Balamuthia mandrillaris trophozoites ingest human neuronal cells via a trogocytosis-independent mechanism.

BACKGROUND: Environmental protozoa need an adaptation mechanism to survive drastic changes in niches in the human body. In the brain parenchyma, Balamuthia mandrillaris trophozoites, which are causative agents of fatal brain damage, must acquire nutrients through the ingestion of surrounding cells. However, the mechanism deployed by the trophozoites for cellular uptake remains unknown. METHODS: Amoebic ingestion of human neural cell components was investigated using a coculture system of clinically isolated B. mandrillaris trophozoites and human neuroblastoma SH-SY5Y cells. Cell-to-cell interactions were visualized in a three-dimensional manner using confocal and holotomographic microscopes. RESULTS: The B. mandrillaris trophozoites first attached themselves to human neuroblastoma SH-SY5Y cells and then twisted themselves around the cytoplasmic bridge. Based on fluorescence-based cell tracking, the B. mandrillaris trophozoites then inserted invadopodia into the cytoplasm of the human cells. Subsequently, the human protein-enriched components were internalized into the trophozoites in the form of nonmembranous granules, whereas the human lipids were dispersed in the cytoplasm. Intervention of trogocytosis, a process involving nibbling on parts of the target cells, failed to inhibit this cellular uptake. CONCLUSIONS: Human cell ingestion by B. mandrillaris trophozoites likely differs from trogocytosis, suggesting that a pathogen-specific strategy can be used to ameliorate brain damage.

Pathogenic free-living amoebic encephalitis in Japan.

Over 600 cases of amoebic encephalitis caused by pathogenic free-living amoebas (Balamuthia mandrillaris, Acanthamoeba spp., and Naegleria fowleri) have been reported worldwide, and in Japan, 24 cases have been reported from the first case in 1976 up to 2018. Among these cases, 18 were caused by B. mandrillaris, four by Acanthamoeba spp., one by N. fowleri, and one was of unknown etiology. Additionally, eight cases were diagnosed with encephalitis due to pathogenic free-living amoebas before death, but only three cases were successfully treated. Unfortunately, all other cases were diagnosed by autopsy. These facts indicate that an adequate diagnosis is difficult, because encephalitis due to pathogenic free-living amoebas does not show typical symptoms or laboratory findings. Moreover, because the number of cases is limited, other cases might have been missed outside of those diagnosed by autopsy. Cases of encephalitis caused by B. mandrillaris have been reported from all over Japan, with B. mandrillaris recently isolated from soil in Aomori prefecture. Therefore, encephalitis caused by pathogenic free-living amoebas should be added to the differential diagnosis of encephalitis patients.

Novel culture medium for the axenic growth of Balamuthia mandrillaris.

Until now, for axenic cultivation of Balamuthia mandrillaris, the BM-3 culture medium and the Modified Chang's special medium have been the only ones recommended, but they have some disadvantages, as both require many components and their preparations are laborious. Therefore, we developed a novel culture medium for B. mandrillaris axenic cultivation. Each one of the 11 components of BM-3 was combined with Cerva's medium as basal culture medium. Ten strains of B. mandrillaris including the reference strain CDC:V039 and 9 environmental isolates were used during trials. After testing all combinations, the basal medium complemented with 10× Hank's balanced salt solution was the only one that supported confluent growth of B. mandrillaris. Cell shape and motility of trophozoites were normal. This developed medium is as useful as BM-3 for axenization. The development of a cheaper and easy-to-prepare medium for B. mandrillaris opens the possibility of increasing its study.

Resistance to intranasal infection with Balamuthia mandrillaris amebae is T-cell dependent.

T and B cell-deficient BALB/c SCID mice become severely ill and die of amebic encephalitis after intranasal infection with Balamuthia mandrillaris, while adult immunocompetent BALB/c wild-type (WT) mice are resistant. To further investigate the role of lymphocytes in protection from Balamuthia amebic encephalitis (BAE), SCID mice were reconstituted with and WT mice selectively depleted of lymphocytes before infection. Reconstitution of SCID mice with whole spleen cells from WT mice rendered the recipients as resistant to BAE as WT mice. SCID mice that had received spleen cells depleted of CD4(+) T cells remained susceptible. When adult WT mice were depleted of both CD4(+) and CD8(+) T cells or of CD4(+) T cells alone, these mice also became susceptible to BAE. Depletion of CD8(+) T cells alone increased susceptibility only marginally. All morbidity and mortality data were underpinned by histological analysis of the brain.

Atomic force microscopic imaging of Acanthamoeba castellanii and Balamuthia mandrillaris trophozoites and cysts.

