1

Amoeba-resisting bacteria found in multilamellar bodies secreted by Dictyostelium 1

discoideum: social amoebae can also package bacteria 2

3

Valérie E. Paquet1,2 and Steve J. Charette1,2,3* 4

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1. Institut de Biologie Intégrative et des Systèmes, Pavillon Charles-Eugène-Marchand, 6

Université Laval, Quebec City, QC, Canada 7

2. Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de 8

Québec, Hôpital Laval, Quebec City, QC, Canada 9

3. Département de biochimie, de microbiologie et de bio-informatique, Faculté des 10

sciences et de génie, Université Laval, Quebec City, QC, Canada 11

12

*Corresponding author: 13

Steve J. Charette, 1030 avenue de la medicine, Pavillon Marchand, local 4245, Université 14

Laval, Quebec City, QC, Canada, G1V 0A6, telephone: 1-418-656-2131, ext. 6914, fax: 15

1-418-656-7176, email: steve.charette@bcm.ulaval.ca 16

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Running title (60 characters with space): Packaging of amoeba-resisting bacteria by D. 18

discoideum 19

20

Keywords (6): Multilamellar bodies; Dictyostelium discoideum; packaged bacteria, 21

amoeba-resisting bacteria, Cupriavidus, Rathayibacter 22

23

2

ABSTRACT 24

Many bacteria can resist phagocytic digestion by various protozoa. Some of these 25

bacteria (all human pathogens) are known to be packaged in multilamellar bodies 26

produced in the phagocytic pathway of the protozoa and that are secreted into the 27

extracellular milieu. Packaged bacteria are protected from harsh conditions, and the 28

packaging process is suspected to promote bacterial persistence in the environment. To 29

date, only a limited number of protozoa, belonging to free-living amoebae and ciliates, 30

have been shown to perform bacteria packaging. It is still unknown if social amoebae can 31

do bacteria packaging. The link between the capacity of 136 bacterial isolates to resist the 32

grazing of the social amoeba Dictyostelium discoideum and to be packaged by this 33

amoeba was investigated in the present study. The 45 bacterial isolates displaying a 34

resisting phenotype were tested for their capacity to be packaged. A total of seven isolates 35

from Cupriavidus, Micrococcus, Microbacterium, and Rathayibacter genera seemed to 36

be packaged and secreted by D. discoideum based on immunofluorescence results. 37

Electron microscopy confirmed that the Cupriavidus and Rathayibacter isolates were 38

formally packaged. These results show that social amoebae can package some bacteria 39

from the environment revealing a new aspect of microbial ecology. 40

41

42

3

INTRODUCTION 43

Free-living amoebae (FLAs) like Acanthamoeba spp. are mobile unicellular 44

protozoa that live in aquatic environments and feed on bacteria, fungi, and algae 45

(Rodriguez-Zaragoza, 1994). FLAs can colonize many man-made infrastructures that 46

provide a favorable environment for the proliferation of microorganisms, especially 47

where high bacterial population densities are found. Cooling towers (Pagnier et al., 48

2009), air conditioners (Walker et al., 1986), and drinking water distribution systems 49

(Thomas & Ashbolt, 2011) are a few examples of man-made infrastructures where FLAs 50

grow (reviewed in (Siddiqui & Khan, 2012, Cateau et al., 2014) and regulate bacterial 51

population densities. 52

53

FLAs capture bacteria by phagocytosis and transfer them to lysosomal 54

compartments in the phagocytic pathway where they are usually digested by enzymes 55

(Siddiqui & Khan, 2012). However, some bacteria referred to as amoebae-resisting 56

bacteria (ARBs) are able to avoid or withstand enzymatic degradation in the phagocytic 57

pathway through various mechanisms and can survive amoeba predation and lodge inside 58

amoebae (Loret et al., 2008). ARBs include human pathogenic bacteria such as 59

Legionella, Chlamydia, and Mycobacteria. It has also recently been shown that the ARB 60

group includes non-pathogenic bacteria (Kebbi-Beghdadi & Greub, 2014). 61

62

ARBs can survive and grow within amoebae and may then escape by cell lysis or 63

exocytosis as free bacteria, or by being packaged in fecal pellets, which are usually 64

4

several concentric layers of lipid membranes known as multilamellar bodies (MLBs). The 65

secretion of packaged bacteria has been confirmed only for a number of human pathogens 66

