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1. Thermodynamics of calcium binding to heparin: Implications of solvation and water structuring for polysaccharide biofunctions

   https://doi.org/10.1073/pnas.2504348122  

   Knight, Brenna M; Gallagher, Connor_M B; Schulz, Michael D; Edgar, Kevin J; McNaul, Caylyn D; McCutchin, Christina A; Dove, Patricia M (September 2025, Proceedings of the National Academy of Sciences)

   Heparan sulfates are found in all animal tissues and have essential roles in living systems. This family of biomacromolecules modulates binding to calcium ions (Ca²⁺) in low free energy reactions that influence biochemical processes from cell signaling and anticoagulant efficacy to biomineralization. Despite their ubiquity, the thermodynamic basis for how heparans and similarly functionalized biomolecules regulate Ca²⁺interactions is not yet established. Using heparosan (Control) and heparins with different positions of sulfate groups, we quantify how SO₃⁻and COO⁻content and SO₃⁻position modulate Ca²⁺binding by isothermal titration calorimetry. The free energy of all heparin-Ca²⁺interactions (ΔG_rxn) is dominated by entropic contributions due to favorable water release from polar, hydrophilic groups. Heparin with both sulfate esters (O-SO₃⁻) and sulfamides (N-SO₃⁻) has the strongest binding to Ca²⁺compared to heparosan and to heparin with onlyO-SO₃⁻groups (~3X). By linking Ca²⁺binding thermodynamics to measurements of the interfacial energy for calcite (CaCO₃) crystallization onto polysaccharides, we show molecule-specific differences in nucleation rate can be explained by differences in water structuring during Ca²⁺interactions. A large entropic term (-TΔS_rxn) upon Ca²⁺–polysaccharide binding correlates with high interfacial energy to CaCO₃nucleation. Combining our measurements with literature values indicates many Ca²⁺–polysaccharide interactions have a shared thermodynamic signature. The resulting enthalpy–entropy compensation relationship suggests these interactions are generally dominated by water restructuring involving few conformational changes, distinct from Ca²⁺–protein binding. Our findings quantify the thermodynamic origins of heparin-specific interactions with Ca²⁺and demonstrate the contributions of solvation and functional group position during biomacromolecule-mediated ion regulation. 

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2. Metabolites derived from bacterial isolates of the human skin microbiome inhibit Staphylococcus aureus biofilm formation

   https://doi.org/10.1128/spectrum.01306-25  

   Le, Viet Hoang; King, Tetyana; Wuerzberger, Breanna; Bauer, Olivia R; Carver, Megan N; Chan, Tiffany S; Henson, Annabeth L; Hubbard, Grace K; Kopadze, Tamar; Patterson, Claire F; et al (September 2025, Microbiology Spectrum)

   Claesen, Jan (Ed.)

   ABSTRACT The human skin microbiome is a diverse ecosystem that can help prevent infections by producing biomolecules and peptides that inhibit growth and virulence of bacterial pathogens.Staphylococcus aureusis a major human pathogen responsible for diseases that range from acute skin and soft tissue infections to life-threatening septicemia. Its ability to form biofilms is a key virulence factor contributing to its success as a pathogen as well as to its increased antimicrobial resistance. Here, we investigated the ability of bacterial skin commensals to produce molecules that inhibitS. aureusbiofilm formation. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identified 77 human skin microbiome bacterial isolates fromStaphylococcusandBacillusgenera. Metabolites from cell-free concentrated media (CFCM) from 26 representative isolates were evaluated for their ability to inhibit biofilm formation by both methicillin-resistant (MRSA) and methicillin-sensitive (MSSA)S. aureusstrains. CFCM, derived from most of the isolates, inhibited biofilm formation to varying extents but did not inhibit planktonic growth ofS. aureus. Size fractionation of the CFCM of threeS.epidermidisisolates indicated that they produce different bioactive molecules. Cluster analysis, based on either MALDI-TOF mass spectra or whole-genome sequencing draft genomes, did not show clear clusters associated with levels of biofilm inhibition amongS. epidermidisstrains. Finally, similar biosynthetic gene clusters were detected in allS. epidermidisstrains analyzed. These findings indicate that several bacterial constituents of the human skin microbiome display antibiofilmin vitroactivity, warranting further investigation on their potential as novel therapeutic agents. IMPORTANCEThe skin is constantly exposed to the environment and consequently to numerous pathogens. The bacterial community that colonizes healthy skin is thought to play an important role in protecting us against infections.S. aureusis a leading cause of death worldwide and is frequently involved in several types of infections, including skin and soft tissue infections. Its ability to adhere to surfaces and produce biofilms is considered an important virulence factor. Here, we analyzed the activity of different species of bacteria isolated from healthy skin onS. aureusbiofilm formation. We found that some species ofStaphylococcusandBacilluscan reduceS. aureusbiofilm formation, although a generally lower level of inhibitory activity was observed compared toS. epidermidisisolates. AmongS. epidermidisisolates, strength of activity was dependent on the strain. Our data highlight the importance of mining the skin microbiome for isolates that could help combat skin pathogens. 

