The Role of AcrA in Antibiotic Resistance and Virulence of - eTheses

279 Pages·2010·5.6 MB·English

by  Jessica Blair

The Role of AcrA in Antibiotic Resistance and Virulence of
Salmonella enterica serovar Typhimurium

By

Jessica Mary Alice Blair

A thesis submitted to the University of Birmingham for the
degree of DOCTOR OF PHILOSOPHY

Antimicrobial Agents Research Group
School of Immunity and Infection
College of Medical and Dental Sciences
University Of Birmingham

January 2010
University of Birmingham Research Archive

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Abstract
AcrA is the periplasmic adaptor protein component of the major efflux system AcrB-TolC of
Salmonella Typhimurium. AcrA of S. Typhimurium SL1344 was inactivated and the mutant
phenotype characterised. RT-PCR and western blotting were used to confirm expression of
acrB/AcrB was retained in the acrA mutant. The AcrA mutant was hyper-susceptible to a range of
antimicrobials and was more susceptible to some agents than strains lacking AcrB and TolC. This is
partially explained by the increase in accumulation of Hoechst H33342, a fluorescent substrate of
AcrAB-TolC, indicating that the inactivation of acrA resulted in reduced efflux activity. Lack of AcrA
also attenuated the ability of S. Typhimurium to infect INT-407 and RAW 264.7 cells in vitro as
previously published for AcrB and TolC mutants. The virulence defect of the mutants could not be
rescued by addition of supernatant from an infection of INT-407 cells with SL1344 or addition of
media conditioned by uninfected INT-407 cells. However, addition of media conditioned by
overnight growth of SL1344 was able to ameliorate the virulence defect of the mutants. This
suggests that AcrAB-TolC of SL1344 exports a factor/s required for virulence which the mutants are
unable to export and that exogenous addition of this factor can restore the virulent phenotype.
Inactivation of acrA conferred a phenotype distinct to that of inactivation of acrB or tolC indicating a
role for AcrA distinct to that of other protein partners in both efflux of substrates and virulence.
For Jonathan
And My Parents
Acknowledgements
I am extremely grateful to my supervisor Professor Laura Piddock for all the help, advice and support
over the last three years. I would also like to thank all the past and present members of the ARG for
their support during the last three years and for making my PhD experience so enjoyable. Particular
thanks go to Mark Webber for knowing the answers to all my stupid questions but not laughing at
me (too much) when I asked them; to Dr Vito Ricci, Dr Andrew Bailey, Dr Mark Garvey and Dr Jon
Caddick for their excellent post-doctoral support and for always making me laugh; and to Leanne
and Jen for always being ready with a cup of tea and an emergency Twix in a crisis.
I would also like to thank Roberto La Ragione and Martin Woodward from the Veterinary
Laboratories Agency for hosting me at the VLA so that I could learn the tissue culture techniques and
for all their help and advice. Also Elena Tikhonova and Helen Zgurskaya for providing the antibodies
for the Western Blotting and for their technical expertise.
Finally, I would like to thank my parents and all my family for their constant support and for always
believing in me; my friends (old and new) for helping me through and taking my mind off it all and
most of all JJ, for……….everything.
Clarification of Contribution to Collaborative work
The following sections or figures were the result of collaborations. Details of the collaboration and
the contribution by both parties is clarified below.
Figure 3.11 Transmission Electron Microscopy to examine bacterial structure
Jessica Blair was responsible for setting up and growing the bacterial cultures at the Veterinary
Laboratories Agency, Weybridge. The samples were then fixed and electron microscopy images
taken by Bill Cooley at the Veterinary Laboratories Agency, Weybridge.

2.11.1 Constructing Green Florescent Protein (GFP) marked strains
The gfp encoding pUA66pacpP plasmid was a gift from Neil Burton at the University of Birmingham.
Jessica Blair was responsible only for transformation of this plasmid into the strains of interest.

