Role of Streptococcal Pyrogenic Exotoxins A and B (SpeA and SpeB) Mutation in Streptococcus pyogenes Virulence Public
Bushman, Summer (Spring 2022)
Published
Abstract
Streptococcus pyogenes (also referred to as group A Streptococcus, or GAS) causes about 18.1 million cases of severe infection annually, resulting in 500,000 deaths worldwide. It is important to study the interactions of GAS with its human host because there is no vaccine, and the only treatment is antibiotics, to which resistance is rapidly increasing. This project looked at two specific streptococcal pyrogenic exotoxin (Spe) proteins, SpeA and SpeB. SpeA is a superantigen associated with cases of streptococcal toxic shock-like syndrome (STSS). SpeB is a protease that degrades immunoglobulins and cytokines. Using sequences that encode for SpeA and SpeB to identify naturally occurring synonymous and nonsynonymous polymorphisms, we found that SpeA is undergoing diversifying selection and SpeB is undergoing purifying selection. Nonsynonymous mutations have the potential to alter protein function, so these mutations within SpeB were further examined. Site-directed mutagenesis was used to change the sites of three natural nonsynonymous polymorphisms (G17, E130, and D154) within speB to alanine via a pET expression plasmid. After sequence confirmation, the DNA was transformed into E. coli where the mutated proteins were expressed. The proteins were subsequently extracted and purified. The activity of each mutant protein was examined in vitro using fluorescent activity assays to quantify the degradation of known SpeB substrate and fluorophore, substrate 73. Non-mutant reference-type SpeB from strain M1T1 5448 served as positive controls; Strains with empty pET vectors served as negative controls. SpeB with mutation G17A was found to have similar activity to wildtype SpeB, indicating no alternation in conformation or functional ability. SpeB with mutations E130A or D154A was found to have a slight reduction in activity, indicating potential interference with binding sites. This data can be used to determine the functional consequences of heterogeneity within speB. In future studies, this data can be applied more broadly to help develop effective public health policies and identify important and conserved proteins to target for the development of a vaccine, which is critically lacking for GAS.
Table of Contents
Table of Contents
Chapter 1: Introduction ………………………………………………………………………… 1
Chapter 2: Examining the Heterogeneity of SpeA and SpeB ………………….………………. 4
Methods ……………………………………...………….….…………….…..………… 5
Results ………………………………………………….………………………………. 7
Figure 1 …...……….…………………………………………………………… 7
Figure 2 …...……………………………………………….…………………… 9
Figure 3 …...…………………………………………………………………… 11
Discussion ………………………………………….………………………………….. 12
Chapter 3: Examining the Functional Consequences of Heterogeneity in SpeB ……………… 13
Methods ……………………………………………….……………………………….. 14
Figure 4 …………………………………….……...…………………………… 14
Results ………………….………………………….……………..……………………. 18
Figure 5 ……………………………………………….……………………….. 18
Figure 6 …………………..………………………….………………………… 20
Figure 7 …………………………..……………………….…………………… 21
Discussion ……………………………………………………………………………... 22
Future Directions and Conclusion …………….………………….……………………….…… 23
References ………………………………………………….………….………………………. 26
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