Anncaliia Algerae Research Articles

The intracellular parasite Anncaliia algerae induces a massive miRNA down-regulation in human cells.

Anncaliia algerae belongs to microsporidia, a group of obligate intracellular parasites related to fungi. These parasites are largely spread in water and food-webs and can infect a wide variety of hosts ranging from invertebrates to vertebrates including humans. In humans, microsporidian infections are mainly opportunistic as immunocompetent hosts can clear parasites naturally. Recent studies however have reported persistent microsporidian infections and have highlighted them as a risk factor in colon cancer. This may be a direct result of cell infection or may be an indirect effect of the infectious microenvironment and the host's response. In both cases, this raises the question of the effects of microsporidian infection at the host and host-cell levels. We aimed to address the question of human host intracellular response to microsporidian infection through a transcriptomic kinetic study of human foreskin fibroblasts (HFF) infected with A.algerae, a human infecting microsporidia with an exceptionally wide host range. We focused solely on host response studying both coding and small non-coding miRNA expression. Our study revealed a generalized down-regulation of cell miRNAs throughout infection with up to 547 different miRNAs downregulated at some timepoints and also transcriptomic dysregulations that could facilitate parasite development with immune and lipid metabolism genes modulation. We also hypothesize possible small nucleic acid expropriation explaining the miRNA downregulation. This work contributes to a better understanding of the dialogue that can occur between an intracellular parasite and its host at the cellular level, and can guide future studies on microsporidian infection biology to unravel the mode of action of these minimalist parasites at the tissue or host levels.We have also generated a kinetic and comprehensive transcriptomic data set of an infectious process that can help support comparative studies in the broader field of parasitology. Lastly, these results may warrant for caution regarding microsporidian exposure and persistent infections.

Identification and localization of polar tube proteins in the extruded polar tube of the microsporidian Anncaliia algerae

Microsporidia are obligate intracellular parasites able to infect a wide range of hosts from invertebrates to vertebrates. The success of their invasion process is based on an original organelle, the polar tube, which is suddenly extruded from the spore to inoculate the sporoplasm into the host cytoplasm. The polar tube is mainly composed of proteins named polar tube proteins (PTPs). A comparative analysis allowed us to identify genes coding for 5 PTPs (PTP1 to PTP5) in the genome of the microsporidian Anncaliia algerae. While PTP1 and PTP2 are found on the whole polar tube, PTP3 is present in a large part of the extruded polar tube except at its end-terminal part. On the contrary, PTP4 is specifically detected at the end-terminal part of the polar tube. To complete PTPs repertoire, sequential sporal protein extractions were done with high concentration of reducing agents. In addition, a method to purify polar tubes was developed. Mass spectrometry analysis conducted on both samples led to the identification of a PTP3-like protein (PTP3b), and a new PTP (PTP7) only found at the extremity of the polar tube. The specific localization of PTPs asks the question of their roles in cell invasion processes used by A. algerae.

Anncaliia algerae Microsporidiosis Diagnosed by Metagenomic Next-Generation Sequencing, China

We report a case of Anncaliia algerae microsporidia infection in an immunosuppressed kidney transplant recipient in China. Light microscopy and transmission electron microscopy initially failed to identify A. algerae, which eventually was detected by metagenomic next-generation sequencing. Our case highlights the supporting role of metagenomic sequencing in early identification of uncommon pathogens.

Ricin B lectin-like proteins of the microsporidian Encephalitozoon cuniculi and Anncaliia algerae are involved in host-cell invasion.