(Legionella pneumophila, Salmonella enterica, Listeria monocytogenes, Helicobacter 67

pylori, and Escherichia coli O157:H7), but this process has been studied only with FLAs 68

and protozoa of the ciliate group (reviewed by Denoncourt et al. 2014). 69

70

Packaging provides bacteria with a number of advantages in unfavorable 71

conditions (Berk et al., 1998; Brandl et al., 2005, Gourabathini et al., 2008, Raghu 72

Nadhanan and Thomas, 2014). For example, Salmonella enterica bacteria packaged in 73

MLBs by the ciliate Tetrahymena are more resistant to low concentrations of calcium 74

hypochlorite than when they are in the planktonic state (Brandl et al., 2005). S. enterica 75

can even multiply inside pellets. 76

77

The social amoeba Dictyostelium discoideum is a bacterial predator that lives in 78

damp forest floors. The virulence traits and host-pathogen relationships of more than 20 79

pathogenic bacterial species have been studied using this amoeba as a model (Cosson & 80

Soldati, 2008, Bonifait et al., 2011, Dallaire-Dufresne et al., 2011). D. discoideum is 81

often compared to a macrophage-like organism that shares many proteins, such as 82

lysosomal hydrolases involved in intracellular killing, that are found in specialized 83

phagocytic cells in mammals (Cosson & Lima, 2014). D. discoideum produces (Mercanti 84

et al., 2006) and secretes large amounts of MLBs when fed digestible bacteria (Paquet et 85

al., 2013). While no studies on bacteria packaging by D. discoideum have been 86

5

published, inert polystyrene beads can be packaged in D. discoideum MLBs in presence 87

of digestible bacteria (Denoncourt et al., 2014). 88

89

We propose that D. discoideum has also the capacity to package ARBs in MLBs. 90

In the present study, 136 bacterial strains of various genera and environments were tested 91

for their capacity to resist D. discoideum predation and to determine whether these newly 92

identified ARBs are packaged in expelled MLBs. As expected, some ARBs were 93

packaged in D. discoideum MLBs and were secreted into the extracellular milieu. 94

95

6

MATERIALS AND METHODS 96

Amoebae 97

D. discoideum DH1-10 cells (Cornillon et al., 2000) were grown at 21°C in HL5 98

medium supplemented with 15 µg/mL of tetracycline (Mercanti et al., 2006). The cells 99

were subcultured twice a week in fresh medium to prevent the cultures from reaching 100

confluence. They were also grown on bacterial lawns as described below. 101

102

Bacteria 103

Klebsiella aerogenes was a kind gift from Pierre Cosson (Geneva University, 104

Switzerland), 19 bacterial isolates were provided by Martin Filion (Moncton University, 105

Canada) (Filion et al., 2004), and 78 bacterial isolates were provided by Janet Martha 106

Blatny et al. (Norwegian University of Science and Technology, Norway) (Dybwad et 107

al., 2012). All the other isolates used in the present study were from a drinking water 108

distribution network model (Berthiaume et al., 2014) or were obtained from ATCC or 109

USDA. Stock cultures were stored at -80°C in LB (EMD, Canada) supplemented with 110

15% glycerol. As needed, the stock cultures were thawed and were inoculated on Tryptic 111

Soy Agar (TSA) (EMD, Canada) plates, which were incubated at 25°C, typically for two 112

days, before being used for the experiments. 113

114

Predation resistance assay 115

Bacterial isolates grown on TSA plates were resuspended in 3 mL of LB, and the 116

OD at 595 nm was adjusted to 1. The resuspended bacteria (300 µL) were plated on three 117

different nutrient media (HL5: bacto peptone (Oxoid) 14.3 g L-1, yeast extract 7.15 g L-1, 118

7

maltose monohydrate 18 g L-1, Na2HPO4.2H2O 0.65 g L-1, KH2PO4 0.5 g L-1, and bacto 119

agar 20 g L-1); SM: bacto peptone 10 g L-1, yeast extract 1 g L-1, KH2PO4 2.2 g L-1, 120

K2HPO4 1 g L-1, MgSO4 1 g L-1, and bacto agar 20 g L-1); or SM1/10 (the ingredients for 121

SM were all diluted 1/10 except for the bacto agar). The plates were allowed to dry under 122

sterile conditions to obtain bacterial lawns. 123

124

The tetracycline from the amoeba cell culture maintenance was removed by 125

medium replacement, and the D. discoideum cells were resuspended in fresh HL5 with no 126

antibiotic before counting them in a hemacytometer chamber. Serial dilutions were 127

prepared in HL5 medium to obtain the following D. discoideum cell concentrations: 128

