2 discoideum: social amoebae can also package bacteria ARB... · 1 1 Amoeba-resisting bacteria...
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Amoeba-resisting bacteria found in multilamellar bodies secreted by Dictyostelium 1
discoideum: social amoebae can also package bacteria 2
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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
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*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: [email protected] 16
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Running title (60 characters with space): Packaging of amoeba-resisting bacteria by D. 18
discoideum 19
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Keywords (6): Multilamellar bodies; Dictyostelium discoideum; packaged bacteria, 21
amoeba-resisting bacteria, Cupriavidus, Rathayibacter 22
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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
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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
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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
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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
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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
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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
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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
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published, inert polystyrene beads can be packaged in D. discoideum MLBs in presence 87
of digestible bacteria (Denoncourt et al., 2014). 88
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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RESULTS AND DISCUSSION 187
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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
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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
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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
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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
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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
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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
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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
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Again, no packaged bacteria were seen because active vegetative D. discoideum cells are 256
required for the packaging process to occur. 257
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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
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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