σ(P)-NagA-L1/L2 Regulatory Circuit Involved in ΔompA(299-356)-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 β-barrel structure embedded in the outer membrane and a C-terminal domain collocated in the periplasm. KJΔOmpA(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. KJΔOmpA(299-356) was more susceptible to β-lactams than wild-type KJ. We aimed to elucidate the mechanism underlying the ΔompA(299-356)-mediated increase in β-lactam susceptibility (abbreviated as “ΔOmpA(299-356) phenotype”). KJΔOmpA(299-356) displayed a lower ceftazidime (CAZ)-induced β-lactamase activity than KJ. Furthermore, KJ2, a L1/L2 β-lactamases-null mutant, and KJ2ΔOmpA(299-356), a KJ2 mutant with truncated OmpA devoid of299 to 356 aa, had comparable β-lactam susceptibility. Both lines of evidence indicate that decreased β-lactamase activity contributes to the ΔOmpA(299-356) phenotype. We analyzed the transcriptome results of KJ and KJΔOmpA(299-356), focusing on PG homeostasis-associated genes. Among the 36 genes analyzed, the nagA gene was upregulated 4.65-fold in KJΔOmpA(299-356). Deletion of the nagA gene from the chromosome of KJΔOmpA(299-356) restored β-lactam susceptibility and CAZ-induced β-lactamase activity to wild-type levels, verifying that nagA-upregulation in KJΔOmpA(299-356) contributes to the ΔOmpA(299-356) phenotype. Furthermore, transcriptome analysis revealed that rpoE (Smlt3555) and rpoP (Smlt3514) were significantly upregulated in KJΔOmpA(299-356). The deletion mutant construction, β-lactam susceptibility, and β-lactamase activity analysis demonstrated that σ(P), but not σ(E), was involved in the ΔOmpA(299-356) phenotype. A real-time quantitative (qRT-PCR) assay confirmed that nagA is a member of the σ(P) regulon. The involvement of the σ(P)-NagA-L1/L2 regulatory circuit in the ΔOmpA(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 β-lactam susceptibility increase was demonstrated. The underlying mechanism revealed that a novel σ(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.