500,000; 50,000; 5,000; 500, 50, and 5 cells per 5 µL. The bacterial lawns were spotted 129

with 5 µL of the serial D. discoideum dilutions. The plates were allowed to dry and were 130

incubated at 21°C for 7 days. They were examined visually for plaque formation on days 131

1, 3, and 7. The isolates that did not allow the growth of amoebae were considered as 132

ARBs. 133

134

Bacteria/amoebae co-cultures 135

The identified ARBs were co-cultured alone or were mixed in a final volume of 136

300 µL with digestible K. aerogenes (Ka), which is known to stimulate the production of 137

MLBs (Paquet et al., 2013), and with 30 prewashed D. discoideum cells. The mixtures 138

were spread on SM agar plates. Serial Ka:ARB ratios ([99:1], [9:1] [1:1], [1:9], and 139

[1:99], in a total volume of 300 µL), based on an OD adjusted to 1, were used to 140

determine the best conditions for D. discoideum growth on bacterial co-cultures. The 141

8

plates were incubated at 21°C for 14 days and were examined visually for phagocytic 142

plaque formation, bacterial colonies within the phagocytic plaques, or all other 143

anomalous growth on days 3, 9, and 14. 144

145

Production of packaged and secreted ARBs 146

Potential packaged bacteria deduced from the bacteria/amoebae co-culture results 147

were mixed in a final volume of 300 µL with Ka using the best ratio determined from 148

previous experiments and were plated on SM1/10 agar. Drops (5 µL) containing 100,000 149

D. discoideum cells were spotted on the bacterial lawns. The plates were allowed to dry 150

and were incubated for 3 or 4 days at 21°C to obtain large phagocytic plaques. Samples 151

from the peripheries of the phagocytic plaques were collected using sterile tips. The 152

samples were gently diluted in fresh SM1/10 medium and were processed for 153

immunofluorescence (IF) or transmission electron microscopy (TEM) as described 154

below. 155

156

Immunofluorescence 157

The samples containing suspended cells and material from the peripheries of 158

phagocytic plaques were allowed to adhere to glass coverslips for 3 h and were then fixed 159

in 4% paraformaldehyde for 30 min. The coverslips were rinsed with PBS 1X (1.9 mM 160

NaH2PO4 + H2O; 8.1 mM Na2HPO4 + 2 H2O; 154 mM NaCl, pH 7.4) containing 40 mM 161

NH4Cl to stop the fixation and then with PBS 1X. The cells were permeabilized for 2 min 162

with methanol at -20 °C, and the coverslips were rinsed with PBS 1X and then with PBS 163

1X containing 0.2 % bovine serum albumin (PBS-BSA) at room temperature for at least 5 164

9

min to block non-specific binding sites. The adherent cells were then incubated for 45 165

min with the H36 antibody (Mercanti et al., 2006) diluted 1:1000 in PBS-BSA and then 166

with Alexa 568-coupled anti-mouse IgG secondary antibody (diluted 1:400; Invitrogen, 167

Canada) and 2.5 µg/mL of DAPI (4,6-di-amidino-2-phenylindole diluted in PBS-BSA) 168

for 30 min at room temperature in the dark. The coverslips were washed at least three 169

times with PBS-BSA between each step. The coverslips were mounted on glass slides 170

using Prolong Gold (Invitrogen). Images were acquired using an Axio Observer Z1 171

microscope equipped with an Axiocam camera (Carl Zeiss, Canada). 172

173

Transmission electron microscopy 174

Samples from the bacteria/amoebae co-cultures and material from the peripheries 175

of the phagocytic plaques were collected using sterile tips and were fixed for 3 h in 0.1 M 176

sodium cacodylate buffer (pH 7.3) containing 2 % glutaraldehyde and 0.3 % osmium 177

tetroxide. They were washed three times with sodium cacodylate buffer and were 178

dehydrated for 5 min in 30 % ethanol, 5 min in 50 % ethanol, 5 min in 70 % ethanol, 10 179

min in 95 % ethanol, and 1 h in 100 % ethanol. The samples were then embedded in 180

Epon resin and were incubated overnight at 37 °C followed by 3 days at 60 °C. Very thin 181

slices (60 to 80 nm) were cut and were stained for 8 min with 0.1 % lead citrate and then 182

for 5 min with 3 % uranyl acetate. They were then examined using a transmission 183

electron microscope (JEOL 1230) at 80 kV. 184

185

186

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RESULTS AND DISCUSSION 187

188

Predation resistance assay 189

D. discoideum is probably the simplest system for assessing bacterial virulence 190

(Hilbi et al., 2007, Froquet et al., 2009). Because medium richness may have an impact 191

on the results of predation resistance assays (Froquet et al., 2007, Filion & Charette, 192

