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    The Class A -Lactamase, FTU-1, Endemic in Francisella tularensis. 1

    FTU-1 -lactamase 2

    Nuno T. Antunes, Hilary Frase, Marta Toth and Sergei B. Vakulenko* 3

    Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, IN 46556 4

    5

    *Corresponding Author Mailing Address: Department of Chemistry and Biochemistry, University of Notre Dame, 6

    417 Nieuwland Science Hall, Notre Dame, IN, 46556; Phone: (574) 631-2935; Fax: (574) 631-6652; E-mail: 7

    svakulen@nd.edu 8

    9

    Copyright 2011, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.Antimicrob. Agents Chemother. doi:10.1128/AAC.05305-11 AAC Accepts, published online ahead of print on 14 November 2011

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    ABSTRACT 10

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    The class A -lactamase FTU-1 produces resistance to penicillins and ceftazidime but not to any other -lactam 12

    antibiotics tested. FTU-1 hydrolyzes penicillin antibiotics with catalytic efficiencies of 105 106 M-1 s-1, 13

    cephalosporins and carbapenems with catalytic efficiencies of 102 103 M-1 s-1, but the monobactam aztreonam and 14

    the cephamycin cefoxitin are not substrates for the enzyme. FTU-1 shares 21-34% amino acid sequence identity 15

    with other class A -lactamases and harbors two cysteine residues conserved in all class A carbapenemases. FTU-1 16

    is the first weak class A carbapenemase that is endemic in Francisella tularensis. 17

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    INTRODUCTION 19

    Since their discovery more than a half-century ago, -lactams continue to be the major class of antibiotics used 20

    in the treatment of infectious diseases (31). Their intensive use has resulted in the selection of microorganisms that 21

    produce -lactamases, enzymes which hydrolyze -lactam antibiotics, thus significantly narrowing our therapeutic 22

    options (14). Based on their amino acid sequence identity and mechanism of catalysis, -lactamases are subdivided 23

    into four major classes; A, B, C, and D. -Lactamases from class A, C and D employ an active-site serine while 24

    class B enzymes utilize metal, usually Zn2+, for catalysis (4, 14). 25

    Shortly after the introduction of -lactams into clinical use, class A -lactamases were identified which were so-26

    called non-extended-spectrum enzymes, only capable of hydrolyzing penicillins and early-generation cephalosporins 27

    (35). In response to the subsequent introduction of third-generation cephalosporins and -lactamase inhibitors, 28

    extended spectrum (ESBL) and inhibitor resistant (IRT) enzymes have been selected (5, 22, 35). These enzymes 29

    evolved from their non-extended-spectrum progenitors as a result of only a few mutations, some with just one, 30

    widening their substrate specificity (27). Currently ESBLs are widely distributed among Gram-negative clinical 31

    isolates and are capable of inactivating the majority of available -lactam antibiotics, with the notable exception of 32

    carbapenems (25, 35). 33

    While use of carbapenems in the treatment of serious infections caused by ESBLs and IRTs failed to select for 34

    carbapenem-resistant variants of these enzymes, novel class A -lactamases of unknown origin and capable of 35

    hydrolyzing carbapenem antibiotics, have been selected (35, 42). These enzymes have relatively low amino acid 36

    identity with classical class A -lactamases and harbor two characteristic cysteine residues at Ambler positions 69, 37

    directly preceding the active site serine, and 238 which follows two resides after the conserved KTG motif (42). 38

    These cysteine residues form a disulfide bridge which serve to connect the two domains of enzyme. Currently, 39

    several subfamilies of class A enzymes containing these characteristic cysteine residues have been described. They 40

    include the IMI/NMC-A, GES-, KPC-, BEL- and SME-type enzymes (36, 37, 41), as well as SFC-1 (26) and BIC-1 41

    (21). Though structurally all these enzymes belong to the class A carbapenemase family, some of them, such as 42

    GES-1 and BEL-1, do not elevate the MICs of carbapenem antibiotics and are devoid of or exhibit very low 43

    carbapenemase activity (36, 38). Here we report a class A -lactamase from Francisella tularensis subsp. holarctica 44

    LVS, which we call FTU-1, that contains the conserved cysteine residues characteristic of the class A 45

    carbapenemase family. Unlike all previously reported class A carbapenemases which are found only sporadically in 46

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    various clinical and environmental microorganisms, FTU-1 is an endemic enzyme encoded by a chromosomally-47

    located gene in all Francisella tularensis strains. 48

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    MATERIALS AND METHODS 50

