Cationic antimicrobial peptide resistant sub-population in P. luminescens TT01

24 Bacteria live in environments that undergo perpetual alterations and in which they are 25 challenged by antibiotics, bacteriophages, mutagens, toxins and more. Which strategies do 26 bacteria use to optimize their chance of surviving? Among mechanisms used by bacteria to 27 survive, there are two key strategies: phase and antigenic variation (Van der Woude 2011) 28 that corresponds to genetic alterations and the bistability generated by epigenetic mechanisms 29 on clonal population (Dubnau et Losick 2006). Both of these strategies lead to bacterial 30 heterogeneity. Bacterial population has been traditionally seen as an isogenic and clonal 31 population genetically and phenotipically identical. The development of single cell 32 technology such as cytometry and fluorescence microscopy allowed the development of 33 studies showing heterogeneous gene expression in bacterial cells (Smits, Kuipers, et Veening 34 2006). Heterogeneity is found in various bacteria like Salmonella in Salmonella-containing 35 vacuoles (Helaine et Holden 2013) or Photorhabdus during the colonization of nematodes 36 (Somvanshi et al. 2012). 37 38 Photorhabdus luminescens subsp laumondii TT01 is an entomopathogenic bacterium 39 (Enterobacteriaceae) living in a symbiotic association with the nematode Heterorhabditis. The 40 bacteria-nematode complex invades insect larvae and the nematode regurgitates its bacterial 41 symbiont directly into the hemolymph, the insect blood. The bacteria can overcome the insect 42 immune system and colonize the insect body cavity leading to lethal septicemia (Waterfield, 43 Ciche, and Clarke 2009). Bacterial virulence factors and insecticidal toxins also participate to 44 the insect death (Silva et al. 2002 ; Nielsen-LeRoux et al. 2012). Once the insect host is dead, 45 bacteria bioconvert the tissues, digest the content of the cadaver and the nematode feeds on it 46 as a food source while reproduction occurs through several generations (Clarke 2014). 47 According to its dual lifestyle, Photorhabdus is a good model to study bacteria-insects and 48 bacteria-nematode interactions. Recently, it has been demonstrated that the mad genes 49 expression is under the control of a genetic switch of the promoter region. In the ON state 50 mad genes can be transcribed and the bacteria are covered with fimbriae. This form of 51 Photorhabdus is called the M form (for mutualistic form) because it is found only during the 52 symbiosis with the nematode. But, when the mad promoter is in the OFF conformation, no 53 fimbriae are produced on the bacterial cell surface and only the P form (for pathogenic form) 54 of Photorhabdus is found. Only the P form, can multiply and kill the insect and can support 55 nematode growth (Somvanshi et al. 2012). 56 Cationic antimicrobial peptides (CAMPs) are produced following insect infection (Bang et al. 57 2012 ; Haine et al. 2008) and act in complement of cellular immunity to fight against bacterial 58 invasion. CAMPs are small amphipathic basic peptides from 15-40 amino acids (for review 59 see (Bulet and Stöcklin 2005)) secreted by a large number of organisms including plants, 60 animals and microbes and present a large variety of structure. However, the two prominent 61 classes involved alpha-helical and beta-sheet peptides (Tossi, Sandri, and Giangaspero 2000). 62 CAMPs have antibacterial activity acting through charge interactions with the anionic 63 bacterial surface, predominantly binding to the acidic lipid A moiety of the LPS (Rana et al. 64 1991 ; Srimal et al. 1996). 65 PhoPQ has been extensively studied in Salmonella where it controls about 3% of Salmonella 66 gene expression either in a direct or indirect pathway (Kato, Groisman, and Howard Hughes 67 Medical Institute 2008). Among genes regulated by PhoPQ, virulence factors or genes 68 implicated in LPS modifications are found such as pagP and pbgPE. pagP is directly 69 regulated by PhoP in Salmonella spp, and responsible for addition of palmitate residue on 70 lipid A of the LPS. This lipid A palmitoylation confers resistance towards cationic 71 antimicrobial peptides (CAMPs) (Guo et al. 1998) and reduce bacterial recognition by 72 immune system in a TLR4 dependant pathway (Kawasaki, Ernst, et Miller 2004). Another 73 modification involved in LPS modification is the addition of an amino-arabinose on lipid A 74 core of the LPS (Gunn et Miller 1996). This modification modifies the global net charge of 75 bacterial cell membrane from negative to positive conferring resistance towards CAMPs 76 (Gunn et al. 1998). In Salmonella the pbgPE operon codes for enzymes responsible for 77 amino-arabinose addition on lipid A. PhoP indirectly regulates pbgPE expression via another 78 two component system PmrAB (Gunn et Miller 1996). pbgPE expression is activated at low.

