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1. Quantitative confocal microscopy and calibration for measuring differences in cyclic-di-GMP signalling by bacteria on biomedical hydrogels

   https://doi.org/10.1098/rsos.201453  

   Blacutt, Jacob; Lan, Ziyang; Cosgriff-Hernandez, Elizabeth M.; Gordon, Vernita D. (January 2021, Royal Society Open Science)

   null (Ed.)

   The growth of bacterial biofilms on implanted medical devices causes harmful infections and device failure. Biofilm development initiates when bacteria attach to and sense a surface. For the common nosocomial pathogen Pseudomonas aeruginosa and many others, the transition to the biofilm phenotype is controlled by the intracellular signal and second messenger cyclic-di-GMP (c-di-GMP). It is not known how biomedical materials might be adjusted to impede c-di-GMP signalling, and there are few extant methods for conducting such studies. Here, we develop such a method. We allowed P. aeruginosa to attach to the surfaces of poly(ethylene glycol) diacrylate (PEGDA) hydrogels. These bacteria contained a plasmid for a green fluorescent protein (GFP) reporter for c-di-GMP. We used laser-scanning confocal microscopy to measure the dynamics of the GFP reporter for 3 h, beginning 1 h after introducing bacteria to the hydrogel. We controlled for the effects of changes in bacterial metabolism using a promoterless plasmid for GFP, and for the effects of light passing through different hydrogels being differently attenuated by using fluorescent plastic beads as ‘standard candles’ for calibration. We demonstrate that this method can measure statistically significant differences in c-di-GMP signalling associated with different PEGDA gel types and with the surface-exposed protein PilY1. 

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2. Activation of Gingival Fibroblasts by Bacterial Cyclic Dinucleotides and Lipopolysaccharide

   https://doi.org/10.3390/pathogens9100792  

   Elmanfi, Samira; Sintim, Herman O.; Zhou, Jie; Gürsoy, Mervi; Könönen, Eija; Gürsoy, Ulvi K. (October 2020, Pathogens)

   null (Ed.)

   Human gingival fibroblasts (HGFs) recognize microbe-associated molecular patterns (MAMPs) and respond with inflammatory proteins. Simultaneous impacts of bacterial cyclic di-guanosine monophosphate (c-di-GMP), cyclic di-adenosine monophosphate (c-di-AMP), and lipopolysaccharide (LPS) on gingival keratinocytes have been previously demonstrated, but the effects of these MAMPs on other periodontal cell types, such as gingival fibroblasts, remain to be clarified. The present aim was to examine the independent and combined effects of these cyclic dinucleotides and LPS on interleukin (IL) and matrix metalloproteinase (MMP) response of HGFs. The cells were incubated with c-di-GMP and c-di-AMP, either in the presence or absence of Porphyromonas gingivalis LPS, for 2 h and 24 h. The levels of IL-8, -10, and -34, and MMP-1, -2, and -3 secreted were measured by the Luminex technique. LPS alone or together with cyclic dinucleotides elevated IL-8 levels. IL-10 levels were significantly increased in the presence of c-di-GMP and LPS after 2 h but disappeared after 24 h of incubation. Concurrent treatment of c-di-AMP and LPS elevated MMP-1 levels, whereas c-di-GMP with LPS suppressed MMP-2 levels but increased MMP-3 levels. To conclude, we produce evidence that cyclic dinucleotides interact with LPS-mediated early response of gingival fibroblasts, while late cellular response is mainly regulated by LPS. 

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3. Quantitative input–output dynamics of a c-di-GMP signal transduction cascade in Vibrio cholerae

   https://doi.org/10.1371/journal.pbio.3001585  

   Bridges, Andrew A.; Prentice, Jojo A.; Fei, Chenyi; Wingreen, Ned S.; Bassler, Bonnie L. (March 2022, PLOS Biology)

   Waldor, Matthew K. (Ed.)

   Bacterial biofilms are multicellular communities that collectively overcome environmental threats and clinical treatments. To regulate the biofilm lifecycle, bacteria commonly transduce sensory information via the second messenger molecule cyclic diguanylate (c-di-GMP). Using experimental and modeling approaches, we quantitatively capture c-di-GMP signal transmission via the bifunctional polyamine receptor NspS-MbaA, from ligand binding to output, in the pathogen Vibrio cholerae . Upon binding of norspermidine or spermidine, NspS-MbaA synthesizes or degrades c-di-GMP, respectively, which, in turn, drives alterations specifically to biofilm gene expression. A long-standing question is how output specificity is achieved via c-di-GMP, a diffusible molecule that regulates dozens of effectors. We show that NspS-MbaA signals locally to specific effectors, sensitizing V . cholerae to polyamines. However, local signaling is not required for specificity, as changes to global cytoplasmic c-di-GMP levels can selectively regulate biofilm genes. This work establishes the input–output dynamics underlying c-di-GMP signaling, which could be useful for developing bacterial manipulation strategies. 

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4. Spectrophotometric Method for the Quantification and Kinetic Evaluation of in vitro c-di-GMP Hydrolysis in the Presence and Absence of Oxygen

   https://doi.org/10.1007/978-1-0716-3080-8_7  

   Patterson, Dayna C.; Weinert, Emily E. (April 2023, Methods in molecular biology)

   A spectrophotometric method to measure hydrolysis of the bacterial second messenger cyclic dimeric guanosine monophosphate is described for characterization of enzymes under aerobic and anaerobic conditions. The method allows for obtaining all necessary data to calculate KM and kcat from reactions within a single 96-well plate that be can measured using a standard plate reader. The spectrophotometric assay has been used to measure the rates and obtain Michaelis-Menten for the c-di-GMP phosphodiesterase DcpG with the sensor domain in various ligation states. 

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5. Regulation of Biofilm Exopolysaccharide Production by Cyclic Di-Guanosine Monophosphate

   https://doi.org/10.3389/fmicb.2021.730980  

   Poulin, Myles B.; Kuperman, Laura L. (September 2021, Frontiers in Microbiology)

   Many bacterial species in nature possess the ability to transition into a sessile lifestyle and aggregate into cohesive colonies, known as biofilms. Within a biofilm, bacterial cells are encapsulated within an extracellular polymeric substance (EPS) comprised of polysaccharides, proteins, nucleic acids, lipids, and other small molecules. The transition from planktonic growth to the biofilm lifecycle provides numerous benefits to bacteria, such as facilitating adherence to abiotic surfaces, evasion of a host immune system, and resistance to common antibiotics. As a result, biofilm-forming bacteria contribute to 65% of infections in humans, and substantially increase the energy and time required for treatment and recovery. Several biofilm specific exopolysaccharides, including cellulose, alginate, Pel polysaccharide, and poly- N -acetylglucosamine (PNAG), have been shown to play an important role in bacterial biofilm formation and their production is strongly correlated with pathogenicity and virulence. In many bacteria the biosynthetic machineries required for assembly of these exopolysaccharides are regulated by common signaling molecules, with the second messenger cyclic di-guanosine monophosphate (c - di-GMP) playing an especially important role in the post-translational activation of exopolysaccharide biosynthesis. Research on treatments of antibiotic-resistant and biofilm-forming bacteria through direct targeting of c-di-GMP signaling has shown promise, including peptide-based treatments that sequester intracellular c-di-GMP. In this review, we will examine the direct role c-di-GMP plays in the biosynthesis and export of biofilm exopolysaccharides with a focus on the mechanism of post-translational activation of these pathways, as well as describe novel approaches to inhibit biofilm formation through direct targeting of c-di-GMP. 

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