Metabolic evolutionary roots of the macrophage immune response in amoeba-bacteria interactions: The conserved role of hypoxia-induced Factor and AMP kinase.

The bacteria Legionella, being able to infect both macrophages and protozoans, reduce oxidative phosphorylation and induce glycolysis, which allows pathogens to grow and replicate in these cells. In amoeba-like inflammatory macrophages (M1), the phagocytizing cells of the primary immune defense, an increase in the rate of glycolysis is followed by a decrease of oxidative phosphorylation. The opposite takes place in anti-inflammatory macrophages (M2). They change from glycolysis to oxidative metabolism when AMP-dependent kinase (AMPK) is activated by a high ratio of AMP/ATP. Stimulation of macrophages with anti-inflammatory cytokines causes activation of AMPK. Infection of macrophages with the parasitic flagellate Leishmania infantum induces a switch from an initial glycolytic phase to oxidative phase with the essential role of AMPK in this change. Activated AMPK induces catabolic pathways effectively producing ATP as well as processes requiring the energy supply. AMPK regulates the migration of cells and enhances the phagocytic activity of macrophages. In macrophages, bacterial products activate TLRs and NF-κB signaling, causing an increase of transcription of hypoxia-induced factor HIF-1α (a subunit of HIF-1). This brings about induction of the enzyme and transporter expression essential for glycolysis and the pentose phosphate pathway to proceed and makes biosynthetic processes and ROS production in macrophages possible. Hypoxia augments macrophage phagocytosis in a HIF-1α-dependent manner. Multicellular parasites experience changes in the availability of oxygen in their life cycle. In the nematode Ascaris suum, HIF participates in the pre-adaptation to hypoxic conditions after infection of their hosts. Also, the freshwater and marine invertebrates meet changes of oxygen concentrations. In the anaerobic branch of the respiratory chain of these invertebrates, fumarate serves as the terminal electron acceptor that is reduced to succinate in complex II of the ETC. In mammalian cells, accumulation of succinate under hypoxic conditions suggests that the mammalian complex II may reduce fumarate to succinate, too. The data reviewed here show that the ability to shift the cell metabolism towards glycolysis observed in activated macrophages can be traced back in evolution to metabolic changes characterizing protozoans infected with bacteria. Anabolic needs of multiplying bacteria direct host metabolism to glycolysis that produces, aside from ATP, precursors of the amino acids used by the pathogen for its protein synthesis. M1-activated mammalian macrophages behave in the same way. Regulation of metabolism in M1 and M2 macrophages is further enhanced by HIF-1 and AMPK, respectively. These archaic functions of AMPK and HIF, important also to control phagocytosis and cell migration were extended to embryonic development in multicellular organisms.

Legionnaires' Disease: State of the Art Knowledge of Pathogenesis Mechanisms of Legionella.

Legionella species are environmental gram-negative bacteria able to cause a severe form of pneumonia in humans known as Legionnaires' disease. Since the identification of Legionella pneumophila in 1977, four decades of research on Legionella biology and Legionnaires' disease have brought important insights into the biology of the bacteria and the molecular mechanisms that these intracellular pathogens use to cause disease in humans. Nowadays, Legionella species constitute a remarkable model of bacterial adaptation, with a genus genome shaped by their close coevolution with amoebae and an ability to exploit many hosts and signaling pathways through the secretion of a myriad of effector proteins, many of which have a eukaryotic origin. This review aims to discuss current knowledge of Legionella infection mechanisms and future research directions to be taken that might answer the many remaining open questions. This research will without a doubt be a terrific scientific journey worth taking.

Characterisation of ZBTB46 and DC-SCRIPT/ZNF366 in rainbow trout, transcription factors potentially involved in dendritic cell maturation and activation in fish.

