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Review
. 2020 Jun 22;88(7):e00964-19.
doi: 10.1128/IAI.00964-19. Print 2020 Jun 22.

Bacterial Defense Systems against the Neutrophilic Oxidant Hypochlorous Acid

Sadia Sultana  1 , Alessandro Foti  2 , Jan-Ulrik Dahl  3
Affiliations
Review

Bacterial Defense Systems against the Neutrophilic Oxidant Hypochlorous Acid

Sadia Sultana et al. Infect Immun. .

Abstract

Neutrophils kill invading microbes and therefore represent the first line of defense of the innate immune response. Activated neutrophils assemble NADPH oxidase to convert substantial amounts of molecular oxygen into superoxide, which, after dismutation into peroxide, serves as the substrate for the generation of the potent antimicrobial hypochlorous acid (HOCl) in the phagosomal space. In this minireview, we explore the most recent insights into physiological consequences of HOCl stress. Not surprisingly, Gram-negative bacteria have evolved diverse posttranslational defense mechanisms to protect their proteins, the main targets of HOCl, from HOCl-mediated damage. We discuss the idea that oxidation of conserved cysteine residues and partial unfolding of its structure convert the heat shock protein Hsp33 into a highly active chaperone holdase that binds unfolded proteins and prevents their aggregation. We examine two novel members of the Escherichia coli chaperone holdase family, RidA and CnoX, whose thiol-independent activation mechanism differs from that of Hsp33 and requires N-chlorination of positively charged amino acids during HOCl exposure. Furthermore, we summarize the latest findings with respect to another bacterial defense strategy employed in response to HOCl stress, which involves the accumulation of the universally conserved biopolymer inorganic polyphosphate. We then discuss sophisticated adaptive strategies that bacteria have developed to enhance their survival during HOCl stress. Understanding bacterial defense and survival strategies against one of the most powerful neutrophilic oxidants may provide novel insights into treatment options that potentially compromise the ability of pathogens to resist HOCl stress and therefore may increase the efficacy of the innate immune response.

Keywords: N-chlorination; biofilm; biofilm formation; chaperedoxin; disulfide bond formation; hypochlorous acid; molecular chaperone; motility; neutrophilic oxidants; oxidative burst; oxidative stress; polyphosphate; posttranslational modifications; protein aggregation.

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Figures

FIG 1
FIG 1
Neutrophil-mediated RO/CS formation. Neutrophils represent the first line of defense of the innate immune system. To kill invading bacteria, they generate powerful antimicrobials in a multistep process. (Upper panel) First, neutrophils sense and bind to the invading microbe (step 1), which results in the ingestion of the microbe as well as in granule-phagosome fusion events (step 2). Subsequently, granular proteins and RO/CS are released into the phagosome (step 3), where myeloperoxidase catalyzes the production of RO/CS. (Lower panel) Superoxide (O2•−) is produced by activated NADPH oxidases (NOX2) located in the phagosome membrane. The charge is balanced through proton channels (gray). O2•− is mainly dismutated into peroxide (H2O2) by myeloperoxidase (MPO), an enzyme that is released from azurophil granules. Moreover, MPO catalyzes the reaction of H2O2 with physiological concentrations of chloride or thiocyanate to yield hypochlorous acid (HOCl) or hypothiocyanous acid (HOSCN), respectively. The rate of production of HOCl is determined by the availability of chloride, as superoxide competes with chloride for MPO and thus controls the rate of production of HOCl. All neutrophilic RO/CS are produced in the phagosomal space, and some readily penetrate into the bacterial cell.
FIG 2
FIG 2
Reversible cysteine oxidation causes conditional disorder to activate Hsp33. One way for bacteria to fight the proteotoxic effects of HOCl stress is to employ the stress-specific molecular chaperone Hsp33, which is present as a chaperone-inactive monomer under nonstress conditions. Reduced Hsp33 is well folded due to the coordination of a Zn2+ ion by four highly conserved cysteine residues in the C-terminal domain. Upon exposure to protein-unfolding conditions, such as HOCl stress, the cysteines are oxidized, resulting in the formation of two disulfide bonds, zinc release, and massive structural rearrangements. Similarly to its client proteins, Hsp33 partially unfolds during HOCl stress; however, the disordered conformation is essential for the chaperone activity of Hsp33. The disulfide bonds are reduced upon return to nonstress conditions, and client proteins are transferred to the ATP-dependent DnaK/DnaJ/GrpE system for refolding.
FIG 3
FIG 3
N-chlorination turns RidA and CnoX into powerful chaperone holdases. Similarly to Hsp33, CnoX (upper panel) and RidA (lower panel) serve as powerful chaperone holdases in E. coli that prevent unfolded proteins from aggregation during HOCl stress. HOCl-mediated N-chlorination increases the surface hydrophobicity of both proteins and is required for activation of their chaperone function. N-chlorination is reversible, and at least RidA forms oligomers when present in its chlorinated form (lower panel). Once nonstress conditions are restored, CnoX (upper panel) transfers the substrate proteins to ATP-dependent chaperone foldase systems such as DnaK/DnaJ/GrpE or GroEL/ES and initiates refolding of the substrate proteins. Moreover, CnoX protects the substrate from overoxidation through the formation of a mixed disulfide. While reduced Trx-SH or GSH can reverse the chlorination of RidA (lower panel), the foldase system(s) required for refolding of the client proteins has not yet been identified.
FIG 4
FIG 4
Conversion of ATP into protein-scaffold polyphosphate. Under HOCl stress conditions, bacteria convert significant amounts of ATP into long chains of polyphosphate (polyP), a reaction catalyzed by the bacterium-specific enzyme polyP kinase (PPK). The reversible oxidative inactivation of the polyP-degrading enzyme exopolyphosphatase (PPX) further contributes to the accumulation of polyP in the cell. PolyP functions as a protein-stabilizing scaffold that binds protein unfolding intermediates, stabilizes them in a soluble β-sheet-rich conformation, and effectively prevents protein aggregation both in vitro and in vivo. Once nonstress conditions are restored, chaperone foldases such as the DnaK/DnaJ/GrpE system take over to refold polyP-bound client proteins.
FIG 5
FIG 5
HOCl sensing affects biofilm formation and chemotaxis in pathogenic bacteria. (Upper panel) Sublethal concentrations of HOCl turn planktonic P. aeruginosa into sessile biofilms. Upon exposure to HOCl, transcription of the P. aeruginosa PA3177 gene encoding a diguanylate cyclase is induced via an as-yet-unknown regulatory pathway. This results in elevated c-di-GMP levels (black arrow) and subsequent induction of exopolysaccharide Pel and Psl production (dotted arrow). The increased hydrophobicity triggers the surface attachment and ultimately results in the switch from planktonic growth to nonmotile biofilm formation. (Lower panel) Cytosolic transducer-like protein D (TlpD) serves as a redox sensor in H. pylori to translate changes in HOCl concentrations. In the absence of HOCl, TlpD’s conserved cysteine in the chemoreceptor zinc-binding (CZB) domain is reduced and coordinates a Zn2+ ion. In its reduced state, TlpD induces the autophosphorylation of chemotaxis phosphorelation system CheA/CheY. CheY-Pi directly interacts with the flagellar rotor (labeled yellow), resulting in a temporary reversal of flagellar rotation (as indicated by arrows). In the presence of HOCl, the conserved cysteine is oxidized, Zn2+ is released, and the signaling is reversibly inactivated, resulting in a smooth swimming behavior (chemoattraction).
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