1 Amoeba-resisting bacteria found in multilamellar bodies secreted by Dictyostelium 2 discoideum: social amoebae can also package bacteria 3 4 Valérie E. Paquet1,2 and Steve J. Charette1,2,3* 5 6 1. Institut de Biologie Intégrative et des Systèmes, Pavillon Charles-Eugène-Marchand, 7 Université Laval, Quebec City, QC, Canada 8 2. Centre de recherche de l’Institut universitaire de cardiologie et de pneumologie de 9 Québec, Hôpital Laval, Quebec City, QC, Canada 10 3. Département de biochimie, de microbiologie et de bio-informatique, Faculté des 11 sciences et de génie, Université Laval, Quebec City, QC, Canada 12 13 *Corresponding author: 14 Steve J. Charette, 1030 avenue de la medicine, Pavillon Marchand, local 4245, Université 15 Laval, Quebec City, QC, Canada, G1V 0A6, telephone: 1-418-656-2131, ext. 6914, fax: 16 1-418-656-7176, email: [email protected] 17 18 Running title (60 characters with space): Packaging of amoeba-resisting bacteria by D. 19 discoideum 20 21 Keywords (6): Multilamellar bodies; Dictyostelium discoideum; packaged bacteria, 22 amoeba-resisting bacteria, Cupriavidus, Rathayibacter 23 1 24 ABSTRACT 25 Many bacteria can resist phagocytic digestion by various protozoa. Some of these 26 bacteria (all human pathogens) are known to be packaged in multilamellar bodies 27 produced in the phagocytic pathway of the protozoa and that are secreted into the 28 extracellular milieu. Packaged bacteria are protected from harsh conditions, and the 29 packaging process is suspected to promote bacterial persistence in the environment. To 30 date, only a limited number of protozoa, belonging to free-living amoebae and ciliates, 31 have been shown to perform bacteria packaging. It is still unknown if social amoebae can 32 do bacteria packaging. The link between the capacity of 136 bacterial isolates to resist the 33 grazing of the social amoeba Dictyostelium discoideum and to be packaged by this 34 amoeba was investigated in the present study. The 45 bacterial isolates displaying a 35 resisting phenotype were tested for their capacity to be packaged. A total of seven isolates 36 from Cupriavidus, Micrococcus, Microbacterium, and Rathayibacter genera seemed to 37 be packaged and secreted by D. discoideum based on immunofluorescence results. 38 Electron microscopy confirmed that the Cupriavidus and Rathayibacter isolates were 39 formally packaged. These results show that social amoebae can package some bacteria 40 from the environment revealing a new aspect of microbial ecology. 41 42 2 43 INTRODUCTION 44 Free-living amoebae (FLAs) like Acanthamoeba spp. are mobile unicellular 45 protozoa that live in aquatic environments and feed on bacteria, fungi, and algae 46 (Rodriguez-Zaragoza, 1994). FLAs can colonize many man-made infrastructures that 47 provide a favorable environment for the proliferation of microorganisms, especially 48 where high bacterial population densities are found. Cooling towers (Pagnier et al., 49 2009), air conditioners (Walker et al., 1986), and drinking water distribution systems 50 (Thomas & Ashbolt, 2011) are a few examples of man-made infrastructures where FLAs 51 grow (reviewed in (Siddiqui & Khan, 2012, Cateau et al., 2014) and regulate bacterial 52 population densities. 53 54 FLAs capture bacteria by phagocytosis and transfer them to lysosomal 55 compartments in the phagocytic pathway where they are usually digested by enzymes 56 (Siddiqui & Khan, 2012). However, some bacteria referred to as amoebae-resisting 57 bacteria (ARBs) are able to avoid or withstand enzymatic degradation in the phagocytic 58 pathway through various mechanisms and can survive amoeba predation and lodge inside 59 amoebae (Loret et al., 2008). ARBs include human pathogenic bacteria such as 60 Legionella, Chlamydia, and Mycobacteria. It has also recently been shown that the ARB 61 group includes non-pathogenic bacteria (Kebbi-Beghdadi & Greub, 2014). 62 63 ARBs can survive and grow within amoebae and may then escape by cell lysis or 64 exocytosis as free bacteria, or by being packaged in fecal pellets, which are usually 3 65 several concentric layers of lipid membranes known as multilamellar bodies (MLBs). The 66 secretion of packaged bacteria has been confirmed only for a number of human pathogens 67 (Legionella pneumophila, Salmonella enterica, Listeria monocytogenes, Helicobacter 68 pylori, and Escherichia coli O157:H7), but this process has been studied only with FLAs 69 and protozoa of the ciliate group (reviewed by Denoncourt et al. 2014). 70 71 Packaging provides bacteria with a number of advantages in unfavorable 72 conditions (Berk et al., 1998; Brandl et al., 2005, Gourabathini et al., 2008, Raghu 73 Nadhanan and Thomas, 2014). For example, Salmonella enterica bacteria packaged in 74 MLBs by the ciliate Tetrahymena are more resistant to low concentrations of calcium 75 hypochlorite than when they are in the planktonic state (Brandl et al., 2005). S. enterica 76 can even multiply inside pellets. 