ZBTB46 and DC-SCRIPT/ZNF366 are two zinc finger transcription factors that play important roles in regulating differentiation of dendritic cells in mammals. In this study, the ZBTB46 and DC-SCRIPT/ZNF366 homologues were identified in rainbow trout Oncorhynchus mykiss and their expression analysed in vivo and in vitro. As transcription factors, they are well conserved in sequence, genomic organisation and gene synteny. Their expression was differentially modulated by bacterial and viral PAMPs in the monocyte/macrophage-like cell line RTS-11, in primary head kidney (HK) macrophages, and in HK macrophages cultured with IL-4/13A. In the RTS-11 cells and primary HK macrophages, all the ZBTB46 and DC-SCRIPT/ZNF366 homologues were down-regulated by interferon gamma (type II IFN) but unaffected by IFN2 (type I IFN), administered as recombinant proteins to cell cultures. In fish gills, infection with amoebae (Paramoebae perurans) resulted in reduction of ZBTB46 and DC-SCRIPT/ZNF366 expression in Atlantic salmon Salmo salar, whilst infection with Yersinia ruckeri induced gene expression in rainbow trout.

Immunity to amoeba.

Amoebic infections in fish are most likely underestimated and sometimes overlooked due to the challenges associated with their diagnosis. Amoebic diseases reported in fish affect either gills or internal organs or may be systemic. Host response ranges from hyperplastic response in gill infections to inflammation (including granuloma formation) in internal organs. This review focuses on the immune response of Atlantic salmon to Neoparamoeba perurans, the causative agent of Amoebic Gill Disease (AGD).

TM9 and cannibalism: how to learn more about cancer by studying amoebae and invertebrates.

Phagocytosis is used not only for immune defense but may also be used to obtain nutrients, as observed when tumor cells feed upon neighboring cells during "tumor cell cannibalism". The TM9 protein TM9SF4, important in canonical phagocytosis, is involved in this cannibalism by metastatic cells and may represent a novel therapeutic target.

Mucosal delivery of ACNPV baculovirus driving expression of the Gal-lectin LC3 fragment confers protection against amoebic liver abscess in hamster.

Mucosal vaccination against amoebiasis using the Gal-lectin of E. histolytica has been proposed as one of the leading strategies for controlling this human disease. However, most mucosal adjuvants used are toxic and the identification of safe delivery systems is necessary. Here, we evaluate the potential of a recombinant Autographa californica baculovirus driving the expression of the LC3 fragment of the Gal-lectin to confer protection against amoebic liver abscess (ALA) in hamsters following oral or nasal immunization. Hamsters immunized by oral route showed complete absence (57.9%) or partial development (21%) of ALA, resulting in some protection in 78.9% of animals when compared with the wild type baculovirus and sham control groups. In contrast, nasal immunization conferred only 21% of protection efficacy. Levels of ALA protection showed lineal correlation with the development of an anti-amoebic cellular immune response evaluated in spleens, but not with the induction of seric IgG anti-amoeba antibodies. These results suggest that baculovirus driving the expression of E. histolytica vaccine candidate antigens is useful for inducing protective cellular and humoral immune responses following oral immunization, and therefore it could be used as a system for mucosal delivery of an anti-amoebic vaccine.

Importance of nonenteric protozoan infections in immunocompromised people.

There are many neglected nonenteric protozoa able to cause serious morbidity and mortality in humans, particularly in the developing world. Diseases caused by certain protozoa are often more severe in the presence of HIV. While information regarding neglected tropical diseases caused by trypanosomatids and Plasmodium is abundant, these protozoa are often not a first consideration in Western countries where they are not endemic. As such, diagnostics may not be available in these regions. Due to global travel and immigration, this has become an increasing problem. Inversely, in certain parts of the world (particularly sub-Saharan Africa), the HIV problem is so severe that diseases like microsporidiosis and toxoplasmosis are common. In Western countries, due to the availability of highly active antiretroviral therapy (HAART), these diseases are infrequently encountered. While free-living amoebae are rarely encountered in a clinical setting, when infections do occur, they are often fatal. Rapid diagnosis and treatment are essential to the survival of patients infected with these organisms. This paper reviews information on the diagnosis and treatment of nonenteric protozoal diseases in immunocompromised people, with a focus on patients infected with HIV. The nonenteric microsporidia, some trypanosomatids, Toxoplasma spp., Neospora spp., some free-living amoebae, Plasmodium spp., and Babesia spp. are discussed.

Assessment of Balamuthia mandrillaris-specific serum antibody concentrations by flow cytometry.

