Inter- and Intra-specific Interactions of Staphylococcus aureus Modeling Cystic Fibrosis Infection Restricted; Files Only
Alexander, Ashley (Fall 2023)
Published
Abstract
The diversity that exists among and between species is a captivating wonder of the living world. At the microbial scale, competition, mutualism, and commensalism between populations drives the evolution of traits like pathogenesis, cross-feeding, and bactericidal warfare. In the case of opportunistic pathogens like Staphylococcus aureus inter- and intra-specific interactions can have major impacts on the emergence of pathogenic traits. For individuals with the genetic disease cystic fibrosis (CF), complex communities of microbes can cause severe and long-term disease that is difficult to treat. In the last several decades S. aureus has become the prominent pathogen in CF related respiratory infections. The research presented in this thesis explores how coexisting populations influence the evolution or emergence of traits that are important factors in cystic fibrosis associated infections like the ability to coexist with P. aeruginosa and the ability to evolve and maintain antimicrobial resistance. In the introduction chapter 1, I frame this work in the context of previous findings on interactions between bacteria and research on cystic fibrosis associated pathogens. I also expand on the utility of experimental evolution approaches in studying complex traits within and between populations. In chapter 2 I present the findings of an evolution experiment and the resulting identification of a genetic signature that had not previously been observed in S. aureus – P. aeruginosa interactions. I demonstrated how the disruption of the amino acid transporter, gltT enables S. aureus to outcompete wild-type S. aureus under specific nutrient conditions and how that relates to its survival in the presence of P. aeruginosa. In chapter 3, I summarize findings about a set of clinical isolates taken from fresh CF sputum. I discuss the intraspecific diversity among single colonies and pool samples and the observed interactions between co-isolated strains. Altogether, the research presented here adds important insight into the role that diversity plays in the evolution of pathogenic traits in the important opportunistic pathogen S. aureus.
Table of Contents
CHAPTER 1 – INTRODUCTION ........................................................................................................................... 1
WHAT MICROBES HAVE TAUGHT US ABOUT ECOLOGY AND EVOLUTION ...................................................................................... 1
Figure 1. Clonal interference and its impact on population structure over time. ............................................................... 2
USING ECOLOGY AND EVOLUTION TO GAIN A PATHOGEN’S PERSPECTIVE – BEYOND KOCH’S POSTULATES ......................................... 4
CYSTIC FIBROSIS – AN IMPORTANT DISEASE THAT LENDS FUNDAMENTAL OBSERVATIONS ABOUT MICROBIAL ECOLOGY AND EVOLUTION.. 7
Figure 2. Model of Monoinfections and Coinfections with S. aureus and P. aeruginosa in CF lungs. ................................ 8
STAPHYLOCOCCUS AUREUS ............................................................................................................................................... 9
General history and physiology ............................................................................................................................. 9
Key traits and adaptive strategies....................................................................................................................... 10
Patterns in interspecific interactions with S. aureus ........................................................................................... 12
PSEUDOMONAS AERUGINOSA.......................................................................................................................................... 14
A useful foe.......................................................................................................................................................... 14
S. aureus and P. aeruginosa – a model system for interspecific interactions in infections................................. 15
EXPERIMENTAL EVOLUTION AS A TOOL FOR STUDYING COMPLEX TRAITS................................................................................... 16
GOALS OF THIS THESIS.................................................................................................................................................... 18 REFERENCES............................................................................................................................................................. 21
CHAPTER II - EXPERIMENTALLY EVOLVED STAPHYLOCOCCUS AUREUS SURVIVES IN THE PRESENCE OF PSEUDOMONAS AERUGINOSA BY ACQUIRING MUTATIONS IN THE AMINO ACID TRANSPORTER, GLTT ............. 27
AUTHOR CONTRIBUTIONS ............................................................................................................................................... 28
ABSTRACT ................................................................................................................................................................... 29
IMPORTANCE ............................................................................................................................................................... 29 INTRODUCTION............................................................................................................................................................. 30
METHODS ................................................................................................................................................................... 32
Bacterial Strains................................................................................................................................................... 32
Table 1. List of Staphylococcus aureus strains used in this study. ..................................................................................... 34 Media................................................................................................................................................................... 35
Experimental evolution........................................................................................................................................ 35
Whole genome sequencing ................................................................................................................................. 36
Complementation of gltT..................................................................................................................................... 37
Analysis of variation of the S. aureus gltT gene .................................................................................................. 37
Phenotypic testing for P. aeruginosa tolerance .................................................................................................. 38
Murine acute pneumonia model ......................................................................................................................... 39
Competitive fitness assay .................................................................................................................................... 40
Amino acid utilization.......................................................................................................................................... 40
Single cell imaging............................................................................................................................................... 40
Image analysis ..................................................................................................................................................... 41
RESULTS...................................................................................................................................................................... 42
gltT truncation in S. aureus is an adaptation to P. aeruginosa tolerance........................................................... 42
Figure 1. Experimental evolution with Staphylococcus aureus USA300 JE2 generates populations that are tolerant to Pseudomonas aeruginosa in coculture.............................................................................................................................. 44
Figure 2. gltT truncation enhances S. aureus recovery after coculture with PAO1........................................................... 46
JE2 gltT::Tn outcompetes wild-type S. aureus in CDMG without glutamate ...................................................... 47
Figure 3. JE2 gltT::Tn outcompetes wild-type JE2 in CDMG when glutamate is limiting and aspartate is in excess. ....... 48
Growth rate differences, as measured by single cell microscopy, are not responsible for the P. aeruginosa- tolerant phenotype.............................................................................................................................................. 49
Figure 4. Growth rates of wild-type JE2 and JE2 gltT::Tn in coculture with PAO1 in rich and depleted media................ 50
gltT disruption alters amino acid uptake in S. aureus strains ............................................................................. 51
