cell envelope phospholipid content of Brucella 1 melitensis , contributing to polymyxin resistance and pathogenicity 2 3 Running title : B . melitensis Phospholipase A 1 alters cell envelope phospholipids

16 A subset of bacterial pathogens, including the zoonotic Brucella species, are highly 17 resistant against polymyxin antibiotics. Bacterial polymyxin resistance has been attributed 18 primarily to modification of lipopolysaccharide, however it is unknown what additional 19 mechanisms mediate high-level resistance against this class of drugs. This work identified a role 20 for the B. melitensis gene bveA (BMEII0681), encoding a predicted esterase, in the resistance 21 of B. melitensis to polymyxin B. Characterization of the enzymatic activity of BveA demonstrated 22 that it is a Phospholipase A1 with specificity for phosphatidylethanolamine (PE). Further, 23 lipidomic analysis of B. melitensis revealed an excess of PE lipids in bacterial membranes 24 isolated from the bveA mutant. These results suggest that by lowering the PE content of the cell 25 envelope, BveA increases resistance of B. melitensis to polymyxin B. BveA was required for 26 survival and replication of B. melitensis in macrophages, as well as for persistent infection in 27 mice. BveA-family esterases are encoded in the genomes of alphaproteobacterial species that 28 co-exist with polymyxin-producing bacteria in the rhizosphere, suggesting that maintaining a low 29 PE content of the bacterial cell envelope may be a shared persistence strategy for association 30 with plant and mammalian hosts. 31 32 INTRODUCTION 33 The polymyxin class of antibiotics, produced by soil bacteria such as Paenibacillus 34 polymyxa (formerly Bacillus polymyxa) was abandoned for therapy in the 1970’s in favor of 35 newer drugs. However, with emerging multi-drug resistance bacteria such as Acinetobacter 36 baumanii and Klebsiella pneumoniae (1), rehabilitation of these drugs, most notably Polymyxin 37 B (PmB) and Colistin (Polymyxin E) has been proposed as a last line treatment for infections 38 with antibiotic-resistant Gram-negative bacteria especially in critical care settings (2-4). 39 40 on M ay 9, 2021 by gest httpaac.asm .rg/ D ow nladed fom Polymyxins act at the cell envelope: their initial association with outer membrane is 41 dependent on displacing divalent cations (Mg and Ca) from lipopolysaccharide (LPS). 42 Subsequent association with the cytoplasmic membrane results in insertion of PmB, leading to 43 formation of pore-like structures and membrane permeabilization. The ensuing disruption of the 44 cytoplasmic membrane leads to inhibition of bacterial respiration via loss of proton-motive force 45 and, consequently, to growth inhibition (5). 46 Several bacterial species are inherently resistant to polymyxins, including Burkholderia, 47 Proteus, Neisseria, and Brucella spp., and this characteristic is actually utilized for primary 48 isolation of Brucella spp. from clinical samples (6, 7). Understanding the basis for this 49 resistance is important, as this knowledge may inform the design of novel therapies against 50 drug-resistant organisms. Here, we identify a new mechanism for increased resistance to this 51 class of drugs in the bacterial pathogen Brucella melitensis and show that this represents part of 52 its adaptation to causing infection in mammalian hosts. 53 54 MATERIALS AND METHODS 55 56 Bacterial growth conditions: E. coli was grown in LB and B. melitensis in tryptic soy broth. For 57 mouse infections B. melitensis was cultured on tryptic soy agar plus 5% blood for 3 days (8). S. 58 Typhimurium strain IR715 was cultured under low magnesium conditions (10 μM MgCl2) in 59 chemically defined M9 minimal medium supplemented with 1% glucose following Groisman et 60 al. (9). When needed, nalidixic acid at 25 μg/ml, ampicillin at 250 μg/ml, kanamycin at 100 61 μg/ml, or gentamicin at 50 μg/ml was added to media. Work with B. melitensis wild type and 62 mutant strains was performed at biosafety level 3 and was approved by the Institutional 63 Biosafety Committee at the University of California, Davis. For expression of proteins an 64 overnight culture was grown to an optical density of 0.4 in LB with glucose and the expression 65 on M ay 9, 2021 by gest httpaac.asm .rg/ D ow nladed fom was induced with 0.5 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 6 h at 37oC with 66 continuous shaking. 67 68 DNA manipulation, construction of mutants and complementation 69 Strains and plasmids used in this study are listed in in Table 1. The bveA gene (BMEII0681) 70 was identified in a bioinformatic screen for cell envelope and Type IV secretion system-related 71 functions (published in part in(10)). It originally came to our attention, because of its similarity to 72 two proteins in Agrobacterium tumefaciens, AcvB and VirJ, which are required for transfer of the 73 Ti plasmid (11). However, closer analysis showed it to have a lipase domain, therefore, we 74 hypothesized a role for this protein related to membrane biogenesis. The bveA mutant was 75 generated by allelic exchange. Regions up and downstream of BMEII0681 (bveA) were 76 amplified using the primer sets H1-For (5’-CTGCAGATAGCTGCGCTCCTGA -3’), H1-Rv (5’77 TCTAGAGCTGCGCACTGTCTTCTATGG-3’), H2-Fw (5’-GTCGACCTTCCTGATCAGTGC-3’) 78 and H2-Rv (5’-CTGCAGGCAAATCACATGCCGT-3’), and cloning of the amplicons into pCR2.179 TOPO (Invitrogen). The Tn903 kanamycin resistance cassette (KSAC from pUC4-KSAC) was 80 introduced between the upand downstream fragments to generate plasmid pCR105 (Table 1). 