MECHANISMS OF ANTIBIOTIC RESISTANCE AND PATHOGENESIS IN Acinetobacter baumannii ...

284 Pages·2012·6.77 MB·English

by  Indriati Hood

MECHANISMS OF ANTIBIOTIC RESISTANCE AND
PATHOGENESIS IN Acinetobacter baumannii
By
Marguerite Indriati Hood

Dissertation
Submitted to the Faculty of the
Graduate School of Vanderbilt University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
in
Microbiology and Immunology
August, 2012
Nashville, Tennessee
Approved: Date:
_Timothy Cover, Chair_______________________________ __5/16/2012_________
_Timothy Blackwell_________________________________ __5/16/2012_________
_Terence Dermody__________________________________ __5/16/2012_________
_Dean Ballard______________________________________ __5/16/2012_________
_Eric Skaar________________________________________ __5/16/2012_________
ACKNOWLEDGEMENTS

Throughout my graduate career I have been extremely fortunate to work
alongside outstanding colleagues in the laboratory, in the Department of Pathology,
Microbiology and Immunology, in the Medical Scientist Training Program and in the
Vanderbilt community as a whole.
I must begin by thanking my mentor, Eric Skaar, for his overwhelming support
and guidance throughout my graduate career. Eric, you have been at various times a
cheerleader, a mentor, an advocate and a friend. Thank you for giving me the opportunity
to pursue a project that was at many times uncertain, but always challenging and exciting.
Thank you also for providing a positive and stimulating work environment and for
always challenging me to reach my full potential. Finally, thank you for being a positive
example and showing me how much fun a career in science can be.
Thanks to all of the members of the Skaar lab with whom I have had the pleasure
of interacting over the last five years. Thanks to Amanda McCoy, Keith Adams and Jeff
Mason who each contributed directly to my thesis work. Thank you, Victor Torres for
helping me get oriented to the lab in the first few months of my rotation and for all of the
helpful and entertaining discussions we enjoyed when we shared “The Island”. Thanks to
former members Michelle Reniere, Devin Stauff and Ahmed Attia and current members,
Gleb Pishchany, Cathy Wakeman, Allison Farrand, Thomas Kehl-Fie, Kate Haley, Laura
Anzaldi, Neal Hammer, Jim Cassat, Kyle Becker, Lorenzo Olive and Brittany Mortenson
for your friendship, discussions and feedback. Thank you all for making the Skaar lab
such fun and stimulating place to work. Thank you, Thomas Kehl-Fie, for all of your help
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with infections and in getting started working with calprotectin. Thanks to Ahmed Attia
for helpful discussions. Thanks to Kyle Becker, Brittany Mortenson and Mike Nodo for
your willingness to carry on the Acinetobacter work after I leave.
I am thankful to my numerous collaborators both at Vanderbilt and at other
institutions. Thank you to Paul Dunman and his graduate student, Anna Jacobs and post
doctoral fellow, Christelle Roux, for their assistance with transcriptional analyses of A.
baumannii. I also thank Anna for helpful discussions regarding the generation of deletion
mutants in A. baumannii. Thank you to Lisa Zimmerman of the Ayers Institute for her
assistance with proteomic data analyses. Thanks also to Rosemary Verrall of the
Vanderbilt Clinical Microbiology Laboratory for providing assistance with interpretation
of MIC values for A. baumannii. I would like to thank Drs. Anthony Campagnari (State
Univerisity of New York at Buffalo), Robert Bonomo (Case Western Reserve University)
and Herbert Schweizer (Colorado State University) for providing A. baumannii clinical
strains and plasmids for generating knockouts. I also thank Luis Actis (Miami University)
for providing plasmids for complementation and GFP expression as well as for helpful
discussions. Thank you Carl Johnson for helpful advice and for allowing us to borrow
your dark box for the circadian rhythm project. Thanks to Sebastian Joyce an Charles
Spencer for providing the MyD88-/- mice.
I am also grateful for the support and guidance I have received from the members
of my thesis committee, Timothy Cover (chair), Timothy Blackwell, Terry Dermody and
Dean Ballard. Thanks in particular to Timothy Blackwell for helpful discussions and for
providing mice and reagents for numerous inflammation-related projects. Thanks to Tim
Cover for helpful advice and discussion regarding whole genome sequencing. Thanks to
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Terry also for all of the opportunities and support you have provided through the MSTP.
Thank you to the staff of the Department of Pathology, Microbiology and Immunology
and the former Department of Microbiology and Immunology for your administrative
support.
Thank you to my family for always supporting me. I could not have made it this
far without you. Thank you to all of my friends in Nashville, at Houghton and back home
in Nova Scotia. You have each helped me in getting to where I am today.
This work was supported in part by the National Institutes of Health [NIAID
AI091771 to E.P.S.]; a Howard Hughes Medical Institute International Student Research
Fellowship to M.I.H.; a Burroughs Wellcome Fund grant to E.P.S.; and the National
Institute of General Medical Studies [T32 GM07347 to Vanderbilt University Medical
Scientist Training Program].

