The Lysozyme Inhibitor Thionine Acetate Is Also an Inhibitor of the Soluble Lytic Transglycosylase Slt35 from Escherichia coli

Lytic transglycosylases such as Slt35 from E. coli are enzymes involved in bacterial cell wall remodelling and recycling, which represent potential targets for novel antibacterial agents. Here, we investigated a series of known glycosidase inhibitors for their ability to inhibit Slt35. While glycosidase inhibitors such as 1-deoxynojirimycin, castanospermine, thiamet G and miglitol had no effect, the phenothiazinium dye thionine acetate was found to be a weak inhibitor. IC50 values and binding constants for thionine acetate were similar for Slt35 and the hen egg white lysozyme. Molecular docking simulations suggest that thionine binds to the active site of both Slt35 and lysozyme, although it does not make direct interactions with the side-chain of the catalytic Asp and Glu residues as might be expected based on other inhibitors. Thionine acetate also increased the potency of the beta-lactam antibiotic ampicillin against a laboratory strain of E. coli.

Effect of Lipidation on the Localization and Activity of a Lysozyme Inhibitor in Neisseria gonorrhoeae

ABSTRACT The Gram-negative pathogen Neisseria gonorrhoeae (gonococcus [Gc]) colonizes lysozyme-rich mucosal surfaces. Lysozyme hydrolyzes peptidoglycan, leading to bacterial lysis. Gc expresses two proteins, SliC and NgACP, that bind and inhibit the enzymatic activity of lysozyme. SliC is a surface-exposed lipoprotein, while NgACP is found in the periplasm and also released extracellularly. Purified SliC and NgACP similarly inhibit lysozyme. However, whereas mutation of ngACP increases Gc susceptibility to lysozyme, the sliC mutant is only susceptible to lysozyme when ngACP is inactivated. In this work, we examined how lipidation contributes to SliC expression, cellular localization, and resistance of Gc to killing by lysozyme. To do so, we mutated the conserved cysteine residue (C18) in the N-terminal lipobox motif of SliC, the site for lipid anchor attachment, to alanine. SliC(C18A) localized to soluble rather than membrane fractions in Gc and was not displayed on the bacterial surface. Less SliC(C18A) was detected in Gc lysates compared to the wild-type protein. This was due in part to some release of the C18A mutant, but not wild-type, protein into the extracellular space. Surprisingly, Gc expressing SliC(C18A) survived better than SliC (wild type)-expressing Gc after exposure to lysozyme. We conclude that lipidation is not required for the ability of SliC to inhibit lysozyme, even though the lipidated cysteine is 100% conserved in Gc SliC alleles. These findings shed light on how members of the growing family of lysozyme inhibitors with distinct subcellular localizations contribute to bacterial defense against lysozyme. IMPORTANCE Neisseria gonorrhoeae is one of many bacterial species that express multiple lysozyme inhibitors. It is unclear how inhibitors that differ in their subcellular localization contribute to defense from lysozyme. We investigated how lipidation of SliC, an MliC (membrane-bound lysozyme inhibitor of c-type lysozyme)-type inhibitor, contributes to its localization and lysozyme inhibitory activity. We found that lipidation was required for surface exposure of SliC and yet was dispensable for protecting the gonococcus from killing by lysozyme. To our knowledge, this is the first time the role of lipid anchoring of a lysozyme inhibitor has been investigated. These results help us understand how different lysozyme inhibitors are localized in bacteria and how this impacts resistance to lysozyme.

The structure of the proteinaceous inhibitor PliI fromAeromonas hydrophilain complex with its target lysozyme

Recent microbiological data have revealed that Gram-negative bacteria are able to protect themselves against the lytic action of host lysozymes by secreting proteinaceous inhibitors. Four distinct classes of such inhibitors have been discovered that specifically act against c-type, g-type and i-type lysozymes. Here, the 1.24 Å resolution crystal structure of the periplasmic i-type lysozyme inhibitor fromAeromonas hydrophila(PliI-Ah) in complex with the i-type lysozyme fromMeretrix lusoriais reported. The structure is the first to explain the inhibitory mechanism of the PliI family at the atomic level. A distinct `ridge' formed by three exposed PliI loops inserts into the substrate-binding groove of the lysozyme, resulting in a complementary `key–lock' interface. The interface is principally stabilized by the interactions made by the PliI-Ah residues Ser104 and Tyr107 belonging to the conserved SGxY motif, as well as by the other conserved residues Ser46 and Asp76. The functional importance of these residues is confirmed by inhibition assays with the corresponding point mutants of PliI-Ah. The accumulated structural data on lysozyme–inhibitor complexes from several classes indicate that in all cases an extensive interface of either a single or a double `key–lock' type is formed, resulting in highly efficient inhibition. These data provide a basis for the rational development of a new class of antibacterial drugs.

