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1. Sustained Release of a Synthetic Autoinducing Peptide Mimetic Blocks Bacterial Communication and Virulence In Vivo .

   https://doi.org/10.1002/anie.202201798  

   West, Korbin H. J. ; Gahan, Curran G. ; Kierski, Patricia R. ; Calderon, Diego F. ; Zhao, Ke ; Czuprynski, Charles J. ; McAnulty, Jonathan F. ; Lynn, David M. ; Blackwell, Helen E. ( April 2022 , Angewandte Chemie International Edition)

   There is significant interest in approaches to the treatment of bacterial infections that block virulence without creating selective pressures that lead to resistance. Here, we report the development of an “anti‐virulence” strategy that exploits the activity of potent synthetic inhibitors of quorum sensing (QS) inStaphylococcus aureus. We identify peptide‐based inhibitors of QS that are resistant to sequestration or degradation by components of murine tissue and demonstrate that encapsulation of a lead inhibitor in degradable polymer microparticles provides materials that substantially inhibit QSin vitro. Using a murine abscess model, we show that this inhibitor attenuates methicillin‐resistantS. aureus(MRSA) skin infectionsin vivo, and that sustained release of the inhibitor from microparticles significantly improved outcomes compared to mice that received a single‐dose bolus. Our results present an effective and modular approach to controlling bacterial virulencein vivoand could advance the development of new strategies for skin infection control.

    

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2. Murine Models for Staphylococcal Infection

   https://doi.org/10.1002/cpz1.52  

   Klopfenstein, Nathan ; Cassat, James E. ; Monteith, Andrew ; Miller, Anderson ; Drury, Sydney ; Skaar, Eric ; Serezani, C. Henrique ( March 2021 , Current Protocols)

   Staphylococcus aureusis a Gram‐positive bacterium that colonizes almost every organ in humans and mice and is a leading cause of diseases worldwide.S. aureusinfections can be challenging to treat due to widespread antibiotic resistance and their ability to cause tissue damage. The primary modes of transmission ofS. aureusare via direct contact with a colonized or infected individual or invasive spread from a colonization niche in the same individual.S. aureuscan cause a myriad of diseases, including skin and soft tissue infections (SSTIs), osteomyelitis, pneumonia, endocarditis, and sepsis.S. aureusinfection is characterized by the formation of purulent lesions known as abscesses, which are rich in live and dead neutrophils, macrophages, and surrounded by a capsule containing fibrin and collagen. Different strains ofS. aureusproduce varying amounts of toxins that evade and/or elicit immune responses. Therefore, animal models ofS. aureusinfection provide a unique opportunity to understand the dynamics of organ‐specific immune responses and modifications in the pathogen that could favor the establishment of the pathogen. With advances in in vivo imaging of fluorescent transgenic mice, combined with fluorescent/bioluminescent bacteria, we can use mouse models to better understand the immune response to these types of infections. By understanding the host and bacterial dynamics within various organ systems, we can develop therapeutics to eliminate these pathogens. This module describes in vivo mouse models of both local and systemicS. aureusinfection. © 2021 Wiley Periodicals LLC.

   This article was corrected on 20 July 2022. See the end of the full text for details.

   Basic Protocol 1: Murine model ofStaphylococcus aureussubcutaneous infection

   Alternate Protocol: Murine tape stripping skin infection model

   Basic Protocol 2: Sample collection to determine skin structure, production of inflammatory mediators, and bacterial load

   Basic Protocol 3: Murine model of post‐traumaticStaphylococcus aureusosteomyelitis

   Basic Protocol 4: Intravenous infection of the retro‐orbital sinus

   Support Protocol: Preparation of the bacterial inoculum

    

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3. The histidine kinase NahK regulates pyocyanin production through the PQS system

   https://doi.org/10.1128/jb.00276-23  

   Mendoza, Alicia G. ; Guercio, Danielle ; Smiley, Marina K. ; Sharma, Gaurav K. ; Withorn, Jason M. ; Hudson-Smith, Natalie V. ; Ndukwe, Chika ; Dietrich, Lars E. ; Boon, Elizabeth M. ( January 2024 , Journal of Bacteriology)

   Bondy-Denomy, Joseph (Ed.)

   Many bacterial histidine kinases work in two-component systems that combine into larger multi-kinase networks. NahK is one of the kinases in the GacS Multi-Kinase Network (MKN), which is the MKN that controls biofilm regulation in the opportunistic pathogenPseudomonas aeruginosa. This network has also been associated with regulating many virulence factorsP. aeruginosasecretes to cause disease. However, the individual role of each kinase is unknown. In this study, we identify NahK as a novel regulator of the phenazine pyocyanin (PYO). Deletion ofnahKleads to a fourfold increase in PYO production, almost exclusively through upregulation of phenazine operon two (phz2). We determined that this upregulation is due to mis-regulation of allP. aeruginosaquorum-sensing (QS) systems, with a large upregulation of thePseudomonasquinolone signal system and a decrease in production of the acyl-homoserine lactone-producing system,las. In addition, we see differences in expression of quorum-sensing inhibitor proteins that align with these changes. Together, these data contribute to understanding how the GacS MKN modulates QS and virulence and suggest a mechanism for cell density-independent regulation of quorum sensing.

   Pseudomonas aeruginosais a Gram-negative bacterium that establishes biofilms as part of its pathogenicity.P. aeruginosainfections are associated with nosocomial infections. As the prevalence of multi-drug-resistantP. aeruginosaincreases, it is essential to understand underlying virulence molecular mechanisms. Histidine kinase NahK is one of several kinases inP. aeruginosaimplicated in biofilm formation and dispersal. Previous work has shown that the nitric oxide sensor, NosP, triggers biofilm dispersal by inhibiting NahK. The data presented here demonstrate that NahK plays additional important roles in theP. aeruginosalifestyle, including regulating bacterial communication mechanisms such as quorum sensing. These effects have larger implications in infection as they affect toxin production and virulence.

    

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4. 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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5. Real‐Time Monitoring of Bacteria Clearance From Blood in a Murine Model

   https://doi.org/10.1002/cyto.a.23925  

   Harrington, Walter N. ; Nolan, Jacqueline ; Nedosekin, Dmitry A. ; Smeltzer, Mark S. ; Zharov, Vladimir P. ( November 2019 , Cytometry Part A)

   Bloodstream infections, especially those that are antibiotic resistant, pose a significant challenge to health care leading to increased hospitalization time and patient mortality. There are different facets to this problem that make these diseases difficult to treat, such as the difficulty to detect bacteria in the blood and the poorly understood mechanism of bacterial invasion into and out of the circulatory system. However, little progress has been made in developing techniques to study bacteria dynamics in the bloodstream. Here, we present a new approach using anin vivoflow cytometry platform for real‐time, noninvasive, label‐free, and quantitative monitoring of the lifespan of green fluorescent protein‐expressingStaphylococcus aureusandPseudomonas aeruginosain a murine model. We report a relatively fast average rate of clearance forS. aureus(k= 0.37 ± 0.09 min⁻¹, half‐life ~1.9 min) and a slower rate forP. aeruginosa(k= 0.07 ± 0.02 min⁻¹, half‐life ~9.6 min). We also observed what appears to be two stages of clearance forS. aureus, whileP. aeruginosaappeared only to have a single stage of clearance. Our results demonstrate that an advanced research tool can be used for studying the dynamics of bacteria cells directly in the bloodstream, providing insight into the progression of infectious diseases in circulation. © 2019 International Society for Advancement of Cytometry

    

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