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    Strains and Plasmids. The open reading frame encoding a putative class A -lactamase from F. tularensis 52

    subsp. holarctica LVS was used to construct a synthetic gene for the FTU-1 enzyme. The nucleotide sequence of the 53

    synthetic gene was optimized for expression in Escherichia coli and the predicted leader sequence replaced by that 54

    for the outer membrane protein OmpA (12). MIC determinations were performed using E. coli JM83 carrying the 55

    FTU-1 gene cloned between the unique NdeI and HindIII sites of pHF016 (16). The enzyme was expressed and 56

    purified from E. coli BL21(DE3) harboring the FTU-1 gene cloned between the unique NdeI and HindIII sites of 57

    pET24a(+) (Invitrogen). 58

    Antimicrobial Susceptibility Testing. The susceptibility to several -lactam antimicrobials and their 59

    combinations with -lactamases inhibitors was determined by the broth microdilution method as described by the 60

    Clinical and Laboratory Standards Institute guidelines (8). The MICs were determined in triplicate using Mueller-61

    Hinton II broth (Difco) and a bacterial inoculum of 5 105 CFU/ml. The plates were incubated at 37C for a period 62

    of 16-20 h before the results were interpreted. 63

    Enzyme Purification. E. coli BL23(DE3) carrying the gene for FTU-1 cloned into pET24a(+) was grown at 64

    37C with shaking (180 rpm) in LB broth supplemented with 0.5 M sorbitol, 25 mM betaine and 60 g/ml 65

    kanamycin. When the bacterial culture reached an optical density of 0.8 at 600 nm, isopropyl--D-66

    thiogalactopyranoside was added to a final concentration of 0.4 mM and the bacteria further grown for 20 h at 30C 67

    with shaking. The culture was pelleted by centrifugation at 20000 g and 4C for 30 min. The broth was recovered 68

    and concentrated by centrifugal filtration using a Centricon Plus 70 concentrator (Millipore) with a molecular mass 69

    cut off of 10 kDa. After overnight dialysis into 20 mM MES (pH 6.0), the sample was loaded onto a DEAE anion-70

    exchange column (Bio-Rad) equilibrated with the same buffer. At this pH, FTU-1 does not bind to the resin and can 71

    be recovered in the flow-through. Two additional column volumes of 20 mM MES (pH 6.0) were added and the 72

    flow-through collected in 10 ml fractions. The fractions containing FTU-1 were identified by their activity against 73

    the chromogenic -lactam nitrocefin and combined. After concentration, the enzyme was dialyzed against 20 mM 74

    Tris (pH 8.0) and loaded onto a MacroPrep S cation-exchange column (Bio-Rad), equilibrated in the same buffer. 75

    The column was washed with three column volumes of 20 mM Tris (pH 8.0) and FTU-1 eluted using a linear 76

    gradient of NaCl (0 to 500 mM). The fractions containing the enzyme were identified by their activity towards 77

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    nitrocefin and their purity assessed by SDS-PAGE prior to dialysis into 20 mM HEPES (pH 7.5) for storage. The 78

    protein concentration was determined using the predicted extinction coefficient (280 = 30495 cm-1 M-1) (20). 79

    Determination of Steady-State Kinetic Parameters. All spectrophotometric data were collected on a Cary 50 80

    spectrophotometer (Varian). The reactions were performed at room temperature in 50 mM sodium phosphate (pH 81

    7.0). The following extinction coefficients and wavelengths were used: ampicillin (235 = -670 cm-1 M-1), 82

    benzylpenicillin (232 = -1096 cm-1 M-1), oxacillin (260 = +440 cm-1 M-1), cefotaxime (265 = -6643 cm-1 M-1), 83

    cefoxitin (265 = -7507 cm-1 M-1), ceftazidime (260 = -10500 cm-1 M-1), cefuroxime (262 = -7800 cm-1 M-1), 84

    cephalothin (262 = -8610 cm-1 M-1), aztreonam (318 = -640 cm-1 M-1), imipenem (297 = -10930 cm-1 M-1), 85

    meropenem (298 = -7200 cm-1 M-1), doripenem (299 = -11460 cm-1 M-1) and ertapenem (295 = -9970 cm-1 M-1). 86

    The steady-state velocities were determined from the linear phase of the reaction time courses and plotted as a 87

    function of -lactam concentration to allow determination of both kcat and Km by nonlinear regression using the 88

    Michaelis-Menten equation and Prism 5 (GraphPad Software, Inc). The half-life of a -lactam was evaluated as the 89

    ln 2/kcat. When Km was high and saturation could not be reached, the ratio kcat/Km