σP-NagA-L1/L2 Regulatory Circuit Involved in ΔompA299-356-Mediated Increase in β-Lactam Susceptibility in Stenotrophomonas maltophilia

OmpA, the most abundant porin in Stenotrophomonas maltophilia KJ, exists as a two-domain structure with an N-terminal domain of beta-barrel structure embedded in the outer membrane and a C-terminal domain collocated in the periplasm. KJ Delta OmpA(299-356), an ompA mutant of S. maltophilia KJ with a truncated OmpA devoid of 299 to 356 amino acids (aa), was able to stably embed in the outer membrane. KJ Delta OmpA(299-356) was more susceptible to beta-lactams than wild-type KJ. We aimed to elucidate the mechanism underlying the Delta ompA(299-356)-mediated increase in beta-lactam susceptibility (abbreviated as Delta OmpA(299-356) phenotype). KJ Delta OmpA(299-356) displayed a lower ceftazidime (CAZ)-induced beta-lactamase activity than KJ. Furthermore, KJ2, a L1/L2 beta-lactamases-null mutant, and KJ2 Delta OmpA(299-356), a KJ2 mutant with truncated OmpA devoid of299 to 356 aa, had comparable beta-lactam susceptibility. Both lines of evidence indicate that decreased beta-lactamase activity contributes to the Delta OmpA(299-356) phenotype. We analyzed the transcriptome results of KJ and KJ Delta OmpA(299-356), focusing on PG homeostasis-associated genes. Among the 36 genes analyzed, the nagA gene was upregulated 4.65-fold in KJ Delta OmpA(299-356). Deletion of the nagA gene from the chromosome of KJ Delta OmpA(299-356) restored beta-lactam susceptibility and CAZ-induced beta-lactamase activity to wild-type levels, verifying that nagA-upregulation in KJ Delta OmpA(299-356) contributes to the Delta OmpA(299-356) phenotype. Furthermore, transcriptome analysis revealed that rpoE (Smlt3555) and rpoP (Smlt3514) were significantly upregulated in KJ Delta OmpA(299-356). The deletion mutant construction, beta-lactam susceptibility, and beta-lactamase activity analysis demonstrated that sigma(P), but not sigma(E), was involved in the Delta OmpA(299-356) phenotype. A real-time quantitative (qRT-PCR) assay confirmed that nagA is a member of the sigma(P) regulon. The involvement of the sigma(P)-NagA-L1/L2 regulatory circuit in the Delta OmpA(299-356) phenotype was manifested.IMPORTANCE Porins of Gram-negative bacteria generally act as channels that allow the entry or extrusion of molecules. Moreover, the structural role of porins in stabilizing the outer membrane by interacting with peptidoglycan (PG) and the outer membrane has been proposed. The linkage between porin deficiency and antibiotic resistance increase has been reported widely, with a rationale for blocking antibiotic influx. In this study, a link between porin defects and beta-lactam susceptibility increase was demonstrated. The underlying mechanism revealed that a novel sigma(P)-NagA-L1/L2 regulatory circuit is triggered due to the loss of the OmpA-PG interaction. This study extends the understanding on the porin defect and antibiotic susceptibility. Porin defects may cause opposite impacts on antibiotic susceptibility, which is dependent on the involvement of the defect. Blocking the porin channel role can increase antibiotic resistance; in contrast, the loss of porin structure role may increase antibiotic susceptibility. Porins of Gram-negative bacteria generally act as channels that allow the entry or extrusion of molecules. Moreover, the structural role of porins in stabilizing the outer membrane by interacting with peptidoglycan (PG) and the outer membrane has been proposed.

Automated summary

This study reveals a link between porin defects and increased susceptibility to beta-lactam antibiotics. The mechanism involves the loss of OmpA-PG interaction, triggering a novel sigma(P)-NagA-L1/L2 regulatory circuit. This research expands our understanding of porin defects and antibiotic susceptibility.