Abstract
Lytic transglycosylases (LT) are enzymes involved in peptidoglycan (PG) remodeling. However, their contribution to cell-wall-modifying complexes and their potential as antimicrobial drug targets remains unclear. Here, we determined a high-resolution structure of the LT, an outer membrane lipoprotein from Neisseria species with a disordered active site helix (alpha helix 30). We show that deletion of the conserved alpha-helix 30 interferes with the integrity of the cell wall, disrupts cell division, cell separation, and impairs the fitness of the human pathogen Neisseria meningitidis during infection. Additionally, deletion of alpha-helix 30 results in hyperacetylated PG, suggesting this LtgA variant affects the function of the PG de-O-acetylase (Ape 1). Our study revealed that Ape 1 requires LtgA for optimal function, demonstrating that LTs can modulate the activity of their protein-binding partner. We show that targeting specific domains in LTs can be lethal, which opens the possibility that LTs are useful drug-targets.
Highlights
Lytic transglycosylases (LTs) degrade peptidoglycan (PG) to produce N-acetylglucosamine (GlcNAc)À1,6-anhydro-N-acetylmuramic acid (MurNAc)-peptide (G-anhM-peptide), a key cytotoxic elicitor of harmful innate immune responses (Viala et al, 2004)
Consistent with the structural data showing the role of alpha helix 30 in substrate/product binding, a heterologously expressed and purified LtgAD30 showed severely diminished PG-binding capabilities when compared to wild-type LtgA or mutants of residues involved in the catalytic mechanism or substrate binding (E481A, E580) of LtgA (Figure 1—figure supplement 2)
Given the important structural role of LtgA alpha helix 30, we investigated its functional role in vivo by engineering the following constructs in N. meningitidis: i) an LtgA knockout strain (DltgA), ii) a knockout strain complemented at an ectopic locus on the meningococcal chromosome with the wild-type gene, or iii) complemented with alpha helix 30 deletion
Summary
Lytic transglycosylases (LTs) degrade peptidoglycan (PG) to produce N-acetylglucosamine (GlcNAc)À1,6-anhydro-N-acetylmuramic acid (MurNAc)-peptide (G-anhM-peptide), a key cytotoxic elicitor of harmful innate immune responses (Viala et al, 2004). LTs have been classified into four distinct families based on sequence similarities and consensus sequences. LTs belonging to family 1 of the glycoside hydrolase (GH) family 23 share sequence similarity with the goose-type lysozyme (Blackburn and Clarke, 2001). 1 can be further subdivided into five subfamilies, 1A through E, which are all structurally distinct (Blackburn and Clarke, 2001). Despite the overall structural differences among LTs, their active sites, enzymatic activities and substrate specificities are fairly well conserved
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