Microfilaments in Naegleria fowleri amoebae.

Examination by electron microscopy has revealed 2 types of microfilament in the cytoplasm of 3 strains of axenically grown Naegleria fowleri amoebae. Thin, actin-like microfilaments 5-7 nm in diameter are randomly oriented in the nonmotile amoebae, and are concentrated near the plasma membrane. In the actively motile amoebae these microfilaments aggregate to form colateral bundles in close proximity to the plasma membrane. Thick, myosin-like microfilaments 17-19 nm in diameter also occur in the amoebae cytoplasm. The significance of these 2 kinds of microfilament in amoeboid motion is discussed.     

Naegleria fowleri amoebae express a membrane-associated calcium-independent phospholipase A2

Naegleria fowleri, a free-living amoeba, is the causative agent of primary amoebic meningoencephalitis. Previous reports have demonstrated that N. fowleri expresses one or more forms of phospholipase A₂ (PLA₂) and that a secreted form of this enzyme is involved in pathogenesis. However, the molecular nature of these phospholipases remains largely unknown. This study was initiated to determine whether N. fowleri expresses analogs of the well-characterized PLA₂s that are expressed by mammalian macrophages. Amoeba cell homogenates contain a PLA₂ activity that hydrolyzes the substrate that is preferred by the 85 kDa calcium-dependent cytosolic PLA₂, cPLA₂. However, unlike the cPLA₂ enzyme in macrophages, this activity is largely calcium-independent, is constitutively associated with membranes and shows only a modest preference for phospholipids that contain arachidonate. The amoeba PLA₂ activity is sensitive to inhibitors that block the activities of cPLA₂-α and the 80 kDa calcium-independent PLA₂, iPLA₂, that are expressed by mammalian cells. One of these compounds, methylarachidonyl fluorophosphonate, partially inhibits the constitutive release of [³H]arachidonic acid from pre-labeled amoebae. Together, these data suggest that N. fowleri expresses a constitutively active calcium-independent PLA₂ that may play a role in the basal phospholipid metabolism of these cells.     

Naegleria fowleri amoebae express a membrane-associated calcium-independent phospholipase A(2)

Naegleria fowleri, a free-living amoeba, is the causative agent of primary amoebic meningoencephalitis. Previous reports have demonstrated that N. fowleri expresses one or more forms of phospholipase A(2) (PLA(2)) and that a secreted form of this enzyme is involved in pathogenesis. However, the molecular nature of these phospholipases remains largely unknown. This study was initiated to determine whether N. fowleri expresses analogs of the well-characterized PLA(2)s that are expressed by mammalian macrophages. Amoeba cell homogenates contain a PLA(2) activity that hydrolyzes the substrate that is preferred by the 85 kDa calcium-dependent cytosolic PLA(2), cPLA(2). However, unlike the cPLA(2) enzyme in macrophages, this activity is largely calcium-independent, is constitutively associated with membranes and shows only a modest preference for phospholipids that contain arachidonate. The amoeba PLA(2) activity is sensitive to inhibitors that block the activities of cPLA(2)-alpha and the 80 kDa calcium-independent PLA(2), iPLA(2), that are expressed by mammalian cells. One of these compounds, methylarachidonyl fluorophosphonate, partially inhibits the constitutive release of [(3)H]arachidonic acid from pre-labeled amoebae. Together, these data suggest that N. fowleri expresses a constitutively active calcium-independent PLA(2) that may play a role in the basal phospholipid metabolism of these cells.     

Amebostomes of Naegleria fowleri

The strain of ameba, culture incubation temperature, and phase of ameba growth affected the number of amebostomes present on amebae of Naegleria fowleri. Serial passage of N. fowleri through mice decreased the average number of amebostomes. Amebostomes were shown to be functional by their ability to engulf yeast cells.     

Amebostomes of Naegleria fowleri1

The strain of ameba, culture incubation temperature, and phase of ameba growth affected the number of amebostomes present on amebae of Naegleria fowleri. Serial passage of N. fowleri through mice decreased the average number of amebostomes. Amebostomes were shown to be functional by their ability to engulf yeast cells.     