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TABLE OF CONTENTS

PAGE
ACKNOWLEDGEMENTS ................................................................................................ ii
LIST OF TABLES ............................................................................................................. iii
LIST OF FIGURES ........................................................................................................... X
LIST OF ABBREVIATIONS .......................................................................................... XII
I. INTRODUCTION ......................................................................................................... 14
ANTIBIOTIC RESISTANCE MECHANISMS UTILIZED BY A. BAUMANNII ............................... 15
A. BAUMANNII PATHOGENESIS AND HOST RESPONSE........................................................ 17
NUTRITIONAL IMMUNITY: EXPLOITING ESSENTIALITY AND TOXICITY............................ 22
AT THE PATHOGEN-HOST INTERFACE .............................................................................. 22
II. ACINETOBACTER BAUMANNII INCREASES TOLERANCE TO ANTIBIOTICS
IN RESPONSE TO EXTRACELLULAR NACL ............................................................ 50
INTRODUCTION .............................................................................................................. 50
MATERIALS AND METHODS ........................................................................................... 51
Bacterial strains, media and antibiotics .................................................................... 51
SDS-PAGE analysis of supernatant proteins ............................................................ 52
Protein sample preparation for proteomic analysis ................................................... 52
LC-MS-MS Analysis and Protein Identification ...................................................... 53
Growth conditions for bacterial RNA isolation ........................................................ 55
GeneChip® Analyses ................................................................................................ 56
Quantitative RT-PCR confirmation of microarray results ........................................ 57
Growth curve and MIC analyses of antimicrobial resistance ................................... 58
RESULTS ........................................................................................................................ 59
A. baumannii secretes antibiotic resistance determinants and virulence factors in
response to NaCl. ...................................................................................................... 59
NaCl induces up-regulation of putative efflux transporters. ..................................... 63
NaCl induces tolerance to distinct classes of antibiotics. ......................................... 67
Inhibition of efflux reduces NaCl-induced resistance to amikacin and
levofloxacin............................................................................................................... 71
NaCl induces tolerance to colistin in multidrug resistant clinical isolates of A.
baumannii. ................................................................................................................ 73
DISCUSSION ................................................................................................................... 76
III. GENETIC DETERMINANTS OF INTRINSIC COLISTIN TOLERANCE IN
ACINETOBACTER BAUMANNII ..................................................................................... 83
INTRODUCTION .............................................................................................................. 83
METHODS ...................................................................................................................... 85
Bacterial strains and reagents.................................................................................... 85
Transposon library screen and mutant identification ................................................ 85
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Metabolic pathway analysis ...................................................................................... 86
LPS analysis .............................................................................................................. 86
Complementation of the LPS synthesis defect in 5A7 ............................................. 87
Site-directed insertional mutagenesis of lpsB ........................................................... 87
Microarray analysis. .................................................................................................. 88
Preparation of bacterial cultures for in vivo studies .................................................. 89
A. baumannii pneumonia model ............................................................................... 89
RESULTS ........................................................................................................................ 90
Identification of genes involved in NaCl-induced colistin tolerance........................ 90