Microsporidia are obligate intracellular pathogens capable of infecting a wide variety of hosts ranging from invertebrates to vertebrates. The infection process requires a step of prior adherence of Microsporidia to the surface of host cells. A few studies demonstrated the involvement of proteins containing a ricin-B lectin (RBL) domain in parasite infection. In this study Anncalia algerae and Encephalitozoon cuniculi genomes were screened by bioinformatic analysis to identify proteins with an extracellular prediction and possessing RBL-type carbohydrate-binding domains, being both potentially relevant factors contributing to host cell adherence. Three proteins named AaRBLL-1 and AaRBLL-2 from A. algerae and EcRBLL-1 from E. cuniculi, were selected and comparative analysis of sequences suggested their belonging to a multigenic family, with a conserved structural RBL domain despite a significant amino acid sequence divergence. The production of recombinant proteins and antibodies against the three proteins allowed their subcellular localization on the spore wall and/or the polar tube. Adherence inhibition assays based on pre-treatments with recombinant proteins or antibodies highlighted the significant decrease of the proliferation of both E. cuniculi and A. algerae, strongly suggesting that these proteins are involved in the infection process.

Microsporidia Have a Peculiar Outer Membrane with Exterior Cytoplasmic Proteins.

Most eukaryotic cells have a plasma membrane with glycoproteins on the outer leaflet and cytoplasmic proteins on the inner leaflet. However, the microsporidians examined have a peculiar outer membrane with cytoplasmic proteins on the surface. His study was to determine if this is true and identify the presence of key cytoplasmic proteins on the exterior of the sporoplasm and spore stages. Specific probes including GFP-labeled genes, antibodies, and electron microscopy were applied to studies on the sporoplasm and spore stages of different microsporidian species. Cytoplasmic membrane proteins cadherin, intermediate filaments, dynactin p150Glued and tubulin were identified on the outer leaflet of the exterior membrane of the sporoplasm or spore stages of Spraguea americanus, Anncaliia algerae, Ameson michaelis and Thelohania sp. The invasive sporoplasm stage of microsporidians acquires an outer membrane from a cytoplasmic organelle and it has been shown that the outside surface of this membrane bears cytoplasmic proteins. This membrane retains these cytoplasmic proteins even on the spore stage. For an intracellular parasite that locates directly within the cytoplasm of a host cell, the presenceof these cytoplasmic proteins on the surface of the parasite appears to have a significant effect on the host's response.

Anncaliia algerae

Anncaliia algerae belongs to the microsporidia, a group of obligate intracellular pathogens originally classified as early branching 'primitive' protozoa but now understood to be related to the Cryptomycota as a basal branch in the fungal kingdom. A. algerae has emerged as a rare opportunistic human pathogen in immune compromised patients such as those taking immune suppressive medications for arthritis, hematologic malignancy, or organ transplantation. It was originally identified as a pathogen of mosquitoes and is probably transmitted to humans by food or water through ingestion, inhalation, or contamination of ocular tissue or wounds with environmental spores. A. algerae infection primarily causes myositis; however, vocal cord, skin, corneal (in immune competent hosts), and disseminated infections have been reported. Human infection has also been reported with other members of the Anncaliia genus: Anncaliia vesicularum in a HIV patient, and Anncaliia connori in an infant with thymic dysplasia.

Cells without an External Plasma Membrane

Nearly all eukaryotes have an exterior plasma membrane with glycoproteins on the outer leaflet however. The exception may be the intracellular microsporidian parasites which have an outer membrane with an exterior leaflet bearing only cytoplasmic proteins. How can this be? The initial stage, the sporoplasm, is injected into a host cell and its outer membrane appears to be derived from a cytoplasmic organelle referred to as the extrusion apparatus. The introduced microsporidian cell retains this organelle membrane with its outer leaflet of cytoplasmic proteins throughout the remainder of its life cycle. A conventional plasma membrane does appear but only during spore wall formation and this membrane remains interior to the microsporidian while it is engaged with the development of the external wall whereas, the outside cytoplasmic membrane remains exterior to the developing spore wall. The intracellular microsporidia, which locate within the host cytoplasm, has its external membrane leaflet in direct contact with the host cytoplasm. All the cytoplasmic proteins on this exterior membrane of the microsporidian are believed to be of host origin and, therefore it is not surprising that the parasitic cell does not yield a detectable reaction either by the host cell or the host organism. This study is based on observations on the microsporidians Spraguea amricanus, Anncaliia algerae, Ameson Michaelis, and Thelohania sp.

