Crippling the bacterial cell wall molecular machinery

Abstract

34Lytic transglycosylases (LT) are redundant enzymes that play a critical role in 35 peptidoglycan (PG) recycling and metabolism. LT(s) role in cell wall-modifying 36 complexes and usefulness as antimicrobial drug targets remain elusive. We determined at 37 high-resolution a structure of the membrane-bound homolog of the soluble LT from 38 Neisseria species with a disordered active site helix (alpha helix 30). Alpha helix 30 is 39 crucial for binding PG during catalysis 1 . Here we show using an alpha helix 30 deletion 40 strain that LT (LtgA) determines the integrity of the cell wall, participates in cell division 41 and separation, and can be manipulated to impair the fitness of the human pathogen 42 Neisseria meningitidis during infection. Characterization of ltgA helix deleted strain 43 interactome identified glycan chain remodeling enzymes whose function appear to be 44 modulated by LTs. Targeting LTs can disrupt the PG machinery, which is fatal for the 45 bacterium, a new approach for antibiotic development. 46 47 65 alpha helices (α 28, 29, 30, 31, 32, 33, 34, 35, 36, 37), with a six-alpha-helix bundle (α 66 29, 30, 31, 32, 33, 34) constituting the core of the active site that firmly secures the 67 glycan chain ( Fig. 1a). 68 LTs utilize a single catalytic residue, either a glutamate or aspartate, which plays 69 the role of an acid and then that of a base 7-11 . In our recent study, active LtgA was 70 monitored for the first time in the crystalline state, and the residues involved in the 71 substrate and product formation steps were identified. Globally, conformational changes 72 occurred in three domains, the U, C and L domains, between native LtgA and LtgA 73 bound to the product 5 . Substantial conformational changes were observed in the active 74 site, for example, during the product formation step, the active site adopted a more open 75 conformation 5 . 76 Many Gram-negative bacteria have multiple and redundant LTs; for example, 77 Escherichia coli has eight (MltA, MltB, MltC, MltD, MltE, MltF, MltG and Slt70), and 78 4 Neisseria species encodes 5 (LtgA, LtgB, LtgC, LtgD, and LtgE), suggesting that LT 79 activity is vital to the life cycle of the cell 12 . Because the activity of LTs is redundant, the 80 loss of one or more LTs in E. coli leads to no observable growth defects. When genes for 81 6 LTs were deleted from E. coli, a mild chaining phenotype was observed 13 . However, 82 despite lack of strong observable phenotypic changes, it has been suggested that LTs may 83 have well-defined roles in the cell. For example, the deletion of ltgA and ltgD in 84 Neisseria gonorrhoeae eliminates the release of cytotoxic PG monomers suggesting the 85 activity of LtgA and LtgD are redundant. Moreover, LtgA primarily localizes at the 86 septum, indicating a role in the divisome machinery; whereas, LtgD is distributed along 87 the entire cell surface 14 . 88 The activities of LTs are known to be inhibited by β-hexosaminidase inhibitors 89 (for example, NAG-thiazoline); bulgecins A, B and C;...

Lytic transglycosylases RlpA and MltC assist in Vibrio cholerae daughter cell separation

“…RlpA is required under low‐salt conditions, but not during growth in standard laboratory media, suggesting P. aeruginosa encodes at least one redundant septal LTG (Jorgenson et al , ). Salmonella enterica similarly appears to require LTGs MltC and MltE for proper daughter cell separation in low‐salt conditions (Monteiro et al ., ) and in Neisseria gonorrhoeae , mutating the LTGs LtgA or LtgC result in daughter cell separation defects (Cloud and Dillard, ; Williams et al , ). Thus, LTGs appear to play important and often redundant, but poorly understood roles in septal cleavage in diverse bacteria.…”