Naegleria fowleri

A Microbial Biorealm page on the genus Naegleria fowleri

Classification

Higher order taxa

Eukaryota, Percoloza, Heterolobosea, Schizopyrenida, Vahlkampfiidae, Naegleria, fowleri

Species

NCBI: Taxonomy

Naegleria fowleri

Description and significance

Naegleria fowleri are found in warm bodies of water across the globe. Its abundance on the earth and its severely toxic result on human hosts are of concern in the medical field. N. fowleri is responsible for causing over 300 cases of primary amoebic meningoencephalitis, or PAM, of which there are only seven survivors though exact numbers of cases and survivors vary by report. Those who fall victim to an infection of N. fowleri will usually die within two weeks of the initial infection. Its ability to transform into a bi-flagellate stage has also increased interest in this microorganism and it's use as a model for differentiation of eukaryotic cells (3).

Genome structure

Few studies have been done involving the genome of Naegleria fowleri, though some important information has been discovered. Although it is known that N. fowleri is polyploidy,the exact number of genome copies is unknown (E). Its genome size was orginally estimated at over 70.6 kbp, obtained by summing the 16.5kpb ribosomal DNA and its 54.1kpb mitochondrial DNA (A). Both the rDNA and mtDNA sizes were calculated by restriction endonuclease fragments. EcoRI and/or HindIII are effective restricition enzymes for creating restriction fragment length polymorphisms of Naegleria fowleri by gel electrophoresis (A). Further research has discovered a 104 kbp genome with 23 chromosomes (G). The rDNA is kept on extrachomosonal, circular plasmids called episomes ranging in size from 14kpb to 17kbp. As the entire genome is polyploidy, these eposomes are multicopy episomes up to 4000 copies.

Species classification is done using either 5.8s rDNA or SSUrDNA (G). Interestingly, in any Naegleria species that has been sequenced, including N. fowleri, standard introns are not few, with only two introns both existing within the same gene (H). A group 1 intron in the SSUrDNA is a property of the species, allowing the small subunit ribosomal DNA to be used as a species classification. It seems likely that this intron was first obtained by horizontal gene transfer to the large subunit ribosomal DNA, and later to the SSUrDNA by vertical transfer (H). Other special traits of Naegleria species include the presence of a phosphate-dependant phosphoftuctokinace, a rare glycolytic enzyme in eukaryotes (XX).

Life Cycle

Naegleria fowleri exist in three stages; cyst, amoebiod trophozoites and flagellated trophozoite. At less than ideal conditions (below 27^oC), the amoeba forms a spherical cyst, approximately 7-14μm. In the cyst stage, the single nucleus is protected by a dense cell wall and a a very granular cytosol. The cell wall generally contains two pores which are plugged with mucus until trophozoite stage is induced (C). When the cyst is activated, binary fission occurs into the trophozoite stage. The trophozoite stage is the only stage in which N. fowleri can grow. Although infection can be caused by cysts in dust, the trophozoite stage is the major active and infectious stage of the life cycle (B). In this primary trophozoite stage, the cells are between 10-30 mu;m characterized by a dark, central nucleus (C). Many vacuoles are seen in the cytoplasm which aid in the cell's movement by lobopodia (D). Stained cultures of trophozoic N. fowleri show the mechanism its slow movements by visualizing the extended and retracted psuedopods (B). An intermediate flagellate trophozoite stage is also observed in some, but not all strains of N. fowleri. These pear shaped, lophotichous cells use 2 flagella on one end of the cell for movement in finding a suitable host. This transformation into the flagellated stage can be induced by changing the environment of the bacteria, even as simply as being placed in distilled water (C).

Cell structure and metabolism

Naegleria fowleri is a aerobic heterotrophic organism. In studies, it if often given the bacteria Xanthomonas matophilia to feed on by phagocytosis (F). It only feeds in the active ameobiod trophozoite stage, using its psuedopods to engulf and partially digest its microorganism food source. Although they can live in an anaerobic environment, N. fowleri do posses their own mitochondria, and as a result are facilitative aerobic organisms. This preference for highly oxygenated environments increase its ability to infect the brain, an oxygen rich tissue (C). It's cells are characterized by large, dark, semi-central karyosomes and numerous vaculols in its very granular cytosol. Surrounding the active cells is a slightly porous cell membrane with selective permeability. When the organism encysts, the cell wall becomes dense with two pores (C).

Ecology

Naegleria fowleri are found world wide in warm freshwater, either naturally geothermic pools or warm bodies of water created by industry such as industrial cooling water (H). Although they ideally grow at 37^oC, the temperature of their human hosts, below 27^oC, N. fowleri are able to survive by encysting. The amoeba is able to grow in environments up to 45^oC (D). Although it is found all across the globe, the only place where N. fowleri contaminates public drinking water is in South Australia (H). It has even been found in poorly chlorinated pools in southern regions of the United States and other warm locales. This widespread finding of N. fowleri and the low number of cases of N. fowleri related PAM lead to the belief that many more humans are exposed to N. fowleri than actually become infected.

