PROTEOMIC ANALYSIS OF TWO DIFFERENT STATES OF NAEGLERIA FOWLERI

Naegleria fowleri are free-living ameboflagellates found in soil and freshwater habitats throughout the world that cause a fatal disease in humans called Primary Amoebic Meningoencephalitis (PAM). Mechanisms of host resistance or susceptibility to infection have not been fully elucidated, and possible treatment methods are still sub optimal. The disease is diagnosed using specific laboratory tests available in only a few laboratories in the United States. Because of the rarity of infection and difficulty in initial detection, more than often PAM is misdiagnosed. Therefore, it is very important to find causative marker for early detection of an infection. The purpose of this study is to create a proteomic signature map using two-dimensional gel electrophoresis (2-D gel) and recommend a subset of proteins that may be directly linked to the pathogenic state of N. fowleri. Replicates of 2-D Gels were created for both strains of N. fowleri and the proteomic templates from these gels were compared with each other. Scatter Plots were generated measuring the density of protein spots from 2-D gels being analyzed for each study. For each strains of N. fowleri, the 2-D gels from each study were compared within and compared between the two studies for reproducibility in data. The resulting correlation values for all of the Scatter Plots were greater or equal to 0.90. Finally, the representative proteomic template for axenically grown N. fowleri and mouse passaged N. fowleri were compared and the correlation value of 0.60 was observed. This confirmed our theory that these two strains or states of N. fowleri have very different protein expressions, and we were able to identify a subset of proteins, both over expressed and newly synthesized, that may be linked to the highly pathogenic state of N. fowleri

Identification of Naegleria fowleri proteins linked to primary amoebic meningoencephalitis

Naegleria fowleri (N. fowleri) causes primary amoebic meningoencephalitis, a rapidly fatal disease of the central nervous system. N. fowleri can exist in cyst, flagellate or amoebic forms, depending on environmental conditions. The amoebic form can invade the brain following introduction into the nasal passages. When applied intranasally to a mouse model, cultured N. fowleri amoebae exhibit low virulence. However, upon serial passage in mouse brain, the amoebae acquire a highly virulent state. In the present study, a proteomics approach was applied to the identification of N. fowleri amoeba proteins whose expression was associated with the highly virulent state in mice. Mice were inoculated intranasally with axenically cultured amoebae or with mouse-passaged amoebae. Examination by light and electron microscopy revealed no morphological differences. However, mouse-passaged amoebae were more virulent in mice as indicated by exhibiting a two log10 titre decrease in median infective dose 50 (ID50). Scatter plot analysis of amoebic lysates revealed a subset of proteins, the expression of which was associated with highly virulent amoebae. MS-MS indicated that this subset contained proteins that shared homology with those linked to cytoskeletal rearrangement and the invasion process. Invasion assays were performed in the presence of a select inhibitor to expand on the findings. The collective results suggest that N. fowleri gene products linked to cytoskeletal rearrangement and invasion may be candidate targets in the management of primary amoebic meningoencephalitis

The Genesis of the “Brain-Eating” Amoeba

The Creator made all creatures, including amoebas, with variation and plasticity in their genome for global differences of climate, terrain, and environment. God made all creatures to multiply and fill the earth. Diversification and speciation from the original kind would be normal even in a perfect world. Naegleria fowleri is a global amoeba pathogen that is the causative agent of primary amoebic meningoencephalitis (PAM), thus termed “brain-eating.” Many people are evidently exposed, based upon antibody levels, to this amoeba while swimming in warm waters. Only a small percentage succumb to the germ, but once invasion happens, only about 1% survive. Because it is such a devastating disease, most people do not survive the infection. This common pathogen is a free-living ameboflagellate present in very warm water during the summer time

The in vitro and in vivo testing of chemotherapeutic agents against pathogenic free living amebae : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand.