Identification of Naegleria fowleri and other Naegleria spp. (free-living amoebae) using cellulose acetate membrane electrophoresis of glucose phosphate isomerase

Abstract A simple isoenzyme cellulose acetate membrane electrophoresis method with respect to glucose phosphate isomerase (GPI) was developed for the differentiation of the human pathogenic free-living amoeba Naegleria fowleri from other Naegleria spp. A single GPI band was detected in all the species tested, the relative mobility of which could be used to identify N. fowleri . Of the other Naegleria spp., only N. italica and N. jadini shared a common GPI mobility. No intraspecies variation in GPI profile was detected, regardless of whether the strains were cultured in monoxenic or axenic media. The technique is proposed as a useful means of identifying N. fowleri soon after isolation from the environment.     

Activated macrophages demonstrate direct cytotoxicity, antibody-dependent cellular cytotoxicity, and enhanced binding of Naegleria fowleri amoebae

Macrophages activated in vivo by injection of Corynebacterium parvum or bacillus Calmette-Guérin caused direct cytolysis of the pathogenic free-living amoeba, Naegleria fowleri, in vitro. Amoebicidal activity was time and cell density-dependent but was not dependent on the presence of specific antibody. Antibody-dependent cellular cytotoxicity for amoebae was also expressed by activated macrophages. Resident and thioglycolate-elicited macrophages demonstrated low cytolytic activity under all conditions tested. From scanning electron microscopy it appears that the degree of target cell binding is directly related to the degree of cytolysis expressed by the macrophage populations. Cell-cell contact was required for cytolysis of amoebae by activated macrophages since cytolysis did not occur when contact was blocked by a porous filter. For each macrophage population, the levels of amoebicidal activity and tumoricidal activity were comparable.     

Natural killer cell activity in Naegleria fowleri infected mice]

The natural killer (NK) cell activity of splenocytes and recycling capacity of NK cells were observed by combining the 51Cr-release cytotoxicity assay and single cell cytotoxicity assay against YAC-1. The ICR mice were infected intranasally with Naegleria fowleri, that is a pathogenic free-living amoeba. The mice infected with 1 x 10(5) trophozoites showed mortality rate of 76.7% and mean survival time of 12.9 days. The cytotoxic activity of NK cells in infected mice was significantly higher than that of non-infected mice during the period between 12 hours and day 3 after infection, and highest on day 1. The target-binding capacity of NK cells in infected mice was not different from that of non-infected ones. Maximal killing potential and maximal recycling capacity were remarkably increased in infected mice as compared with the control. The results obtained in this observation indicated that elevated NK cell activity in mice infected with N. fowleri was not due to target-binding capacity of NK cells but due to the increased activity of NK cells and increased recycling capacity of individual NK cells.     

Ultrastructure of Naegleria fowleri enflagellation.

Amoebae of Naegleria fowleri nN68 became elongated flagellated cells 150 to 180 min after subculture to non-nutrient buffer. N. fowleri NF69 did not become elongated or flagellated under these conditions. Electron microscopic examination of N. fowleri confirmed that it is a typical eucaryotic protist with a distinct nuclear envelope and prominent nucleolus, numerous vacuoles and cytoplasmic inclusions, pleomorphic mitochondria, and some rough endoplasmic reticulum. During incubation in non-nutrient buffer, both strains lost ultraviolet-absorbing material to the medium, and the number of vacuoles decreased. In strain nN68, basal bodies, a rootlet, and flagella are formed quickly after an initial lag of 90 min. Initially, the rootlet is not associated with the nucleus but they become associated subsequent at the leading end of the elongated cell. In elongated cells, the rootlet lies in a furrow or groove extending the length of the nucleus. Flagella of N. fowleri nN68 exhibit the typical 9 + 2 arrangement of filaments and are surrounded by a sheath which is continuous with the plasma membrane. The enflagellation process in N. fowleri can be manipulated reproducibly.     

