Peptide Methionine Sulfoxide Reductase from Haemophilus influenzae Is Required for Protection against HOCl and Affects the Host Response to Infection

Abstract

Peptide methionine sulfoxide reductases (Msrs) are enzymes that repair ROS-damage to sulfur-containing amino acids such as methionine, ensuring functional integrity of cellular proteins. Here we have shown that unlike the majority of pro-and eukaryotic Msrs, the peptide methionine sulfoxide reductase (MsrAB) from the human pathobiont Haemophilus influenzae (Hi) is required for the repair of hypochlorite damage to cell envelope proteins, but more importantly, we were able to demonstrate that MsrAB plays a role in modulating the host immune response to Hi infection. Loss of MsrAB resulted in >1000-fold increase in sensitivity of Hi to HOClmediated killing, and also reduced biofilm formation and in-biofilm survival. Expression of msrAB was also induced by hydrogen peroxide and paraquat, but a Hi2019 ΔmsrAB strain was not susceptible to killing by these ROS in vitro. Hi2019 ΔmsrAB fitness in infection models was low, with a 3-fold reduction in intracellular survival in bronchial epithelial cells, increased susceptibility to neutrophil killing, and a 10-fold reduction in survival in a mouse model of lung infection. Interestingly, infection with Hi2019 ΔmsrAB led to specific changes in the antibacterial response of human host cells, with genes encoding antimicrobial peptides (BPI, CAMP) upregulated between 4 and 9 fold compared to infection with Hi2019 WT , and reduction in expression of two proteins with antiapoptotic functions (BIRC3, XIAP). Modulation of host immune responses is a novel role for an enzyme of this type and provides first insights into mechanisms by which MsrAB supports Hi survival in vivo.

The DmsABC Sulfoxide Reductase Supports Virulence in Non-typeable Haemophilus influenzae

“…One of the most common types of biomolecular damage caused by oxidative stress is the oxidation of sulfur compounds including amino acids. Both cysteine and methionine are highly susceptible to oxidative damage, and there is increasing evidence that both thiol-based and molybdenum-containing methionine sulfoxide reductases protect bacterial pathogens from hostinduced oxidative stress and thus support bacterial virulence (Ezraty et al, 2005(Ezraty et al, , 2017Denkel et al, 2013;Nasreen et al, 2020;Zhong et al, 2020).…”

“…We have previously characterized two methionine sulfoxide reductases from the human-adapted respiratory pathogen Haemophilus influenzae (Dhouib et al, 2016;Nasreen et al, 2020), a human pathobiont that asymptomatically colonizes and persists in the human nasopharynx. H. influenzae can migrate to other niches in the upper and lower respiratory tracts where it can cause both acute diseases such as otitis media, sinusitis, and pneumonia or exacerbate existing chronic lung conditions such as chronic obstructive pulmonary disease (COPD), bronchiectasis, asthma, and cystic fibrosis (Eldere et al, 2014;King and Sharma, 2015).…”

“…The two H. influenzae methionine sulfoxide reductases, MtsZ and MsrAB, are both part of the bacterium's periplasmic defenses against oxidative stress (Dhouib et al, 2016;Nasreen et al, 2020). MsrAB is a peptide-sulfoxide reductase that can repair oxidative damage to H. influenzae cell envelope proteins using thiol redox chemistry.…”

“…MsrAB is a peptide-sulfoxide reductase that can repair oxidative damage to H. influenzae cell envelope proteins using thiol redox chemistry. MsrAB is required for resistance to hypochlorite and was also shown to modulate host responses to H. influenzae infection (Nasreen et al, 2020). The protein-repairing activity of MsrAB is complemented by the activity of the molybdenumcontaining MtsZ methionine sulfoxide reductase that reduces free methionine sulfoxide, thereby increasing the bioavailability of methionine for uptake by bacteria (Dhouib et al, 2016).…”

Access to highly specialized growth substrates and production of epithelial immunomodulatory metabolites determine survival of Haemophilus influenzae in human airway epithelial cells

“…Giemsa staining of mouse immune cells in BALF revealed that, compared to Hi2019 WT , influx of neutrophils was reduced during infection with Hi2019 Δ lldD while macrophage numbers showed statistically significant increases throughout ( S4 Fig ). Expression of genes encoding key cytokines (IL-6, TNFα, IL-1β) and BIRC3, which is involved in innate immune signalling and has been shown to increase in expression during infections of human cells with NTHi [ 48 ], were similar in mice infected with Hi2019 Δ lldD and Hi2019 WT at 6 h post-infection, while at 24 h and 48 h post-infection gene expression levels were reduced between 2.5- and 11-fold ( S5 Fig , 2-Way ANOVA, p<0.05) in mice infected with Hi2019 Δ lldD , in keeping with the reduction of Hi2019 Δ lldD CFUs recovered at these timepoints. Despite this difference in cytokine gene expression, levels of TNFα & IL-1β in BALF were generally similar for the Hi2019 WT and Hi2019 Δ lldD strain except for IL-1β levels at 6 h post-infection where lower levels were observed for infections with the mutant strain.…”