Article

A brief historical and evolutionary perspective on the origin of cellular research

SOLDATI, Thierry, CARDENAL-MUNOZ, Elena

Abstract

Integrated with both a historical perspective and an evolutionary angle, this opinion article presents a brief and personal view of the emergence of cellular microbiology research. From the very first observations of phagocytosis by Goeze in 1777 to the exhaustive analysis of the cellular defence mechanisms performed in modern laboratories, the studies by and microbiologists have converged into an integrative research field distinct from, but fully coupled to immunity: cellular microbiology. In addition, this brief article is thought as a humble patchwork of the motivations that have guided the research in my group over a quarter century.

Reference

SOLDATI, Thierry, CARDENAL-MUNOZ, Elena. A brief historical and evolutionary perspective on the origin of cellular microbiology research. Cellular Microbiology, 2019, vol. 21, no. 11

DOI : 10.1111/cmi.13083

Available at: http://archive-ouverte.unige.ch/unige:136128

Disclaimer: layout of this document may differ from the published version.

1 / 1 Cellular Microbiology

A brief historical and evolutionary perspective on the origin of cellular microbiology research

Journal: Cellular Microbiology

Manuscript ID CMI-19-0138.R1 Manuscript Type:ForSpecial Peer Issue - Review Review Date Submitted by the 02-Jul-2019 Author:

Complete List of Authors: Soldati, Thierry; Université de Genève, Département de Biochimie Cardenal-Munoz, Elena; University of Geneva,

macrophage, innate immunity, Dictyostelium discoideum, Mycobacterium Key Words: marinum, - interactions, model organisms

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1 2 3 A brief historical and evolutionary perspective on the origin of cellular 4 1 5 6 2 microbiology research 7 8 9 3 10 11 4 Thierry Soldati and Elena Cardenal-Muñoz 12 13 5 14 15 6 Department of Biochemistry, Faculty of Science, Sciences II, University of Geneva, Geneva, 16 17 7 Switzerland 18 19 8 20 21 9 For Peer Review 22 23 24 10 25 26 11 Abstract 27 28 12 Integrated with both a historical perspective and an evolutionary angle, this opinion article presents a 29 30 13 brief and personal view of the emergence of cellular microbiology research. From the very first 31 32 14 observations of phagocytosis by Goeze in 1777 to the exhaustive analysis of the cellular defence 33 34 15 mechanisms performed in modern laboratories, the studies by cell biologists and microbiologists have 35 36 16 converged into an integrative research field distinct from, but fully coupled to immunity: cellular 37 38 17 microbiology. In addition, this brief article is thought as a humble patchwork of the motivations that 39 40 18 have guided the research in my group over a quarter century. 41 42 43 19 44 45 20 46 47 21 48 49 22 50 51 23 52 53 24 Keywords: macrophage, innate immunity, Dictyostelium discoideum, Mycobacterium marinum, host- 54 55 25 pathogen interactions, model organisms, phagocytosis, , cell-autonomous defense, autophagy, 56 57 26 metal poisoning, nutritional immunity 58 59 60 Cellular Microbiology Page 2 of 14

