High-Throughput Screen for Inhibitors of the Type IV Pilus Assembly ATPase PilB.

Keane J Dye,Zhaomin Yang,Nancy J Vogelaar,Pablo Sobrado,Paul Dunman

doi:10.1128/msphere.00129-21

Abstract

ABSTRACTThe bacterial type IV pilus (T4P) is a prominent virulence factor in many significant human pathogens, some of which have become increasingly antibiotic resistant. Antivirulence chemotherapeutics are considered a promising alternative to antibiotics because they target the disease process instead of bacterial viability. However, a roadblock to the discovery of anti-T4P compounds is the lack of a high-throughput screen (HTS) that can be implemented relatively easily and economically. Here, we describe the first HTS for the identification of inhibitors specifically against the T4P assembly ATPase PilB in vitro. Chloracidobacterium thermophilum PilB (CtPilB) had been demonstrated to have robust ATPase activity and the ability to bind its expected ligands in vitro. We utilized CtPilB and MANT-ATP, a fluorescent ATP analog, to develop a binding assay and adapted it for an HTS. As a proof of principle, we performed a pilot screen with a small compound library of kinase inhibitors and identified quercetin as a PilB inhibitor in vitro. Using Myxococcus xanthus as a model bacterium, we found quercetin to reduce its T4P-dependent motility and T4P assembly in vivo. These results validated our HTS as effective in identifying PilB inhibitors. This assay may prove valuable in seeking leads for the development of antivirulence chemotherapeutics against PilB, an essential and universal component of all bacterial T4P systems.IMPORTANCE Many bacterial pathogens use their type IV pili (T4P) to facilitate and maintain infection of a human host. Small chemical compounds that inhibit the production or assembly of T4P hold promise in the treatment and prevention of infections, especially in the era of increasing threats from antibiotic-resistant bacteria. However, few chemicals are known to have inhibitory or anti-T4P activity. Their identification has not been easy due to the lack of a method for the screening of compound collections or libraries on a large scale. Here, we report the development of an assay that can be scaled up to screen compound libraries for inhibitors of a critical T4P assembly protein. We further demonstrate that it is feasible to use whole cells to examine potential inhibitors for their activity against T4P assembly in a bacterium.

Highlights

The bacterial type IV pilus (T4P) is a prominent virulence factor in many significant human pathogens, some of which have become increasingly antibiotic resistant
We examined the binding between Chloracidobacterium thermophilum PilB (CtPilB) and MANT-ATP as described in Materials and Methods (M&M)
A binding isotherm fitted to this data set produced a dissociation constant (KD) of 0.29 6 0.01 mM for the binding of these two partners. These results indicate that the fluorescence of MANT-ATP can be used to analyze its binding with CtPilB

Summary

Introduction

The bacterial type IV pilus (T4P) is a prominent virulence factor in many significant human pathogens, some of which have become increasingly antibiotic resistant. Using Myxococcus xanthus as a model bacterium, we found quercetin to reduce its T4P-dependent motility and T4P assembly in vivo These results validated our HTS as effective in identifying PilB inhibitors. The T4P facilitates the adherence of a bacterium to the surface of host cells or a medical device to initiate infections [6,7,8] In some cases, this leads to the development of biofilms, which can. The recurrent cycles of T4P extension and retraction catalyzed by PilB and PilT can result in motility in bacteria such as Neisseria, P. aeruginosa, and Myxococcus xanthus [22,23,24] This form of motility is known as bacterial twitching or M. xanthus social (S) gliding [23, 25]. The convergence of their effects on the T4P strongly substantiated the T4PM as a valuable target for the development of antivirulence chemotherapeutics [5]

Methods

Results

Conclusion