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1. 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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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. 

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3. Identification of a hormogonium polysaccharide‐specific gene set conserved in filamentous cyanobacteria

   https://doi.org/10.1111/mmi.14566  

   Zuniga, Esthefani G. ; Boateng, Kelvin K. A. ; Bui, Nhi U. ; Kurnfuli, Shadi ; Muthana, Sawsan M. ; Risser, Douglas D. ( July 2020 , Molecular Microbiology)

   Cyanobacteria comprise a phylum defined by the capacity for oxygenic photosynthesis. Members of this phylum are frequently motile as well. Strains that display gliding or twitching motility across semisolid surfaces are powered by a conserved type IV pilus system (T4P). Among the filamentous, heterocyst‐forming cyanobacteria, motility is usually confined to specialized filaments known as hormogonia, and requires the deposition of an associated hormogonium polysaccharide (HPS). The genes involved in assembly and export of HPS are largely undefined, and it has been hypothesized that HPS exits the outer membrane via an atypical T4P‐driven mechanism. Here, several novelhpsloci, primarily encoding glycosyl transferases, are identified. Mutational analysis demonstrates that the majority of these genes are essential for both motility and production of HPS. Notably, most mutant strains accumulate wild‐type cellular levels of the major pilin PilA, but not extracellular PilA, indicating dysregulation of the T4P motors, and, therefore, a regulatory interaction between HPS assembly and T4P activity. A co‐occurrence analysis of Hps orthologs among cyanobacteria identified an extended set of putative Hps proteins comprising most components of a Wzx/Wzy‐type polysaccharide synthesis and export system. This implies that HPS may be secreted through a more canonical pathway, rather than a T4P‐mediated mechanism.

    

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4. Phototaxis in Cyanobacteria: From Mutants to Models of Collective Behavior

   https://doi.org/10.1128/mBio.02398-21  

   Menon, Shakti N. ; Varuni, P. ; Bunbury, Freddy ; Bhaya, Devaki ; Menon, Gautam I. ( December 2021 , mBio)

   Harwood, Caroline S. (Ed.)

   ABSTRACT Cyanobacteria rely on photosynthesis, and thus have evolved complex responses to light. These include phototaxis, the ability of cells to sense light direction and move towards or away from it. Analysis of mutants has demonstrated that phototaxis requires the coordination of multiple photoreceptors and signal transduction networks. The output of these networks is relayed to type IV pili (T4P) that attach to and exert forces on surfaces or other neighboring cells to drive “twitching” or “gliding” motility. This, along with the extrusion of polysaccharides or “slime” by cells, facilitates the emergence of group behavior. We evaluate recent models that describe the emergence of collective colony-scale behavior from the responses of individual, interacting cells. We highlight the advantages of “active matter” approaches in the study of bacterial communities, discussing key differences between emergent behavior in cyanobacterial phototaxis and similar behavior in chemotaxis or quorum sensing. 

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5. 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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