Microsporidia Can Acquire Lamin-like Intermediate Filaments and Cell Adhesion Catenin-cadherin Complexes from the Host (?).

On their spore surfaces, Microsporidia often develop a canopy of filaments with characteristics of intermediate filaments (IF), as we demonstrated in previous studies on Thelohania sp., Ameson michaelis, and Spraguea lophii. Genomic studies indicate that among invertebrates, lamins that may localize in the cytoplasm or nucleus, are the only known IF type. These IFs can bind to the substrate containing cell adhesion molecules (CAMs) cadherins, associated with β and γ catenins. The objects of this study were to determine whether microsporidia have CAMs with the attached IFs on their envelopes and to find out if these proteins are provided by the host. An examination was made for localization of lamins and CAMs on the spores of the mentioned above species and Anncaliia algerae, plus in the host animals. Then, we determined whether the spores of A.michaelis and A.algerae could bind vertebrate nuclear lamin onto the spore surface. We also tested transgenic Drosophila melanogaster stocks bearing cadherin-GFP to see whether developing A.algerae parasites in these hosts could acquire host CAMs. The tests were positive for all these experiments. We hypothesize that microsporidia are able to acquire host lamin IFs and cell adhesion catenin-cadherin complexes from the host.

445. A Case of Disseminated Microsporidia Manifesting as Skin Lesions in a Patient with Acute Lymphoblastic Leukemia

BackgroundWe present a case of a 65-year-old male with a history of acute B-cell lymphoblastic leukemia (ALL) who presented with fevers and skin lesions. The patient achieved remission after induction chemotherapy. Nineteen months after diagnosis, while on maintenance therapy, the patient presented as noted above. He was instructed to hold maintenance therapy and sent to the hospital.Methods ResultsOn examination, the patient was febrile. He had papules present on the forehead, chest, arms, legs, and back. Physical examination was otherwise unremarkable. Labs were notable for a white blood cell count of 3600/uL (absolute neutrophil 3100/uL) and creatine kinase (CK) of 593 U/L. Blood and urine cultures, Histoplasma, Varicella, Toxoplasma, HIV, and an acute hepatitis panel were negative. CT of the chest, abdomen, and pelvis was unremarkable. He was empirically started on micafungin and valacyclovir. However, he continued to have fevers and myalgias. Wound culture was negative for fungus after four weeks of growth. Skin biopsy immunostains were consistent with an unknown protozoan. Specimens were sent to an outside facility and yielded a diagnosis of Anncaliia algerae. Antibiotics were changed to albendazole and voriconazole, with subsequent improvement in all symptoms.ConclusionMicrosporidia most commonly infect immunocompromised hosts. Clinical manifestations of microsporidiosis are extremely diverse. Oftentimes, symptoms are not present in those found to be infected with Microsporidia. A disseminated disease has been identified but remains rare. Although Microsporidia have been identified as a cause of infection in immunocompromised patients, there are few reports of infection in those diagnosed with cancer, and only a few cases have been due to Anncaliia algerae. In studies pertaining to ALL patients, roughly one-fifth of patients were identified as being infected with Microsporidia, but most did not have symptoms. This is the first reported case of disseminated Microsporidia in a patient with ALL and the first disseminated infection presenting as a skin manifestation. Of identified cases of disseminated Microsporidial infection, mortality was high. Thus, prompt recognition of Microsporidia as a cause of infection in patients with ALL is of utmost importance. Disclosures All authors: No reported disclosures.

A Fatal Case of Disseminated Microsporidiosis Due to Anncaliia algerae in a Renal and Pancreas Allograft Recipient.

Microsporidiosis is an emerging opportunistic infection in immunocompromised patients. We report a case of fatal disseminated Anncaliia algerae infection in a profoundly immunosuppressed pancreas and kidney transplant recipient.

Development of a continuous cell line from larval Atlantic cod (Gadus morhua) and its use in the study of the microsporidian, Loma morhua.