Figure 5. gltT is required for S. aureus glutamate uptake and aspartate and glutamate are greatly reduced when PAO1
is present. ........................................................................................................................................................................... 52
gltT disruption does not impact S. aureus host colonization .............................................................................. 53
Figure 6. Colonization ability of S. aureus is not impacted by gltT genotype.................................................................... 54
gltT disruption was rare in diverse S. aureus genomes ....................................................................................... 55
DISCUSSION ................................................................................................................................................................. 55
Impact of inactivation of S. aureus gltT gene in S. aureus-P. aeruginosa interactions....................................... 55
The role of aspartate and glutamate in P. aeruginosa tolerance ....................................................................... 57
Experimental evolution as a useful tool for studying pathogens ........................................................................ 58
Acknowledgements ............................................................................................................................................. 59
REFERENCES ........................................................................................................................................................ 60
SUPPLEMENTARY METHODS ........................................................................................................................................... 65
Growth in chemically defined media................................................................................................................... 65
SUPPLEMENTARY RESULTS.............................................................................................................................................. 65
gltT disruption does not alter S. aureus growth in chemically defined media .................................................... 65
Supplementary Table 1. ..................................................................................................................................................... 66
Supplementary Table 2 ...................................................................................................................................................... 67
Supplementary Figure 1 ..................................................................................................................................................... 68
Supplementary Figure 2. .................................................................................................................................................... 69
Supplementary Figure 3 ..................................................................................................................................................... 70
Supplementary Figure 4 ..................................................................................................................................................... 71
SUPPLEMENTARY REFERENCES ........................................................................................................................... 72
CHAPTER III- INTRASPECIFIC DIVERSITY AND DYNAMICS OF STAPHYLOCOCCUS AUREUS POPULATIONS ISOLATED FROM CYSTIC FIBROSIS RESPIRATORY INFECTIONS.......................................................................................... 73
AUTHOR CONTRIBUTIONS ............................................................................................................................................... 74
ABSTRACT ................................................................................................................................................................... 75 INTRODUCTION............................................................................................................................................................. 75
METHODS ................................................................................................................................................................... 76
Sample collection................................................................................................................................................. 76
Figure 1. Sampling methods............................................................................................................................................... 78
Phenotyping......................................................................................................................................................... 79
Table 1. Compilation of genotypes and phenotypes of CF clinical isolates ....................................................................... 80
Whole genome sequencing ................................................................................................................................. 81
Analysis of Genomic Diversity ............................................................................................................................. 81
Relative fitness of co-isolates .............................................................................................................................. 82
Co-isolated strain growth in the presence of antibiotics..................................................................................... 83
Experimental evolution of co-isolated strains in the presence of antibiotics...................................................... 83
Clinical data collection......................................................................................................................................... 84
RESULTS...................................................................................................................................................................... 85
Phenotypic analysis of all isolates ....................................................................................................................... 85
Genotypic analysis............................................................................................................................................... 86
Figure 2. Genetic distances across all patient isolates....................................................................................................... 88
Figure 3. Antimicrobial resistance genetic signatures ....................................................................................................... 90
Clinical testing and metadata ............................................................................................................................. 91
Table 2. Clinical Antibiotic Resistance report of S. aureus isolates ................................................................................... 92
Relative fitness of MRSA and MSSA co-isolates .................................................................................................. 93
Figure 4. Relative fitness of co-isolated MRSA and MSSA. ................................................................................................ 95
Growth in antibiotics of co-isolated MRSA and MSSA ........................................................................................ 96
Figure 5. Growth of co-isolated MRSA and MSSA populations with antibiotics. .............................................................. 97
Experimental evolution of co-isolates in the presence of antibiotics .................................................................. 98
Figure 6. Stepwise experimental evolution of co-isolates alone and cocultured with oxacillin and vancomycin ............ 99
DISCUSSION ............................................................................................................................................................... 100
Observed diversity of S. aureus patient isolates................................................................................................ 100 ACKNOWLEDGEMENTS................................................................................................................................................. 103 REFERENCES........................................................................................................................................................... 104
CHAPTER IV - DISCUSSION AND CONCLUSIONS .............................................................................................. 107
EXPERIMENTAL EVOLUTION – AN EFFECTIVE TOOL WITH IMPORTANT LIMITATIONS................................................................... 108
INFECTIONS AS EVOLVING ECOSYSTEMS ........................................................................................................................... 111
FUTURE DIRECTIONS.................................................................................................................................................... 114 REFERENCES........................................................................................................................................................... 117
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