Light microscopy and electron microscopy have been successfully used in the study of microbes, as well as free-living protists. Unlike light microscopy, which enables us to observe living organisms or the electron microscope which provides a two-dimensional image, atomic force microscopy provides a three-dimensional surface profile. Here, we observed two free-living amoebae, Acanthamoeba castellanii and Balamuthia mandrillaris under the phase contrast inverted microscope, transmission electron microscope and atomic force microscope. Although light microscopy was of lower magnification, it revealed functional biology of live amoebae such as motility and osmoregulation using contractile vacuoles of the trophozoite stage, but it is of limited value in defining the cyst stage. In contrast, transmission electron microscopy showed significantly greater magnification and resolution to reveal the ultra-structural features of trophozoites and cysts including intracellular organelles and cyst wall characteristics but it only produced a snapshot in time of a dead amoeba cell. Atomic force microscopy produced three-dimensional images providing detailed topographic description of shape and surface, phase imaging measuring boundary stiffness, and amplitude measurements including width, height and length of A. castellanii and B. mandrillaris trophozoites and cysts. These results demonstrate the importance of the application of various microscopic methods in the biological and structural characterization of the whole cell, ultra-structural features, as well as surface components and cytoskeleton of protist pathogens.

Balamuthia mandrillaris in South America: an emerging potential hidden pathogen in Perú.

Balamuthia mandrillaris is a free living amoeba that can be isolated from soil. It is an emerging pathogen causing skin lesions as well as CNS involvement with a fatal outcome if untreated. Further, infections can sometimes can also appear in peripheral areas such as extremities (usually knee), or trunk. Moreover, it often progresses to an infiltrative lesion that occasionally becomes ulcerated. In countries like Peru, a skin lesion will precede other symptoms. This primary cutaneous lesion can be present for weeks or even months. However, the appearance of neurological disease predicts a poor prognosis. Diagnosis requires a high level of suspicion.

Balamuthia mandrillaris: in vitro interactions with selected protozoa and algae.

Although Balamuthia mandrillaris was identified more than two decades ago as an agent of fatal granulomatous encephalitis in humans and other animals, little is known about its ecological niche, biological behavior in the environment, food preferences and predators, if any. When infecting humans or other animals, Balamuthia feeds on tissues; and in vitro culture, it feeds on mammalian cells (monkey kidney cells, human lung fibroblasts, and human microvascular endothelial cells). According to recent reports, it is believed that Balamuthia feeds on small amebae, for example, Acanthamoeba that are present in its ecological niche. To test this hypothesis, we associated Balamuthia on a one-on-one basis with selected protozoa and algae. We videotaped the behavior of Balamuthia in the presence of a potential prey, its ability to hunt and attack its food, and the time required to eat and cause damage to the target cell by direct contact. We found that B. mandrillaris ingested trophozoites of Naegleria fowleri, Naegleria gruberi, Acanthamoeba spp., Trypanosoma cruzi epimastigotes, Toxoplasma gondii tachyzoites, and Giardia. However, it did not feed on Acanthamoeba cysts or algae. Balamuthia caused cytolysis of T. cruzi epimastigotes and T. gondii tachyzoites by direct contact. Balamuthia trophozoites and cysts were, however, eaten by Paramecium sp.

Autopsy case of amebic granulomatous meningoencephalitis caused by Balamuthia mandrillaris in Japan.

Balamuthia mandrillaris is a free-living ameba that causes amebic encephalitis. Herein, we report an autopsy case of Balamuthia encephalitis proven with polymerase chain reaction (PCR) and immunohistochemistry from paraffin-embedded brain biopsy specimens. A 68-year-old Japanese male presented at a hospital with progressive right hemiparesis approximately 3 months before his death. An open-brain biopsy specimen showed diffuse meningitis with massive coagulative necrosis. The perivascular spaces contained numerous lymphocytes, histiocytes and giant cells, although the etiology was not determined. The patient deteriorated into coma and died from cerebral herniation. Autopsy revealed abundant trophozoites and cysts in the subarachnoid and Virchow-Robin's spaces. Electron-micrographs of the amebic cysts showed a characteristic triple-walled envelope. The amebas were identified as Balamuthia mandrillaris based on immunohistochemical analysis from the autopsy and biopsy specimens. Primer sets designed to amplify approximately 200 bp bands of mitochondrial 16S rRNA gene of Balamuthia by PCR produced positive results from the biopsy specimens but negative results from the autopsy specimens. In summary, PCR to amplify shorter segments of DNA may be of diagnostic value in detecting suspected cases of balamuthiasis in formalin-fixed, paraffin-embedded specimens. Increased awareness and timely diagnosis of Balamuthia encephalitis might lead to earlier initiation of therapy and improved outcome.