The Alternative Sigma Factor RpoE2 Is Involved in the Stress Response to Hypochlorite and in vivo Survival of Haemophilus influenzae

Abstract

Extracytoplasmic function (ECF) sigma factors underpin the ability of bacteria to adapt to changing environmental conditions, a process that is particularly relevant in human pathogens that inhabit niches where human immune cells contribute to high levels of extracellular stress. Here, we have characterized the previously unstudied RpoE2 ECF sigma factor from the human respiratory pathogen H. influenzae (Hi) and its role in hypochlorite-induced stress. Exposure of H. influenzae to oxidative stress (HOCl, H2O2) increased rpoE2 gene expression, and the activity of RpoE2 was controlled by a cytoplasmic 67-aa anti-sigma factor, HrsE. RpoE2 regulated the expression of the periplasmic MsrAB peptide methionine sulfoxide reductase that, in H. influenzae, is required for HOCl resistance, thus linking RpoE2 to HOCl stress. Interestingly, a HiΔrpoE2 strain had wild-type levels of resistance to oxidative stress in vitro, but HiΔrpoE2 survival was reduced 26-fold in a mouse model of lung infection, demonstrating the relevance of this sigma factor for H. influenzae pathogenesis. The HiRpoE2 system has some similarity to the ECF sigma factors described in Streptomyces and Neisseria sp. that also control the expression of msr genes. However, HiRpoE2 regulation extended to genes encoding other periplasmic damage repair proteins, an operon containing a DoxX-like protein, and also included selected OxyR-controlled genes. Based on our results, we propose that the highly conserved HiRpoE2 sigma factor is a key regulator of H. influenzae responses to oxidative damage in the cell envelope region that controls a variety of target genes required for survival in the host.

The DmsABC S-oxide reductase is an essential component of a novel, hypochlorite-inducible system of extracellular stress defense in Haemophilus influenzae

“…Traditionally, this type of enzyme has been associated with bacterial energy generation under anaerobic conditions as catalysis uses quinols that mediate electron transfer between respiratory chain complexes as the electron donors ( Kappler and Schaefer, 2014 ; Kappler et al, 2019 ). At present, only a single hypochlorite-responsive regulator, the RpoE2 extracytoplasmic function—sigma factor, has been identified in H. influenzae ( Nasreen et al, 2021 ), and this regulator might mediate the hypochlorite-based induction of dmsABC expression.…”

“…S-oxide reductases are enzymes that can reverse sulfoxide-formation on biomolecules, including proteins, sulfur-containing amino acids and vitamins such as methionine, cysteine and biotin that are highly susceptible to oxidative damage ( Baltes et al, 2003 ; Denkel et al, 2013 ; Dhouib et al, 2016 , 2021 ; Ezraty et al, 2017 ; Kappler et al, 2019 ; Zhong et al, 2020 ; Nasreen et al, 2021 ). S-oxide reductases are emerging as essential components for virulence and fitness of bacterial pathogens such as Escherichia coli , Salmonella sp., Actinobacillus pleuropneumoniae , and Haemophilus influenzae ( Baltes et al, 2003 ; Denkel et al, 2013 ; Dhouib et al, 2016 , 2021 ; Ezraty et al, 2017 ; Kappler et al, 2019 ; Zhong et al, 2020 ; Nasreen et al, 2021 ), where a loss of S-oxide reduction causes a reduction of survival in infection models.…”

The Peptide Methionine Sulfoxide Reductase (MsrAB) of Haemophilus influenzae Repairs Oxidatively Damaged Outer Membrane and Periplasmic Proteins Involved in Nutrient Acquisition and Virulence

“…Expression of both mtsZ and msrAB was induced by exposure of the bacteria to hypochlorite and is controlled by an extracytoplasmic function sigma factor, RpoE2, that is conserved in H. influenzae strains. In keeping with this observation, MsrAB is required for the hypochlorite resistance of H. influenzae [ 16 , 18 ]. MsrAB was also involved in host immune response modulation, where in response to infections with an H. influenzae msrAB strain, higher levels of expression of antimicrobial peptides were observed, while expression of the antiapoptotic XIAP protein was reduced [ 18 ].…”

“…We have recently characterized three enzymes involved in MetSO reduction in the H. influenzae periplasm, the molybdenum enzymes DmsABC and MtsZ and the MsrAB methionine sulfoxide reductase that contains both an MsrA and MsrB domain [ 16 , 17 , 18 , 19 ]. Both of the molybdenum-containing enzymes were able to reduce free MetSO; however, expression levels of DmsABC were significantly lower than for MtsZ, which appears to be the major molybdenum-dependent methionine sulfoxide reductase in H. influenzae strain Hi2019 [ 17 , 19 ].…”

Hfe Permease and Haemophilus influenzae Manganese Homeostasis

“…Studies investigating H. influenzae oxidative stress resistance have identified numerous enzymes that contribute to protection that include catalase ( hktE ) and peroxiredoxin ( pgdX ), which degrade H 2 O 2 and alkyl hydroperoxides; the DNA-binding ferritin-like protein ( dps ); three periplasmic methionine sulfoxide reductases ( torZ , msrAB, dmsABC ); and a manganese (Mn) superoxide dismutase ( sodA ). − In addition, key regulators that control responses to oxidative stress have also been identified that include those for H 2 O 2 exposure (OxyR), perturbation of iron (Fe) homeostasis (Fur, ferric uptake regulator), and reactive chlorine species-induced stress, such as HOCl (alternative sigma factor RpoE2). − Nevertheless, there remain unexplored aspects of H. influenzae oxidative stress tolerance such as Mn homeostasis, which has a prominent contribution to bacterial stress responses and has recently been shown to have important roles in H.…”