In vitro cell culture methods are crucial for the isolation, purification and mass propagation of intracellular pathogens of aquatic organisms. Cell culture infection models can yield insights into infection mechanisms, aid in developing methods for disease mitigation and prevention, and inform commercial-scale cultivation approaches. This study details the establishment of a larval cell line (GML-5) from the Atlantic cod (Gadus morhua) and its use in the study of microsporidia. GML-5 has survived over 100 passages in 8years of culture. The line remains active and viable between 8 and 21°C in Leibovitz-15 (L-15) media with 10% foetal bovine serum and exhibits a myofibroblast phenotype as indicated by immuno-positive results for vimentin, α-smooth muscle actin, collagen I and S-100 proteins, while being desmin-negative. GML-5 supports the infection and development of two microsporidian parasites, an opportunistic generalist (Anncaliia algerae) and cod-specific Loma morhua. Using GML-5, spore germination and proliferation of L.morhua was found to require exposure to basic pH and cool incubation temperatures (8°C), in contrast to A.algerae, which required no cultural modifications. Loma morhua-associated xenoma-like structures were observed 2weeks postexposure. This invitro infection model may serve as a valuable tool for cod parasitology and aquaculture research.

Successful Treatment of Disseminated Anncaliia algerae Microsporidial Infection With Combination Fumagillin and Albendazole.

Anncaliia algerae myositis is a life-threatening, emerging microsporidiosis among immunocompromised hosts. We report a case of disseminated A algerae infection in a man previously treated with alemtuzumab. Due to failure of albendazole-based therapy, fumagillin was added as a novel approach to management, with a good clinical response and patient survival.

A Novel Fluorescent Labeling Method Enables Monitoring of Spatio-Temporal Dynamics of Developing Microsporidia.

The microsporidium, Anncaliia algerae (Brachiola algerae), is a eukaryotic obligate intracellular parasite first isolated from mosquitoes and is an important opportunistic human pathogen that can cause morbidity and mortality among immune-compromised individuals including patients with AIDS and those undergoing chemotherapy. There is little known about the Microsporidia-host cell interface in living host cells, due to current approaches being limited by the lack of fluorescent reporters for detecting the parasite lifecycle. Here, we have developed and applied novel vital fluorescent parasite labeling methodologies in conjunction with fluorescent protein-tagged reporters to track simultaneously the dynamics of both parasite and host cell specific components, including the secretory and endocytic trafficking pathways, during the entire infection time period. We have found dramatic changes in the dynamics of host secretory trafficking organelles during the course of infection. The Golgi compartment is gradually disassembled and regenerated into mini-Golgi structures in parallel with cellular microtubule depolymerization. Importantly, we find that Microsporidia progeny are associated with these de novo formed mini-Golgi structures. These host structures appear to create a membrane bound niche environment for parasite development. Our studies presented here provide novel imaging tools and methodologies that will facilitate in understanding the biology of microsporidial parasites in the living host.

Analysis of the beta-tubulin gene and morphological changes of the microsporidium Anncaliia algerae both suggest albendazole sensitivity.

The Microsporidium, Anncaliia algerae, an obligate intracellular parasite, has been identified as an opportunistic human pathogen, but treatment has not been evaluated for infections with this organism. Albendazole, an antitubulin polymerization drug used against parasitic worm infections, has been the medication of choice used to treat some microsporidial infections affecting humans, with varying results ranging from clearing infection (Encephalitozoon) to resistance (Enterocytozoon). This study illustrates the effect of albendazole treatment on A. algerae infection in Rabbit Kidney (RK13) cells and Human Fetal Lung (HFL-1) fibroblasts. Albendazole appears to have an attenuating effect on A. algerae infection and albendazole's IC50 in RK13 cells is 0.1μg/ml. Long-term treatment inhibits up to 98% of spore production, but interrupting treatment reestablishes the infection without new exposure to the parasite as supported by microscopic observations. The parasite's beta-tubulin gene was purified, cloned, and sequenced. Five of the six specific amino acids, associated with benzimidazole sensitivity, are conserved in A. algerae. These findings suggest that A. algerae is sensitive to albendazole; however, the organism is not completely cleared from cultures.