2014), our assays were performed using three different media of varying composition and 193

richness (HL5, SM, SM1/10). 194

195

Phagocytic plaques, which are bacteria-free zones due to amoeba grazing, are 196

produced when amoebae are spotted on lawns of digestible bacteria (Figure 1). 197

Phagocytic plaques were not observed in the presence of ARBs or were observed only for 198

the highest D. discoideum cell concentrations (Figure 1C and D) (Filion & Charette, 199

2014). Ka is used routinely in many phagocytic experiments to feed D. discoideum, 200

which is why we used it as a positive control for amoeba predation (Figure 1B) (Froquet 201

et al., 2009). 202

203

We considered that the isolates were ARBs when 500 or fewer D. discoideum 204

cells were unable to produce phagocytic plaques on the bacterial lawn for at least one of 205

the media tested. For example, it is the case for Cupriavidus sp. and Microbacterium sp. 206

isolates shown in Figure 1C and D. Isolates that allowed the growth of the amoebae with 207

an initial inoculum of 500 D. discoideum cells per drop or less were considered sensitive 208

to amoeba predation and were rejected for subsequent experiments. 209

11

A total of 136 bacterial isolates were screened with the amoeba predation assay to 210

identify those that were potential ARBs. All the experiments were performed twice, and 211

45 isolates were considered as D. discoideum resisting bacteria and, as such, potential 212

candidates for the packaging process (see Table S1). 213

The newly discovered ARBs were not specific to one phylum but belonged to various 214

clades distributed throughout the prokaryotes, which was in agreement with a study by 215

Moliner et al. (Moliner et al., 2010). Table 1 presents the ARBs discovered in the present 216

study. Our results suggested that the adaptation of bacteria to avoid digestion during 217

phagocytosis is widespread in bacteria. Moreover, the term ARB cannot be generalized 218

and be applied to an entire genus or species since bacteria from the same genus or species 219

did not display the same resistance to predation (Table 1). 220

221

Triple co-cultures 222

The 45 newly identified ARBs were co-cultured with digestible bacteria (Ka) and 223

D. discoideum. The goal of this experiment was to assess the growth of amoebae on 224

digestible bacteria (Ka) in the presence of ARBs to determine whether the ARBs were 225

toxic for the amoebae, making it impossible for them to produce packaged bacteria. All 226

the phagocytic plaques with a profile similar to the positive control, that is, with a large 227

bacteria-free zone (black arrow, Figure 2A) due to extensive amoeba growth, were 228

rejected. Similarly, co-cultures where no amoeba growth occurred, as for the negative 229

control, were also rejected. For example, all the Ka:Luteibacter anthropic ratios produced 230

small phagocytic plaques compared to the plaques produced by amoebae grown only on 231

Ka, suggesting that L. anthropic was toxic to the amoebae or markedly limited their 232

12

growth (black arrow, Figure 2B). Conversely, the presence of bacterial colonies in the 233

middle of grazing plaques (black arrow at top, Figure 2C) or substantial growth of the 234

ARB around phagocytic plaques (black arrow at the bottom, Figure 2C) indicated that the 235

ARB was resistant to predation and had no obvious toxicity for D. discoideum. One 236

possibility is that the bacteria passed through the phagocytic pathway and were expelled 237

as packaged bacteria, which then began to grow and form colonies. Three Cupriavidus 238

and 17 other isolates displayed this profile (Table 2). Thus based on the unusual growth 239

pattern of amoebae on their lawns, 20 isolates were considered as ARBs and were 240

retained in order to determine whether they were packageable. 241

242

Bacteria packaging by D. discoideum 243

The next step was to determine whether D. discoideum cells were able to package 244

ARBs. Based on previous packaging assays by Gourabathini et al. with E. coli O157:H7 245

and the ciliate Tetrahymena pyriformis (Gourabathini et al., 2008), packaged bacteria 246

released on a rich medium are able to grow inside the package and break out. Indeed, 247

packaged bacteria are likely a transitory state, allowing the bacteria to survive in harsh 248

conditions (Berk et al., 1998, Marciano-Cabral & Cabral, 2003) until they are released 249

into an environment that is more favorable for bacterial growth. Packaged ARBs were not 250

observed during the triple co-culture experiments using rich medium even after a long 251

period of time probably due to growth of potentially packaged bacteria. On the other 252

hand, starvation media (Smith et al., 2010), which contains only few nutriments to 253

prevent bacterial growth have been also tried, but they induce the multicellular 254

development of amoebae despite the presence of digestible bacteria (data not shown). 255