Bacterial strains, plasmids, and growth conditions

The strains and plasmids used in this study are listed in Table S1. P. luminescens strains were routinely grown at 28°C in Luria-Bertani (LB) or Mueller Hinton broth (Biokar), nutrient agar medium (Difco), NBTA agar (Brunel et al. 1997). Photorhabdus was also grown in M9 liquid medium supplemented with 0.1 % casamino acids, 0.41 mM nicotinic acid, 9.1 mM 1sodium pyruvate, 0.1 mM CaCl2 and 0.2 % glycerol with different concentrations of MgSO4 (10 µM and 10 mM). When required, antibiotics were used at the following final 123 Hinton broth (Biokar) following incubation at 28°C for 48 h. The microtiter plates were read 124 by visual observation. 125 126 127 Protein PhoP-His purification protocol The entire coding region of phoP gene from TT01 strain was amplified by PCR and digested 130 by NdeI and BamHI (Table S2). The ligation of the PCR product obtained was performed into 131 the same site of the expression vector, pETPhos (Pfaffl, Horgan, et Dempfle 2002) inserting a 132 His-tag in N-term part of proteins thereby generating PT7PhoP-His. The recombinant plasmid 133 encoding a PhoP-His fusion protein was transformed into E. coli BL21 (DE3) pLysS cells. At 134 an OD between 0.5-0.8, the expression of PhoP-His was induced by adding isopropyl-beta-D- 135 thiogalactoside at 0.5 mM, then an overnight induction was performed at 18°C. Bacterial 136 culture was centrifuged at 7,000 x g for 15 min at 4°C, washed twice in resuspension buffer 137 (Tris 5 mM pH 7.5, NaCl 300 mM, Glycerol 10 %, Imidazole 10 mM) and pellet was frozen 138 at -80°C for 30 min. Pellet was then suspended in 5 ml resuspension buffer and lysed by 139 sonication during 10 min at 4°C. Lysis products were centrifuged at 10,000 x g during 30 min 140 at 4°C. 500 µ L of pre-equilibrated beads of Ni-NTA agarose (Qiagen) in the wash buffer (Tris 141 5 mM pH 7.5, NaCl 300 mM, glycerol 10 %, Imidazole 15 mM) were added to the 142 supernatant fraction and incubated during 45 min with shaking at 4°C. The fraction was 143 centrifuged at 500 x g during 2 min at 4°C and wash 5 times with wash buffer. Protein was 144 eluted twice in 1 mL elution buffer (Tris 5 mM pH 7.5, NaCl 300 mM, glycerol 10%, 145 Imidazole 200 mM). Concentration of recombinant protein was assessed by Bradford assay 146 and controlled by SDS-page gel. Recombinant proteins were conserved at -80°C until use. 147 148 149 Electrophoretic mobility-shift assays (EMSA) 150 151 The promoter of ail1Pl was amplified by PCR from the genomic DNA of TT01 strain using 152 primers (Table S2) and purified using the High Pure PCR Product Purification kit (ROCHE).