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Trends in Parasitology

Forum Interplay between introduction of H. meleagridis into a poul- agreement with studies on the homolo- try flock (Figure 1). gous protozoan parasite T. vaginalis [7]. Consequently, (i) why does H. meleagridis and Bacteria: Mutualistic Initial studies set up to investigate the need bacteria for its survival and growth, – parasite–bacteria interaction aimed to and (ii) is this interaction mutualistic or or Predator Prey? resolve the aetiology of histomonosis. For rather of a predator–prey nature as outlined Ivana Bilic1,* and Michael Hess1,2 this purpose, sterilised eggs below and summarised in Figure 2? harbouring H. meleagridis were combined with selected bacteria in vitro and in Bacteria as a Food Source for the gnotobiotic and turkeys. As a Parasite Histomonas meleagridis is an extra- general outcome, it was noted that Experiments feeding an H. meleagridis cellular protozoan parasite and the the pathogenicity of the parasite is not culture with dead instead of live bacteria aetiological agent of histomonosis, influenced by the microflora; however, resulted in the death of the parasite, an important disease whose its replication relies on the presence of suggesting that killed bacteria are not impact is greatly accentuated by bacteria, which differ in their level of sup- an essential nutrient source. However, inaccessibility of any treatment. A port. Establishing a system based on a bacteria are taken up into food vacuoles special feature of the parasite is monoxenic clonal culture of H. meleagridis and metabolised by the parasite. In agree- its intricate interplay with bacteria enabled the targeted exchange of the ment with this, peptidoglycan-degrading fi in vitro and in vivo, the focus of cocultivated bacterial strain [4]. It was enzymes were identi ed in the tran- confirmed by in vitro experiments that scriptome, proteome and exoproteome this article. Escherichia coli was superior to other of H. meleagridis, suggesting their involve- bacterial . Growth of the parasite in ment in the destruction of cocultivating or Histomonosis: A Bacteria-Driven a monoxenic background deferred the accompanying bacteria [8–10]. The acqui- Parasitic Disease onset of histomonosis as compared with in- sition of these bacterial genes involved in The protozoan Histomonas meleagridis fection with a xenic culture, indicating a de- degradation of the bacterial cell envelope is the causative agent of histomonosis layed adaptation of H. meleagridis to caecal into the parasite’s genome allegedly by (synonyms blackhead disease and conditions and a subsequent infection [4]. lateral gene transfer is not new and was ) in gallinaceous [1]. Dysbiosis, modulation of mucosal immunity reported for the closely related parasite The disease was controlled for decades and disruption of the epithelial barrier in the T. vaginalis [11]. This could be beneficial using chemotherapeutics as preventive context of bacteria–parasite interaction, for the protozoan in respect to nutrient and curative drugs, but changes in drug has been reported for other extracellular acquisition and to control the presence or legislation in the EU and the USA have parasites, such as Entamoeba histolytica abundance of certain bacteria. Selective led to its reappearance [2]. The devastat- and Trichomonas vaginalis [5,6]. Such predation of bacteria by protozoans was ing outcome of histomonosis is obvious studies highlight the importance of the implicated in the regulation of bacterial in turkeys, in which it can lead to mortality microflora in the context of infection and strain composition and was shown to be of up to 100%, whereas in chickens the clinical consequences. Special attention abundant, involving both parasitic and disease is less severe [1]. The essential has to be given to the immune and physio- free-living species [12]. Considering that presence of live bacteria either to propa- logical reactions of the and its nutrition H. meleagridis prefers certain bacterial gate H. meleagridis in vitro or to success- in order to fully elucidate the complete background [4], it is possible that the fullyestablishaninfectioninitshost interaction triangle. parasite exploits a similar process. questioned the aetiology of histomonosis Altogether, it appears that H. meleagridis for a long time [3]. The infection biology E. coli, and bacteria in general, are found phagocytises bacteria for nutrient acquisi- of H. meleagridis is complicated by attached to the H. meleagridis surface tion but, at the same time, relies on proteins the fact that the Heterakis but also enclosed in the protozoan cell. and metabolites released during bacterial gallinarum, an intermediate host and However, it is unclear whether all these replication. most important vector, also needs a bacteria die upon ingestion. Observations certain bacterial flora to complete its life during monoxenisation and maintenance Bacteria as Regulators of the cycle in birds. Due to the low tenacity of of monoxenic cultures suggest that some Parasite’s Environment the protozoan parasite, Heterakis eggs ingested bacteria survive the enclosure The need for live bacteria in the are considered a crucial vector for initial into the H. meleagridis cell [4], which is in H. meleagridis environment advocates for