Edwardsiella tarda MliC, a Lysozyme Inhibitor That Participates in Pathogenesis in a Manner That Parallels Ivy

Edwardsiella tarda, a bacterial pathogen to farmed fish as well as humans, possesses the genes of two lysozyme inhibitors,ivyandmliC(ivyEtandmliCEt). We recently studied IvyEtand found it to be implicated inE. tardavirulence. In the present study, we characterized MliCEtin comparison with IvyEtin a turbot model. MliCEtcontains the FWSKG motif and two cysteines (C33 and C98) that are highly conserved in subgroup 1 MliCs but are of unknown functional importance. To examine the essentialness of these conserved structural features, recombinant MliCEt(rMliC) and its mutants bearing C33S and W79A (of the FWSKG motif) substitutions were prepared. Subsequent analysis showed that rMliC (i) inhibited lysozyme-induced lysis of a Gram-positive bacterium, (ii) reduced serum-facilitated lysozyme killing ofE. tarda, and (iii) when introduced into turbot, promoted bacterial dissemination in fish tissues. The C33S mutation had no influence on the activity of rMliC, while the W79A mutation slightly but significantly enhanced the activity of rMliC. Knockout strains of eithermliCEtorivyEtwere severely attenuated for the ability of tissue invasion, host lethality, serum survival, and intracellular replication. The lost virulence of themliCtransformant (TXΔmliC) was restored by complementation with an introducedmliCEtgene. Compared to the ΔivyEtor ΔmliCEtsingle-knockout strains, the ΔmliCEtΔivyEtdouble-knockout strain was significantly impaired in most of the virulence features. Together, these results provide the first evidence that the conserved cysteine is functionally dispensable to a subgroup 1 MliC and that as a virulence factor, MliCEtmost likely works in a concerted and parallel manner with Ivy.

Edwardsiella tarda Ivy, a Lysozyme Inhibitor That Blocks the Lytic Effect of Lysozyme and Facilitates Host Infection in a Manner That Is Dependent on the Conserved Cysteine Residue

ABSTRACTEdwardsiella tardais a Gram-negative bacterial pathogen with a broad host range that includes fish and humans. In this study, we examined the activity and function of the lysozyme inhibitor Ivy (named IvyEt) identified in the pathogenicE. tardastrain TX01. IvyEtpossesses the Ivy signature motif CKPHDC in the form of82CQPHNC87and contains several highly conserved residues, including a tryptophan (W55). For the purpose of virulence analysis, an isogenic TX01 mutant, TXivy, was created. TXivy bears an in-frame deletion of theivyEtgene. A live infection study in a turbot (Scophthalmus maximus) model showed that, compared to TX01, TXivy exhibited attenuated overall virulence, reduced tissue dissemination and colonization capacity, an impaired ability to replicate in host macrophages, and decreased resistance against the bactericidal effect of host serum. To facilitate functional analysis, recombinant IvyEt(rIvy) and three mutant proteins, i.e., rIvyW55A, rIvyC82S, and rIvyH85D, which bear Ala, Ser, and Asp substitutions at W55, C82, and H85, respectively, were prepared.In vitrostudies showed that rIvy, rIvyW55A, and rIvyH85D were able to block the lytic effect of lysozyme on a Gram-positive bacterium, whereas rIvyC82S could not do so. Likewise, rIvy, but not rIvyC82S, inhibited the serum-facilitated killing effect of lysozyme onE. tarda.In vivoanalysis showed that rIvy, but not rIvyC82S, restored the lost pathogenicity of TXivy and enhanced the infectivity of TX01. Together these results indicate that IvyEtis a lysozyme inhibitor and a virulence factor that depends on the conserved C82 for biological activity.