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    -lactam resistance in methicillin-resistant 3

    Staphylococcus aureus USA300 is increased by 4

    inactivation of the ClpXP protease 5

    Kristoffer T. Bk1, Angelika Grndling2, Ren G. Mogensen1, Louise Thgersen1, 6

    Andreas Petersen3, Wilhelm Paulander1, and Dorte Frees1# 7

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    1) Faculty of Health and Medical sciences, Department of Veterinary Disease 10

    Biology, University of Copenhagen, Frederiksberg, Denmark, 2) Section of 11

    Microbiology and MRC Centre for Molecular Bacteriology and Infection, Imperial 12

    College London, London, United Kingdom, 3) Statens Serum Institut, 13

    Copenhagen, Denmark 14

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    Running title: ClpXP protease and -lactam resistance in MRSA 16

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    # Corresponding author. Mailing address: Department of Veterinary Disease Biology, 18

    University of Copenhagen, Stigbjlen 4, DK-1870 Frederiksberg C, Denmark. E-mail: 19

    df@sund.ku.dk; Tel: (+45) 3533 2719; Fax: (+45) 3533 2757. 20

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    AAC Accepts, published online ahead of print on 27 May 2014Antimicrob. Agents Chemother. doi:10.1128/AAC.02802-14Copyright 2014, American Society for Microbiology. All Rights Reserved.

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    Abstract 22

    Methicillin resistant Staphylococcus aureus (MRSA) have acquired the mecA 23

    gene encoding a peptidoglycan transpeptidase PBP2a that has a decreased affinity 24

    for -lactams. Fast spreading and highly virulent community-acquired (CA) MRSA 25

    strains have recently emerged as a frequent cause of infection in individuals without 26

    exposure to the healthcare system. In this study, we found that inactivation of the 27

    components of the ClpXP protease substantially increased the -lactam resistance 28

    level of a CA-MRSA USA300 strain, suggesting that ClpXP proteolytic activity 29

    controls one or more pathways modulating -lactam resistance. These pathways do 30

    not involve control of mecA expression as the cellular level of PBP2a was unaltered 31

    in the clp mutants. Analysis of the cell envelope properties of the clpX and clpP 32

    mutants revealed a number of distinct phenotypes that may contribute to the 33

    enhanced -lactam tolerance. Both mutants displayed significantly thicker cell walls, 34

    increased peptidoglycan cross-linking, and altered composition of monomeric 35

    muropeptides species. Moreover, changes in Sle1-mediated peptidoglycan 36

    hydrolysis and altered processing of the major autolysin Atl were observed in the clp 37

    mutants. In conclusion, the presented results point to an important role for the ClpXP 38

    protease in controlling cell wall metabolism and add novel insights into the molecular 39

    factors that determine strain-dependent -lactam resistance. 40

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    Introduction 41

    The rapid spread of methicillin-resistant Staphylococcus aureus (MRSA) has 42

    made treatment of staphylococcal infections increasingly difficult (1, 2). Community-43

    acquired (CA) MRSA strains of the USA300 type cause particular concern because 44

    of their frequent isolation and severity of infection (3). Methicillin and other -lactam 45

    antibiotics inhibit growth of S. aureus by covalently binding to the transpeptidase 46

    domain of penicillin binding proteins (PBPs), which cross-link the polypeptide chains 47

    of the cell wall component peptidoglycan. The resistance of MRSA strains is caused 48

    by acquisition of the mecA gene encoding the alternative transpeptidase PBP2a with 49

    very low affinity for almost all -lactam antibiotics (4-7). Recently, anti-MRSA -50

    lactams such as ceftaroline and ceftobiprole with stronger binding to PBP2a have 51

    been discovered. 52

    Clinical MRSA isolates exhibit highly variable resistance levels towards 53

    methicillin with minimal inhibitory concentrations (MICs) ranging from < 3 g/ml 54

    (being comparable to those of susceptible strains) to 1600 g/ml in highly resistant 55

    strains (8). The mechanisms underlying this variation remain poorly understood, but 56

    the lack of correlation between resistance level and the level of mecA expression, 57

    suggests that factors other than PBP2a modulate the strain-specific level of -lactam 58

    resistance (8-11). Indeed, genetic screens have identified a number of auxiliary 59

    factors in addition to PBP2a that are critical for the resistance to -lactam antibiotics 60

    (12, 13). Examples include cell division proteins, endogenous PBPs, and enzymes 61

    involved in the synthesis of teichoic acids and peptidoglycan precursors (5, 14-21). 62