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TrendsTrends inin ParasitologyParasitology Figure 1. Life Cycle of Histomonas meleagridis. (A) H. meleagridis appears in different forms in vivo and in vitro. (Cultures are shown with bacteria and rice starch.) (B) The nematode Heterakis gallinarum cohabitates the caecum of and incorporates the protozoan into its eggs, supplying long-term protection to the highly vulnerable flagellate. (C–E) Heterakis eggs (C) are taken up by the host as the most efficient way of infection or (D) reside in as a paratenic host and might be taken up by snails or (E) are mechanically carried by insects. (F) Following introduction into a flock, H. meleagridis can spread directly between birds, and turkeys display severe clinical signs before death. After evasion from the gut, the flagellum is lost, and H. meleagridis appears in spherical form in various organs of diseased birds, predominantly the caecum and liver. roles of bacterial support other than as a organism by nature, has its energy produc- in oxidative stress management and by nutrition source. Experiments with various tion based in hydrogenosomes, whose doing so stimulating its intrinsic ability to monoxenic cultures suggested that the in- major enzymes are inhibited by the pres- cope with an intoxicating environment. teraction might also involve other indirect ence of oxygen, and its survival is strictly Support for this comes from E. histolytica, effects, such as adjusting environmental dependant on its effective removal [8]. Bac- where a direct bacterial effect on the regu- conditions by regulating the pH and de- terial species, identified as most supportive lation of parasite’sgeneswasdemon- creasing the availability of free oxygen [4]. of H. meleagridis growth, are themselves strated [13]. Assuming that the intrinsic As Histomonas favours a neutral pH, buff- able to use both aerobic and anaerobic capacity of H. meleagridis to cope with ered media are used for in vitro growth to respiration to obtain the energy [4]. The oxidative stress is provided [8], such direct optimise cell yields. Similarly, a neutral pH high division rate of cocultivating bacteria effects of bacteria might be feasible. in the caecum as the target organ and en- effectively consumes oxygen from the envi- trance gate of the parasite should favour ronment and improves the conditions Impact of H. meleagridis on replication and infection. Such conditions for anaerobic metabolism of the parasite Bacteria are noted in gnotobiotic turkeys; yet, an [4]. Another possible, and more direct, So far, only very few relevant studies are infection could not be established. Aside bacterial effect would be influencing the available, but molecular data point to- from this, H. meleagridis, a microaerophilic expression of parasite’s genes involved wards an intricate relationship between

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TrendsTrends inin ParasitologyParasitology Figure 2. Potential Aspects of the ‘Eu-prokaryotic’ Interaction. The cocultivating bacteria or intestinal prokaryotes invoke an indirect influence on Histomonas meleagridis by maintaining optimal environmental conditions (e.g., regulation of pH or reduction of oxygen), shown in the violet rectangles. A direct effect of bacteria on H. meleagridis and vice versa is shown in the blue rectangles: bacteria acting as a food source; bacteria invoking protection of H. meleagridis against oxidative stress by stimulating the expression of the parasite’s antioxidant proteins; bacteria and H. meleagridis influencing components of substrates enabling mutual nutrient supply; and H. meleagridis providing protection of bacteria by allocating them into its vacuoles. Live and dead bacteria are shown in red and blue, respectively. Green and black arrows indicate direct and indirect effects, respectively. Solid arrows show processes reported in the literature, whereas dashed arrows mark processes speculated here.

H. meleagridis and bacteria. On the basis is remarkable and thought-provoking. divergence in E. coli exoproteins suggests of proteomic analyses of H. meleagridis, Especially the exoproteome study demon- that, during cultivation, E. coli relies on the it seems that the presence of the parasite strated substantial changes in the ex- consumption of bioproducts from the itself substantially influences the expres- pression of E. coli proteins. Considering parasite’s metabolism, indicating a mutual sion pattern of genes of cocultivated bac- that about one-third of bacterial proteins role as nutrient supply [9]. However, the teria [9,10,14]. Due to the monoxenic undertake their function outside the data also reveal a potential resilience of nature of the H. meleagridis in vitro culture, cytoplasm, it seems unsurprising that prokaryotes to predation by the parasite. the detection of a few E. coli proteins was major differences in the exoproteome Whether bacteria themselves acquire anticipated. However, a substantial varia- were of E. coli origin, whereas variations some advantage from this tight interaction tion of their abundance, which could be in protozoal exoproteins were almost remains hypothetical and needs to be associated with the parasite’s phenotype, nonexistent [9,15]. Analysis of detected investigated in more detail. The obvious