1 2 3 27 The phagocytic cells of the innate immune system (i.e. mononuclear macrophages and dendritic 4 5 28 cells, as well as various polymorphonuclear cells such as neutrophils) monitor blood and tissue for the 6 7 29 presence of microbes, acting as a first line of defence against infection. One of the major defence 8 9 10 30 mechanisms of these cells against microbial intruders is phagocytosis, a process consisting in the 11 12 31 engulfment and digestion of extracellular particles within the phasogome. During evolution, 13 14 32 phagocytosis, originally intended for nutrient acquisition (Cavalier-Smith, 2009), expanded to fulfil 15 16 33 new and complex tasks that are still central in the cell-autonomous defence of multicellular 17 18 34 against the prokaryotes. As a consequence, the basic components of phagocytosis are conserved 19 20 35 between immune cells of metazoa and bacterivorous single-celled eukaryotes (Boulais et al., 2010; 21 For Peer Review 22 36 Gaudet et al., 2016; Randow et al., 2013). 23 24 37 The phagosome is thus one of the primary battlefield between prokaryotes and their predators. 25 26 38 Interestingly, from phagotrophy, where single cells such as amoebae feed by engulfing and 27 28 digesting them inside the phagolysosome, to adaptive immunity in complex organisms, where the 29 39 30 31 40 defence responses are multifaceted and adjustable to the different intruders, the core machinery of the 32 33 41 phagosome is highly conserved (Boulais et al., 2010). Upon fusion with lysosomes, the phagosome 34 35 42 becomes a strongly acidic and hydrolytic compartment where intruders are killed and digested. 36 37 43 Equipped with NOX enzymes for local production of toxic ROS compounds, the phagosome also 38 39 44 harbours metal transporters and metal-binding proteins that induce the intoxication of the microbe or 40 41 45 deplete its environment from transition metal ions (Flannagan et al., 2009). The phagosome is therefore 42 43 46 a selective environment for prokaryotes to evolve countermeasures to resist predation, giving rise to an 44 45 47 evolutionary arms race. This explains why many, perhaps most, virulence traits did not arise originally 46 47 to damage the host and create disease, but rather as a means to compete with other microbes, to prevent 48 48 49 50 49 predation and, presumably, finally also to obtain nutrients from the host (Casadevall and Pirofski, 2007; 51 52 50 Ehrlich et al., 2008). Therefore, arguably, the most successful of these prokaryotes have given rise to 53 54 51 intracellular that are able to manipulate the phagosome and divert it from its bactericidal 55 56 52 , even transforming into Trojan horses where bacteria, protected from humoral 57 58 53 immune defences, can multiply and spread (Cossart et al., 1996; Cossart and Sansonetti, 2004). The 59 60 54 realisation that these complex phenomena result from the co-evolution of host and pathogens, and Page 3 of 14 Cellular Microbiology

1 2 3 55 eventually drive disease and pathogenesis, lead to the emergence in the early 90's of the field of 4 5 56 "Cellular Microbiology". The "scientific soil" was rich and fertile due to the preparatory work of a select 6 7 57 group of cell biologists and microbiologists around the world, including some originally trained as 8 9 10 58 medical doctors. Therefore, the seed planted by Philippe J. Sansonetti and colleagues at the Pasteur 11 12 59 Institute in Paris grew quickly, wild and strong.The main innovations of this new scientific discipline 13 14 60 were not only to dissect pathogenicity in the context of its host cell environment, but to use pathogens 15 16 61 as perturbations and tools to uncover key mechanisms in the biology of immune and other cells (Cossart 17 18 62 et al., 1996). But in fact, did it really emerge from scratch in the 1990s? 19 20 63 The very first documented observations of phagocytosis were made in 1777 by the Lutheran 21 For Peer Review 22 64 pastor Johann August Ephraim Goeze [(Goeze, 1777), translated and commented in (Stossel, 1999)]. 23 24 65 Goeze described infusoria (aquatic ) eating particles and other infusoria (Fig. 1, left top 25 26 66 panel), and Stossel noted "Goeze's vivid and anthropomorphic depiction of the predatory behaviour of 27 28 his microorganisms" and commented that "this attitude is particularly interesting, in light of 29 67 30 31 68 Metchnikoff's later imaging of pitched battles between phagocytes and microbes, a metaphor about 32 33 69 which much has been made regarding its influence on the way we think about health and disease". 34 35 70 Almost at the same time as Goeze's observations, the first investigations and experimental 36 37 71 manipulations of phagocytosis can be assigned to the Baron Wilhelm Friedrich Freiherr von Gleichen- 38 39 72 Russworm, who developed in 1778 a technique to "feed" microorganisms with the colloidal stains 40 41 73 indigo and carmine [retrieved from (Danchin)]. He drew the protists that had ingested the carmine 42 43 74 particles and described that his "expectations were fulfilled in that (he) was convinced not only that the 44 45 75 meal had been swallowed but that (he) could learn more about the viscera of these animals" [(Gleichen- 46 47 Russwurm, 1799), translated and commented in (Stossel, 1999)] (Fig. 1, right panel). But, as for Goeze, 48 76 49 50 77 there was no parallel proposed with the predatory behaviour of animal phagocytes. Almost a century 51 52 78 later, the surgeon Sir William Osler was studying autopsy material from miner's lungs (Fig. 1, left 53 54 79 bottom panels), when he described the presence of corpuscles containing coal residues in alveolar cells. 55 56 80 Even though he did not discuss the mechanism by which these cells had incorporated this particulate 57 58 81 material, this description is possibly the first documented report that phagocytosis also occurred in 59 60 82 human cells [(Osler, 1875), mentioned and commented in (Ambrose, 2006)]. Cellular Microbiology Page 4 of 14

