More Like this

1. Surface Hydrophilicity Promotes Bacterial Twitching Motility

   https://doi.org/10.1101/2024.03.23.586342  

   O’Hara, Megan T; Shimozono, Tori M; Dye, Keane J; Harris, David; Yang, Zhaomin (March 2024, bioRxiv)

   Abstract Twitching motility is a form of bacterial surface translocation powered by the type IV pilus (T4P). It is frequently analyzed by interstitial colony expansion between agar and the polystyrene surfaces of Petri dishes. In such assays, the twitching motility of Acinetobacter nosocomialis was observed with MacConkey but not Luria-Bertani (LB) agar media. One difference between these two media is the presence of bile salts as a selective agent in MacConkey but not in LB. Here, we demonstrate that the addition of bile salts to LB allowed A. nosocomialis to display twitching. Similarly, bile salts enhanced the twitching of Acinetobacter baumannii and Pseudomonas aeruginosa in LB. These observations suggest that there is a common mechanism whereby bile salts enhance bacterial twitching and promote interstitial colony expansion. Bile salts disrupt lipid membranes and apply envelope stress as detergents. Surprisingly, their stimulatory effect on twitching appears not to be related to a bacterial physiological response to stressors. Rather it is due to their ability to alter the physicochemical properties of a twitching surface. We observed that while other detergents promoted twitching like bile salts, stresses applied by antibiotics, including the outer membrane-targeting polymyxin B, did not enhanced twitching motility. More importantly, bacteria displayed increased twitching on hydrophilic surfaces such as those of glass and tissue culture-treated polystyrene plastics, and bile salts no longer stimulated twitching on these surfaces. Together, our results show that altering the hydrophilicity of a twitching surface significantly impacts T4P functionality. 

   more » « less
2. Discovery of Two Inhibitors of the Type IV Pilus Assembly ATPase PilB as Potential Antivirulence Compounds

   https://doi.org/10.1128/spectrum.03877-22  

   Dye, Keane J.; Vogelaar, Nancy J.; O’Hara, Megan; Sobrado, Pablo; Santos, Webster; Carlier, Paul R.; Yang, Zhaomin; Lostroh, Phoebe (December 2022, Microbiology Spectrum)

   Visca, Paolo (Ed.)

   ABSTRACT 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. IMPORTANCE 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. Here, we report the development and implementation of a method to identify anti-T4P chemicals from compound libraries by high-throughput screen. This led to the identification and validation of two T4P inhibitors both in the test tubes and in bacteria. The discovery and validation pipeline reported here as well as the confirmation of two anti-T4P inhibitors provide new venues and leads for the development of chemotherapeutics against antibiotic-resistant infections. 

   more » « less
3. 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. 

   more » « less
4. 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. 

   more » « less
5. Removal of Pseudomonas type IV pili by a small RNA virus

   https://doi.org/10.1126/science.adl0635  

   Thongchol, Jirapat; Yu, Zihao; Harb, Laith; Lin, Yiruo; Koch, Matthias; Theodore, Matthew; Narsaria, Utkarsh; Shaevitz, Joshua; Gitai, Zemer; Wu, Yinghao; et al (April 2024, Science)

   The retractile type IV pilus (T4P) is important for virulence of the opportunistic human pathogenPseudomonas aeruginosa. The single-stranded RNA (ssRNA) phage PP7 binds to T4P and is brought to the cell surface through pilus retraction. Using fluorescence microscopy, we discovered that PP7 detaches T4P, which impairs cell motility and restricts the pathogen’s virulence. Using cryo–electron microscopy, mutagenesis, optical trapping, and Langevin dynamics simulation, we resolved the structure of PP7, T4P, and the PP7/T4P complex and showed that T4P detachment is driven by the affinity between the phage maturation protein and its bound pilin, plus the pilus retraction force and speed, and pilus bending. Pilus detachment may be widespread among other ssRNA phages and their retractile pilus systems and offers new prospects for antibacterial prophylaxis and therapeutics. 

   more » « less