Date of Completion

4-12-2018

Embargo Period

4-11-2020

Keywords

TWO COMPONENT SYSTEM, ANTIBIOTIC RESISTANCE

Major Advisor

Xiaohui Zhou

Co-Major Advisor

Mazhar Khan

Associate Advisor

Kumar Venkitanarayanan

Associate Advisor

NA

Field of Study

Pathobiology

Degree

Doctor of Philosophy

Open Access

Open Access

Abstract

In this study, we report two previously unidentified mechanisms that governs β-lactam

and antimicrobial peptide resistance.

In Gram-negative bacteria, production of β-lactamases is often induced in response to

the antibiotic- associated damage to the cell wall. Here, we have identified a previously

unidentified mechanism that governs β-lactamase production. In the Gram-negative

enteric pathogen Vibrio parahaemolyticus, we found a histidine kinase/response

regulator pair (VbrK/VbrR) that controls expression of a β-lactamase. Mutants lacking

either VbrK or VbrR do not produce the β-lactamase and are no longer resistant to β-

lactam antibiotics. Notably, VbrK autophosphorylation is activated by β-lactam

antibiotics, but not by other lactams. However, single amino acid substitutions in the

putative periplasmic binding pocket of VbrK leads its phosphorylation in response to

both β-lactam and other lactams, suggesting that this kinase is a β-lactam receptor that

can directly detect β-lactam antibiotics instead of detecting the damage to cell wall

resulting from β-lactams. In strong support of this idea, we found that purified

periplasmic sensor domain of VbrK binds penicillin, and that such binding is critical for

VbrK autophosphorylation and β lactamase production. Direct recognition of β-lactam

antibiotics by a histidine kinase receptor may represent an evolutionarily favorable

mechanism to defend against β-lactam antibiotics.

Antimicrobial peptide is amphipathic peptide that inserts into phospholipid bilayer and

disrupt the integrity of bacterial cell membrane. Many bacteria employ the strategy of

LPS modification to resist attack from AMPs. Here, we have identified a mechanism that

governs LPS modification that has never been reported in Vibrio parahaemolyticus

before. We identified a histidine kinase/response regulator pair (PvrK/PvrR) that

controls expression a lipid A ethanolamine phosphotransferase (PEA transferase), an

enzyme adds positively charged phosphoethanolamine (PEA) to the core of lipid A.

Resistance to AMPs or the expression of PEA transferase drastically decrease in

mutants lacking either PvrK or PvrR. The autophosphorylation of PvrK can be

stimulated through either low Mg2+ concentration or the presence of AMPs, which

resembles the host environment when Vibrio parahaemolyticus invades the host.

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