1 2 3 83 As textbooks then remind us, further developments in the field waited until 1884 when the 4 5 84 zoologist Elie Metchnikoff presented the first recorded event of phagocytosis during a host-pathogen 6 7 85 interaction. He studied in detail how the ameboid motile blood cells from the planktonic crustacean 8 9 10 86 Daphnia surrounded and killed the spores of a parasitic fungus named by him Monospora bicuspidata 11 12 87 (Fig. 2, left and mid panels). In his article A disease of Daphnia caused by a yeast (Metchnikoff, 1884), 13 14 88 he highlighted that "blood corpuscles attach to the fungus spore, in order to begin the battle against the 15 16 89 intruder", but "blood cells can also be affected by the parasites". He was the first to name these "eating 17 18 90 cells" "phagocytes", and to recognise the roles of circulating "microphages" and tissue-resident 19 20 91 "macrophages" in the uptake and bactericidal control of invading pathogens (Metchnikoff, 1887). 21 For Peer Review 22 92 Similarly to his comment on the "inflammation" occurring at the infected area of Daphnia's 23 24 93 body cavity, Metchnikoff also described the reaction of phagocytes to injury in the caudal fin of a 25 26 94 transparent Triton embryo (Fig. 2, right panel), characterizing the progressive appearance of these cells 27 28 to the site of inflammation caused by cauterization (Metchnikoff, 1893). Extension of these 29 95 30 31 96 observations to animal and human diseases, from thyphus to malaria, including for example the 32 33 97 experimentation with bacteria inoculations in the eye ball of rabbits, allowed Metchnikoff, his 34 35 98 colleagues and followers to experimentally test their hypotheses and make rapid and relevant progress. 36 37 99 As a result, Metchnikoff is considered the father of "cellular immunity". His concept of innate immunity 38 39 100 contradicted the theory of humoral immunity defended by the German Paul Ehrlich. In Ehrlich's theory, 40 41 101 the antibodies helped by the complement, and not the phagocytes, were responsible for the 42 43 102 neutralization and lysis of intruding bacteria. Both scientists shared the Nobel Prize for or 44 45 103 Medicine in 1908 for their complementary contributions to the understanding of immunity (Kaufmann, 46 47 2008; Tauber, 2003). 48 104 49 50 105 Because of his excellent knowledge of the infusoria fights and his studies on transparent 51 52 106 metazoa, from sponges and Triton larvae to infected Daphnia, we can also claim that Metchnikoff is 53 54 107 the precursor of the use of "model organisms" in host-pathogen interactions research and also was 55 56 108 responsible for the first implementation of the 3Rs principle (replacement, reduction and refinement). 57 58 109 At the heart of this is Metchnikoff's faith in the fact that evolutionary conservation, and hence 59 60 110 homologies between mechanisms and processes in protozoan, animals and even humans predominate Page 5 of 14 Cellular Microbiology