A flow cytometry (fluorescence-activated cell sorter)-based assay was adapted to detect and quantify antibodies to Balamuthia mandrillaris, a causative agent of fatal amoebic encephalitis (BAE), and to Acanthamoeba species. With sera from BAE patients for positive and a group of inconspicuous volunteers for negative reference, most of the 237 sera from random blood donors, patients with atypical encephalitis, atypical pneumonitis, visceral amoebiasis and toxoplasmosis and from subjects working with primates and other mammals were rated negative, 19% elevated and of these 2% high. In comparison, 23 of 25 West Africans living in rural areas revealed elevated, of these 15 high, and one very high B. mandrillaris-binding antibody titers, the latter well in the range of BAE patients. To date, none of the tested individuals have developed symptoms indicative of BAE. Criss-cross analysis with rabbit hyper immune sera against B. mandrillaris, Acanthamoeba comandoni (group 1), Acanthamoeba castellanii (group 2) and Acanthamoeba lenticulata (group 3) confirmed that cross-reactivity between B. mandrillaris and Acanthamoeba sp. is negligible while accentuating antigenic differences between the three morphological groups of Acanthamoeba.

Seroprevalence of anti-amoebic antibody among blood donors by indirect hemeagglutination assay.

The screening for anti-amoebic antibody among a group of donors was to obtain negative control serum samples for an on-going antigen development assay in diagnosis of amoebic liver abscess. Out of 200 samples, 125 (62.5%) were negative, whereas 44 (21.5%) had IHA titer of less than 1:128 and 31 (16.0%) of the samples had significant IHA titers of 1:128 or more, in which 2 serum samples gave titers of 1:4096.

Evolutionary origin of the immune system: a dialogue with Rod Langman.

The need of a specific defence mechanism against intracellular pathogens is proposed to have arisen very early in evolution, perhaps already in protozoa, e.g. amoebae. The phagocytic machinery of amoebae lends itself as a possible starting point for the evolution of such a mechanism. The hypothetical evolutionary pathway described here has been constructed to demonstrate the feasibility of developing a defence system against pathogens in the amoeba, which bears resemblances to contemporary cell-mediated immunity, and can thus be considered as its ancestor.

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.

Quantitation of immune response gene expression and cellular localisation of interleukin-1beta mRNA in Atlantic salmon, Salmo salar L., affected by amoebic gill disease (AGD).

The characterisation of selected immune response genes during amoebic gill disease (AGD) in Atlantic salmon, Salmo salar L., was performed using semi-quantitative RT-PCR, quantitative real-time RT-PCR (qRT-PCR), and in situ hybridisation (ISH). The immune response genes of interest were interleukin-1beta (IL-1beta), inducible nitric oxide synthase (iNOS), serum amyloid A (SAA), and serum amyloid P-like pentraxin (SAP). Atlantic salmon were inoculated with the ectoparasite Neoparamoeba sp., the causative agent of AGD, and gill, liver and anterior kidney tissue sampled at 0, 7 and 14 d post-inoculation (p.i.). Semi-quantitative RT-PCR was performed on the tissue samples to identify up/down-regulated mRNA expression relative to uninfected control fish and normalised to the housekeeping gene, beta-actin. Interleukin-1beta (IL-1beta) was the only immune response gene of those investigated whose mRNA was differentially regulated in any of the tissues and was found to be up-regulated in the gills by semi-quantitative RT-PCR. Increased gill IL-1beta mRNA expression was then accurately quantitated and confirmed using probe-based qRT-PCR. The cellular localisation of the IL-1beta mRNA expression in the gills of uninfected and infected fish was then determined by ISH using an IL-1beta-specific biotinylated cRNA probe. Expression of IL-1beta mRNA was localised to filament and lamellar epithelium pavement cells in gills of uninfected and infected Atlantic salmon. These data implicate the involvement of IL-1beta at the site of infection, the gills, of Atlantic salmon during AGD. This work supports previous studies that suggest IL-1beta is important in the regulation of the fish immune response to parasitic infection but additionally shows the cellular localisation of fish IL-1beta mRNA expression during infection.

Using non-mammalian hosts to study fungal virulence and host defense.

