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1. Discovery of Two Inhibitors of the Type IV Pilus Assembly ATPase PilB as Potential Antivirulence Compounds

   Keane J Dye, Nancy J (December 2022, Microbiology spectrum)

   Paolo Visca (Ed.)

   With the pressing antibiotic resistance pandemic, antivirulence has been increasingly explored as an alternative strategy against bacterial infections. The bacterial type IV pilus (T4P) is a well-documented virulence factor and an attractive target for small molecules for antivirulence purposes. The PilB ATPase is essential for T4P biogenesis because it catalyzes the assembly of monomeric pilins into the polymeric pilus filament. Here, we describe the identification of two PilB inhibitors by a high-throughput screen (HTS) in vitro and their validation as effective inhibitors of T4P assembly in vivo. We used Chloracidobacterium thermophilum PilB as a model enzyme to optimize an ATPase assay for the HTS. From a library of 2,320 compounds, benserazide and levodopa, two approved drugs for Parkinson’s disease, were identified and confirmed biochemically to be PilB inhibitors. We demonstrate that both compounds inhibited the T4P-dependent motility of the bacteria Myxoccocus xanthus and Acinetobacter nosocomialis. Additionally, benserazide and levodopa were shown to inhibit A. nosocomialis biofilm formation, a T4P-dependent process. Using M. xanthus as a model, we showed that both compounds inhibited T4P assembly in a dose-dependent manner. These results suggest that these two compounds are effective against the PilB protein in vivo. The potency of benserazide and levodopa as PilB inhibitors both in vitro and in vivo demonstrate potentials of the HTS and its two hits here for the development of anti-T4P chemotherapeutics. 

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

   https://doi.org/10.1128/mSphere.00129-21  

   Dye, Keane J.; Vogelaar, Nancy J.; Sobrado, Pablo; Yang, Zhaomin (April 2021, mSphere)

   Dunman, Paul (Ed.)

   ABSTRACT 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 ( Ct PilB) had been demonstrated to have robust ATPase activity and the ability to bind its expected ligands in vitro. We utilized Ct PilB 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. 

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3. A Phage‐Based Approach to Identify Antivirulence Inhibitors of Bacterial Type IV Pili

   https://doi.org/10.1111/1751-7915.70081  

   Shimozono, Tori_M; Vogelaar, Nancy_J; O'Hara, Megan_T; Yang, Zhaomin (January 2025, Microbial Biotechnology)

   ABSTRACT The increasing threat of antibiotic resistance underscores the urgent need for innovative strategies to combat infectious diseases, including the development of antivirulants. Microbial pathogens rely on their virulence factors to initiate and sustain infections. Antivirulants are small molecules designed to target virulence factors, thereby attenuating the virulence of infectious microbes. The bacterial type IV pilus (T4P), an extracellular protein filament that depends on the T4P machinery (T4PM) for its biogenesis, dynamics and function, is a key virulence factor in many significant bacterial pathogens. While the T4PM presents a promising antivirulence target, the systematic identification of inhibitors for its multiple protein constituents remains a considerable challenge. Here we report a novel high‐throughput screening (HTS) approach for discovering T4P inhibitors. It usesPseudomonas aeruginosa,a high‐priority pathogen, in combination with its T4P‐targeting phage, φKMV. Screening of a library of 2168 compounds using an optimised protocol led to the identification of tuspetinib, based on its deterrence of the lysis ofP. aeruginosaby φKMV. Our findings show that tuspetinib also inhibits two additional T4P‐targeting phages, while having no effect on a phage that recognises lipopolysaccharides as its receptor. Additionally, tuspetinib impedes T4P‐mediated motility inP. aeruginosaandAcinetobacterspecies without impacting growth or flagellar motility. This bacterium‐phage pairing approach is applicable to a broad range of virulence factors that are required for phage infection, paving ways for the development of advanced chemotherapeutics against antibiotic‐resistant infections. 

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4. Myxococcus xanthus PilB interacts with c-di-GMP and modulates motility and biofilm formation

   https://doi.org/10.1128/jb.00221-23  

   Dye, Keane J; Salar, Safoura; Allen, Uvina; Smith, Wraylyn; Yang, Zhaomin (September 2023, Journal of Bacteriology)

   Galperin, Michael Y (Ed.)

   ABSTRACT The regulation of biofilm and motile states as alternate bacterial lifestyles has been studied extensively in flagellated bacteria, where the second messenger cyclic-di-GMP (cdG) plays a crucial role. However, much less is known about the mechanisms of such regulation in motile bacteria without flagella. The bacterial type IV pilus (T4P) serves as a motility apparatus that enables Myxococcus xanthus to move on solid surfaces. PilB, the T4P assembly ATPase, is, therefore, required for T4P-dependent motility in M. xanthus. Interestingly, T4P is also involved in the regulation of exopolysaccharide as the biofilm matrix material in this bacterium. A newly discovered cdG-binding domain, MshE_N, is conserved in the N-terminus of PilB (PilB_N) in M. xanthus and other bacteria. This suggests that cdG may bind to PilB to control the respective outputs that regulate biofilm development and T4P-powered motility. In this study, we aimed to validate M. xanthus PilB as a cdG effector protein. We performed a systematic mutational analysis of its cdG-binding domain to investigate its relationship with motility, piliation, and biofilm formation. Excluding those resulting in low levels of PilB protein, all other substitution mutations in PilB_N resulted in pilB mutants with distinct and differential phenotypes in piliation and biofilm levels in M. xanthus. This suggests that the PilB_N domain plays dual roles in modulating motility and biofilm levels, and these two functions of PilB can be dependent on and independent of each other in M. xanthus. 

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5. Identification of small molecule inhibitors of the Chloracidobacterium thermophilum type IV pilus protein PilB by ensemble virtual screening

   https://doi.org/10.1016/j.abb.2024.110127  

   McDonald-Ramos, Jay S; Hicklin, Ian K; Yang, Zhaomin; Brown, Anne M (October 2024, Archives of Biochemistry and Biophysics)

   Antivirulence strategy has been explored as an alternative to traditional antibiotic development. The bacterial type IV pilus is a virulence factor involved in host invasion and colonization in many antibiotic resistant pathogens. The PilB ATPase hydrolyzes ATP to drive the assembly of the pilus filament from pilin subunits. We evaluated Chloracidobacterium thermophilum PilB (CtPilB) as a model for structure-based virtual screening by molecular docking and molecular dynamics (MD) simulations. A hexameric structure of CtPilB was generated through homology modeling based on an existing crystal structure of a PilB from Geobacter metallireducens. Four representative structures were obtained from molecular dynamics simulations to examine the conformational plasticity of PilB and improve docking analyses by ensemble docking. Structural analyses after 1 μs of simulation revealed conformational changes in individual PilB subunits are dependent on ligand presence. Further, ensemble virtual screening of a library of 4234 compounds retrieved from the ZINC15 database identified five promising PilB inhibitors. Molecular docking and binding analyses using the four representative structures from MD simulations revealed that top-ranked compounds interact with multiple Walker A residues, one Asp-box residue, and one arginine finger, indicating these are key residues in inhibitor binding within the ATP binding pocket. The use of multiple conformations in molecular screening can provide greater insight into compound flexibility within receptor sites and better inform future drug development for therapeutics targeting the type IV pilus assembly ATPase. 

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