1 2 3 111 over the divergence and specific . Humbly following in Metchnikoff's tracks, my group uses 4 5 112 the social amoeba and professional Dictyostelium discoideum as a versatile model organism 6 7 113 to study phagocytosis and host-pathogen interactions. 8 9 10 114 D. discoideum has a haploid genome, harbouring approximately 12,000 predicted protein- 11 12 115 coding genes. It diverged from the path leading to the crown groups of metazoa and fungi, shortly after 13 14 116 the ramification of plants. Importantly for its role as a model organism relevant for human research, the 15 16 117 D. discoideum proteins are more similar to their mammalian counterparts than those in yeasts (Eichinger 17 18 118 et al., 2005). In addition, D. discoideum possesses an ancestral set of the animal armament for cell- 19 20 119 autonomous defence (e.g., scavenger receptors, metal pumps, NADPH-oxidases, phagolysosomes, 21 For Peer Review 22 120 autophagy, etc.). Although infection of amoebae cannot completely recapitulate the complexities of 23 24 121 host-pathogen interactions in metazoa, we can consider the amoeba D. discoideum as a proto- 25 26 122 macrophage relevant to study both the conserved innate immune responses and the microbial 27 28 mechanisms to resist killing, as witnessed by a steady stream of articles emanating from the work of 29 123 30 31 124 about a dozen groups world-wide [reviewed in (Cosson and Soldati, 2008; Dunn et al., 2017; Erken et 32 33 125 al., 2013; Hilbi et al., 2007; Pan et al., 2011; Pukatzki et al., 2002; Pukatzki et al., 2006; Soldati and 34 35 126 Neyrolles, 2012)]. Following this line of research, D. discoideum has been used as a host cell model to 36 37 127 study the cell-intrinsic mechanisms active during infection by various animal and human pathogens 38 39 128 such as Mycobacterium spp (Fig. 3, bottom left panel), Legionella pneumophila, enterica, 40 41 129 Pseudomonas aeruginosa, Vibrio cholera, etc. (reviewed in (Dunn et al., 2017)). 42 43 130 One of the additional features that makes D. discoideum so interesting for immunity studies is 44 45 131 its capacity to switch from a solitary life, where it defends itself from infection in a cell-autonomous 46 47 mode, to a multicellular cycle where "altruistic" innate immunity takes place (Fig. 3, top panel). Upon 48 132 49 5 50 133 starvation, approximately 10 cells migrate toward the chemoattractant cyclic AMP, aggregate into a 51 52 134 mound that gets enclosed within an extracellular sheath of cellulose and mucopolysaccharide. During 53 54 135 this aggregation phase, D. discoideum uses a combination of cell-autonomous defence mechanisms to 55 56 136 ensure that only non-infected cells form the compact streams. In a final effort to generate a multicellular 57 58 137 structure free of infection, the few remaining infected cells are physically excluded from the mound by 59 60 138 a still mysterious mechanism (López-Jiménez et al., 2019).The so-formed mound then develops into a Cellular Microbiology Page 6 of 14

1 2 3 139 slug that uses phototaxis and thermotaxis to migrate to the surface of the soil to ultimately increase 4 5 140 spore dissemination. The developmental cycle culminates with the formation of a fruiting body, which 6 7 141 consists of a spore-containing sorus resting upon a stalk of dead cells. Under propitious conditions of 8 9 10 142 heat, light and food, spores germinate into growing cells, which will again kill and extract nutrients 11 12 143 from soil bacteria by phagocytosis (Dunn et al., 2017). The migrating slug can be considered as a 13 14 144 "facultative metazoan". It possesses a rudimentary immune system of specialized Sentinel cells that 15 16 145 protect and cure the slug from infection during migration (Chen et al., 2007). These Sentinel cells patrol 17 18 146 the slug, phagocytose bacteria and , and are shed along with the cellulose sheath as the slug moves 19 20 147 (Fig. 3, bottom right panel). In response to bacteria, apart from employing intracellular killing 21 For Peer Review 22 148 mechanisms, Sentinel cells also release DNA extracellular traps (ETs, derived from mitochondrial DNA 23 24 149 and decorated with anti-microbial proteins) via a mechanism involving NOX-generated ROS (Zhang 25 26 150 and Soldati, 2016; Zhang et al., 2016). In this way, Sentinel cells sterilize the slug, evoking an ancestral 27 28 innate immune system designed a billion years ago. 29 151 30 31 152 Very interestingly, D. discoideum not only helps us deciphering the mechanisms of defence 32 33 153 against pathogens, but it also possibly contribute to reveal the origins of microbiota, another field in 34 35 154 which both Elie Metchnikoff [reviewed in (Mackowiak, 2013) and (Podolsky, 2012)] and Philippe 36 37 155 Sansonetti have excelled. Humans are complex ecological systems where cells not only face occasional 38 39 156 contact with pathogens but they live in peace and harmony with beneficial microbes. In other words, a 40 41 157 healthy human body represents the assemblage of approximately 3 x 1012 nucleated cells co-habiting 42 43 158 with 3.8 x 1013 bacteria and 1 x 1015 viruses, mainly bacteriophages (Sender et al., 2016). All these cells 44 45 159 and organisms are subjected to coevolutionary pressures inside the human body. In a surprisingly 46 47 similar way, bacteria of the Burkholderia family have been shown to induce a so-called farming 48 160 49 50 161 symbiosis in D. discoideum (DiSalvo et al., 2015). As a result of this stable association, Burkholderia 51 52 162 induces their own carriage from generation to generation of D. discoideum, serving both food and 53 54 163 protection purposes (Brock et al., 2016). This interaction even turns out to induce a “ménage à trois”, 55 56 164 in which Burkholderia not only forces the otherwise bactivorous amoebae to carry them in their spores, 57 58 165 but to also become “tolerant” to food bacteria that are normally, in absence of Burkholderia, rapidly 59 60 166 killed and degraded in their phagosomes (Shu et al., 2018). Extended studies now show that several Page 7 of 14 Cellular Microbiology