Microsporidian genomes harbor a diverse array of transposable elements that demonstrate an ancestry of horizontal exchange with metazoans.

Microsporidian genomes are the leading models to understand the streamlining in response to a pathogenic lifestyle; they are gene-poor and often possess small genomes. In this study, we show a feature of microsporidian genomes that contrasts this pattern of genome reduction. Specifically, genome investigations targeted at Anncaliia algerae, a human pathogen with a genome size of 23 Mb, revealed the presence of a hitherto undetected diversity in transposable elements (TEs). A total of 240 TE families per genome were identified, exceeding that found in many free-living fungi, and searches of microsporidian species revealed that these mobile elements represent a significant portion of their coding repertoire. Their phylogenetic analysis revealed that many cases of ancestry involve recent and bidirectional horizontal transfers with metazoans. The abundance and horizontal transfer origin of microsporidian TEs highlight a novel dimension of genome evolution in these intracellular pathogens, demonstrating that factors beyond reduction are at play in their diversification.

Hijacking of host cellular functions by an intracellular parasite, the microsporidian Anncaliia algerae.

Intracellular pathogens including bacteria, viruses and protozoa hijack host cell functions to access nutrients and to bypass cellular defenses and immune responses. These strategies have been acquired through selective pressure and allowed pathogens to reach an appropriate cellular niche for their survival and growth. To get new insights on how parasites hijack host cellular functions, we developed a SILAC (Stable Isotope Labeling by Amino Acids in Cell culture) quantitative proteomics workflow. Our study focused on deciphering the cross-talk in a host-parasite association, involving human foreskin fibroblasts (HFF) and the microsporidia Anncaliia algerae, a fungus related parasite with an obligate intracellular lifestyle and a strong host dependency. The host-parasite cross-talk was analyzed at five post-infection times 1, 6, 12 and 24 hours post-infection (hpi) and 8 days post-infection (dpi). A significant up-regulation of four interferon-induced proteins with tetratricopeptide repeats IFIT1, IFIT2, IFIT3 and MX1 was observed at 8 dpi suggesting a type 1 interferon (IFN) host response. Quantitative alteration of host proteins involved in biological functions such as signaling (STAT1, Ras) and reduction of the translation activity (EIF3) confirmed a host type 1 IFN response. Interestingly, the SILAC approach also allowed the detection of 148 A. algerae proteins during the kinetics of infection. Among these proteins many are involved in parasite proliferation, and an over-representation of putative secreted effectors proteins was observed. Finally our survey also suggests that A. algerae could use a transposable element as a lure strategy to escape the host innate immune system.

Anncaliia algeraeMicrosporidial Myositis

The insect microsporidian Anncaliia algerae was first described in 2004 as a cause of fatal myositis in an immunosuppressed person from Pennsylvania, USA. Two cases were subsequently reported, and we detail 2 additional cases, including the only nonfatal case. We reviewed all 5 case histories with respect to clinical characteristics, diagnosis, and management and summarized organism life cycle and epidemiology. Before infection, all case-patients were using immunosuppressive medications for rheumatoid arthritis or solid-organ transplantation. Four of the 5 case-patients were from Australia. All diagnoses were confirmed by skeletal muscle biopsy; however, peripheral nerves and other tissues may be infected. The surviving patient received albendazole and had a reduction of immunosuppressive medications and measures to prevent complications. Although insects are the natural hosts for A. algerae, human contact with water contaminated by spores may be a mode of transmission. A. algerae has emerged as a cause of myositis, particularly in coastal Australia.