13

Again, no packaged bacteria were seen because active vegetative D. discoideum cells are 256

required for the packaging process to occur. 257

258

The stimulation of bacteria packaging and secretion was also studied using diluted 259

nutrient agar (SM1/10) to avoid rapid bacterial growth following exocytosis that could 260

break up the packages. We observed amoebae on mixed bacterial lawns of digestible 261

bacteria and ARBs (see ratios and strains in Table 2). Samples collected at the peripheries 262

of the phagocytic plaques were examined by IF with the H36 antibody (Mercanti et al., 263

2006) and by TEM. 264

265

A sample containing potential packaged bacteria had to display combined DAPI and 266

H36 antibody-positive staining for structures smaller than amoebae but bigger than free-267

living bacteria (data not shown) due to packaging of bacteria. DAPI would reveal the 268

presence of bacteria in the structures. On its side, H36 antibody has been shown in a 269

previous study to be a specific marker of MLBs by binding to a protein still not 270

characterized (Paquet et al, 2013). The magenta arrows in Figure 3 point to bacteria 271

packages measuring 2 to 3 µm in diameter, and the black arrow indicates a D. discoideum 272

cell. Of the 20 potential candidates tested by IF, three Cupriavidus isolates, two 273

Micrococcus luteus isolates, and one isolate each of Rathayibacter tritici and 274

Microbacterium oxydans presented features suggesting that they were packaged by D. 275

discoideum. 276

277

14

The same co-culture protocol was performed on several samples to formally confirm 278

the presence of expelled packaged bacteria by TEM. For the control condition shown on 279

Figure 4, D. discoideum produced (white arrow, Figure 4B) and secreted empty MLBs 280

(black arrow, Figure 4C) in the presence of digestible bacteria on SM1/10. However, 281

D. discoideum produced fewer MLBs on SM1/10 than on rich HL5 medium (Paquet et al., 282

2013). Despite this, Cupriavidus sp. and R. tritici were found inside secreted MLBs when 283

they were co-cultured with amoeba and digestible bacteria (Figure 4E, F, and I). The 284

TEM observations revealed that some of the tested bacteria could be packaged by D. 285

discoideum. 286

287

Interestingly, R. tritici accumulated inside the amoebae, with up to 50 undigested 288

bacteria visible inside each D. discoideum cell (Figure 4H). It is not clear whether the 289

accumulation was due to rapid bacterial growth inside the amoebae, the inhibition of the 290

exocytic process, or a combination of both. While the mechanism involved is not known, 291

this result suggested that bacteria can also survive in harsh environments by residing 292

inside amoebae. The intracellular survival in protozoa of many bacteria has been 293

described in the past (reviewed in Denoncourt et al., 2014). Many bacteria of the genus 294

Rathayibacter are phytopathogens of terrestrial plants (Hahn et al., 2003, Schaad & 295

Schuenzel, 2010), and it is likely that amoebae and these soil bacteria interact. 296

297

We showed that the packaging of bacteria is possible by D. discoideum amoeba model 298

and that the phenomenon is not restricted to specific genera. Indeed, both Gram-negative 299

and -positive bacteria from various environments, including soil and water, were trapped 300

15

inside the MLBs. Moreover, the outcome of various isolates from a same genera or even 301

a same species regarding packaging is fairly variable. For example, 23 strains of M. 302

luteus were tested using the predation assay and 9 were identified as ARBs, two of which 303

were packaged in MLBs based on the IF results. Thirteen Pseudomonas strains were also 304

tested using the predation assay. While 4 displayed an ARB phenotype, none was 305

packaged in MLBs. These results indicated that bacterial adaptive evolution with respect 306

to protozoa is complex, as has been shown by the farming of different strains of 307

Burkholderia sp. by non-farmer D. discoideum (DiSalvo et al., 2015). Given this, it 308

would be difficult to predict whether a given bacterial isolate can be packaged or can 309

resist predation by a specific protozoan without in vitro testing. It would thus be 310

interesting to determine whether the same ARBs are packaged by different wild-type 311

strains of D. discoideum or other protozoa. 312

313

Lastly, the present study showed that some ARBs are packaged in MLBs and are 314

secreted by D. discoideum in laboratory conditions. Amoeba/bacteria interactions are 315

ubiquitous in natural as well as in man-made environments such as in municipal drinking 316

water storage tank sediments (Lu et al., 2015), the floating and fixed biofilms of spring 317

recreation areas (Hsu et al., 2011), and the surface water of warm water systems and 318

cooling towers (Kuiper et al., 2006). As such, it is likely that bacteria packaging occurs in 319

real conditions, not just in the laboratory. 320

16

CONCLUSION 321

The resistance to predation of 136 bacterial isolates was assessed using a standardized 322