Non-mammalian hosts have been used to study host-fungal interactions. Hosts such as Drosophila melanogaster, Caenorhabditis elegans, Acathamoeba castellanii, Dictyostelium discoideum, and Galleria mellonella have provided means to examine the physical barriers, cellular mechanisms and molecular elements of the host response. The Drosophila host-response to fungi is mediated through the Toll pathway, whereas in C. elegans the host-response is TIR-1-dependent. Virulence traits that are involved in mammalian infection are important for the interaction of fungi with these hosts. Screening of fungal virulence traits using mutagenized fungi to determine changes in fungal infectivity of non-mammalian hosts has been used to identify novel virulence proteins used to infect C. elegans such as Kin1 (a serine/threonine protein kinase) and Rom2 (a Rho1 guanyl-nucleotide exchange factor) from Cryptococcus neoformans. These heterologous non-mammalian hosts highlight the similarities and differences between different hosts in fungal pathogenesis and they complement studies in mammalian systems and those using other genetic approaches.

MHC class II+ cells in the gills of Atlantic salmon (Salmo salar L.) affected by amoebic gill disease.

Amoebic gill disease (AGD) is characterised by the association of Neoparamoeba sp. with hyperplastic gill tissue of affected fishes, however, the identity and role of host cells associated with AGD lesions are not known. Here, we investigated cells with an immunological role that were associated with AGD lesions by locating cellular MHC class II beta chain. A tank housing Atlantic salmon (Salmo salar) was inoculated with Neoparamoeba sp., and MHC class II beta chain expression in the gills was qualitatively assessed by immunohistochemistry. In AGD-naïve control fish, MHC class II+ cells were detected basolateral to the interlamellar epithelium as well as upon the interlamellar and secondary epithelium. In the gills of AGD affected fish MHC class II+ cells were observed in both affected and unaffected tissue. Within AGD lesions, numerous MHC class II+ cells were present and these cells exhibited variable levels of expression suggesting that like mammals, MHC class II expression is highly regulated. The presence of MHC class II+ cells within gill lesions is indicative of immune cell trafficking and these cells could contribute in an antigen presentation capacity to the development of an antibody response in fish chronically affected by AGD.

Changes in antigenic profile during culture of Neoparamoeba sp., causative agent of amoebic gill disease in Atlantic salmon.

Amoebic gill disease (AGD), the most serious infectious disease affecting farmed salmon in Tasmania, is caused by free-living marine amoeba Neoparamoeba sp. The parasites on the gills induce proliferation of epithelial cells initiating a hyperplastic response and reducing the surface area available for gaseous exchange. AGD can be induced in salmon by exposure to freshly isolated Neoparamoeba from AGD infected fish, however cultured Neoparamoeba are non-infective. We describe here antigenic differences between freshly isolated and in vitro cultured parasites, and within individual isolates of the parasite cultured under different conditions. Immunoblot analysis using polyclonal antisera, revealed differences in the antigen profiles of two cultured isolates of Neoparamoeba sp. when they were grown on agar versus in liquid medium. However, the antigen profiles of the two isolates were very similar when they were grown under the same culture conditions. Comparison of these antigen profiles with a preparation from parasites freshly isolated from infected gills revealed a very limited number of shared antigens. In addition monoclonal antibodies (mAbs) raised against surface antigens of cultured parasites were used in an indirect immunofluorescence assay to assess the expression of specific surface antigens of Neoparamoeba sp. after various periods in culture. Significant changes in antigen expression of freshly isolated parasites were observed after 15 days of in vitro culture. The use of mAb demonstrated progressive exposure/expression of individual antigens on the surface of the freshly isolated parasites during the period in culture.

Immunodetection and intracellular localization of caldesmon-like proteins in Amoeba proteus.

