Poster Presentation BACPATH 2017

Methionine sulfoxide reductase confers resistance to oxidative stress caused by HOCl in haemophilus influenzae   (#151)

Marufa Nasreen 1 , Rabeb 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. The cytoplasmic defence strategies in NTHi to deal with host-induced oxidative stress are well studied, but little is known about oxidative stress defences in the periplasm. This project investigates the role of the periplasmic peptide methionine sulfoxide reductase (Msr), which in NTHi is a fusion of MsrA and MsrB domains, in oxidative stress defence. In NTHi, the msrAB gene forms an operon with genes encoding a thioredoxin (trx) and a protein related to the CcdA cytochrome-c-type biogenesis protein (ccdA), which are likely involved in electron transfer to the MsrAB enzyme. We created a non-polar mutation in msrAB. This Hi2019∆msrAB strain had no growth phenotype in vitro and showed no difference in its ability to use different carbon, nitrogen, or sulfur sources, or in its responses to pH change and osmolytes. Compared to the Hi2019WT, Hi2019msrAB produced slightly less biofilm (15% and 17% under aerobic and microaerophilic conditions, (p≤0.0001)). However, a challenge with 150µM HOCl, Hi2019msrAB resulted in 50% less viable cells while the WT showed little sensitivity. We also found that exposure to HOCl increased the expression of msrAB in Hi2019WT, indicating that gene expression responds to oxidative stress caused by HOCl. To confirm MsrAB properties, we have optimized the expression of Hi2019 MsrAB, Trx and thioredoxin reductase for development of an activity assay. Interestingly, in co-culture with 16HBE14 bronchial cells, there were less Hi2019msrAB detected intracellularly compared to Hi2019WT. We therefore 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.