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3. The iron-regulated small regulatory RNA IsrR modulates expression of genes utilized for dioxygen metabolism and heme synthesis in Staphylococcus aureus

   https://doi.org/10.1128/mbio.01415-25  

   Rios-Delgado, Gustavo; McFarlane, Riley; Zheng, Vincent; Kim, Jisun; Parker, Dane; Kehl-Fie, Thomas; Lalaouna, David; Boyd, Jeffrey M (November 2025, mBio)

   Darwin, K Heran (Ed.)

   ABSTRACT Bacterial regulatory RNAs (sRNAs) are commonly short non-coding RNAs that function as pleiotropic regulators by post-transcriptionally impacting mRNA stability and/or translation. They play significant roles in bacterial physiology and are typically expressed in response to specific environmental stimuli such as nutrient limitation. The bacterial pathogenStaphylococcus aureusfaces decreased access to essential metal ions, including iron, in the mammalian host via a process called nutritional immunity. In response to host-mediated iron limitation,S. aureusexpresses the sRNA IsrR, which coordinates an iron-sparing response by downregulating the expression of mRNAs coding for iron-requiring proteins or processes. Herein, we utilized MS2-Affinity Purification coupled with RNA Sequencing (MAPS) to reveal thein vivoIsrR interaction network. Analysis of co-purified RNAs revealed previously unpredicted putative IsrR targets coding for proteins associated with iron-requiring processes. We validated that IsrR directly interacts with nine targetsin vitro. We demonstrate physiological roles for IsrR in mediating heme biosynthesis, aerobic respiration, and the detoxification of oxygen radicals. These activities are critical for pathogenesis, and this study establishes howS. aureusleverages these processes to adapt to iron scarcity, which is commonly encountered in the mammalian host. IMPORTANCEStaphylococcus aureuscauses numerous and varied infections in mammals, making it a significant public health burden and concern. The prevalence ofS. aureusinfections is due to its robust repertoire of virulence factors and its ability to adapt to host microenvironments. Elucidation of the metabolic processes and pathways that promote adaptation to host-promoted stressors provides information about host-pathogen interactions. It could also aid the development of new antimicrobials or unveil treatment and prevention strategies. One common stress bacteria encounter within the mammalian hosts is limited access to iron. In response to iron scarcity,S. aureusexpresses the regulatory sRNA IsrR. Here, we identified mRNAs that associate with IsrR. We verified that IsrR targets mRNAs that code for proteins involved in aerobic respiration, the metabolism of reactive oxygen species, and heme synthesis. This work provides significant insight into howS. aureusresponds to host-mediated iron starvation. 

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4. Probiotic colonization of Xenopus laevis skin causes short-term changes in skin microbiomes and gene expression

   https://doi.org/10.1128/iai.00569-24  

   Madison, Joseph D; Osborne, Owen G; Ellison, Amy; Garvey_Griffith, Christina N; Gentry, Lindsey; Gross, Harald; Gratwicke, Brian; Grayfer, Leon; Muletz-Wolz, Carly R (May 2025, Infection and Immunity)

   Raffatellu, Manuela (Ed.)