Identification of two new polar tube proteins related to polar tube protein 2 in the microsporidianAntonospora locustae

Microsporidia are obligate intracellular eukaryotic parasites with a broad host spectrum characterized by a unique and highly sophisticated invasion apparatus, the polar tube (PT). In a previous study, two PT proteins, named AlPTP1 (50 kDa) and AlPTP2 (35 kDa), were identified in Antonospora locustae, an orthoptera parasite that is used as a biological control agent against locusts. Antibodies raised against AlPTP2 cross-reacted with a band migrating at ~70 kDa, suggesting that this 70-kDa antigen is closely related to AlPTP2. A blastp search against the A. locustae genome database allowed the identification of two further PTP2-like proteins named AlPTP2b (568 aa) and AlPTP2c (599 aa). Both proteins are characterized by a specific serine- and glycine-rich N-terminal extension with elastomeric structural features and share a common C-terminal end conserved with AlPTP2 (~88% identity for the last 250 aa). MS analysis of the 70-kDa band revealed the presence of AlPTP2b. Specific anti-AlPTP2b antibodies labelled the extruded PTs of the A. locustae spores, confirming that this antigen is a PT component. Finally, we showed that several PTP2-like proteins are also present in other phylogenetically related insect microsporidia, including Anncaliia algerae and Paranosema grylli.

Molecular characterization of human-pathogenic microsporidia and Cyclospora cayetanensis isolated from various water sources in Spain: a year-long longitudinal study.

Recent studies suggest the involvement of water in the epidemiology of Cyclospora cayetanensis and some microsporidia. A total of 223 samples from four drinking water treatment plants (DWTPs), seven wastewater treatment plants (WWTPs), and six locations of influence (LI) on four river basins from Madrid, Spain, were analyzed from spring 2008 to winter 2009. Microsporidia were detected in 49% of samples (109/223), Cyclospora spp. were detected in 9% (20/223), and both parasites were found in 5.4% (12/223) of samples. Human-pathogenic microsporidia were detected, including Enterocytozoon bieneusi (C, D, and D-like genotypes), Encephalitozoon intestinalis, Encephalitozoon cuniculi (genotypes I and III), and Anncaliia algerae. C. cayetanensis was identified in 17 of 20 samples. To our knowledge, this is the first study that shows a year-long longitudinal study of C. cayetanensis in drinking water treatment plants. Additionally, data about the presence and molecular characterization of the human-pathogenic microsporidia in drinking water, wastewater, and locations of influence during 1 year in Spain are shown. It is noteworthy that although the DWTPs and WWTPs studied meet European and national regulations on water sanitary quality, both parasites were found in water samples from these plants, supporting the idea that new and appropriate controls and regulations for drinking water, wastewater, and recreational waters should be proposed to avoid health risks from these pathogens.

G.P.68 Survival versus a fatal outcome in two cases of Anncaliia algerae microsporidial myositis

Abstract Two cases of Anncaliia (previously Brachiola, previously Nosema) algerae microsporidial parasitic myositis in patients with rheumatoid arthritis (RA) treated with immunosuppression are presented with one surviving; the factors that may have contributed to survival are discussed. A 66 year old man with RA and renal impairment treated with methotrexate; the prednisone dose had recently increased as high as 50mg daily for increasing muscle pain over a month. He was admitted febrile with a peak CK 6630U/L ( Brachiola algerae were demonstrated on electron microscopy of biopsied muscle, and confirmed on PCR. A 68year old man with RA treated with etanercept and leflunomide; these were sequentially ceased for diarrhoea several weeks prior to admission; the prednisone dose had recently increased to 35mg daily for increasing muscle pain over this month. He was admitted afebrile with a peak CK of 1141. A muscle biopsy was immediately performed for infective myositis as the critical diagnosis of exclusion. This demonstrated microsporidial structures later confirmed as Brachiola algerae . Prednisone was weaned to 5mg daily whereupon he became febrile, leflunomide was washed out. Albendazole, pyrimethamine and sulfadiazine were administered. He reached a clinical nadir after a further week and recovered over two months. This report is of the third and fourth cases of Anncaliia algerae myositis. The second case appears to the first patient to survive Anncaliia algerae myositis, and one of the few to survive any parasitic myositis with systemic infection. We suspect that the combination of rapid diagnosis before the patient was moribund, rapid reversal of immunosuppression and early administration of albendazole and other antibiotics are likely to have contributed to the patient surviving.