Pathology

Although humans are a known host of N. fowleri, since infections are so rare, it is expected there is an alternative, primary host such as fish, amphibians or reptiles. N. fowleri are known parasitize fish and antibodies to different Naegleria species have been found in many wild animals (G). In humans, N. fowleri enters the olfactory mucosa through the nose either by splashing or diving into contaiminated waters (C). It crosses barriers and invades the subarachnoid space where it focuses on the olfactory bulb and nerves connected to the brain, causing an infection of the central nervous system. The resulting primary amoebic meningoencephalitis, or PAM, is generally fatal. The trophozotic stage is actively growing and diving, it's mitochondrial activity encouraged by the high oxygen concentration in the brain (C). Once in the brain, N. fowleri phagocytizes red blood cells and produces an amebostome to feed off of brain tissue. Along with the ameobostome, N. fowleri secretes hydrolases and phospholases to begin digestion of the brain tissue before phagocytizing it. This highly active phagocytotic activity causes severe hemorraging (C).

Clinical symptoms of PAM mimic that of any other menitigis including headache, fever, nausea and vomiting (C). As stated before, infection of N. fowleri is toxic over 90% of the time. Even f medical care is sought early enough, within a day or two of exposure and infection, the chance of correct diagnosis of PAM as caused by N. fowleri is rare. The most rapid and accurate way to diagnose an N. fowleri is to examine a fresh cerebrospinal tap sample under a light microscope. For future investigation,cultures can be grown and brain tissue can be stained with hematoxylin and eosin, however this culturing and staining process is too lengthy to be used in regards to patient care. The only effective drug against the amoeba is amphotericin B, though other sulfa drugs have been used in collaboration with amphotericin B for a slightly better response.

Current Research

Because of its extreme effects on human hosts and its high mortality rate, most current research on N. fowleri involves the immune system. A group of researchers in Mexico is examining the amoeba's ability to evade the host's innate immune response. A primary defense of the human immune system is the use of mucus to prevent the invader from adhering. As the report by Cervantes-Sandoval et al. investigates, it seems as though N. fowleri is able to overcome this mucosa inhibition quicker than any non-pathogenic strain of Naegleria. It does so by producing enzymes to digest the mucosa, called mucinolytic activity. This begins the all too familiar arms race of the immune system, with the mucosa production being pitted against the mucinolytic enzymes of N. fowleri. This competition for success of the host's immune system helps to explain the low number of infections regardless of the high numbers of exposure to N. fowleri (I).

At the Ajou University School of Medicine, researchers Kim et al. have been conducting numerous studies to discover methods of treating primary amoebic meningoencephalitis caused by Naegleria fowleri. The current drug of choice, should the cause of PAM be discovered quickly enough, is amphotericin B. This polyene (primarily) antifungal drug has an extensive list of severe side effects, from organ damage up to drug related fatality. Because of the toxicity of the drug, Kim et al has been doing multiple studies to investigate alternative treatment. One study tested the effectiveness of miltefosine, origninally developed for HIV infections and chlorpromazine, an antipsychotic. The study found both drugs to reduce infection, however chlorpromazine was discovered to be even more effective than amphotericin B both in vitro and in vivo (J). A second, concurrent study performed by Kim et al tested the effect of various more common antibacterial drugs such as clarithromysin, erthromycin, neomycin and rokitamycin. These drugs were selected for their less severe side effects and more in depth research on their function and mechanism. This second study discovered that rokitamycin, similar to chlorpromazine, was an effective inhibitor of N. fowleri both in vivo and in vitro (K). The results of their research branch the way into developing less toxic but highly effective antibiotic drugs for more microorganisms than simply N. fowleri.

References

1.) Bennett, N.J., Domachowske, J., Khan, A., King, J., and Cross, J.T. 2008. Naegleria. The Medscape Journal of Medicine. 2807.

2.) Cervantes-Sandoval, I., Serrano-Luna, J., Garcia-Latorre, E., Tsutsumi, V., and Shibayama, M. 2008. Mucins in the host defence against Naegleria fowleri and mucinolytic activity as a possible means of evasion.

3.) Clark, C.H. 1990. Genome Structure and Evolution of Naegleria and its Relatives. The Journal of Eukaryotic Microbiology. v.37(4). p.2s-6s.

4.) De Jonckheere, J.F. 2002. A Century of Research on the Amoeboflagellate Genus Naegleria. Acta Protozoologica. v.41. p.309-342.

5.) John, D.T. 1982. Primary amebic meningoencephalitis and the biology of Naegleria fowleri. Annual Review of Microbiology. v.36. p.101-123.

6.) Kilvington, S. and Beeching, J. 1995. Identification and Epidemiological Typic of Naegleria fowleri with DNA Probes.Applied and Environmental Microbiology. v.61(6). p.2071-2078.

7.) Kim, J.H., Jung, S.Y., Lee, Y.J., Song, K.J., Kwon, D., Kim, K., Park, S., Im, K.I., and Shin, H.J. 2008. Effect of theraputic chemical agents in vitron and on experimental meningoencephalitis due to Naegleria fowleri. Antimicrobial Agents and Chemotherapy. v.52(11). p.4010-4016.

8.) Kim, J.H., Lee, Y.J., Sohn, H.J., Song, K.J., Kwon, D., Kwon, M.H., Im, K.I., and Shin, H.J. 2008. Theraputic effect of rokitamycin in vitro and on experimental meningoencephalitis due to Naegleria fowleri. International Journal of Antimicrobial Agents. v.32(5). p. 411-417.

9.) Martinez, A.J. 1996. Free-Living Amebas: Naegleria, Acanthamoeba and Balamuthia. In: Baron, S. ed. Medical Microbiology. Section 4. Chapter 81.

10.) Mertens, E. De Jonckheere, J., and Van Schaftingen, E. 1993. Pyrophoshate-dependent phosphofructokinase from Naegleria fowleri: and AMP-sensitive enzyme. Biochemical Journal. v.292. p.797-803.