Doctoral Dissertations

Date of Award

5-2017

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Major

Comparative and Experimental Medicine

Major Professor

Stephen A. Kania

Committee Members

David A. Bemis, Rebecca P. Wilkes, Doris H. D'Souza

Abstract

Staphylococcus pseudintermedius is an opportunistic pathogen in dogs and is the most frequent cause of canine pyoderma. The recent emergence of methicillin resistance and multidrug resistance has made it increasingly difficult to treat infections using conventional antibiotics. There is an urgent need for alternative strategies to prevent S. pseudintermedius infections. The development of a successful vaccine against S. pseudintermedius is complicated by several virulence and immune evasion factors. The objective of this project was to identify new targets for treatment and prophylaxis of methicillin-resistant and multidrug-resistant S. pseudintermedius infection. This was achieved using two strategies (a) understanding the roles of Protein A and Sortase A during pathogenesis, along with exploring Sortase A inhibition as an alternate treatment strategy (b) analyzing the surface-associated and secreted proteins in S. pseudintermedius to identify novel vaccine targets.

In this study, a 3-D homology model for Sortase A was generated using molecular dynamic simulation. Using this model, drug databases were screened in silico and potential inhibitors of Sortase A were identified and tested in cell-free and functional whole-cell assays. Four promising compounds were identified using this strategy. Sortase A anchors proteins to the bacterial surface. These include potent virulence and immune evasion factors such as Protein A. Both cell wall-associated and secreted Protein A in S. pseudintermedius was characterized and its role in immune evasion was examined. Since Sortase A inhibition alone may not be an effective treatment option, the entire secretome and the surface-associated proteins in S. pseudintermedius were analyzed by mass spectrometry. Eight orthologous proteins across several S. pseudintermedius isolates were identified that could serve as potential vaccine targets. Ultimately, Sortase A inhibition along with a multicomponent vaccine containing a non-toxigenic version of Protein A and other potential B-cell/T-cell epitopes may be effective against S. pseudintermedius infection.

This study is an important step in (i) understanding the nature of immunoglobulin binding onto Protein A and (ii) the inhibition of Sortase A as an alternate treatment strategy in S.pseudintermedius, and may ultimately pave the way towards the development of a successful vaccine against S. pseudintermedius infection.

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