   ABSTRACT Probiotic therapies have been suggested for amelioration efforts of wildlife disease such as chytridiomycosis caused byBatrachochytriumspp. in amphibians. However, there is a lack of information on how probiotic application affects resident microbial communities and immune responses. To better understand these interactions, we hypothesized that probiotic application would alter microbial community composition and host immune expression inXenopus laevis. Accordingly, we applied three amphibian-derived and anti-Batrachochytriumbacteria strains (twoPseudomonasspp. and oneStenotrophomonassp.) toX. laevisin monoculture and also as a cocktail. We quantified microbial community structure using 16S rRNA gene sequencing. We also quantified genes involved inX. laevisimmune responses using quantitative reverse transcriptase polymerase chain reaction (RT-qPCR) and skin transcriptomics over 1 and 3-week periods. All probiotic treatments successfully colonizedX. laevisskin for 3 weeks, but with differential amplicon sequence variant (ASV) sequence counts over time. Bacterial community and immune gene effects were most pronounced at week 1 post-probiotic exposure and decreased thereafter. All probiotic treatments caused initial changes to bacterial community alpha and beta diversity, including reduction in diversity from pre-exposure anti-Batrachochytriumbacterial ASV relative abundance. Probiotic colonization byPseudomonasprobiotic strain RSB5.4 reduced expression of regulatory T cell marker (FOXP3,measured with RT-qPCR) and caused the greatest gene expression changes detected by transcriptomics. Single bacterial strains and mixed cultures, therefore, altered amphibian microbiome-immune interactions. This work will help to improve our understanding of the role of the microbiome-immune interface underlying both disease dynamics and emergent eco-evolutionary processes.IMPORTANCEAmphibian skin microbial communities have an important role in determining disease outcomes, in part through complex yet poorly understood interactions with host immune systems. Here we report that probiotic-induced changes to theXenopus laevisfrog skin microbial communities also result in significant alterations to these animals’ immune gene expression. These findings underscore the interdependence of amphibian skin immune-microbiome interactions. 

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5. Dissecting the role of flagellar subunits in C. difficile mucosal colonization

   https://doi.org/10.1128/jb.00428-25  

   Biswas, Baishakhi; Do, Thi_Van Thanh; Perdomo, Justin G; Auchtung, Jennifer M; Piepenbrink, Kurt H (December 2025, Journal of Bacteriology)

   Champion, Patricia A (Ed.)

   ABSTRACT Clostridioides difficileis a common cause of acute gastrointestinal (GI) inflammation in mammals, which can have detrimental effects on host health.C. difficile-associated disease requires the secretion of high-molecular-weight toxins after colonization of the GI tract. The molecular mechanisms of GI colonization byC. difficileinclude potential interactions with host cells and the mucus layer formed from secreted mucin glycoproteins.C. difficileassociates with the mucus layerin vivoand will associate with both epithelial cells and mucosal surfacesin vitro. Previously, we found a substantial defect in binding to mucosal surfaces for mutants of the major flagellar subunit,fliC, while mutation of the major subunit of type IV pili,pilA1, showed increased adhesion. To elucidate the mechanisms by whichC. difficileinteracts withex vivomucosal surfaces, we have measured swimming motility, mucosal adhesion, and levels of flagellation by transmission electron microscopy for mutants of flagellar and T4P genes inC. difficileR20291. We discovered that thepilA1mutant showed increased flagellation, while decreases in flagellation were found forfliC, fliD,andflg-Δ3 OFF (a phase-locked mutant with low transcription of the F3 flagellar operon), which were associated with both low swimming motility and low adhesion to mucosal surfaces. However, the reversedflg-Δ3 ON mutant showed increased flagellation without a significant increase in adhesion. We also found that thefliCmutant was defective in binding to mucus-secreting HT-29 MTX cells, and that decreased binding was not observed for other mutants with reduced flagellation. These results imply that at least two molecular pathways contribute toC. difficilemucosal adhesion. In addition to their direct roles encoding T4P and flagellar subunits,pilA1andfliCmay contribute to regulatory networks governing other proteins relevant to mucosal adhesion.IMPORTANCEIn the context of previous work onClostridioides difficilehost adhesion by our groups and others, our results suggest that (i) at least two mechanisms exist for mucosal adhesion byC. difficile, potentially direct adhesion by flagella and another lectin-like adhesion regulated by flagellar components; (ii) mucosal binding can also contribute toC. difficileadhesion in 2D cell culture and could explain previous defects fromfliCmutants; and (iii) levels of flagellation are largely insensitive tofliCtranscription, implying that other factors limit flagellar production and that FliC levels may be regulated independently as part of regulatory networks withinC. difficile. 

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