The Interaction of Naegleria fowleri Amoebae with Murine Macrophage Cell Lines

The present study was undertaken to determine whether murine macrophage cell lines exhibited in vitro amoebicidal activity comparable to that elicited by activated murine peritoneal macrophages. Peritoneal macrophages activated in vivo by bacillus Calmette-Guérin or Propionibacterium acnes demonstrated significant cytolysis of Naegleria fowleri amoebae. The macrophage cell line RAW264.7 also effected cytolysis of amoebae, but to a lesser extent than that elicited by activated peritoneal macrophages. However, the macrophage cell lines, J774A.1 and P388D₁, did not exhibit amoebicidal activity. Macrophage conditioned medium prepared from RAW264.7 macrophages mediated cytolysis of L929 tumor cells but had no effect on N. fowleri amoebae. In addition, neither recombinant tumor necrosis factor nor recombinant interleukin-1 exhibited amoebicidal activity. Scanning electron microscopy of co-cultures revealed that N. fowler bound to activated peritoneal macrophages and RAW264.7 macrophages. These results suggest that RAW264.7 macrophages treated in vitro with lipopolysaccharide are similar to macrophages activated in vivo in that they effect contact-dependent cytolysis of Naegleria fowleri amoebae. The RAW264.7 macrophages are unlike primary macrophage cultures in that they either do not release soluble amoebicidal factors into the conditioned medium or they release insufficient quantities.     

Cell surface differences of Naegleria fowleri and Naegleria lovaniensis exposed with surface markers

Differences in the distribution of diverse cell surface coat markers were found between Naegleria fowleri and Naegleria lovaniensis. The presence of carbohydrate-containing components in the cell coat of the two species was detected by selective staining with ruthenium red and alcian blue. Using both markers, N. fowleri presented a thicker deposit than N. lovaniensis. The existence of exposed mannose or glucose residues was revealed by discriminatory agglutination with the plant lectin Concanavalin A. These sugar residues were also visualized at the cell surface of these parasites either by transmission electron microscopy or by fluorescein-tagged Concanavalin A. Using this lectin cap formation was induced only in N. fowleri. The anionic sites on the cell surface detected by means of cationized ferritin were more apparent in N. fowleri. Biotinylation assays confirmed that even though the two amoebae species have some analogous plasma membrane proteins, there is a clear difference in their composition.     

Infection of murine macrophage cell lines by Legionella pneumophila

Legionella pneumophila causes pneumonia by infecting alveolar macrophages. Although several model systems have been used for L. pneumophila virulence studies, no detailed comparisons have been made between them. An ideal in vitro virulence model should be cost-effective, easy to obtain in large amounts and as relevant as possible to the actual disease. We compared the MH-S cell line to human peripheral blood monocyte-derived macrophages and the J774A.1 cell line. We found that the interactions of L. pneumophila with MH-S at the cellular level resemble those of human primary monocyte-derived macrophages, suggesting that these cells provide a valuable model for this bacterial pathogen.     

Susceptibility of wild mammals to infection with Naegleria fowleri

Animals of 4 families of small wild mammals were live-trapped and inoculated intranasally with Naegleria fowleri to determine patterns of susceptibility. Of the 7 species of animals examined, only rodents were susceptible to N. fowleri. Susceptible animals were eastern gray squirrel, hispid cotton rat, muskrat, and house mouse. Mammals that were not susceptible at a dose of 10(6) were opossum, raccoon, and eastern cottontail rabbit. Perhaps rodents and humans share a common anatomical or physiological determinant that makes them susceptible to infection with N. fowleri.     

Serology of Naegleria fowleri and Naegleria lovaniensis in a hospital survey

An avidin-biotin horseradish peroxidase method was used to detect antibodies to Naegleria fowleri and N. lovaniensis in human serum samples. Antibodies were detected in 101 specimens from 115 hospital patients ranging in age from 15 to 98 years. Class-specific anti-immunoglobulins identified antibodies as IgG and IgM. IgG antibody titers to both species ranged from 1:20 to 1:640. Seven of 15 serum samples collected from newborn infants also demonstrated IgG antibodies to these organisms with a titer range of 1:20 to 1:80. The immunoperoxidase test and Western blot analysis of selected serum samples demonstrated a close similarity in serological results between N. fowleri and N. lovaniensis.     

Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea

Among the many genera of free-living amoebae that exist in nature, members of only four genera have an association with human disease: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri and Sappinia diploidea. Acanthamoeba spp. and B. mandrillaris are opportunistic pathogens causing infections of the central nervous system, lungs, sinuses and skin, mostly in immunocompromised humans. Balamuthia is also associated with disease in immunocompetent children, and Acanthamoeba spp. cause a sight-threatening infection, Acanthamoeba keratitis, mostly in contact-lens wearers. Of more than 30 species of Naegleria, only one species, N. fowleri, causes an acute and fulminating meningoencephalitis in immunocompetent children and young adults. In addition to human infections, Acanthamoeba, Balamuthia and Naegleria can cause central nervous system infections in animals. Because only one human case of encephalitis caused by Sappinia diploidea is known, generalizations about the organism as an agent of disease are premature. In this review we summarize what is known of these free-living amoebae, focusing on their biology, ecology, types of disease and diagnostic methods. We also discuss the clinical profiles, mechanisms of pathogenesis, pathophysiology, immunology, antimicrobial sensitivity and molecular characteristics of these amoebae.     

Preparation of lymphocyte-activating factor from continuous murine macrophage cell lines

Lymphocyte-activating factor (LAF), a mitogen for thymocytes and T lymphocytes, is released into the culture medium by human mononuclear cells and mouse peritoneal exudate cells following treatment with various macrophage stimulants. Experiments were performed to determine if recently described mouse macrophage cell lines released LAF in response to stimulation with bacterial lipopolysaccharide. Four continuous cell lines (P388-D₁, J774, WEHI 3, and PU5-1.8) were found to release LAF in serum-free medium following endotoxin stimulation. The results of partial purification indicated that LAF obtained from cell lines had a higher molecular weight and lower isoelectric point than LAF from human mononuclear cells.     

Production of prostaglandin D2 by murine macrophage cell lines

Several tumor-derived murine macrophage cell lines were evaluated in vitro as cloned prototypes of tissue macrophages for their ability to metabolize arachidonic acid. Unexpectedly, two cell lines, J774A.1 and WR19M.1, rapidly converted exogenous 14C-arachidonic acid (AA) to a single major prostaglandin metabolite. The compound, PGD2, was positively identified by TLC, HPLC, and GC-MS. The enzymatic formation of the PGD2 was shown by inhibition of its formation by indomethacin and reduced formation of 14C-PGD2 from 14C-PGH2 in boiled cells. When J774A.1 cells were prelabeled with 3H-AA, cultured for 24 hours, and stimulated with lipopolysaccharide (LPS), PGD2 was again the predominant product. No other tumor derived cell lines, including several other murine macrophage lines, produced significant amounts of PGD2. Elicited and activated murine peritoneal macrophages produced only small amounts of PGD2, but resident peritoneal macrophages produced modest amounts of PGD2. Exaggerated formation of PGD2 by J774A.1 and WR19M.1 cells may be a consequence of neoplastic transformation or the clonal expansion of a minor subpopulation of normal tissue macrophages.     

Intranasal immunization of mice against Naegleria fowleri

The purpose of this research was to determine whether mice could be protected from lethal challenge with Naegleria fowleri by prior intranasal exposure to pathogenic and nonpathogenic Naegleria. Mortality ranged from 0 to 100% for mice inoculated intranasally (i.n.) with 5 x 10(3) amebae of 13 human isolates of N. fowleri. Mice were immunized and challenged i.n. using live amebae of strains of low, medium, and high virulence. The greatest protection against lethal challenge was afforded by three immunizing doses of 10(3) amebae per dose of the strain of medium virulence. Nonpathogenic N. gruberi also was used to immunize mice i.n. against lethal challenge with N. fowleri. Protection was greater following immunization with N. gruberi than it was after immunization with N. fowleri, suggesting that nonpathogenic N. gruberi may be a better immunogen in protecting mice against lethal naeglerial challenge.