1 2 3 167 bacteria genera are able to evade phagocytosis and persist in D. discoideum cells and spores through 4 5 168 one or more social cycles, creating a form of semi-stable and mutualistic proto-microbiome, which 6 7 169 might play an important role in supporting D. discoideum growth in nutrient-poor conditions (Brock et 8 9 10 170 al., 2018; Haselkorn et al., 2019). 11 12 171 In conclusion, as can be perceived through the brief and humble exposé of Metchnikoff’s 13 14 172 seminal discoveries and his darwinian concept of immunological research, there is a (model) organism 15 16 173 for each and every purpose. This frame of mind has been re-invigorated by the birth of the discipline of 17 18 174 cellular microbiology and the inspiring and constructive guidance of Philippe Sansonetti. Nowadays, 19 20 175 with public pressure exerted on the use of animal models in biomedical research, we should all revisit 21 For Peer Review 22 176 and apply again the motivations of the founding Fathers of cellular immunity and cellular microbiology, 23 24 177 and continue improving our search for the best possible model organisms (throughout evolution) or 25 26 178 model systems (making use of stem cells and organoids). Maybe the last word goes to the evolutionary 27 28 and Russian christian orthodox Theodosius Dobzhansky, who summarised in 1973 his 29 179 30 31 180 understanding of in a famous sentence: "Nothing in biology makes sense except in the light of 32 33 181 evolution" (Dobzhansky, 1973). These thoughts and concepts will continue to help us shed light on the 34 35 182 very complex relationship between a host and its associated commensals and pathogens. 36 37 183 38 39 184 Figure legends 40 41 185 42 43 186 Figure 1. Drawings depicting the first observations of phagocytosis made by JAE Goeze ((Goeze, 44 45 187 1777), left top panel), WFF von Gleichen-Russworm ((Stossel, 1999), right panel) and W Osler 46 47 ((Ambrose, 2006), left bottom panels). 48 188 49 50 189 51 52 190 Figure 2. Left panel: Female adult of D. magna (Watanabe, 2011); Mid and right panels: Metchnikoff's 53 54 191 drawings of M. bicuspidata conidia and spores surrounded by blood cells of D. magna, as well as the 55 56 192 infected abdomen and intestine of D. magna (mid, (Metchnikoff, 1884)) and of Triton phagocytes 57 58 193 reacting to wounding by cauterisation [right, (Metchnikoff, 1893), mentioned in (Tauber, 2003)]. 59 60 194 Cellular Microbiology Page 8 of 14