LpsB contributes to colistin resistance. .................................................................... 94
Adaptation to chronic membrane instability ............................................................. 95
Involvement of LpsB in the pathogenesis of pneumonia.......................................... 96
DISCUSSION ................................................................................................................... 98
IV. ACINETOBACTER BAUMANNII TRANSPOSON MUTANTS REDIRECT
HOST INFLAMMATION TO PROMOTE PATHOGEN CLEARANCE. ................... 102
INTRODUCTION ............................................................................................................ 102
METHODS .................................................................................................................... 103
Bacterial strains ....................................................................................................... 103
Transposon library screen and mutant identification .............................................. 103
Preparation of bacterial cultures for in vivo studies ................................................ 103
A. baumannii pneumonia model ............................................................................. 104
Tissue preparation for histology ............................................................................. 105
RNA isolation and PCR array ................................................................................. 105
Flow cytometric analysis of neutrophils in infected lungs ..................................... 106
Neutrophil depletion experiments ........................................................................... 106
Microarray analysis. ................................................................................................ 107
RESULTS ...................................................................................................................... 109
A. baumannii Tn5A7 attenuates wildtype infection. .............................................. 109
Tn5A7 attenuates wildtype infection by inducing increased bacterial clearance
from the lung. .......................................................................................................... 112
Chemically inactivated Tn5A7 is effective in inhibiting WT A. baumannii infection.
................................................................................................................................. 113
Tn5A7 blunts the inflammatory response to A. baumannii. ................................... 115
Attenuation of WT bacteria by treatment with Tn5A7 does not depend on
neutrophils or macrophages. ................................................................................... 123
TLR4 is not required for the dominant negative effect of the transposon mutants. 125
The attenuating phenotype of Tn5A7 is not due to disruption of lpsB. .................. 126
Transposon mutagenesis induces the attenuating phenotype with a high
frequency................................................................................................................. 127
Attenuating mutants do not share any common secondary mutations. ................... 129
Attenuating mutants share a common pattern of gene dysregulation. .................... 130
The attenuating effect of the transposon mutants requires signaling through
MyD88. ................................................................................................................... 133
DISCUSSION ................................................................................................................. 135
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V. ZNUB CONTRIBUTES TO ZINC ACQUISITION, RESISTANCE TO
CALPROTECTIN AND PATHOGENESIS OF ACINETOBACTER BAUMANNII
INFECTIONS ................................................................................................................. 140
In vitro growth inhibition assays with calprotectin ................................................. 141
A. baumannii infections .......................................................................................... 142
Transposon mutant library screen ........................................................................... 143
Identification of a putative zur-binding consensus sequence and zur-regulated
genes ....................................................................................................................... 143
Construction of znuB ............................................................................................ 144
ICP-MS analyses of intracellular zinc concentrations ............................................ 145
Imipenem inhibition assays..................................................................................... 146
Identification of A. baumannii mutants with altered sensitivity to CP ................... 148
Identification of a Zn uptake system in A. baumannii ............................................ 151
Regulation of znuB in Zn-limiting conditions ........................................................ 154
ZnuB contributes to the pathogenesis of A. baumannii pulmonary infections. ...... 158
