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Midecamycin Is Inactivated by Several Different Sugar Moieties at Its Inactivation Site.

ABSTRACT: Glycosylation inactivation is one of the important macrolide resistance mechanisms. The accumulated evidences attributed glycosylation inactivation to a glucosylation modification at the inactivation sites of macrolides. Whether other glycosylation modifications lead to macrolides inactivation is unclear. Herein, we demonstrated that varied glycosylation modifications could cause inactivation of midecamycin, a 16-membered macrolide antibiotic used clinically and agriculturally. Specifically, an actinomycetic glycosyltransferase (GT) OleD was selected for its glycodiversification capacity towards midecamycin. OleD was demonstrated to recognize UDP-D-glucose, UDP-D-xylose, UDP-galactose, UDP-rhamnose and UDP-N-acetylglucosamine to yield corresponding midecamycin 2'-O-glycosides, most of which displayed low yields. Protein engineering of OleD was thus performed to improve its conversions towards sugar donors. Q327F was the most favorable variant with seven times the conversion enhancement towards UDP-N-acetylglucosamine. Likewise, Q327A exhibited 30% conversion enhancement towards UDP-D-xylose. Potent biocatalysts for midecamycin glycosylation were thus obtained through protein engineering. Wild OleD, Q327F and Q327A were used as biocatalysts for scale-up preparation of midecamycin 2'-O-glucopyranoside, midecamycin 2'-O-GlcNAc and midecamycin 2'-O-xylopyranoside. In contrast to midecamycin, these midecamycin 2'-O-glycosides displayed no antimicrobial activities. These evidences suggested that besides glucosylation, other glycosylation patterns also could inactivate midecamycin, providing a new inactivation mechanism for midecamycin resistance. Cumulatively, glycosylation inactivation of midecamycin was independent of the type of attached sugar moieties at its inactivation site.

SUBMITTER: Lin R

PROVIDER: S-EPMC8657839 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Several successful pathogens have evolved mechanisms to evade host defense, resulting in the establishment of persistent and chronic infections. One such pathogen, Porphyromonas gingivalis, induces chronic low-grade inflammation associated with local inflammatory bone loss and systemic inflammation manifested as atherosclerosis. P. gingivalis expresses an atypical lipopolysaccharide (LPS) structure containing heterogeneous lipid A species, that exhibit Toll-like receptor-4 (TLR4) agonist or antagonist activity, or are non-activating at TLR4. In this study, we utilized a series of P. gingivalis lipid A mutants to demonstrate that antagonistic lipid A structures enable the pathogen to evade TLR4-mediated bactericidal activity in macrophages resulting in systemic inflammation. Production of antagonistic lipid A was associated with the induction of low levels of TLR4-dependent proinflammatory mediators, failed activation of the inflammasome and increased bacterial survival in macrophages. Oral infection of ApoE(-/-) mice with the P. gingivalis strain expressing antagonistic lipid A resulted in vascular inflammation, macrophage accumulation and atherosclerosis progression. In contrast, a P. gingivalis strain producing exclusively agonistic lipid A augmented levels of proinflammatory mediators and activated the inflammasome in a caspase-11-dependent manner, resulting in host cell lysis and decreased bacterial survival. ApoE(-/-) mice infected with this strain exhibited diminished vascular inflammation, macrophage accumulation, and atherosclerosis progression. Notably, the ability of P. gingivalis to induce local inflammatory bone loss was independent of lipid A expression, indicative of distinct mechanisms for induction of local versus systemic inflammation by this pathogen. Collectively, our results point to a pivotal role for activation of the non-canonical inflammasome in P. gingivalis infection and demonstrate that P. gingivalis evades immune detection at TLR4 facilitating chronic inflammation in the vasculature. These studies support the emerging concept that pathogen-mediated chronic inflammatory disorders result from specific pathogen-mediated evasion strategies resulting in low-grade chronic inflammation.

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Midecamycin Is Inactivated by Several Different Sugar Moieties at Its Inactivation Site.

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