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1. A multimodal nonlinear optical microscopy study of the responses of Pseudomonas aeruginosa to blue light and antibiotic treatment

   https://doi.org/10.1002/jbio.202300384  

   Zhang, Chi ; Zaki, Farzana R. ; Won, Jungeun ; Boppart, Stephen A. ( December 2023 , Journal of Biophotonics)

   Pseudomonas aeruginosa(P. aeruginosa) is a multidrug‐resistant human pathogen involved in numerous infections. Understanding the response ofP. aeruginosato various treatments is critical to developing new ways for the antimicrobial susceptibly test and more effective treatment methods. Conventional antimicrobial susceptibility tests lack molecular information at the single bacterium level. In this study, we used label‐free multimodal nonlinear optical microscopy to identify an autofluorescence signal from pyoverdine, a siderophore of the bacteria, for quantification ofP. aeruginosaresponses to antibiotics and blue light treatment. We also discovered that the bleaching of the pyoverdine autofluorescence signals is correlated with the inactivation ofP. aeruginosaand is perhaps one of the mechanisms involved in the blue light inactivation ofP. aeruginosa.

    

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2. Elucidating the molecular programming of a nonlinear non-ribosomal peptide synthetase responsible for fungal siderophore biosynthesis

   https://doi.org/10.1038/s41467-023-38484-8  

   Jenner, Matthew ; Hai, Yang ; Nguyen, Hong H. ; Passmore, Munro ; Skyrud, Will ; Kim, Junyong ; Garg, Neil K. ; Zhang, Wenjun ; Ogorzalek Loo, Rachel R. ; Tang, Yi ( May 2023 , Nature Communications)

   Siderophores belonging to the ferrichrome family are essential for the viability of fungal species and play a key role for virulence of numerous pathogenic fungi. Despite their biological significance, our understanding of how these iron-chelating cyclic hexapeptides are assembled by non-ribosomal peptide synthetase (NRPS) enzymes remains poorly understood, primarily due to the nonlinearity exhibited by the domain architecture. Herein, we report the biochemical characterization of the SidC NRPS, responsible for construction of the intracellular siderophore ferricrocin. In vitro reconstitution of purified SidC reveals its ability to produce ferricrocin and its structural variant, ferrichrome. Application of intact protein mass spectrometry uncovers several non-canonical events during peptidyl siderophore biosynthesis, including inter-modular loading of amino acid substrates and an adenylation domain capable of poly-amide bond formation. This work expands the scope of NRPS programming, allows biosynthetic assignment of ferrichrome NRPSs, and sets the stage for reprogramming towards novel hydroxamate scaffolds.

    

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3. Let-7b-5p in vesicles secreted by human airway cells reduces biofilm formation and increases antibiotic sensitivity of P. aeruginosa

   https://doi.org/10.1073/pnas.2105370118  

   Koeppen, Katja ; Nymon, Amanda ; Barnaby, Roxanna ; Bashor, Laura ; Li, Zhongyou ; Hampton, Thomas H. ; Liefeld, Amanda E. ; Kolling, Fred W. ; LaCroix, Ian S. ; Gerber, Scott A. ; et al ( July 2021 , Proceedings of the National Academy of Sciences)

   Pseudomonas aeruginosais an opportunistic pathogen that forms antibiotic-resistant biofilms, which facilitate chronic infections in immunocompromised hosts. We have previously shown thatP. aeruginosasecretes outer-membrane vesicles that deliver a small RNA to human airway epithelial cells (AECs), in which it suppresses the innate immune response. Here, we demonstrate that interdomain communication through small RNA–containing membrane vesicles is bidirectional and that microRNAs (miRNAs) in extracellular vesicles (EVs) secreted by human AECs regulate protein expression, antibiotic sensitivity, and biofilm formation byP. aeruginosa. Specifically, human EVs deliver miRNA let-7b-5p toP. aeruginosa, which systematically decreases the abundance of proteins essential for biofilm formation, including PpkA and ClpV1-3, and increases the ability of beta-lactam antibiotics to reduce biofilm formation by targeting the beta-lactamase AmpC. Let-7b-5p is bioinformatically predicted to target not only PpkA, ClpV1, and AmpC inP. aeruginosabut also the corresponding orthologs inBurkholderia cenocepacia, another notorious opportunistic lung pathogen, suggesting that the ability of let-7b-5p to reduce biofilm formation and increase beta-lactam sensitivity is not limited toP. aeruginosa. Here, we provide direct evidence for transfer of miRNAs in EVs secreted by eukaryotic cells to a prokaryote, resulting in subsequent phenotypic alterations in the prokaryote as a result of this interdomain communication. Since let-7–family miRNAs are in clinical trials to reduce inflammation and because chronicP. aeruginosalung infections are associated with a hyperinflammatory state, treatment with let-7b-5p and a beta-lactam antibiotic in nanoparticles or EVs may benefit patients with antibiotic-resistantP. aeruginosainfections.

    

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4. 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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5. Modification of narrow‐spectrum peptidomimetic polyurethanes with fatty acid chains confers broad‐spectrum antibacterial activity

   https://doi.org/10.1002/pi.5773  

   Peng, Chao ; Zhang, Tian ; Ortiz‐Ortiz, Deliris N. ; Vishwakarma, Apoorva ; Barton, Hazel A. ; Joy, Abraham ( February 2019 , Polymer International)

   Each year, thousands of patients die from antimicrobial‐resistant bacterial infections that fail to respond to conventional antibiotic treatment. Antimicrobial polymers are a promising new method of combating antibiotic‐resistant bacterial infections. We have previously reported the synthesis of a series of narrow‐spectrum peptidomimetic antimicrobial polyurethanes that are effective against Gram‐negative bacteria, such asEscherichia coli; however, these polymers are not effective against Gram‐positive bacteria, such asStaphylococcus aureus. With the aim of understanding the correlation between chemical structure and antibacterial activity, we have subsequently developed three structural variants of these antimicrobial polyurethanes using post‐polymerization modification with decanoic acid and oleic acid. Our results show that such modifications converted the narrow‐spectrum antibacterial activity of these polymers into broad‐spectrum activity against Gram‐positive species such asS. aureus, however, also increasing their toxicity to mammalian cells. Mechanistic studies of bacterial membrane disruption illustrate the differences in antibacterial action between the various polymers. The results demonstrate the challenge of balancing antimicrobial activity and mammalian cell compatibility in the design of antimicrobial polymer compositions. © 2019 Society of Chemical Industry

    

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