A Novel, Molybdenum-Containing Methionine Sulfoxide Reductase Supports Survival of Haemophilus influenzae in an In vivo Model of Infection

Abstract

Haemophilus influenzae is a host adapted human mucosal pathogen involved in a variety of acute and chronic respiratory tract infections, including chronic obstructive pulmonary disease and asthma, all of which rely on its ability to efficiently establish continuing interactions with the host. Here we report the characterization of a novel molybdenum enzyme, TorZ/MtsZ that supports interactions of H. influenzae with host cells during growth in oxygen-limited environments. Strains lacking TorZ/MtsZ showed a reduced ability to survive in contact with epithelial cells as shown by immunofluorescence microscopy and adherence/invasion assays. This included a reduction in the ability of the strain to invade human epithelial cells, a trait that could be linked to the persistence of H. influenzae. The observation that in a murine model of H. influenzae infection, strains lacking TorZ/MtsZ were almost undetectable after 72 h of infection, while ∼3.6 × 103 CFU/mL of the wild type strain were measured under the same conditions is consistent with this view. To understand how TorZ/MtsZ mediates this effect we purified and characterized the enzyme, and were able to show that it is an S- and N-oxide reductase with a stereospecificity for S-sulfoxides. The enzyme converts two physiologically relevant sulfoxides, biotin sulfoxide and methionine sulfoxide (MetSO), with the kinetic parameters suggesting that MetSO is the natural substrate of this enzyme. TorZ/MtsZ was unable to repair sulfoxides in oxidized Calmodulin, suggesting that a role in cell metabolism/energy generation and not protein repair is the key function of this enzyme. Phylogenetic analyses showed that H. influenzae TorZ/MtsZ is only distantly related to the Escherichia coli TorZ TMAO reductase, but instead is a representative of a new, previously uncharacterized clade of molybdenum enzyme that is widely distributed within the Pasteurellaceae family of pathogenic bacteria. It is likely that MtsZ/TorZ has a similar role in supporting host/pathogen interactions in other members of the Pasteurellaceae, which includes both human and animal pathogens.

Rhodobacter sphaeroides methionine sulfoxide reductase P reduces R- and S-diastereomers of methionine sulfoxide from a broad-spectrum of protein substrates

“…All organisms have to face harmful protein oxidation and almost all possess canonical Msrs that protect proteins by reducing MetO. Bacteria also have molybdoenzymes able to reduce MetO, as a free amino acid for the DMSO reductase [26] or the biotin sulfoxide reductase BisC/Z [24,25], but also included in proteins in the case of MsrP [28,29]. Genetic studies and the conservation of MsrP in most gram-negative bacteria indicate that it is very likely a key player in the protection of periplasmic proteins against oxidative stress [28,29] However, an in-depth characterization of its protein substrate specificity is still lacking.…”

“…In bacteria, several molybdenum cofactor-containing enzymes were also shown to be able to reduce oxidized Met. Particularly, the biotin sulfoxide reductase BisC, or its homolog TorZ/BisZ, specifically reduce the free form of Met-S-O, in the Escherichia coli cytoplasm and the Haemophilus influenza periplasm, respectively [24,25]. Moreover, E. coli DMSO reductase reduces a broad spectrum of substrates, including MetO [26], while the R. sphaeroides homolog was shown to be absolutely stereospecific towards S-enantiomer of several alkyl aryl sulfoxides [27].…”

TheRhodobacter sphaeroidesmethionine sulfoxide reductase MsrP can reduceR- andS-diastereomers of methionine sulfoxide from a broad-spectrum of protein substrates

“…Indeed, to our knowledge, all previously characterized enzymes able to reduce Met sulfoxide or related substrates were shown as absolutely stereospecific. This was the case for the canonical MsrA and MsrB, which reduce only the S -diastereomer and the R-diastereomer, respectively (Ejiri et al, 1979; Grimaud et al, 2001; Kumar et al, 2002; Lowther et al, 2002; Moskovitz et al, 2002; Sharov et al, 1999; Vieira Dos Santos et al, 2005), as well as for the free Met- R -O reductase (Le et al, 2009; Lin et al, 2007) and for the molybdoenzymes DMSO reductase (Abo et al, 1995; Weiner et al, 1988) and BisC/Z (Dhouib et al, 2016; Ezraty et al, 2005). To evaluate the potential lack of stereospecificity of the RsMsrP, we chose to use a strategy different than the one used for E. coli MsrP (Gennaris et al, 2015) and prepared oxidized β-casein containing only one or the other MetO diastereomer using yeast MsrA and MsrB to eliminate the S - and the R -diastereomers, respectively.…”

“…All organisms have to face harmful protein oxidation and almost all possess canonical Msrs that protect proteins by reducing MetO. Bacteria also have molybdoenzymes able to reduce MetO, as a free amino acid for the DMSO reductase (Weiner et al, 1988) or the biotin sulfoxide reductase BisC/Z (Dhouib et al, 2016; Ezraty et al, 2005), but also included in proteins in the case of the MsrP (Gennaris et al, 2015; Melnyk et al, 2015). Genetic studies and the conservation of MsrP in most gram-negative bacteria indicate that it is very likely a key player in the protection of periplasmic proteins against oxidative stress (Gennaris et al, 2015; Melnyk et al, 2015) However, an in-depth characterization of its protein substrate specificity is still lacking.…”

“…Beside these stereotypical MSRs found in all kind of organisms, several bacterial molybdenum cofactor-containing enzymes can reduce MetO. Particularly, the biotin sulfoxide reductase BisC, or its homolog TorZ/BisZ, specifically reduce the free form of Met- S -O, in Escherichia coli cytoplasm (Ezraty et al, 2005) and Haemophilus influenza periplasm, respectively (Dhouib et al, 2016). Moreover, E. coli DMSO reductase reduce a broad spectrum of substrates, among which MetO (Weiner et al, 1988), and the R. sphaeroides homolog was shown as absolutely stereospecific towards S -enantiomer of several alkyl aryl sulfoxides (Abo et al, 1995).…”