Figure 4.4 Confocal microscopy images of INT-407 cells infected with wild-type and efflux mutant
strains containing the GFP encoding pUA66pacpP plasmid.
Jessica Blair was responsible for the culture of eukaryotic cells, preparing eukaryotic cell infection
plates, growing bacterial cultures, infecting the eukaryotic cells and fixing the infections with 1%
Formalin. Dr. Rob Shaw (University of Birmingham)stained the eukaryotic cells and took confocal
microscopy images.
Contents
Section Page
number
1. Introduction 2
1.1 Salmonella 2
1.2 Salmonella Infection 2
1.3. The Salmonella Cell Wall 6
1.3.1 The Inner Membrane 6
1.3.2 The Periplasm 7
1.3.3 The Outer Membrane 7
1.3.4 Protein Secretion Systems 10
1.4 Pathogenesis and Virulence of Salmonella 10
1.4.1 Colonisation and adhesion 10
1.4.2 Invasion 13
1.4.3 Intra-cellular phase of infection 16
1.4.4 Dissemination of infection 19
1.4.5 Model Systems to study Salmonella infection 20
1.5 Treatment of Salmonella Infections 24
1.6 Antibiotic Resistance 25
1.6.1 Mechanisms of Antibiotic Resistance 27
1.6.2 Efflux as a Mechanism of Resistance 29
1.7 The RND Superfamily of Efflux Pumps 31
1.8 The AcrAB-TolC Efflux Pump 32
1.8.1 TolC 36
1.8.2 AcrB 38
1.8.3 AcrA 40
1.8.4 A fourth component: YajC 44
1.9 Interactions between components of AcrAB-TolC 44
1.10 Mechanism of transport by AcrAB-TolC 46
1.10.1 The role of AcrA in transport by AcrAB-TolC 49
1.11 Genetic organisation of AcrAB-TolC 52
1.12 Control of AcrAB-TolC expression 53
1.13 Promiscuity of AcrAB-TolC components 59
1.14 The Physiological Role of AcrAB-TolC 61
Hypotheses 66
Aims and Objectives 67

2. Materials and Methods 68
2.1 Bacterial strains, growth, storage and identification 69
2.2 Disruption of acrA in S. Typhimurium 73
2.2.1 Isolation of the pKD4 plasmid 74
2.2.2 Generation of the PCR product to inactivate acrA 74
2.2.3 Transformation of the recipient Salmonella strain, L642, with the PCR 81
construct
2.2.4 Verification of gene disruption in candidate colonies by PCR 82
2.2.5 Sequencing of candidate acrA mutants 83
2.2.6 Verification that transformants were S. enterica 83
2.3 P22 transduction of the disrupted region 84
2.3.1 Production of the P22 phage 84
2.3.2 P22 Transduction 84
2.4 Removal of the kanamycin resistance gene, aph, using the pCP20 plasmid 85
2.4.1 Treatment of L884 with pCP20 85
2.4.2 Curing the plasmid 86
2.4.3 Confirmation of removal of the aph gene 86
2.5 Complementation of acrA 86
2.5.1 Amplification and restriction digest of the wild type acrA insert 87
2.5.2 Isolation and digestion of pWKS30 plasmid 87
2.5.3 Ligation of acrA insert and pWKS30 plasmid 88
2.5.4 Transformation of DH5α cells with the ligated product 88
2.5.5 Transformation of L884 (acrA::aph) with pWKS30acrA 89
2.6. Measuring transcription and translation of acrA/AcrA and acrB/AcrB 91
2.6.1 Determination of acrA and acrB transcription 91
2.6.1.1 RNA extraction 91
2.6.1.2 cDNA synthesis 92
2.6.1.3 RT-PCR 92
2.6.1.4 PCR product quantification using Denaturing High Pressure Liquid 92
Chromotography (DHPLC)
2.6.2 Western Blotting 93
2.6.2.1 Isolation of membrane proteins 93
2.6.2.2 Western blotting 94
2.7 Phenotypic characterisation of S. Typhimurium acrA::aph 95
2.7.1 Bacterial growth kinetics 95
2.7.2 Determination of the Minimum Inhibitory Concentration (MIC) of 96
antibiotics, dyes and detergents
2.7.3 Accumulation of Hoescht 33342 (Bis-benzamide) 96
2.7.4 Electron microscopy of SL1344 and strains lacking AcrA, AcrB and TolC 97
2.8 Investigating the role of AcrAB-TolC in Adhesion and Invasion 97
2.8.1 Tissue culture 98
2.8.2 Preparation of bacterial strains for infection assays 98
2.8.3 The Association Assay 99
2.8.4 The Invasion Assay 99
2.8.5 Statistical Analyses 100
2.8.5.1 Mean and standard deviation 100
2.8.5.2 Determining the level of Adhesion 100
2.8.5.3 Student’s t-Test 100
2.8.5.4 Calculating the confidence interval for the association level of SL1344 101
2.8.5.5 Multiplicity of Exposure 101
2.9 Competition Assays 102
2.10 Does AcrAB-TolC export a molecule required for invasiveness? 102
2.10.1 The effect of addition of culture supernatant, INT-407 conditioned media 103
or SL1344 conditioned media on the invasive abilities of mutants
2.10.1.1 Generation of culture supernatant from SL1344 infected INT-407 cell 103
monolayers
2.10.1.2 Generation of INT-407 conditioned media 103
2.10.1.3 Generation of SL1344 and L109 (tolC::aph) conditioned media 104
2.10.1.4 Generation of boiled and proteinase K treated bacterially conditioned 104
medium
2.11 Imaging of infected INT-407 cells 105
2.11.1 Construction of GFP marked strains 105
2.11.2 Preparing infection plates, staining and imaging 106
2.11.3 Test to see whether the strains aggregate in broth culture 107
3. Construction and characterisation of mutants lacking AcrA 109
3.1 Background 109
3.2 Aims and Hypotheses 109
3.3 Disruption of acrA 109
3.3.1 Verification of Gene Disruption 110
3.3.2 P22 transduction of the disrupted region 112
3.3.3 Removal of the aph cassette using the pCP20 plasmid 114
3.4 Complementation of acrA in the acrA::aph mutants 114
3.4.1 Construction of the complementation plasmid 116
3.4.2 Transformation of strains with pWKS30acrA 116
3.5 Expression of acrB/AcrB in mutants lacking AcrA 120
3.5.1 Reverse transcriptase (RT) PCR to measure acrA and acrB transcription 120
3.5.2 Western Blotting to detect the presence of AcrA, AcrB and TolC proteins 121
3.6 Phenotypic Characterisation of S. Typhimurium mutants lacking AcrA 124
3.6.1 Bacterial growth kinetics in LB broth 124
3.6.2 Hoechst 33342 (Bis-benzamide) accumulation 124
3.6.3 Antimicrobial Susceptibility 127
3.6.3.1 Antimicrobial Susceptibility of the acrA mutant, the transduced acrA 127
mutant strain (L884) and the acrA mutant with aph removed (L976)
3.6.3.2 Antimicrobial Susceptibility of the strains containing the pWKS30 vector 129
alone (L917, L918 and L996) and strains containing pWKS30 acrA (L980, L975 and
L979)
3.6.4 Transmission electron microscopy 131
3.7 Discussion 131
3.8 Further Work 138
3.9 Key Findings 140