77 78 The social amoeba Dictyostelium discoideum is a bacterial predator that lives in 79 damp forest floors. The virulence traits and host-pathogen relationships of more than 20 80 pathogenic bacterial species have been studied using this amoeba as a model (Cosson & 81 Soldati, 2008, Bonifait et al., 2011, Dallaire-Dufresne et al., 2011). D. discoideum is 82 often compared to a macrophage-like organism that shares many proteins, such as 83 lysosomal hydrolases involved in intracellular killing, that are found in specialized 84 phagocytic cells in mammals (Cosson & Lima, 2014). D. discoideum produces (Mercanti 85 et al., 2006) and secretes large amounts of MLBs when fed digestible bacteria (Paquet et 86 al., 2013). While no studies on bacteria packaging by D. discoideum have been 4 87 published, inert polystyrene beads can be packaged in D. discoideum MLBs in presence 88 of digestible bacteria (Denoncourt et al., 2014). 89 90 We propose that D. discoideum has also the capacity to package ARBs in MLBs. 91 In the present study, 136 bacterial strains of various genera and environments were tested 92 for their capacity to resist D. discoideum predation and to determine whether these newly 93 identified ARBs are packaged in expelled MLBs. As expected, some ARBs were 94 packaged in D. discoideum MLBs and were secreted into the extracellular milieu. 95 5 96 MATERIALS AND METHODS 97 Amoebae 98 D. discoideum DH1-10 cells (Cornillon et al., 2000) were grown at 21°C in HL5 99 medium supplemented with 15 µg/mL of tetracycline (Mercanti et al., 2006). The cells 100 were subcultured twice a week in fresh medium to prevent the cultures from reaching 101 confluence. They were also grown on bacterial lawns as described below. 102 103 Bacteria 104 Klebsiella aerogenes was a kind gift from Pierre Cosson (Geneva University, 105 Switzerland), 19 bacterial isolates were provided by Martin Filion (Moncton University, 106 Canada) (Filion et al., 2004), and 78 bacterial isolates were provided by Janet Martha 107 Blatny et al. (Norwegian University of Science and Technology, Norway) (Dybwad et 108 al., 2012). All the other isolates used in the present study were from a drinking water 109 distribution network model (Berthiaume et al., 2014) or were obtained from ATCC or 110 USDA. Stock cultures were stored at -80°C in LB (EMD, Canada) supplemented with 111 15% glycerol. As needed, the stock cultures were thawed and were inoculated on Tryptic 112 Soy Agar (TSA) (EMD, Canada) plates, which were incubated at 25°C, typically for two 113 days, before being used for the experiments. 114 115 Predation resistance assay 116 Bacterial isolates grown on TSA plates were resuspended in 3 mL of LB, and the 117 OD at 595 nm was adjusted to 1. The resuspended bacteria (300 µL) were plated on three 118 different nutrient media (HL5: bacto peptone (Oxoid) 14.3 g L-1, yeast extract 7.15 g L-1, 6 -1 -1 -1 119 maltose monohydrate 18 g L , Na2HPO4.2H2O 0.65 g L , KH2PO4 0.5 g L , and bacto -1 -1 -1 -1 120 agar 20 g L ); SM: bacto peptone 10 g L , yeast extract 1 g L , KH2PO4 2.2 g L , -1 -1 -1 1/10 121 K2HPO4 1 g L , MgSO4 1 g L , and bacto agar 20 g L ); or SM (the ingredients for 122 SM were all diluted 1/10 except for the bacto agar). The plates were allowed to dry under 123 sterile conditions to obtain bacterial lawns. 124 125 The tetracycline from the amoeba cell culture maintenance was removed by 126 medium replacement, and the D. discoideum cells were resuspended in fresh HL5 with no 127 antibiotic before counting them in a hemacytometer chamber. Serial dilutions were 128 prepared in HL5 medium to obtain the following D. discoideum cell concentrations: 129 500,000; 50,000; 5,000; 500, 50, and 5 cells per 5 µL. The bacterial lawns were spotted 130 with 5 µL of the serial D. discoideum dilutions. The plates were allowed to dry and were 131 incubated at 21°C for 7 days. They were examined visually for plaque formation on days 132 1, 3, and 7. The isolates that did not allow the growth of amoebae were considered as 133 ARBs. 134 135 Bacteria/amoebae co-cultures 136 The identified ARBs were co-cultured alone or were mixed in a final volume of 137 300 µL with digestible K. aerogenes (Ka), which is known to stimulate the production of 138 MLBs (Paquet et al., 2013), and with 30 prewashed D. discoideum cells. The mixtures 139 were spread on SM agar plates. Serial Ka:ARB ratios ([99:1], [9:1] [1:1], [1:9], and 140 [1:99], in a total volume of 300 µL), based on an OD adjusted to 1, were used to 141 determine the best conditions for D. discoideum growth on bacterial co-cultures. The 7 142 plates were incubated at 21°C for 14 days and were examined visually for phagocytic 143 plaque formation, bacterial colonies within the phagocytic plaques, or all other 144 anomalous growth on days 3, 9, and 14.