Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals

Knowledge that free-living amoebae are capable of causing human disease dates back some 50 years, prior to which time they were regarded as harmless soil organisms or, at most, commensals of mammals. First Naegleria fowleri, then Acanthamoeba spp. and Balamuthia mandrillaris, and finally Sappinia diploidea have been recognised as etiologic agents of encephalitis; Acanthamoeba spp. are also responsible for amoebic keratitis. Some of the infections are opportunistic, occurring mainly in immunocompromised hosts (Acanthamoeba and Balamuthia encephalitides), while others are non-opportunistic (Acanthamoeba keratitis, Naegleria meningoencephalitis, and cases of Balamuthia encephalitis occurring in immunocompetent humans). The amoebae have a cosmopolitan distribution in soil and water, providing multiple opportunities for contacts with humans and animals, as evidenced by antibody titers in surveyed human populations. Although, the numbers of infections caused by these amoebae are low in comparison to other protozoal parasitoses (trypanosomiasis, toxoplasmosis, malaria, etc.), the difficulty in diagnosing them, the challenge of finding optimal antimicrobial treatments and the morbidity and relatively high mortality associated with, in particular, the encephalitides have been a cause for concern for clinical and laboratory personnel and parasitologists. This review presents information about the individual amoebae: their morphologies and life-cycles, laboratory cultivation, ecology, epidemiology, nature of the infections and appropriate antimicrobial therapies, the immune response, and molecular diagnostic procedures that have been developed for identification of the amoebae in the environment and in clinical specimens.     

Entamoeba histolytica: Expression and localization of Gal/GalNAc lectin in virulent and non-virulent variants from HM1:IMSS strain

We have purified Gal/GalNAc lectin from Entamoeba histolytica by electroelution. The purified protein was used to immunize rabbits and obtain polyclonal IgG’s anti-lectin. These antibodies were used as tools to analyze the expression and localization of the amoebic lectin in both virulent (vEh) and non-virulent (nvEh) variants of axenically cultured HM1:IMSS strain. vEh is able to induce liver abscesses in hamsters, whereas nvEh has lost this ability. In vitro, amoebic trophozoites from both variants equally express this protein as shown by densitometric analysis of the corresponding band in Western blots from lysates. In both types of trophozoites, the pattern of distribution of the lectin was mainly on the surface. We have also compared by immunohistochemistry the presence and distribution of lectin in the in vivo liver lesions produced in hamsters. In order to prolong the survival of nvEh to analyze both variants in an in vivo model, hamsters inoculated with nvEh were treated with methyl prednisolone. Our results suggest that the Gal/GalNAc lectin is equally expressed in both nvEh and vEh.     

Parasitic diseases in marine cage culture--an example of experimental evolution of parasites?

Rapid development of fish culture in marine cages has been associated with an emergence of parasitic diseases. There is a general trend to an increase in infections with ectoparasites with direct life cycles and a reduced diversity of parasites in aquaculture. Some mariculture creates conditions that are similar to serial passage experiments, which are used to study adaptation during experimental evolution of pathogens. In particular, increased density of fish, repeated introduction of naive hosts, homogenous host populations, fast growth and a potential decrease in genetic diversity are attributes of both aquaculture and serial passage experiments. Some free-living organisms, for example Neoparamoeba spp. and Uronema spp. parasitise fish in culture, but have not been reported from wild populations. Farming fish in marine cages can increase the risk of outbreaks of parasitic diseases, including those caused by opportunistic parasites. However, aquaculture has the potential to control parasitic diseases through selective breeding, vaccination and general fish health management.     

Entamoeba histolytica: Virulence enhancement of isoenzyme-stable parasites

Pathogenic Entamoeba histolytica isolated from patients with clinical amoebiasis can be differentiated from nonpathogenic E. histolytica obtained from asymptomatic carriers on the basis of the electrophoretic pattern of their isoenzymes. Virulence of different strains of axenically grown trophozoites of Entamoeba histolytica, as determined by various laboratory tests, such as damage to tissue culture monolayers, or their ability to cause an hepatic abscess in a hamster, are known to vary considerably. Reassociation of trophozoites of strain HK-9 with certain Escherichia coli strains for short periods of time markedly augmented their virulence, as tested by the above-mentioned methods. The bacterial association, however, did not cause any change in the electrophoretic pattern of amoebic isoenzymes (zymodeme).     

Acanthamoeba culbertsoni: Electron‐Dense Granules in a Highly Virulent Clinical Isolate

The virulence of various amoebic parasites has been correlated with the presence of electron‐dense granules (EDGs) in the cytoplasm of trophozoites. Here, we report the finding by transmission electron microscopy of a large number of EDGs in a recent culture of Acanthamoeba culbertsoni, isolated from a severe case of human keratitis. When this isolate was maintained in culture for 6 mo, the granules almost disappeared. However, after induction of mice brain lesions with the long‐term cultured isolate, recovered amoebas had abundant EDGs. Trophozoites of the original isolate, or those recovered from experimental lesions, secreted EDGs into the medium when incubated with MDCK cells. To analyze a possible cytotoxic effect the conditioned medium was incubated with MDCK monolayers. After 5 h, the media containing EDGs produced opening of the tight junctions; at 24 h, cell viability was compromised, and at 48 h most of the cells were detached from the monolayer. In contrast, trophozoites in long‐term cultures did not release EDGs to the medium during incubation with MDCK cells, and the corresponding conditioned medium did not have any effect on MDCK monolayers. Our observations further support the hypothesis that EDGs play a role in the cytopathogenic mechanisms of A. culbertsoni.     