4. The role of AcrAB-TolC in infection 142
4.1 Background 143
4.2 Aims and Hypothesis 143
4.3 The Effect of Inactivation of acrA on the ability of S. Typhimurium to cause 144
infection
4.4 Confidence Interval for the Level of SL1344 Association 146
4.5 The effect of Triton X-100 on bacterial survival 148
4.6 Confocal microscopy of INT-407 cells infected with SL1344 or mutants lacking 149
AcrA, AcrB or TolC
4.7 Discussion 152
4.8Further work 158
4.9 Key findings 160

5. Does AcrAB-TolC export a molecule required for infection? 162
5.1 Background 162
5.2 Aims and Hypotheses 162
5.3 Can the virulence of the TolC mutant be restored by addition of supernatant 163
from SL1344 infected INT-407 monolayers?
5.4 Can the ability of acrA, acrB or tolC mutants to adhere to and invade INT-407 171
cells be restored by Co-infection with SL1344?
5.4.1 SL1344 and L884 (acrA::aph) 175
5.4.2 SL1344 and L110 (acrB::aph) 175
5.4.3 SL1344 and L109 (tolC::aph) 178
5.5 Is AcrAB-TolC involved in the efflux of a host derived factor which affects 179
adhesion or invasion?
5.6 Is AcrAB-TolC involved in the efflux of a bacterially derived product or products 180
which affect adhesion or invasion?
5.6.1 Can the invasiveness of the TolC mutant be restored by addition of media 180
conditioned by growth of SL1344?
5.6.2 Is the restorative effect of SL1344 conditioned media abolished by pre- 187
boiling the conditioned media?
5.6.3 Is the restorative effect of SL1344 conditioned media abolished by 188
treatment with Proteinase K?
5.6.4 The effect of the bacterially conditioned media on the number and viability 189
of INT-407 cells.
5.7 Discussion 190
5.8 Further Work 200
5.9 Key findings 202

6. Overall Discussion and Conclusions 203

Publications resulting from this study 209

Appendices 212

Appendix 1 Figure 1. The acrA gene with primer locations indicated 212
Appendix 1. Figure 2. The predicted sequence acrA region after insertion of the 213
aph gene by homologous recombination
Appendix 1. Figure 3. Alignment of the expected sequence for the disrupted acrA 214
region with the upstream sequencing data for L823 (acrA::aph)
Appendix 1. Figure 4. Alignment of the expected sequence for the disrupted acrA 215
region with the downstream sequencing data for L823 (acrA::aph)
Appendix 2. The ability of S. Typhimurium SL1344 and various efflux mutants 216
thereof to adhere to and invade mouse macrophages (RAW264.7)

References 217