During the last ten years, there has been an increasing awareness of sporadic cases of Primary Amoebic Meningo-encephalitis (PAM) affecting primarily younger age groups and appearing in an acute fulminant form. The earliest positive case (Willaert, 1974) may have been in England in 1909 which shows that the disease has been with us for a long time. The pathogenic free-living amebae (PFLA), which comprises the genus Naegleria and the genus Acanthamoeba, are the causative organisms of PAM and AM*respectively. PAM is a rapidly fatal disease affecting the central nervous system (CNS),the treatment of which to date has been successful in only a small number of cases, and therefore the continual screening of suitable chemotherapeutic agents against amebae of the Naegleria spp. and Acanthamoeba spp., is of great importance. AM is also essentially confined to the CNS although it may take the form of chronic granulomata in the liver, spleen, uterus and kidneys (Martinez et al., 1977). Six chemotherapeutic agents: Amphotericin B, 5-Fluorocytosine, Kanamycin, Oxytetracycline, Tylosine and Levamisole were tested for activity against a non-pathogenic and a pathogenic species of Naegleria and a non-pathogenic and a pathogenic species of Acanthamoeba in axenic culture. For the Naegleria spp., Amphotericin B and Oxytetracycline were found to be active and the Acanthamoeba spp. were found to be only susceptible to Levamisole. The synergistic combinations of drugs against the amebae were also investigated in axenic culture. In preliminary trials Kanamycin together with Oxytetracycline showed promise against Naegleria fowleri (MsM) but this was later shown not to be the case. Amphotericin B in combination with 5-Fluorocytosine was also shown not to be synergistic, however Amphotericin B in combination with Oxytetracycline proved to be effective against N. fowleri. Amphotericin B was combined with 5-Fluorocytosine against A. culbertsoni (A-1) but was not found to be synergistically active. * Amebic meningitis caused by Acanthamoeba infections. Levamisole was also tested against N. gruberi (P1200f) and A. castellanii (0.1) at various stages in growth of the amebae (i.e. 24, 48 and 72 hour stock cultures) to determine the effect of using aged amebae. It was found that the age of the stock culture bore no relation to the activity of the drug. After axenic culture testing, the susceptibility of the pathogenic N. fowleri (MsM) and A. culbertsoni (A-1) to the agents which showed activity, was investigated in a vero cell culture system. For N. fowleri (MsM) the results of axenic culture testing were confirmed, with Amphotericin B and Oxytetracycline protecting the monolayer from the destructive effects of the amebae, both when used singly and at a greater efficiency when added together as a synergistic combination. Levamisole, although effective to some extent against Acanthamoeba spp. in axenic culture, failed to show any activity against the amebae in vero cell culture testing. In vivo animal protection studies were then performed using drugs that had been shown either in this or other studies to be effective against either Naegleria or Acanthamoeba spp. Chemotherapeutic agents tested on N. fowleri (MsM) included two imidazoles; Miconazole nitrate and Ketoconazole (previously known as R41,400), as well as Amphotericin B. The synergistic combination of Amphotericin B with either Tetracycline or Oxytetracycline was also investigated. For A. culbertsoni (A-1), 5-Fluorocytosine, and Polymyxin B were tried both singly and in combination. These drugs were injected by intraperitoneal (I.P.) and intraventricular (I.vent.) routes. The results were not promising, with none of the drugs offering significant protection even whilst using Amphotericin B which is considered the drug of choice. The question of adequate drug levels reaching the brain was tested out with two imidazoles, Ketoconazole and Miconazole. Serum samples were assayed against Candida pirapsilosis and C. pseudotropicalis respectively at various time intervals after innoculation with the drug, and a gradual increase and breakdown of the drug in the animal system could then be shown. These results showed that based on in vitro results, the levels of the imidazoles obtained in the serum after the first eight hours after injection, should have been sufficiently high to prevent amebic multiplication

A study of various environmental factors on the growth, encystment and survival of free living amoebae : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University

Free-living amoebae (FLA) are soil organisms which have a worldwide distribution. Interest was raised when they were implicated in two fatal and several non-fatal infections in humans. This investigation involved examination of the role and/or effect of several environmental factors on growth, encystment and cyst survival of FLA. The effect of K+, Na+ , Mg+2, Ca+2 and Fe+2/+3 on the growth of four species of amoebae (Naegleria gruberi, Naegleria fowleri, Acanthamoeba culbertsoni and Acanthamoeba castellanii) was investigated. Inhibition of growth rate increased as the cation concentration was increased. The roles of Mg+2 and Ca+2 in encystment were investigated and it was found that rather than being necessary, they were inhibitory. The survival of cysts under low temperatures and high cation concentrations was studied. Acanthamoeba proved to be resistant to adverse conditions once encysted. Naegleria were not affected by high cation levels but were adversely affected by low temperatures. A preliminary identification of two isolates from Ngawha Springs hot pools was undertaken showing both amoebae to be temperature tolerant Naegleria species

Distribution of Naegleria fowleri in Selected Northeast Arkansas Lakes

Seven northeast Arkansas recreational lakes were examined for the presence of pathogenic and nonpathogenic Naegleria fowleri. Cultural differentiation and microscopic morphology were used as species determining tests, while mouse pathogenicity tests were conducted to determine virulence. Only one isolate met all criteria utilized for definite identification of Naegleria fowleri, although Naegleria type organisms were found in all of the lakes. None of the isolates were pathogenic in mice

Environmental factors affecting encystment of P.F.L.A. together with disinfection studies: a thesis is presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University, Palmerston North, New Zealand