    Intriguingly, the realization that -lactam resistance depends on auxiliary factors 63

    opens up new possibilities for the treatment of MRSA infections, as drugs that inhibit 64

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    the function of auxiliary factors are predicted and have been shown to work 65

    synergistically with -lactams to kill MRSA (22, 23). 66

    Intracellular proteolysis carried out by energy-dependent proteases is one of 67

    the most conserved biological processes. During the infection process, bacterial 68

    pathogens depend on energy-dependent proteases both for the general turnover of 69

    damaged and non-functional proteins and for the degradation of short-lived 70

    regulatory proteins (24). Accordingly, the highly conserved ClpXP protease is 71

    essential for the virulence of S. aureus in both systemic and abscess models of 72

    infection (25, 26), and has also been implicated in virulence of other pathogens such 73

    as Listeria monocytogenes, Salmonella, and Bacillus anthracis (24, 27). The ClpXP 74

    protease is composed of proteolytic and ATPase subunits. Fourteen ClpP subunits 75

    constitute a proteolytic chamber that is only accessible through a narrow pore, which 76

    prevents the entrance of native, folded proteins (reviewed in (24)). ClpX serves to 77

    specifically recognize, unfold, and translocate substrates into the ClpP proteolytic 78

    chamber. ClpX belongs to the family of closely related Clp ATPases, and can also 79

    function independently of ClpP as a molecular chaperone (28). Treatment of MRSA 80

    infections with daptomycin in vivo or vancomycin in vitro has been shown to select for 81

    mutants that carry loss-of-function mutations in clpX or clpP, suggesting that ClpX 82

    and ClpP are involved in the response of S. aureus to cell wall acting antibiotics (29-83

    31). This finding prompted us to investigate if inactivation of the components of the 84

    ClpXP protease modulates the susceptibility of S. aureus to antibiotics targeting the 85

    cell wall. We found that inactivation of clpX or clpP increased the level of resistance 86

    to -lactam antibiotics in a CA-MRSA USA300 strain and at the same time affected a 87

    number of cell envelope properties associated with resistance. 88

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    Materials and Methods 90

    Strains and culture conditions 91

    S. aureus strains (Table 1) were cultured in tryptic soy broth (TSB; Oxoid) at 37C 92

    with aeration. JE2 derived strains were obtained from the Network of Antimicrobial 93

    Resistance in Staphylococcus aureus (NARSA) program supported under NIAID/NIH 94

    contract # HHSN272200700055C. 95

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    Construction of S. aureus mutants 97

    USA300 JE2 and COL clpX mutants: The clpX gene was deleted in JE2 and COL 98

    by transduction with bacteriophage 11, using an ermB tagged 8325-4clpX mutant 99

    (KB1257) as donor strain for the transduction. KB1257 was constructed by inserting 100

    an ermB marker 8 kb from clpX between the convergently transcribed genes with 101

    locus tags SAOUHSC_1768 and SAOUHSC_1769. The ermB marker was inserted 102

    into the chromosome as follows: First, two PCR fragments were amplified from 8325-103

    4 using the primer pairs KB65F (5-CATTTCCATTTGCGGTACCTCC-3) / KB65R (5-104

    GCATTAACTTGGATCCTTAACTTATG-3) and KB66F (5-105

    CATAAGTTAAGGATCCAAGTTAATGC-3) / KB67R (5-106

    TTAGGTCTGCAGATCGGATCAAC-3), respectively. Primers KB65R and KB66F are 107

    overlapping and contain substitutions that introduce a BamHI restriction site. The 108

    PCR fragments were joined by the splicing-by-overlap-extension PCR method (32) 109

    using KB65F, introducing a KpnI site, and KB67R, introducing a PstI site, and the 110

    resulting fragment was cloned into the KpnI and PstI sites of the allelic exchange 111

    vector pBT2 (33), resulting in plasmid pKB1217. A BamHI-fragment containing ermB 112

    was excised from the plasmid pAM367 (W.L. Kelley, pers. comm.) and cloned into 113

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    the BamHI site of pKB1217. The resulting plasmid, pKB1221, was then transformed 114

    into S. aureus strain RN4220 and subsequently introduced into 8325-4. The resulting 115

    strain was grown in the presence of erythromycin at the non-permissive temperature 116

    for plasmid replication to select for double cross-over and insertion of the ermB 117

    marker into the chromos