1 2 3 195 Figure 3. Top panel: The D. discoideum life cycle (Dunn et al., 2017). Bottom left panel: electron 4 5 196 micrograph of an amoeboid cell of D. discoideum containing Mycobacterium marinum bacteria within 6 7 197 a specialised phagosome (the mycobacteria-containing vesicle, MCV); Bottom right panel: a moving 8 9 10 198 D. discoideum slug. The ROS-generating Sentinel cells appear in red. 11 12 199 13 14 200 References: 15 16 201 Ambrose, C.T. 2006. The Osler slide, a demonstration of phagocytosis from 1876 Reports of 17 18 202 phagocytosis before Metchnikoff's 1880 paper. Cell Immunol. 240:1-4. 19 20 203 Boulais, J., M. Trost, C.R. Landry, R. Dieckmann, E.D. Levy, T. Soldati, S.W. Michnick, P. Thibault, 21 For Peer Review 22 204 and M. Desjardins. 2010. Molecular characterization of the evolution of phagosomes. Mol Syst 23 24 205 Biol. 6:423. 25 26 206 Brock, D.A., W.E. Callison, J.E. Strassmann, and D.C. Queller. 2016. Sentinel cells, symbiotic bacteria 27 28 and resistance in the social amoeba Dictyostelium discoideum. Proc Biol Sci. 283. 29 207 30 31 208 Brock, D.A., T.S. Haselkorn, J.R. Garcia, U. Bashir, T.E. Douglas, J. Galloway, F. Brodie, D.C. Queller, 32 33 209 and J.E. Strassmann. 2018. Diversity of Free-Living Environmental Bacteria and Their Interactions 34 35 210 With a Bactivorous Amoeba. Front Cell Infect Microbiol. 8:411. 36 37 211 Casadevall, A., and L.A. Pirofski. 2007. Accidental virulence, cryptic pathogenesis, martians, lost hosts, 38 39 212 and the pathogenicity of environmental microbes. Eukaryot Cell. 6:2169-2174. 40 41 213 Cavalier-Smith, T. 2009. Predation and cell origins: a coevolutionary perspective. Int J 42 43 214 Biochem Cell Biol. 41:307-322. 44 45 215 Chen, G., O. Zhuchenko, and A. Kuspa. 2007. Immune-like phagocyte activity in the social amoeba. 46 47 Science. 317:678-681. 48 216 49 50 217 Cossart, P., P. Boquet, S. Normark, and R. Rappuoli. 1996. Cellular microbiology emerging. Science. 51 52 218 271:315-316. 53 54 219 Cossart, P., and P.J. Sansonetti. 2004. Bacterial invasion: the paradigms of enteroinvasive pathogens. 55 56 220 Science. 304:242-248. 57 58 221 Cosson, P., and T. Soldati. 2008. Eat, kill or die: when amoeba meets bacteria. Curr Opin Microbiol. 59 60 222 11:271-276. Page 9 of 14 Cellular Microbiology

