期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2021
卷号：118
期号：35
DOI：10.1073/pnas.2108894118
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Significance
Class A penicillin-binding proteins (aPBPs) assemble the bacterial cell wall and are the targets of penicillin and related β-lactam antibiotics. In gram-negative bacteria, the aPBPs require outer membrane lipoproteins to function. However, little is known about how these proteins promote the activity of their cognate synthases in cells. Here, we show that one of these lipoproteins, called LpoA, has a much more pronounced effect on aPBP activity in cells than anticipated from biochemical studies. It not only modulates the cross-linking of cell wall polymers but is also required for the aPBP to make the polymers in the first place. Our findings therefore provide insights into the regulation of an important class of antibiotic targets in their native cellular context.
A cell wall made of the heteropolymer peptidoglycan (PG) surrounds most bacterial cells. This essential surface layer is required to prevent lysis from internal osmotic pressure. The class A penicillin-binding proteins (aPBPs) play key roles in building the PG network. These bifunctional enzymes possess both PG glycosyltransferase (PGT) and transpeptidase (TP) activity to polymerize the wall glycans and cross-link them, respectively. In
Escherichia coli and other gram-negative bacteria, aPBP function is dependent on outer membrane lipoproteins. The lipoprotein LpoA activates PBP1a and LpoB promotes PBP1b activity. In a purified system, the major effect of LpoA on PBP1a is TP stimulation. However, the relevance of this activation to the cellular function of LpoA has remained unclear. To better understand why PBP1a requires LpoA for its activity in cells, we identified variants of PBP1a from
E. coli and
Pseudomonas aeruginosa that function in the absence of the lipoprotein. The changes resulting in LpoA bypass map to the PGT domain and the linker region between the two catalytic domains. Purification of the
E. coli variants showed that they are hyperactivated for PGT but not TP activity. Furthermore, in vivo analysis found that LpoA is necessary for the glycan synthesis activity of PBP1a in cells. Thus, our results reveal that LpoA exerts a much greater control over the cellular activity of PBP1a than previously appreciated. It not only modulates PG cross-linking but is also required for its cognate synthase to make PG glycans in the first place.