ABSTRACT Free living amoebae from the genera Naegleria and Acanthamoeba have been implicated in fatal and several non-fatal infections of the human central nervous system, and other organs. They can be isolated from a worldwide range of environments. The common occurance of these organisms in nature may be attributed to the ability to form resistant cysts to withstand adverse environmental conditions. Research was performed to determine factors that will promote the encystment of amoebae, particularly Naegleria species. The parameters examined for the induction of encystment were: the type of substrate amoebae were growing on liquid or solid, the presence of bacteria, the cell concentration and nutrient availability and incubation at temperatures other than the optimal growth temperature. Higher percentages of amoebae encysted on solid surface environ­ments in comparison to the liquid media. In liquid media a greater percentage of trophozoites only formed a pre-encystment or roundform stage. The factor required for the complete encyst­ment of roundforms was not present. The encystment of Naegleria fowleri was not significantly influenced by the presence or absence of the bacterial species used (E. cloacae). Encystment of Naegleria spat different cell concentrations, using a nutrient media, a soil extract broth and a non-nutrient media was examined. Complete encystment of cells did not occur where nutrients were either high or absent, and the cell concentration was low. The ability of trophozoites of N. fowerli, N. gruberi, culbertsoni, and A. castellanii to encyst at a range of temperatures from 4°C - 44°C was studied. The trophozoites of Acanthamoeba sp could encyst over a wider temperature range in comparison to the trophozoites of Naegleria sp. The effect of disinfection using Baquacil was studied. Previously isolated strains of baquacil resistant N. fowleri, still had higher disinfection survival rates compared to sensitive strains. Resistance of trophozoites to Baquacil was not affected even after storage as a cyst. Baquacil resistant strains of N. fowleri were still sensitive to chlorination. Disinfection of amoebic cysts using chlorine and Baquacil was investigated. Amoebic cysts require higher levels of disinfection for inactivation in comparison to trophozoites. Acanthamoeba cysts have a greater tolerance to chlorination than Naegleria sp

Primary Amoebic (Naegleria fowleri) Meningoencephalitis Presenting as Status Epilepticus

Primary amebic meningoencephalitis (PAM) is a rare entity. Usual presenting features are fever, headache and seizures with meningeal signs and this disease carries high mortality rate. We present a case report of PAM presenting as status epilepticus

The susceptibility of pathogenic free-living amebae to chemotherapeutic agents : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Microbiology at Massey University

The treatment of infections caused by pathogenic free-living amebae (PFLA) has, until only recently, been far from successful. The continued screening of chemotherapeutic agents against amebae of the genera Naegleria and Acanthamoeba is therefore of the utmost importance. Seven chemotherapeutic agents, amphotericin B, rifampicin, tetracycline, polymyxin B sulphate, 5-fluorocytosine, miconazole and R41,400 were screened for activity against a non-pathogenic and pathogenic species of Naegleria and a non-pathogenic and pathogenic species of Acanthamoeba in axenic culture. For the Naegleria spp. amphotericin B, miconazole and R41,400 were found to be active. Acanthamoebae spp. were found to be susceptible only to 5-fluorocytosine and R41,400. The possible use of combinations of drugs against the amebae was also investigated in axenic culture. For Naegleria fowleri (MsT) amphotericin B with either tetracycline or rifampicin showed a synergistic effect. Polymyxin B sulphate and 5-fluorocytosine showed synergistic activity against Acanthamoeba culbertsoni (A-1) but when polymyxin B was combined with tetracycline or rifampicin no significant additive effect was seen. After axenic culture testing the susceptibility of the pathogenic species, N. fowleri (MsT) and A. culbertsoni (A-1), to the agents which showed activity, was investigated in a Vero cell culture system. For N. fowleri (MsT) the results of axenic testing were confirmed with amphotericin B, miconazole and R41,400 protecting the monolayer from the destructive effects of the amebae. 5-Fluorocytosine inhibited the formation of cytopathic effect (CPE) when the cell cultures were inoculated with A. culbertsoni (A-1) but viable amebae were still present. R41,400 had no effect on A. culbertsoni (A-1) at concentrations at or above those which were cytotoxic to the Vero cells. The use of combinations of drugs was also investigated in Vero cell culture. Amphotericin B and rifampicin showed an antagonistic rather than a synergistic effect when used against N. fowleri (MsT) in cell culture but amphotericin B and tetracycline showed synergistic activity. For A. culbertsoni (A-1) the synergistic activity of polymyxin B and 5-fluorocytosine was confirmed. The lack of an additive effect between polymyxin B and either tetracycline or rifampicin was also shown in cell culture. The new imidazole derivative R41,400, which showed promise against N. fowleri (MsT) in in vitro tests was then tested in the in vivo situation. Mice experimentally infected with N. fowleri (MsT) were treated once or twice daily intraperitoneally with different doses of R41,400. At the higher dosage levels tested the drug appeared to have a deleterious effect, the average time for death being less than that for the controls

Eleutherodactylus fowleri

Number of Pages: 1Integrative BiologyGeological Science