1 2 3 223 Danchin, A. 2018. The time of Enlightment. 4 5 224 DiSalvo, S., T.S. Haselkorn, U. Bashir, D. Jimenez, D.A. Brock, D.C. Queller, and J.E. Strassmann. 6 7 225 2015. Burkholderia bacteria infectiously induce the proto-farming symbiosis of Dictyostelium 8 9 10 226 amoebae and food bacteria. Proc Natl Acad Sci U S A. 112:E5029-5037. 11 12 227 Dobzhansky, T. 1973. Nothing in Biology Makes Sense except in the Light of Evolution. The American 13 14 228 Biology Teacher. 35:125-129. 15 16 229 Dunn, J.D., C. Bosmani, C. Barisch, L. Raykov, L.H. Lefrancois, E. Cardenal-Munoz, A.T. Lopez- 17 18 230 Jimenez, and T. Soldati. 2017. Eat Prey, Live: Dictyostelium discoideum As a Model for Cell- 19 20 231 Autonomous Defenses. Front Immunol. 8:1906. 21 For Peer Review 22 232 Ehrlich, G.D., N.L. Hiller, and F.Z. Hu. 2008. What makes pathogens pathogenic. Genome Biol. 9:225. 23 24 233 Eichinger, L., J.A. Pachebat, G. Glockner, M.A. Rajandream, R. Sucgang, M. Berriman, J. Song, R. 25 26 234 Olsen, K. Szafranski, Q. Xu, B. Tunggal, S. Kummerfeld, M. Madera, B.A. Konfortov, F. Rivero, 27 28 A.T. Bankier, R. Lehmann, N. Hamlin, R. Davies, P. Gaudet, P. Fey, K. Pilcher, G. Chen, D. 29 235 30 31 236 Saunders, E. Sodergren, P. Davis, A. Kerhornou, X. Nie, N. Hall, C. Anjard, L. Hemphill, N. 32 33 237 Bason, P. Farbrother, B. Desany, E. Just, T. Morio, R. Rost, C. Churcher, J. Cooper, S. Haydock, 34 35 238 N. van Driessche, A. Cronin, I. Goodhead, D. Muzny, T. Mourier, A. Pain, M. Lu, D. Harper, R. 36 37 239 Lindsay, H. Hauser, K. James, M. Quiles, M. Madan Babu, T. Saito, C. Buchrieser, A. Wardroper, 38 39 240 M. Felder, M. Thangavelu, D. Johnson, A. Knights, H. Loulseged, K. Mungall, K. Oliver, C. Price, 40 41 241 M.A. Quail, H. Urushihara, J. Hernandez, E. Rabbinowitsch, D. Steffen, M. Sanders, J. Ma, Y. 42 43 242 Kohara, S. Sharp, M. Simmonds, S. Spiegler, A. Tivey, S. Sugano, B. White, D. Walker, J. 44 45 243 Woodward, T. Winckler, Y. Tanaka, G. Shaulsky, M. Schleicher, G. Weinstock, A. Rosenthal, 46 47 E.C. Cox, R.L. Chisholm, R. Gibbs, W.F. Loomis, M. Platzer, R.R. Kay, J. Williams, P.H. Dear, 48 244 49 50 245 A.A. Noegel, B. Barrell, and A. Kuspa. 2005. The genome of the social amoeba Dictyostelium 51 52 246 discoideum. Nature. 435:43-57. 53 54 247 Erken, M., C. Lutz, and D. McDougald. 2013. The rise of pathogens: predation as a factor driving the 55 56 248 evolution of human pathogens in the environment. Microb Ecol. 65:860-868. 57 58 249 Flannagan, R.S., G. Cosio, and S. Grinstein. 2009. Antimicrobial mechanisms of phagocytes and 59 60 250 bacterial evasion strategies. Nat Rev Microbiol. 7:355-366. Cellular Microbiology Page 10 of 14

1 2 3 251 Gaudet, R.G., C.J. Bradfield, and J.D. MacMicking. 2016. Evolution of Cell-Autonomous Effector 4 5 252 Mechanisms in Macrophages versus Non-Immune Cells. Microbiol Spectr. 4. 6 7 253 Gleichen-Russwurm, W.F. 1799. Dissertation sur la génération, les animalcules spermatiques, et ceux 8 9 10 254 d'infusions, avec des observations microscopiques sur le sperme, et sur différentes infusions. 11 12 255 Goeze, J.A.E. 1777. Infusion animals that eat others. Activities of the Berlin Society of Naturalist 13 14 256 Companions. 3:373-384. 15 16 257 Haselkorn, T.S., S. DiSalvo, J.W. Miller, U. Bashir, D.A. Brock, D.C. Queller, and J.E. Strassmann. 17 18 258 2019. The specificity of Burkholderia symbionts in the social amoeba farming symbiosis: 19 20 259 Prevalence, species, genetic and phenotypic diversity. Mol Ecol. 28:847-862. 21 For Peer Review 22 260 Hilbi, H., S.S. Weber, C. Ragaz, Y. Nyfeler, and S. Urwyler. 2007. Environmental predators as models 23 24 261 for bacterial pathogenesis. Environ Microbiol. 9:563-575. 25 26 262 Kaufmann, S.H. 2008. 's foundation: the 100-year anniversary of the Nobel Prize to Paul 27 28 Ehrlich and Elie Metchnikoff. Nat Immunol. 9:705-712. 29 263 30 31 264 López-Jiménez, A.T., M. Hagedorn, M.J. Delincé, J. McKinney, and T. Soldati. 2019. The 32 33 265 developmental cycle of Dictyostelium discoideum ensures curing of a mycobacterial 34 35 266 infection at both cell-autonomous level and by collaborative exclusion. bioRxiv:586263. 36 37 267 Mackowiak, P.A. 2013. Recycling metchnikoff: probiotics, the intestinal microbiome and the quest for 38 39 268 long life. Frontiers in public health. 1:52. 40 41 269 Metchnikoff, E. 1884. A disease of Daphnia caused by a yeast. A contribution to the theory of 42 43 270 phagocytes as agents for attack on disease-causing organisms. Archiv für Pathologische Anatomie 44 45 271 und Physiologie und für Klinische Medicin. 96:177-195. 46 47 Metchnikoff, E. 1887. Sur la lutte des cellules de l’organisme contre l’invasion des microbes. Annales 48 272 49 50 273 de L’Institut Pasteur. 1:321-336. 51 52 274 Metchnikoff, E. 1893. Lectures on the Comparative of Inflammation. 53 54 275 Osler, W. 1875. On the pathology of miner’s lung. Canada Medical and Surgical Journal. 4:145-168. 55 56 276 Pan, Y.-J., T.-L. Lin, C.-R. Hsu, and J.-T. Wang. 2011. Use of a Dictyostelium model for isolation of 57 58 277 genetic loci associated with phagocytosis and virulence in Klebsiella pneumoniae. Infect. Immun. 59 60 278 79:1006. Page 11 of 14 Cellular Microbiology