Caldesmon immunoanalogues were detected in Amoeba proteus cell homogenates by the Western blot technique. Three immunoreactive bands were recognized by polyclonal antibodies against the whole molecule of chicken gizzard caldesmon as well as by a monoclonal antibody against its C-terminal domain: one major and two minor bands corresponding to proteins with apparent molecular masses of 150, 69, and 60 kDa. The presence of caldesmon-like protein(s) in amoebae was revealed as well in single cells after their fixation, staining with the same antibodies, and recording their total fluorescence in a confocal laser scanning microscope. Proteins recognized by the antibodies bind to filamentous actin. This was established by a cosedimentation assay in cell homogenates and by colocalization of the caldesmon-related immunofluorescence with the fluorescence of filamentous actin stained with rhodamine-labelled phalloidin, demonstrated in optical sections of single cells in a confocal microscope. Caldesmon is colocalized with filamentous actin in the withdrawn cell regions where the cortical actomyosin network contracts and actin is depolymerized, in the frontal zone where actin is polymerized again and the cortical cytoskeleton is reconstructed, inside the nucleus and in the perinuclear cytoskeleton, and probably at the cell-to-substratum adhesion sites. The regulatory role of caldesmon in these functionally different regions of locomoting amoebae is discussed.

Serum antibodies to Balamuthia mandrillaris, a free-living amoeba recently demonstrated to cause granulomatous amoebic encephalitis.

Free-living amoebae cause three well-defined disease entities: a rapidly fatal primary meningoencephalitis, a chronic granulomatous amoebic encephalitis (GAE), and a chronic amoebic keratitis. GAE occurs in immunocompromised persons. Recently, another type of free-living amoeba, Balamuthia mandrillaris, has been shown to cause GAE. The finding that this amoeba has caused infection in some healthy children has raised the possibility that humans may lack immunity to B. mandrillaris. Human serum was examined for the presence of surface antibodies specific for this amoeba by immunofluorescence. Sera from adults contained titers of 1/64-1/256 of anti-B. mandrillaris antibodies (IgM and IgG classes), which did not cross-react with other amoebae. Cord blood contained very low antibody levels, but levels similar to those in adults were seen in serum of 1- to 5-year-old children.

Characterization of a monoclonal antibody and a cDNA for polyubiquitin of Amoeba proteus.

A monoclonal antibody was obtained that reacts with many different proteins (14-200 kDa) of Amoeba proteus. By indirect immunofluorescence microscopy we found the antigens to be dispersed throughout the cytoplasm but were more concentrated in the nucleus. The antibody cross-reacted with proteins of Tetrahymena, Xenopus embryo, and mouse macrophages. Using the antibody as a probe we cloned a cDNA of 1.2 kb coding for ubiquitin in five repeats. Amino acid sequences of ameba's polyubiquitin showed the most variations among the nineteen polyubiquitins of other organisms compared. The well-conserved 20Ser and 55Thr residues were replaced with Gly and Ser, respectively. The 28Ala residue found in most organisms was replaced with Gln or Glu in the amoeba. Amoebae contained two ubiquitin-mRNAs that could be detected by Northern blot analysis using the cDNA as a probe. In an analysis for specificity, the antibody reacted with polyubiquitin and ubiquitin-fusion proteins larger than 14 kDa but not with monomeric ubiquitin. The antibody is a useful probe in the detection and characterization of proteins ubiquitinated in response to cellular stresses.

Quantification and isotype analysis of the serum anti-amebic antibody response produced after mucosal and systemic immunization in male and female mice.

It has not been established whether seric antiamebic antibodies are produced after local presence of amebas, or only after they invade the tissues. Therefore, the purpose of this work was to determine if mucosal immunization with glutaraldehyde-fixed trophozoites (GFT) could induce seric anti-amebic antibody responses in mice. We determined by ELISA the quality and quantity of anti-E. histolytica seric antibody responses induced after either a single or multiple doses of GFT by intragastric, rectal and intraperitoneal routes. Seric anti-amebic antibody responses were elicited by both mucosal or systemic immunization although such responses induced via intragastric route were low. However, the responses achieved via rectal route were high, suggesting that the local presence of amebas without mucosal tissue damage is enough to stimulate seric anti-amebic response. The highest seric anti-amebic responses were obtained via intraperitoneal route. The antibody isotype of the induced responses was influenced by the immunization route. The major isotypes produced via intraperitoneal and rectal routes were IgM and IgG. The predominant isotype elicited by intragastric route was IgM whereas the IgA and IgG responses were small and of the same magnitude. The lowest IgA anti-amebic response was produced by intraperitoneal route. In addition, our data show that in general GFT immunization elicits higher anti-amebic antibody responses in female than in male mice.