Background: The Macrophage Infectivity Potentiator (Mip) protein is an established virulence factor in Neisseria gonorrhoeae (Ngo), and inhibitors of Mip have been shown to reduce survival in macrophages. However, there is a lack of data regarding functional redundancy within the FK506-binding protein (FKBP) family. To move Mip-targeted therapies toward the clinic, it is essential to determine whether other gonococcal FKBPs, Trigger Factor (tig; TF), and SlyD can provide "escape routes" for the pathogen and to develop compounds with improved potency.
Aims/Methods: This study aimed to advance gonococcal anti-virulence research by evaluating next-generation Mip inhibitors and defining the functional boundaries of the FKBP machinery. To assess potential pathways for bacterial resistance to our compound, we conducted a comparative phenotypic analysis of single (Ngo∆mip, Ngo∆tig, Ngo∆slyD) and double (Ngo∆tig∆slyD) deletion mutants. Simultaneously, we performed lead optimisation by screening novel small-molecule Mip inhibitors from our library for in vitro potency.
Results: Our phenotypic mapping revealed that gonococcal FKBPs occupy distinct, non-overlapping functional niches. While TF was identified as a driver of thermal proteostasis, with NgoΔtig and NgoΔtigΔslyD exhibiting an ~80% reduction in survival at 42°C, neither protein was associated with defects in host-cell interactions. Conversely, Mip was confirmed as the sole FKBP essential for intracellular macrophage survival and resistance to Cationic Antimicrobial Peptides (CAMPs). This lack of functional overlap suggests that TF and SlyD cannot compensate for the loss of Mip during host-cell infection. Building on this target validation, docking models of first-generation inhibitors guided the SAR-based development of novel chemical scaffolds that successfully maintain high in vitro potency. Treatment with 50 µM of these next-generation inhibitors during macrophage infection assays successfully replicated the Δmip phenotype, resulting in a ≥40% reduction in intracellular survival in wild-type Ngo.
Conclusions: By demonstrating that Mip is a functionally independent target required for host-cell survival, this work significantly advances the development of Mip-targeted therapies. These next-generation inhibitors represent a robust strategy to resensitise multidrug-resistant Ngo to innate immune clearance, bridging the gap between basic FKBP biology and clinical anti-virulence applications.