D. discoideum predation assay. Forty-five of these isolates displayed an ARB phenotype 323

and were co-cultured with digestible bacteria to stimulate MLB production. Twenty 324

potential candidates were retained based on this screening. The bacteria packaging of 325

seven isolates by D. discoideum was suggested by IF and confirmed for two isolates by 326

TEM. This is the first study to show that D. discoideum can package bacteria. These 327

results open the way to a better understanding of the role of ARBs in microbial ecology 328

and their persistence in many environments. 329

330

331

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FUNDING 332

This work was supported by grants to S. J. C. from the Fonds de la Recherche du Québec 333

– Nature et Technologies (FRQNT) [2014-PR-173418], the Chaire de pneumologie de la 334

fondation J.-D. Bégin de l’Université Laval, the Fonds Alphonse L’Espérance de la 335

fondation de l’IUCPQ, and the Establishment of young researchers - Juniors 1 program of 336

the Fonds de la Recherche du Québec en Santé (FRQS) [20004]. 337

338

18

Acknowledgements 339

We are grateful to P. Cosson (University of Geneva, Switzerland) for the antibodies and 340

bacterial strains. We warmly thank the teams of J. M. Blatny (FFI, Norway) and M. 341

Filion (University of Moncton, Canada) as well as the USDA, who provided many 342

bacterial strains. We thank A. Denoncourt and A. Vincent (Université Laval, Canada) for 343

their critical reading of the manuscript and Richard Janvier (Plateforme de microscopie, 344

IBIS, Université Laval, Canada) for acquiring the transmission electron 345

microphotographs. 346

347

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441

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Table 1. Taxonomic grouping of new ARBs identified by the predation assay 442 Gram Class a Order Familia Genera Species No. of

isolates tested

No. of ARB

isolates Positive

Actino

Actinomycetales

Microbacteriaceae Microbacterium Microbacterium sp. 8 3 Rathayibacter Rathayibacter tritici 1 1

Micrococcaceae Kocuria Kocuria sp. 17 2 Micrococcus Micrococcus luteus 23 9

Nocardiaceae Rhodococcus Rhodococcus sp. 6 6 Streptomycetaceae Streptomyces Streptomyces

luridiscabiei 1 1

Micrococcales Promicromono-sporaceae

Cellulosimicrobium Cellulosimicrobium funkei

1 1

Bacilli

Bacillales

Paenibacillaceae Paenibacillus Paenibacillus larvae 1 1 Staphylococcaceae Staphylococcus Staphylococcus sp. 9 1

Lactobacillales Leuconostocaceae Weissella Weissella confusa 1 1 Negative

Alpha

- - - - 1 1 Rhizobiales Rhizobiaceae Sinorhizobium Sinorhizobium sp. 2 1

Beta

Burkholderiales

Burkholderiaceae Burkholderia Burkholderia sp. 3 3 Cupriavidus Cupriavidus sp. 5 4

Comamonadaceae Comamonas Comamonas koreensis

1 1

Oxalobacteraceae Duganella Duganella zoogloeoides

1 1

Gamma

Enterobacteriales

Enterobacteriaceae

Escherichia Escherichia coli 3 2 Serratia Serratia grimesii 1 1

Pseudomonadales Pseudomonadaceae Pseudomonas Pseudomonas sp. 13 4 Xanthomonadales Xanthomonadaceae Luteibacter Luteibacter anthropi 1 1

a Actino = Actinobacteria; Alpha = Alphaproteobacteria; Beta = Betaproteobacteria; Gamma = Gammaproteobacteria 443

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Table 2. ARBs identified after co-culture assays as potential candidates for bacteria 444 packaging. 445 Strains Ratio KA:ARB Observations and comments Cupriavidus basilensis 1:1 Based on the morphology and

color of the colonies at the center and periphery of the phagocytic plaques A few fruiting bodies, with colored spores at the top.