Zn chelation reverses carbapenem resistance in MDR A. baumannii. ................... 158
DISCUSSION ................................................................................................................. 160
VI. CIRCADIAN VARIATION IN SUSCEPTIBILITY TO ACINETOBACTER
BAUMANNII PULMONARY INFECTIONS. ............................................................... 169
INTRODUCTION ............................................................................................................ 169
METHODS .................................................................................................................... 170
A. baumannii infection model and housing conditions ........................................... 170
Infections with A. baumannii blsA ....................................................................... 170
RESULTS AND DISCUSSION ........................................................................................... 171
Mice are most susceptible to infection near the onset of their active period. ......... 171
Contribution of a bacterial light sensing protein to A. baumannii infection ........... 172
VII. SUMMARY AND SIGNIFICANCE ...................................................................... 174
RESTORING THE UTILITY OF CURRENTLY AVAILABLE ANTIBIOTICS BY TARGETING
ANTIBIOTIC RESISTANCE DETERMINANTS IN A. BAUMANNII. .......................................... 175
Targeting intrinsic and inducible resistance in A. baumannii ................................. 175
Targeting Zn acquisition to combat imipenem resistance ...................................... 176
ALTERNATIVE THERAPEUTIC STRATEGIES THAT ENHANCE HOST DEFENSES TO
PREVENT OR REDUCE THE SEVERITY OF A. BAUMANNII INFECTIONS. ............................. 176
Dominant negative mutants of A. baumannii ......................................................... 176
Manipulation of circadian rhythms to reduce susceptibility to A. baumannii
infection. ................................................................................................................. 178
ACINETOBACTER BAUMANNII TRANSPOSON MUTANTS AND IMPLICATIONS FOR
GENETIC MANIPULATION AND INVESTIGATION OF PATHOGENESIS. ............................... 180
VIII. FUTURE DIRECTIONS ....................................................................................... 181
REDIRECTION OF HOST INFLAMMATION TO PROMOTE BACTERIAL CLEARANCE ............ 181
1a. Develop an in vitro system that recapitulates the pattern of attenuation observed in vivo. ..... 182
1b. Identify bacterial products that are recognized by the host upon treatment with the
transposon mutants. ....................................................................................................................... 183
1c. Define the genetic element encoding the bacterial product that elicits the attenuating
immune response........................................................................................................................... 184
2. Elucidate the host factors necessary for recognizing the transposon mutants
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and mediating bacterial clearance. .......................................................................... 185
3. Define the mechanism through which transposon mutagenesis induces the
attenuating phenotype. ............................................................................................ 186
NUTRIENT METAL ACQUISITION AND METAL DEPENDENT PROCESSES IN A. BAUMANNII 190
1. Elucidate the mechanisms for Zn acquisition in A. baumannii. ......................... 190
1a. Define the inner membrane Zn transport machinery in A. baumannii. ................................... 190
1b. Elucidate the mechanism for translocation of Zn across the outer membrane. ....................... 191
2. Define the A. baumannii Zur regulon and the roles for Zur-regulated genes
in Zn uptake and homeostasis. ................................................................................ 192
2a. Elucidate the transcriptional profiles of a zur deletion mutant compared to WT bacteria
in Zn-replete and Zn-deplete conditions. ...................................................................................... 192
2b. Determine the function of Zur-regulated genes and define their roles in Zn homeostasis. ..... 192
3. Elucidate the A. baumannii metalloproteome and define changes to the
metalloproteome as a result of nutrient metal limitation. ....................................... 193
4. Determine the contribution of transition metal intoxication to A. baumannii
pathogenesis. ........................................................................................................... 194
REFERENCES…………………………………………………………………………196
APPENDIX 1. SUPPLEMENTARY TABLES ASSOCIATED WITH CHAPTER II. 232
APPENDIX II. SUPPLEMENTARY TABLES ASSOCIATED WITH CHAPTER
III..................................................................................................................................... 269
APPENDIX III. SUPPLEMENTARY TABLES ASSOCIATED WITH CHAPTER
IV .................................................................................................................................... 280

