Elucidating the evolutionary changes that enable Staphylococcus aureus to overcome the synergistic impact of host-imposed manganese starvation and oxidative stress

Frye, Katie

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https://hdl.handle.net/2142/115879 Copy

Description

Title

Elucidating the evolutionary changes that enable Staphylococcus aureus to overcome the synergistic impact of host-imposed manganese starvation and oxidative stress

Author(s)

Frye, Katie

Issue Date

2022-06-30

Director of Research (if dissertation) or Advisor (if thesis)

Kehl-Fie, Thomas

Doctoral Committee Chair(s)

Kehl-Fie, Thomas

Committee Member(s)

• Imlay, James
• Kuzminov, Andrei
• Whitaker, Rachel

Department of Study

Microbiology

Discipline

Microbiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Metalloenzyme
• Staphylococcus aureus
• superoxide dismutase
• metals

Abstract

Metals are essential to all forms of life, with 30% of all proteins being reliant on a metal for full functionality. The metal used by metalloproteins can change over time in response to various pressures, presumably including that of metal availability. In line with this, the bioavailability of metals changes both across geological time as well as among macro- and micro-environments. One example of this change in metal availability is seen ~2.5 billion years ago during the Great Oxygenation Event. Among other metals, this oxygenation led to a four orders of magnitude decline in the availability of iron (Fe). This decline placed significant pressure on organisms with metalloproteins relying on this metal to switch to a more available metal. Another example of this change in metal availability occurs during infection by a pathogen where the host sequesters metals in a defense termed nutritional immunity. By limiting these essential metals, the host creates a pressure on pathogens to evolve strategies to circumvent this limitation such as metal-independent enzymes or enzymes relying on alternative metals. Together, these observations lead to the hypothesis that metal availability drove metalloenzyme neofunctionalization. Potentially, this neofunctionalization occurred through iterative rounds of single mutations, each providing a slightly more beneficial enzyme, enabling the shift in metal specificity. However, this hypothesis has not been evaluated as there was not a system that provided evolutionary starting and ending points where biologically relevant stressors, like metal limitation, could be imposed. Recently, it has been revealed that the superoxide dismutases (SODs) in the bacterial pathogen Staphylococcus aureus could be a useful tool in the evaluation of the pressures on metalloenzyme neofunctionalization. S. aureus possesses two superoxide dismutases (SODs), one manganese-dependent (SodA) and one capable of functioning with either Mn or Fe (SodM). SODs catalyze the conversion of superoxide to hydrogen peroxide and oxygen, helping to protect the cell from toxic reactive oxygen species. The cambialistic (either of two metals) nature of SodM allows the cell to retain SOD activity even when Mn starved, promoting resistance to nutritional immunity. SodA is present in all staphylococci, while SodM is only present in S. aureus and two closely related species. SodA and SodM are 75% identical at the amino acid level, and they coordinate their metal ions with the same residues. Further analysis suggests that SodM arose following a duplication and subsequent neofunctionalization of sodA. These analyses also proposed that the ancestor of SodM was strictly Mndependent, not cambialistic. This makes the staphylococcal SODs a unique system where there is a predicted evolutionary starting point and a present-day metalloenzyme, allowing for studies of the evolutionary path from one enzyme to the other. Previous work identified three residues in the staphylococcal SODs that, when swapped, enable SodA to use Fe as a cofactor and increase the activity of SodM with Mn. In the studies presented herein, I utilized the system provided by the staphylococcal SODs to investigate how metal availability might drive metalloprotein neofunctionalization. By creating a system that mimics the predicted evolutionary ancestor of S. aureus with a duplicated SodA, the second copy of SodA was manipulated with swapping mutations at the residues previously identified to dictate the Fe or Mn-dependent activity of the SODs. These strains then could act as potential evolutionary intermediates, and the effect of physiologically relevant stresses, such as metal limitation and oxidative stress, was evaluated. Preliminary results suggest that swapping one residue in SodA for the one present in SodM leads to an increased selectivity of SodA for Fe over Mn, indicating that non-coordinating residues within the protein might be involved in metal selectivity of the enzyme. These studies also revealed that the same single residue swap in SodA also led to an increased ability for S. aureus to resist oxidative stress and Mn-limitation. Additionally, S. aureus strains encoding SodA variants with this single residue swap had an increased bacterial burden during infection as compared to a S. aureus strain encoding two copies of wild type SodA (sodA2). Finally, this work also suggests that there is a disconnect between predictions based on in vitro SOD activity and performance of the SodA variant in S. aureus. This disconnect might be driven by a reduction of SOD activity of these SodA variants when they are within S. aureus. These results demonstrate that biochemically, the path from Mn-dependency to cambialism is simple, but biologically that path is much more constrained. Note to the reader: When deciding to stay at the University of Illinois Urbana-Champaign after the departure of my first advisor, Dr. Patrick Degnan, it was upon the mutual agreement of my committee members and the head of the department at the time, Dr. John Cronan, that my contributions to science from both labs would be included in my thesis. Accordingly, Dr. Cronan affectionately refers to my thesis as “Meanderings in Microbiology”. My introduction, Chapter 2, and conclusion will be based on my work performed in Dr. Kehl-Fie’s lab, and Chapter 3 will be the published work from my time in Dr. Degnan’s lab.

Graduation Semester

2022-08

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Katie Frye

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