81 This generated a plasmid that was transferred to B. melitensis 16M wild type by electroporation 82 and allelic exchange mutants of bveA were screened for resistance to kanamycin and loss of 83 the plasmid-encoded ampicillin resistance. The correct position of the insertion was verified by 84 PCR and Southern blotting of a HindIII digested chromosomal preparation of bveA using a 85 bveA-specific probe (data not shown). The correct strain was designated CMR27 (Table 1). 86 To complement the bveA mutant (CMR27), plasmid pCR108 was constructed as follows: 87 primers CR-forward (5’-TCAGCGCGCAGGGCGCGGCGG-3’) and CR-reverse (5’88 CGTATTCTTTATCGTCCTGGGGTTGCG-3’) were used to amplify bveA together with its 89 promoter from the B. melitensis 16M genome. This PCR product was introduced into pCR2.1 by 90 TA-cloning (Invitrogen) and was sub-cloned into the pBBR1MCS4 vector (12) using EcoRI to 91 on M ay 9, 2021 by gest httpaac.asm .rg/ D ow nladed fom obtain the plasmid pCR108. Plasmid pCR108 was introduced into CMR27 to yield strain 92 CMR28. However the expression of bveA in CMR28 was unstable during exposure to 93 polymyxin B. We therefore utilized a previously described strategy for stable, single-copy 94 chromosomal gene expression from the promoter of the secE gene encoding the preprotein 95 translocase (13). To this end, we constructed a suicide plasmid (pTK19) by replacing the 96 mCherry gene of pKSoriT-bla-PsecE-mCherry (kindly provided by X. DeBolle)(13) with an 97 amplicon containing the bveA coding sequence (secE-bveA-Fr 5’98 CCTGATCAGACAGAGTATGAAGAAAGAACGCGTATTCTTTATCGTCCTGGGGTTGG-3’ and 99 secE-bveA-Rv 5’-CCCTGCAGGTCGAGGTCAGCGCGCAGGGCGCGG-3’) from B. melitensis 100 16 via Gibson Assembly cloning (NEB). All cloning steps were verified by sequencing and 101 following electroporation the subsequent insertion of the fusion gene was confirmed by PCR. 102 For overexpression and purification of His-tagged BveA in E. coli we amplified the gene using 103 the primer combination His-681-SalI-For (5’104 ATAGTCGACTCAGTGGTGGTGGTGGTGGTGGCGCGCAGGGCGCGGCGGC -3’) and 0681105 BamHI-Rev (5’ ATAAGGATCCGAAGAAAGAACGCGTATTCTTTATCG -3’). This amplified the 106 bveA gene with an additional in frame 6xHis-tag and two restriction sites (SalI and BamHI) that 107 were used to clone the fragment into the pET25b(+) vector (EMD Millipore) generating the 108 plasmid pTK07. This step fused the BveA-His6 construct to the PelB leader that enables 109 periplasmic localization of the final fusion protein. The construct was verified by sequencing. To 110 express MBP-BveA, we amplified bveA with the primers pMAL-Fr and pMAL-Rv (5’111 GGGATCGAGGGAAGGAAGAAAGAACGCGTATTCTTTATCGTCCTGGGGTTGGC-3’ and 5,112 catggacatatgtgaaatTCAGCGCGCAGGGCGCGG-3’) and cloned the resulting amplicon into 113 pMALp5x (NEB) using the Gibson cloning method, to yield plasmid pTK25. Subsequent 114 sequencing verified the correct sequence of the fusion construct. 115 116 Influence of Polymyxin B on bacterial growth 117 on M ay 9, 2021 by gest httpaac.asm .rg/ D ow nladed fom The bactericidal effect of polymyxin B (Difco) against B. melitensis strains and S. Typhimurium 118 IR715 was tested by incubating 1x10 colony-forming units (CFU) of each strain for 48 h at 37°C 119 with 0 (control), 5, 10, 15, 50 and 150 μg/ml of PmB in TSB (pH 7.4), and subsequent 120 enumeration of CFU on TSA. 121 122 Sensitivity of B. melitensis to antimicrobials 123 The killing ability of several antimicrobial substances, peptides and proteins against B. 124 melitensis was investigated as described by Martinez de Tejada et al. (14) with the exception of 125 using 25 μg of polymyxin B sulfate (Difco), magainin 2 and cecropin P (Sigma-Aldrich). 126 127 Protein expression, periplasmic preparation and purification of BveA 128 C-terminal 6xHis-tagged PelB-BveA protein was expressed in E. coli OrigamiTM 2 (DE3) pLysS 129 (EMD Millipore) and a periplasmic fraction was obtained following the recommendations of 130 Novagen’s pET System Manual 11 edition. Periplasmic fractions were concentrated using spin 131 concentrator units with a cut off of 3 kDa (Millipore). The presence of the BveA fusion protein 132 was verified by Western blot detection using an anti-His antibody (BioRad). An MBP-BveA 133 fusion protein was purified from the periplasm of E. coli OrigamiTM B (DE3) pLysS (EMD 134 Millipore) using amylose resin (NEB). The eluted protein was concentrated using a 50 kDa 135 concentrator unit (Thermo Fisher Scientific) and the purity of the MBP-BveA protein sample was 136 confirmed by SDS-PAGE. 137 138 Esterase activity As