Pathogenic bacteria prime the induction of Toll-like receptor signalling in human colonic cells by the Gal/GalNAc lectin Carbohydrate Recognition Domain of Entamoeba histolytica

In mixed intestinal infections with Entamoeba histolytica trophozoites and enteropathogenic bacteria, which are wide-spread in areas of endemic amoebiasis, interaction between the pathogens could be an important factor in the occurrence of invasive disease. It has been reported that exposure of human colonic cells to enteropathogenic bacteria increased trophozoite adherence to the cells and their subsequent damage. We report here that the Carbohydrate Recognition Domain (CRD) of the amoebic Gal/GalNAc lectin binds to Toll-like receptors TLR-2 and TLR-4 in human colonic cells, activating the “classic” signalling pathway of these receptors. Activation induced expression of TLR-2 and TLR-4 mRNAs and the mRNAs of pro-inflammatory cytokines, as well as an increase in the corresponding proteins. Direct correlation was observed between the increased expression of TLRs and pro-inflammatory cytokines, the enhanced adhesion of trophozoites to the cells and the inflicted cell damage. When cells were exposed to pathogenic bacteria Staphylococcus aureus (Gram+) or Shigella dysenteriae (Gram−), elements of an innate immune response were induced. CRD by itself elicited a similar cell response, while exposure to a commensal Escherichia coli had a null effect. Pre-exposure of the cells to pathogenic bacteria and then to CRD rendered an inflammatory-like microenvironment that after addition of trophozoites facilitated greater cell destruction. Our results suggest that CRD is recognised by human colonic cells as a pathogen-associated-molecular-pattern-like molecule and as such can induce the expression of elements of an innate immune response. In the human host, an exacerbated inflammatory environment, derived from pathogen interplay, may be an important factor for development of invasive disease.     

Neoparamoeba perurans n. sp., an agent of amoebic gill disease of Atlantic salmon (Salmo salar)

Amoebic gill disease (AGD) is a potentially fatal disease of some marine fish. Two amphizoic amoebae Neoparamoeba pemaquidensis and Neoparamoeba branchiphila have been cultured from AGD-affected fish, yet it is not known if one or both are aetiological agents. Here, we PCR amplified the 18S rRNA gene of non-cultured, gill-derived (NCGD) amoebae from AGD-affected Atlantic salmon (Salmo salar) using N. pemaquidensis and N. branchiphila-specific oligonucleotides. Variability in PCR amplification led to comparisons of 18S rRNA and 28S rRNA gene sequences from NCGD and clonal cultured, gill-derived (CCGD) N. pemaquidensis and N. branchiphila. Phylogenetic analyses inferred from either 18S or 28S rRNA gene sequences unambiguously segregated a lineage consisting of NCGD amoebae from other members of the genus Neoparamoeba. Species-specific oligonucleotide probes that hybridise 18S rRNA were designed, validated and used to probe gill tissue from AGD-affected Atlantic salmon. The NCGD amoebae-specific probe bound AGD-associated amoebae while neither N. pemaquidensis nor N. branchiphila were associated with AGD-lesions. Together, these data indicate that NCGD amoebae are a new species, designated Neoparamoeba perurans n.sp. and this is the predominant aetiological agent of AGD of Atlantic salmon cultured in Tasmania, Australia.     

Precision-cut hamster liver slices as an ex vivo model to study amoebic liver abscess

Entamoeba histolytica is the etiological agent of amoebiasis, the second cause of global morbidity and mortality due to parasitic diseases in humans. In approximately 1% of the cases, amoebas penetrate the intestinal mucosa and spread to other organs, producing extra-intestinal lesions, among which amoebic liver abscess (ALA) is the most common. To study ALA, in vivo and in vitro models are used. However, animal models may pose ethical issues, and are time-consuming and costly; and cell cultures represent isolated cellular lineages. The present study reports the infection of precision-cut hamster liver slices with Entamoeba histolytica trophozoites. The infection time-course, including tissue damage, parallels findings previously reported in the animal model. At the same time amoebic virulence factors were detected in the infected slices. This new model to study ALA is simple and reproducible, and employs less than 1/3 of the hamsters required for in vivo analyses.     

Stochastic control of spontaneous signal generation for gradient sensing in chemotaxis

Chemotaxis is characterized by spontaneous cellular behavior. This spontaneity results, in part, from the stochasticity of intracellular reactions. Spontaneous and random migration of chemotactic cells is regulated by spontaneously generated signals, namely transient local increases in the level of phosphoinositol-3,4,5-triphosphate (PIP3 pulses). In this study, we attempted to elucidate the mechanisms that generate these PIP3 pulses and how the pulses contribute to gradient sensing during chemotaxis. To this end, we constructed a simple biophysical model of intracellular signal transduction consisting of an inositol phospholipid signaling pathway and small GTPases. Our theoretical analysis revealed that an excitable system can emerge from the non-linear dynamics of the model, and that stochastic reactions allow the system to spontaneously become excited, which was corresponded to the PIP3 pulses. Based on these results, we framed a hypothesis of the gradient sensing; a chemical gradient spatially modifies a potential barrier for excitation and then PIP3 pulses are preferentially generated on the side of the cell exposed to the higher chemical concentration. We then validated our hypothesis using stochastic simulations of the signal transduction.