The 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.

PilB is the assembly ATPase for the bacterial type IV pilus (T4P), and as a consequence, it is essential for T4P-mediated bacterial motility. In some cases, PilB has been demonstrated to regulate the production of exopolysaccharide (EPS) during bacterial biofilm development independently of or in addition to its function in pilus assembly. While the ATPase activity of PilB resides at its C-terminal region, the N terminus of a subset of PilBs forms a novel cyclic-di-GMP (cdG)-binding domain. This multi-domain structure suggests that PilB binds cdG and adenine nucleotides through separate domains which may influence the functionality of PilB in both motility and biofilm development. Here, Chloracidobacterium thermophilum PilB is used to investigate ligand binding by its separate domains and by the full-length protein. Our results confirm the specificity of these individual domains for their respective ligands and demonstrate communications between these domains in the full-length protein. It is clear that when the N-and the C-terminal domains of PilB bind to cdG and ADP, respectively, they mutually influence each other in conformation and in their binding to ligands. We propose that the interactions between these domains in response to their ligands play critical roles in modulating or controlling the functions of PilB as a regulator of EPS production and as the T4P assembly ATPase.The type IV pilus machinery (T4PM) in Gram-negative bacteria is made of a dozen T4P or Pil proteins including the T4P assembly ATPase PilB (or PilF in T. thermophilus and a few others) [10,[17][18][19]. Most of these proteins form a stable supramolecular complex that spans the bacterial envelope from the cytoplasm to the outer membrane [17]. PilB is proposed to dock with this stable T4PM complex on the cytoplasmic side and hydrolyze ATP to power the assembly of pilin monomers into the base of a growing T4P filament [17,20,21]. PilB and all proteins in this stable complex are required for T4P assembly and deletions of their genes result in a non-piliated and non-motile phenotype [10,18]. The C-terminus of PilB contains the structure of an AAA+ ATPase which crystallizes as a hexamer [22][23][24][25]. Based on the hexameric structures of these ATPase domains, it is proposed that PilB utilizes a symmetric rotary mechanism to hydrolyze ATP [23][24][25][26]. That is, two ATP molecules are hydrolyzed to ADP simultaneously by two protomers at the opposite corners of the hexameric ring. Another pair adjacent to these two would hydrolyze ATP next. It is proposed that the hydrolysis of ATP by the C-terminal ATPase domain of PilB results in the movement of the N-terminal domain which is mechanically coupled to the insertion of pilins into the pilus through other components in the T4PM [22]. More recently, the full-length PilB of Chloracidobacterium thermophilum (CtPilB) was shown to be a hexamer in solution with robust ATPase activity in vitro [27]. All available evidence supports the model that the ATPase activity of PilB endowed by its C terminus ...

Many bacterial pathogens use their type IV pilus (T4P) to facilitate and maintain an infection in a human host. Small-molecule inhibitors of the production or assembly of the T4P are promising for the treatment and prevention of infections by these bacteria, especially in our fight against antibiotic-resistant pathogens.

The bacterium Myxococcus xanthus forms both developmental and vegetative types of biofilms. While the former has been studied on both agar plates and submerged surfaces, the latter has been investigated predominantly on agar surfaces as swarming colonies. Here we describe the development of a microplate-based assay for the submerged biofilms of M. xanthus under vegetative conditions. We examined the impacts of inoculation, aeration, and temperature to optimize the conditions for the assay. Aeration was observed to be critical for the effective development of submerged biofilms by M. xanthus, an obligate aerobic bacterium. In addition, temperature plays an important role in the development of M. xanthus submerged biofilms. It is well established that the formation of submerged biofilms by many bacteria requires both exopolysaccharide (EPS) and the type IV pilus (T4P). EPS constitutes part of the biofilm matrix that maintains and organizes bacterial biofilms while the T4P facilitates surface attachment as adhesins. For validation, we used our biofilm assay to examine a multitude of M. xanthus strains with various EPS and T4P phenotypes. The results indicate that the levels of EPS, but not of piliation, positively correlate with submerged biofilm formation in M. xanthus.