1 2 3 279 Podolsky, S.H. 2012. Metchnikoff and the microbiome. Lancet. 380:1810-1811. 4 5 280 Pukatzki, S., R.H. Kessin, and J.J. Mekalanos. 2002. The human pathogen Pseudomonas aeruginosa 6 7 281 utilizes conserved virulence pathways to infect the social amoeba Dictyostelium discoideum. Proc. 8 9 10 282 Natl. Acad. Sci. U. S. A. 99:3159-3164. 11 12 283 Pukatzki, S., A.T. Ma, D. Sturtevant, B. Krastins, D. Sarracino, W.C. Nelson, J.F. Heidelberg, and J.J. 13 14 284 Mekalanos. 2006. Identification of a conserved bacterial protein system in Vibrio 15 16 285 cholerae using the Dictyostelium host model system. Proc. Natl. Acad. Sci. U. S. A. 103:1528- 17 18 286 1533. 19 20 287 Randow, F., J.D. MacMicking, and L.C. James. 2013. Cellular self-defense: how cell-autonomous 21 For Peer Review 22 288 immunity protects against pathogens. Science. 340:701-706. 23 24 289 Sender, R., S. Fuchs, and R. Milo. 2016. Revised Estimates for the Number of Human and Bacteria 25 26 290 Cells in the Body. PLoS Biol. 14:e1002533. 27 28 Shu, L., D.A. Brock, K.S. Geist, J.W. Miller, D.C. Queller, J.E. Strassmann, and S. DiSalvo. 2018. 29 291 30 31 292 Symbiont location, host fitness, and possible coadaptation in a symbiosis between social amoebae 32 33 293 and bacteria. Elife. 7. 34 35 294 Soldati, T., and O. Neyrolles. 2012. Mycobacteria and the intraphagosomal environment: take it with a 36 37 295 pinch of salt(s)! Traffic. 13:1042-1052. 38 39 296 Stossel, T.P. 1999. The early history of phagocytosis. Advances in Cellular and of 40 41 297 Membranes and Organelles. 5:3-18. 42 43 298 Tauber, A.I. 2003. Metchnikoff and the phagocytosis theory. Nat Rev Mol Cell Biol. 4:897-901. 44 45 299 Watanabe, H. 2011. PLoS Issue Image I. PLoS Genet. 7. 46 47 Zhang, X., and T. Soldati. 2016. Of Amoebae and Men: Extracellular DNA Traps as an Ancient Cell- 48 300 49 50 301 Intrinsic Defense Mechanism. Front Immunol. 7:269. 51 52 302 Zhang, X., O. Zhuchenko, A. Kuspa, and T. Soldati. 2016. Social amoebae trap and kill bacteria by 53 54 303 casting DNA nets. Nat Commun. 7:10938. 55 56 304 57 58 59 60 Cellular Microbiology Page 12 of 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 Figure 1 25 205x107mm (300 x 300 DPI) 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 13 of 14 Cellular Microbiology

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For PeerFigure Review 2 20 21 205x79mm (300 x 300 DPI) 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Cellular Microbiology Page 14 of 14

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 For Peer Review 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 Figure 3 35 36 207x176mm (300 x 300 DPI) 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60