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hypotheses are (i) the protection from of a predator–prey nature. Future re- culture, without influence on its pathogenicity. Int. J. Parasitol. 42, 893–901 external agents or conditions, (ii) the search should focusonresolvingthe 5. Burgess, S.L. and Petri, W.A. (2016) The intestinal bacte- nutrient supply and/or (iii) the use of unknowns of this interaction in order rial microbiome and E. histolytica infection. Curr. Trop. Med. Rep. 3, 71–74 H. meleagridis as a Trojan horse for pro- to elaborate whether a targeted manipu- 6. Hinderfeld, A.S. et al. (2019) Cooperative interactions be- karyotes.Someevidenceforthelasthy- lation of the gut microbiome can be tween Trichomonas vaginalis and associated bacteria en- hance paracellular permeability of the cervicovaginal pothesis comes from experiments achieved in order to minimise clinical con- epithelium by dysregulating tight junctions. Infect. and case reports of histomonosis from sequences. Similarly, such knowledge Immun. 87, e00141-19 fi fi 7. Fichorova, R. et al. (2017) Trichomonas vaginalis infection the eld, which often nd a secondary could also be used to optimise the infec- in symbiosis with Trichomonasvirus and Mycoplasma. E. coli infection [3]. tion of attenuated H. meleagridis strains Res. Microbiol. 168, 882–891 8. Mazumdar, R. et al. (2017) Establishment of a de novo ref- used for vaccination. erence transcriptome of Histomonas meleagridis reveals Future Perspectives basic insights about biological functions and potential pathogenic mechanisms of the parasite. Protist 168, Aside from a nutrition source and/or 663–685 1Clinic for Poultry and Fish Medicine, University of Veterinary 9. Mazumdar, R. et al. (2019) Molecular characterization of the creation of favourable environmental Medicine, Vienna, Austria Histomonas meleagridis exoproteome with emphasis on 2Christian Doppler Laboratory for Innovative Poultry Vaccines conditions, the need for bacteria in protease secretion and parasite-bacteria interaction. (IPOV), University of Veterinary Medicine, Vienna, Austria the / caecum to induce PLoS One 14, e212429 10. Monoyios, A. et al. (2018) An alliance of gel-based and gel- histomonosis might be seen as a cooper- *Correspondence: free proteomic techniques displays substantial insight into ative aid in the disruption of the gut [email protected] (I. Bilic). the proteome of a virulent and an attenuated Histomonas https://doi.org/10.1016/j.pt.2019.12.015 meleagridis strain. Front. Cell. Infect. Microbiol. 8, 407 epithelial barrier. This ‘eu-prokaryotic’ in- 11. Pinheiro, J. et al. (2018) The protozoan Trichomonas teraction can have fatal consequences © 2020 Elsevier Ltd. All rights reserved. vaginalis targets bacteria with laterally acquired NlpC/P60 peptidoglycan hydrolases. mBio 9, e01784-18 for the host, altogether a unique alliance 12. Wildschutte, H. et al. (2004) Protozoan predation, diversi- in medicine. However, the underlying fying selection, and the evolution of antigenic diversity References in Salmonella. Proc.Natl.Acad.Sci.U.S.A.101, functional mechanisms are still to be 1. McDougald, L.R. (2005) Blackhead disease (histomoniasis) 10644–10649 resolved, considering that the host itself in poultry: a critical review. Avian Dis. 34, 462–476 13. Varet, H. et al. (2018) Enteric bacteria boost defences 2. Liebhart, D. et al. (2017) Histomonosis in poultry: previous against oxidative stress in Entamoeba histolytica. Sci. may trigger and contribute certain fea- and current strategies for prevention and therapy. Avian Rep. 8, 9042 tures inducing substantial consequences Pathol. 46, 1–18 14. Monoyios, A. et al. (2018) Unravelling the differences: 3. Hess, M. (2017) Commensal or pathogen – a comparative proteomic analysis of a clonal virulent on the outcome. On the basis of available challenge to fulfilKoch’s Postulates. Br.Poult.Sci. and an attenuated Histomonas meleagridis strain. Int. data, we hypothesise that the parasite– 58, 1–12 J. Parasitol. 48, 145–157 4. Ganas, P. et al. (2012) Escherichia coli strongly supports 15. Steinberg, R. et al. (2018) Co-translational protein bacteria interplay is mutualistic and not the growth of Histomonas meleagridis, in a monoxenic targeting in bacteria. FEMS Microbiol Lett. 365, fny095

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