Cupriavidus sp. 9:1 Micrococcus luteus (Norway) 9:1 Micrococcus luteus US4 1:9 Rathayibacter tritici 9:1 Rhodococcus erythropolis US1 1:9 Rhodococcus erythropolis US2 9:1 Rhodococcus fascians US1 9:1 Rhodococcus fascians US2 1:1 Cupriavidus necator US1 1:1 Several colonies within the

phagocytic plaques. Duganella zoogloeoides 1:1 Kocuria kristinae 1:9 Microbacterium oxydans US1 9:1 Micrococcus luteus 9:1 Micrococcus luteus 8_4_14 x2 1:9 Micrococcus luteus D_1_6 x2 1:9 Micrococcus luteus US3 1:9 Rhodococcus erythropolis 1:1 Rhodococcus pyridinovorans 1:9 Cellulosimicrobium funkei 1:1 Unusual growth on agar. 446 447 448

24

FIGURE LEGENDS 449

Figure 1. Predation resistance assay. A. Serial dilutions of D. discoideum cells 450

(500,000 to 5 cells/5 µL) were spotted counter clockwise on bacterial lawns on HL5 agar 451

plates. The plates were incubated for 7 days. The negative control (HL5 medium only) 452

was spotted in the middle of the lawn. B. Klebsiella aerogenes is sensitive to predation by 453

amoebae. It was used as a positive control for amoeba predation. Cupriavidus sp. (C) and 454

Microbacterium sp. (D) were resistant to predation and were considered as potential 455

ARBs.456

25

Figure 2. Triple co-cultures. Example of potential ARB isolates co-cultured with 457

digestible bacteria (Ka) and 30 D. discoideum cells on SM agar. A. A lawn of Ka was 458

used as positive control for phagocytic plaque formation (clear zones in the bacterial 459

lawn; black arrow). B. A lawn of co-cultured Ka and Luteibacter anthropic [ratio 1:9]. 460

After the same incubation time, the amoebae were unable to farm the bacterial lawn, and 461

the plaques (black arrow) were much smaller than those of the negative control. This 462

bacterial species was not retained for subsequent analyses. C. A lawn of co-cultured Ka 463

and Cupriavidus sp. [ratio 1:9]. Pigmented colonies corresponding to the Cupriavidus sp. 464

can be seen in the middle of the phagocytic plaques (upper black arrow). Pigmented 465

colonies can also seen around the plaques (lower black arrow). This isolate was 466

considered as an ARB. 467

468

26

Figure 3. Immunofluorescence of bacteria packaged by D. discoideum. Material from 469

the peripheries of phagocytic plaques on lawns of co-cultured bacteria (see ratio in Table 470

2) on SM1/10 agar spotted with D. discoideum were processed for IF and were observed 471

under an epifluorescence microscope. For each ARB tested, the differential interference 472

contrast (DIC) is shown on the left while DAPI (blue), which targets the DNA of bacteria 473

and amoebae, and the H36 antibody (red), which targets MLBs and the amoeba 474

membrane, staining are presented on the right. D. discoideum (black arrow in A) 475

produced and secreted a few packaged Cupriavidus sp. (magenta arrow) into the 476

extracellular milieu. The bacteria shown on the images (A and B) were coated and 477

recognized by the H36 antibody. In C and D, only a fraction of the M. luteus and R. tritici 478

cells were in H36-positive structures. 479

480

27

Figure 4. Transmission electron microscopy of bacteria packaged and secreted by D. 481

discoideum. The peripheries of phagocytic plaques from co-cultured bacteria (see ratio in 482

Table 2) on SM1/10 agar spotted with D. discoideum were processed and were observed 483

by TEM. A, D, and G. Bacteria grown alone on rich medium. B and C. D. discoideum 484

produces (white arrow) and secretes (black arrow) MLBs with digestible bacteria on 485

SM1/10. No Ka were seen inside the MLBs. E, F, and I. Cupriavidus sp. and R. tritici 486

were packaged by D. discoideum and were exocytosed into the extracellular milieu. H. 487

More than 50 undigested R. tritici can be seen inside a D. discoideum cell. 488

Table S1. Compilation of the results of the predation resistance assays. One hundred thirty-six soil and water isolates were plated on three types of medium: HL5 = rich medium, SM = nutrient medium, and SM1/10 = nutrient-poor medium. Serial dilutions of D. discoideum cells (500,000 to 5 cells/5 µL) were spotted and spread on bacterial lawns. The plates were incubated for 7 days at 21°C. The phagocytic plaques were counted to determine the resistance of each isolate to predation by D. discoideum. The potential ARBs (underlined in yellow) are located in the magenta spectrum. The predation-sensitive strains are located in the cyan spectrum. A brown box indicates that the bacterial isolate did not grow on that medium after two or more tries.

