane Protein of 30 kDa (MBOMP30). Initially identified through in silico analysis, we experimentally confirmed that MBOMP30 is indeed a ß-barrel protein. Circular dichroism analysis showed MBOMP30 has a predominant ß-sheet structure. Localization to Entamoeba histolytica mitosomes was observed through Percoll-gradient fractionation and immunofluorescence assay. Mitosomal membrane integration was demonstrated by carbonate fractionation, proteinase K digestion, and immunoelectron microscopy. Interestingly, the deletion of the putative ß-signal, a sequence believed to guide ß-barrel outer membrane protein (BOMP) assembly, did not affect membrane integration, but abolished the formation of a ~240 kDa complex. MBOMP30 represents only the seventh subclass of eukaryotic BOMPs discovered to date and lacks detectable homologs outside Entamoeba, suggesting that it may be unique to Entamoeba mitosomes.

Highly divergent mitochondrion-related organelles in anaerobic parasitic protozoa.

The mitochondria have arisen as a consequence of endosymbiosis of an ancestral α-proteobacterium with a methane-producing archae. The main function of the canonical aerobic mitochondria include ATP generation via oxidative phosphorylation, heme and phospholipid synthesis, calcium homeostasis, programmed cell death, and the formation of iron-sulfur clusters. Under oxygen-restricted conditions, the mitochondrion has often undergone remarkable reductive alterations of its content and function, leading to the generation of mitochondrion-related organelles (MROs), such as mitosomes, hydrogenosomes, and mithochondrion-like organelles, which are found in a wide range of anaerobic/microaerophilic eukaryotes that include several medically important parasitic protists such as Entamoeba histolytica, Giardia intestinalis, Trichomonas vaginalis, Cryptosporidium parvum, Blastocystis hominis, and Encephalitozoon cuniculi, as well as free-living protists such as Sawyeria marylandensis, Neocallimastix patriciarum, and Mastigamoeba balamuthi. The transformation from canonical aerobic mitochondria to MROs apparently have occurred in independent lineages, and resulted in the diversity of their components and functions. Due to medical and veterinary importance of the MRO-possessing human- and animal-pathogenic protozoa, their genomic, transcriptomic, proteomic, and biochemical evidence has been accumulated. Detailed analyses of the constituents and functions of the MROs in such anaerobic pathogenic protozoa, which reside oxygen-deprived or oxygen-poor environments such as the mammalian intestine and the genital organs, should illuminate the current evolutionary status of the MROs in these organisms, and give insight to environmental constraints that drive the evolution of eukaryotes and their organelles. In this review, we summarize and discuss the diverse metabolic functions and protein transport systems of the MROs from anaerobic parasitic protozoa.