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LIST OF TABLES

Table 1. Selected bacterial iron uptake systems referenced in the text. ........................... 30
Table 2. Selected bacterial transporters of Mn and Zn. .................................................... 38
Table 3: Proteins identified by LC/MS/MS analysis of supernatants from A. baumannii
cultured in LB or LB + 200 mM NaCl. .................................................................... 61
Table 4: Predicted transporters that were found to be significantly upregulated in
response to NaCl by microarray analysis.................................................................. 66
Table 5: Antibiotic susceptibility profiles of clinical isolates obtained from the University
of Nebraska Medical Center compared to the reference strain ATCC 17978. ......... 73
Table 6: Colistin MIC values determined in MHB (-) or MHB supplemented with 150
mM NaCl (+). ........................................................................................................... 75
Table 7. Primers and plasmids used in this work.............................................................. 85
Table 8. Transposon mutants with reduced NaCl-induced colistin resistance. ................ 93
Table 9. HR-ICP-MS parameters. ................................................................................... 146
Table 10. Transposon mutants with altered sensitive to CP. .......................................... 151
Table 11. Locus tags and descriptions of predicted Zur-regulated genes ....................... 156
Table 12: Primers used for real-time PCR ...................................................................... 232
Table 13: Transcripts that increased significantly upon NaCl exposure as determined by
microarray analysis ................................................................................................. 233
Table 14: Transcripts that decreased significantly upon exposure to NaCl as determined
by microarray analyses ........................................................................................... 237
lpsB compared to WT. ............ 269
lpsB compared to WT ........ 275
Table 17. Genes that are significantly upregulated in Tn5A7 and Tn20A11 compared to
WT and lpsB. ........................................................................................................ 280
Table 18. Genes that are significantly downregulated in Tn5A7 and Tn20A11 compared
to WT and lpsB. .................................................................................................... 282

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LIST OF FIGURES

Figure 1. Schematic diagram of the LPS structure from A. baumannii strain 19606……24
Figure 2. Fe limitation and Fe acquisition during bacterial infections. ............................ 26
Figure 3. Mn and Zn homeostasis at the pathogen-host interface. ................................... 33
Figure 4. New insights into the roles for Cu in innate immunity. .................................... 43
Figure 5. Overview of the impact of CP on S. aureus superoxide defenses. .................... 47
Figure 6. NaCl induces increased release of proteins into culture supernatants. .............. 59
Figure 7: A. baumannii up-regulates putative efflux transporters upon culture in high
NaCl media ............................................................................................................... 67
Figure 8. SDS-PAGE analyses of proteins released into culture supernatants. ................ 68
Figure 9. NaCl induces increased tolerance to distinct classes of antibiotics in A.
baumannii. ................................................................................................................ 69
Figure 10. Effects of KCl on resistance to antibiotics and on release of proteins into
culture media. ............................................................................................................ 71
Figure 11. NaCl-induced resistance to levofloxacin and amikacin is due in part to
increased antibiotic efflux. ........................................................................................ 73
Figure 12. NaCl-induced resistance to colistin is conserved among drug susceptible and
multidrug resistant A. baumannii. ............................................................................. 75
Figure 13. Schematic overview of the transposon library screen to identify genes
involved in NaCl-induced colistin resistance. .......................................................... 91
Figure 14. Metabolic pathways disrupted in colistin-sensitive mutants of A. baumannii. 94
Figure 15. Demonstration of the LPS synthesis defect and colistin sensitivity of 5A7. ... 95
Figure 16. A. baumannii strains lacking LpsB are attenuated for virulence in the lung. .. 97
Figure 17. A. baumannii strains lacking LpsB are attenuated for virulence and promote
bacterial clearance in vivo. ...................................................................................... 112
Figure 18. Application of Tn5A7 to the treatment of A. baumannii pneumonia. ........... 113
Figure 19. Cluster analysis of all genes regulated at least 5-fold in at least one
experimental group compared to uninfected control. ............................................. 116
Figure 20. PCR array data for all genes that demonstrated at least 5-fold regulation
in at least one experimental group compared to uninfected control. .............................. 119
Figure 21. Measurement of the inflammatory response to WT and Tn5A7. .................. 122
Figure 22. Effect of neutrophil depletion on the attenuating effect of Tn5A7. .............. 123
Figure 23. Macrophages contribute to defense against A. baumannii but are not
required for the attenuating phenotype. .......................................................................... 124
Figure 24. TLR4 is not required for the attenuating phenotype of Tn5A7. .................... 126
Figure 25. Disruption of lpsB is not responsible for the attenuating phenotype of
Tn5A7. ............................................................................................................................ 127
Figure 26. Demonstration of the attenuating phenotype in a separate transposon mutant.
................................................................................................................................. 128
Figure 27. Induction of the therapeutic phenotype by transposon mutagenesis. ............ 129
Figure 28. Subtractive microarray defining a conserved pattern of gene expression in
attenuating strains. .................................................................................................. 132
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