Legend: > 500,000 > 50,000 > 5,000 > 500 > 50 > 5

No resistance ARBs No growth

Isolates HL5 SM SM1/10 Aeromonas hydrophila M15918-11

Alcaligenes faecalis

Alphaproteobacterium

Arthrobacter humicola Arthrobacter koreensis

Arthrobacter tumbae

Brevundimonas vesicularis

Burkholderia ambifaria HSJ1 Burkholderia ambifaria variant

Burkholderia thailendensis

Cellulosimicrobium funkei

Clavibacter michiganensis Comamonas koreensis

Corynebacterium callunae

Cupriavidus basilensis

Cupriavidus necator-US1 Cupriavidus necator-US2

Cupriavidus sp.

Curtobacterium pusillum

Dietzia cinnamea Duganella zoogloeoides

Endophytic bacterium

Enhydrobacter aerosaccus

Ensifer adhaerens P43 Erwinia tasmaniensis

Escherichia coli B/R

Escherichia coli BL21

Isolates HL5 SM SM1/10 Escherichia coli MC1061

Exiguobacterium indicum

Flavobacterium sp. Frigoribacterium sp. Ev.-gws-26

Gordonia alkanivorans Janibacter limosus

Klebsiella aerogenes

Kocuria kristinae

Kocuria kristinae-US1 Kocuria kristinae-US2

Kocuria kristinae-US3

Kocuria kristinae-US5

Kocuria kristinae-US6 Kocuria kristinae-US7

Kocuria palustris

Kocuria rosea

Kocuria sp.8_1_14 Kocuria sp.32_3_20

Kocuria sp. D_1_23

Kocuria sp. 56_3_23_x1

Kocuria sp.56_2_16 Kocuria sp. 72_1_15

Kocuria sp. 48_5_11

Kocuria sp. 1_3_18B

Luteibacter anthropi Microbacterium sp. Microbacterium esteraromaticum

Microbacterium hatanonis

Microbacterium lacus Microbacterium oleivorans

Microbacterium oxydans

Microbacterium oxydans-US1

Microbacterium phyllosphaerae Micrococcus luteus

Micrococcus luteus (Norway)

Micrococcus luteus 24_4_18

Micrococcus luteus 8_4_14_X2

Isolates HL5 SM SM1/10 Micrococcus luteus D_1_6_x2

Micrococcus luteus 25_5_4

Micrococcus luteus 1_1_24

Micrococcus luteus 8_4_15_x2 Micrococcus luteus D_3_15

Micrococcus luteus 61_5_26

Micrococcus luteus 37_4_14

Micrococcus luteus 8_5_8 Micrococcus luteus 48_3_19

Micrococcus luteus 48_5_10

Micrococcus luteus 4_3_25

Micrococcus luteus 8_5_6 Micrococcus luteus 4_4_11

Micrococcus luteus 4698

Micrococcus luteus-US1

Micrococcus luteus-US2 Micrococcus luteus-US3

Micrococcus luteus-US4

Micrococcus luteus-US6

Ochrobactrum intermedium Oerskovia paurometabola

Paenibacillus larvae 3558

Paracoccus yeei

Pectobacterium cypripedii Pedobacter agri

Phyllobacterium sp. ORS 1420

Planococcus rifietoensis

Plantibacter flavus Pseudomonas asplenii isolate 1

Pseudomonas asplenii isolate 2

Pseudomonas CT107

Pseudomonas fluorescence Pseudomonas fulva

Pseudomonas koreensis

Pseudomonas poea

Pseudomonas psychrotolerans Pseudomonas putida

Pseudomonas sp.

Isolates HL5 SM SM1/10 Pseudomonas sp. LBUM-636

Pseudomonas sp. LBUM-677

Pseudomonas stutzeri

Ralstonia sp. Rathayibacter tritici

Rhodococcus erythropolis

Rhodococcus erythropolis-US1

Rhodococcus erythropolis-US2 Rhodococcus fascians-US1

Rhodococcus fascians-US2

Rhodococcus pyridinivorans

Rhodospirulum rubrum Roseomonas mucosa

Rothia amarae

Rothia nasimurium

Serratia grimesii Serratia marcescens

Sinorhizobium meliloti

Sphingomonas paucimobilis

Sphingomonas sanguinis Staphylococcus cohnii

Staphylococcus aureus

Staphylococcus epidermidis

Staphylococcus equorum Staphylococcus haemolyticus

Staphylococcus kloosii

Staphylococcus lentus

Staphylococcus saprophyticus Staphylococcus succinus

Streptomyces luridiscabiei

Variovorax paradoxus

Wautersia eutropha Weissella confusa

Yersinia ruckeri RS41