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Title: Response of Pseudomonas aeruginosa to the innate immune system-derived oxidants hypochlorous acid and hypothiocyanous acid
Author: Farrant, Katie Victoria
ISNI:       0000 0004 8504 5194
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2018
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P. aeruginosa is an opportunistic pathogen that causes infections in immunocompromised hosts including chronic infections in the lungs of Cystic Fibrosis (CF) patients, which are ineffectively cleared by the host immune response and associated with a poor prognosis. The host's innate immune cells produce reactive species to target the bacteria, including the neutrophil-derived oxidant hypochlorous acid (HOCl) and the epithelial cell-derived oxidant hypothiocyanous acid (HOSCN). Both systems are implicated in CF due to the proposed ion transport roles of the CF transmembrane conductance regulator (CFTR) channel, which is mutated in CF. CFTR is proposed to transport chloride ions into the phagosome, where they are required for HOCl production and to transport thiocyanate ions into the airway surface liquid where they form HOSCN. Little is known about how P. aeruginosa responds to and avoids being killed by these oxidants, therefore my thesis aimed to address this. A screening approach to determine PA14 transposon mutants with altered HOCl susceptibility was performed to reveal regulators involved in bacterial protection from HOCl. This identified regulators involved in methionine biosynthesis, catabolite repression, and antibiotic resistance, and demonstrated a role for the novel regulator HcsR, a homolog of the E. coli HOCl-sensor RclR, in HOCl protection. Characterisation of this regulator found that it binds to the promoter region of hcsA and responds specifically to HOCl and HOSCN stress, perhaps via oxidation of its redox-active residues; this results in its activation and expression of hcsA encoding a putative peroxiredoxin required for HOCl protection. Investigation of the HcsR regulon revealed that HcsR predominately regulates hcsA expression in response to HOCl. However it positively regulates 132 genes and negatively regulates 213 genes in response to HOSCN, including activation of pyocyanin and denitrification genes and repression of heat shock and Type III secretion system (T3SS) genes. Transcriptome profiling of P. aeruginosa following HOCl or HOSCN exposure revealed significant overlap in the bacterial response to these oxidants, including expression of T3SS, taurine transport systems, the MexEF-OprN multidrug efflux pump, and non-coding RNAs. These data indicate that the bacterial response to HOCl and HOSCN is multifaceted, with HcsR playing an essential role, and additionally involves various mechanisms involved in virulence factor production, metabolism, antibiotic resistance and repairing oxidised macromolecules. This work forms the foundation from which the importance of these mechanisms in protecting P. aeruginosa against these oxidants in the context of CF infection can be further explored.
Supervisor: Williams, Huw Sponsor: Medical Research Council
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral