Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2018

Extracytoplasmic Methionine sulfoxide reductase protects Haemophilus influenzae from oxidative stress caused by HOCl. (#337)

Marufa Nasreen 1 , Rebeb Dhouib 1 , Alastair G McEwan 1 , Ulrike Kappler 1
  1. University of Queensland, St. Lucia, QUEENSLAND, Australia

Non-typeable Haemophilus influenzae (NTHi) is a host-adapted human pathogen that causes diseases of the respiratory tract such as chronic bronchitis and COPD as well as otitis media. The cytoplasmic defense strategies in NTHi to deal with host-induced oxidative stress are well studied, but little is known about oxidative stress defenses in the periplasm.This project investigates the role of peptide-methionine sulfoxide reductase (Msr), which in NTHi is a fusion of MsrA and MsrB domains, in oxidative stress defense. We characterized the effects of an msrAB gene knockout on NTHi physiology and virulence and examined the reductase activity of MsrAB.The msrAB gene is co-transcribed with genes encoding a thioredoxin and a protein related to the cytochrome-c-type-biogenesis protein CcdA which are likely involved in electron transfer to MsrAB enzyme. Expression of msrAB in Hi2019WT increased 45-fold following HOCl challenge, following which the MsrAB protein was present in the periplasmic space and also associated with the outer membrane. An assay for the reductase activity of periplasmic-MsrAB was developed, revealing 0.712U/mg in the presence of methyl-p-tolyl-sulfoxide as substrate.We created a mutation in msrAB by inserting a kanamycin cassette which was non-polar. Hi2019∆msrAB had no growth phenotype in vitro and showed no difference in its ability to use different sources of carbon, nitrogen, or sulfur, or in responses to pH change and osmolytes in phenotypic microarrays. Hi2019∆msrAB consistently produced 15% and 17% less biofilm under aerobic and microaerophilic conditions, (p≤0.0001). However, exposure of Hi2019∆msrAB to 150mM HOCl resulted in 50% less viable cells compared to the WT and this was also observed when Hi2019∆msrAB was exposed to HOCl-producing neutrophils. We propose that Hi-MsrAB is an important enzyme for protecting NTHi against extracellular HOCl stress, a condition that is well described to exist at sites of acute infection and that a secondary role may be in mediating successful host-interactions, especially with neutrophils.