Comparative Sequence, Structure and Redox Analyses of Klebsiella pneumoniae DsbA Show That Anti-Virulence Target DsbA Enzymes Fall into Distinct Classes

Abstract

Bacterial DsbA enzymes catalyze oxidative folding of virulence factors, and have been identified as targets for antivirulence drugs. However, DsbA enzymes characterized to date exhibit a wide spectrum of redox properties and divergent structural features compared to the prototypical DsbA enzyme of Escherichia coli DsbA (EcDsbA). Nonetheless, sequence analysis shows that DsbAs are more highly conserved than their known substrate virulence factors, highlighting the potential to inhibit virulence across a range of organisms by targeting DsbA. For example, Salmonella enterica typhimurium (SeDsbA, 86 % sequence identity to EcDsbA) shares almost identical structural, surface and redox properties. Using comparative sequence and structure analysis we predicted that five other bacterial DsbAs would share these properties. To confirm this, we characterized Klebsiella pneumoniae DsbA (KpDsbA, 81 % identity to EcDsbA). As expected, the redox properties, structure and surface features (from crystal and NMR data) of KpDsbA were almost identical to those of EcDsbA and SeDsbA. Moreover, KpDsbA and EcDsbA bind peptides derived from their respective DsbBs with almost equal affinity, supporting the notion that compounds designed to inhibit EcDsbA will also inhibit KpDsbA. Taken together, our data show that DsbAs fall into different classes; that DsbAs within a class may be predicted by sequence analysis of binding loops; that DsbAs within a class are able to complement one another in vivo and that compounds designed to inhibit EcDsbA are likely to inhibit DsbAs within the same class.

Crystal Structure of the Dithiol Oxidase DsbA Enzyme from Proteus Mirabilis Bound Non-covalently to an Active Site Peptide Ligand

“…These distances are in agreement with those of other reduced DsbA structures (e.g. KpDsbA 3.3-3.8 Å) (18). Overall, there is no apparent change in the active site upon binding of the peptide.…”

“…1F), but more slowly than the specialist reductant EcDsbC. In summary, the redox properties of PmDsbA reported here place it in the same class as EcDsbA and other Enterobacteriaceae DsbAs such as S. enterica DsbA (SeDsbA) and K. pneumoniae DsbA (KpDsbA) (18).…”

“…Thus, inhibiting DsbA/B would not kill bacteria and this property may reduce the selective pressure to develop resistance. Second, structures of several DsbA homologues have been solved (18,28,71), providing a framework for structure-based drug design. Third, DsbA structures and properties are more highly conserved than the structures and sequences of virulence factors across pathogens (18).…”

“…DsbA enzymes from Gram-negative bacteria are targets for the development of anti-virulence drugs that could represent an entirely new class of antimicrobial agents (17,18). Drugs that target bacterial virulence could minimize the selective pressure that generates antibiotic resistance and simultaneously preserve the endogenous host microbiome (19).…”

Structure of the Acinetobacter baumannii Dithiol Oxidase DsbA Bound to Elongation Factor EF-Tu Reveals a Novel Protein Interaction Site

“…In EcDsbA the equivalent acidic surface was identified several decades ago as a potential interaction site (83). The variable molecular landscape and nature of this surface among DsbAs may reflect the extraordinary variability of substrates (61,84). The high resolution structure of the AbDsbA⅐EcEF-Tu complex supports the idea that the pronounced non-catalytic surface groove of class I DsbA enzymes serves as an additional protein interaction site.…”