It is demonstrated that SGO_2031, but not SGO_2030, seems to contribute to biofilm formation by modulating the abundance of extracellular polysaccharides within the biofilm matrix, and proposed naming this enzyme Streptococcal Lysine Acetyltransferase A (SktA) after Streptococcus gordonii.
Abstract
ABSTRACT Streptococcus gordonii is a gram-positive oral bacterium capable of adhering to a variety of biotic and abiotic surfaces and forming biofilms. To characterize physiological changes associated with biofilm formation in S. gordonii, we investigated the roles of two putative GCN5-related N-acetyltransferases (GNATs), SGO_2030 and SGO_2031, in in vitro biofilm formation on saliva-coated surfaces using the laboratory strain DL1. Our results demonstrate that SGO_2031, but not SGO_2030, seems to contribute to biofilm formation by modulating the abundance of extracellular polysaccharides within the biofilm matrix. This defect in biofilm formation observed by the deletion of SGO_2031 resulted in a significant fitness disadvantage during colonization of the murine oral cavity compared to the wild-type parent strain. Consistent with the role of S. gordonii as an early colonizer of tooth surfaces that influences oral biofilm community structure, inoculation with either the wild-type or the SGO_2031 mutant strain led to distinct alterations in the murine oral microbiome composition. Deletion of SGO_2031 also resulted in changes in protein acetylation patterns, as assessed by Western immunoblot analysis, supporting the role of this enzyme as an acetyltransferase. Given that SGO_2031 is conserved and widely distributed among streptococci, we propose naming this enzyme Streptococcal Lysine Acetyltransferase A (SktA). IMPORTANCE Protein acetylation is a common posttranslational modification conserved across all domains of life. In bacteria, protein acetylation is carried out by homologs of the GCN5-related N-acetyltransferase (GNAT) family. GNATs catalyze the transfer of an acetyl group from acetyl-CoA to the ε-amino group of lysine residues on proteins. This process changes the charge and length of lysine residues, resulting in changes to protein function. Streptococcus gordonii is predicted to encode 17 GNAT homologs. Here, we report that one of them, SGO_2031 (SktA), plays an important role in S. gordonii biofilms. Protein acetylation is a common posttranslational modification conserved across all domains of life. In bacteria, protein acetylation is carried out by homologs of the GCN5-related N-acetyltransferase (GNAT) family. GNATs catalyze the transfer of an acetyl group from acetyl-CoA to the ε-amino group of lysine residues on proteins. This process changes the charge and length of lysine residues, resulting in changes to protein function. Streptococcus gordonii is predicted to encode 17 GNAT homologs. Here, we report that one of them, SGO_2031 (SktA), plays an important role in S. gordonii biofilms.
Klebsiella pneumoniae (K. pneumoniae) is a major opportunistic pathogen associated with a broad spectrum of hospital-and community-acquired infections. Biofilm formation contributes to bacterial persistence, stress tolerance, and host immune clearance. In this study, we constructed a transposon mutant library of the clinical K. pneumoniae strain KP20 using a mariner-based mutagenesis system and screened for mutants exhibiting defective biofilm formation. Targeted knockout of thrA significantly reduced biofilm formation and attenuated the adhesion and invasion of K. pneumoniae with respect to human airway epithelial Calu-3 cells and lung adenocarcinoma A549 cells. These phenotypic changes are associated with altered transcription of the gene clusters responsible for type I and type III fimbria biosynthesis. Moreover, deletion of thrA compromised bacterial tolerance to acidic and alkaline stresses and increased susceptibility to phagocytosis by dendritic cells. In a murine infection model, thrA deletion significantly attenuated the virulence of K. pneumoniae and decreased bacterial colonization in target organs. Collectively, these findings indicate that thrA substantially contributes to biofilm formation, pH stress tolerance, host–cell interaction, and virulence in K. pneumoniae.
An electron cryomicroscopy structure of the 60 kDa CdrA adhesive N-terminus is reported, which combined with electron cryotomography of focused-ion beam milled specimens, allows for a complete in situ model of the native adhesin and reveals a small adhesive domain (called ADEPT) at the distal tip of CdrA that is nearly perfectly conserved across the P. aeruginosa pangenome.
Olivia E. R. Smith, Camila M. Clemente, Antonina Andreeva et al.· bioRxiv· 0 citations
Sortase A (SrtA) enzymes covalently anchor surface proteins to Gram-positive bacterial cell walls, promoting colonization and virulence. In Streptococcus pneumoniae, previous studies identified both a domain-swapped dimer and an active refolded monomer, but the active enzyme has not been characterized at the structural and residue-specific level. Here, we performed quantitative proteomic comparisons of wild-type and SrtA knockout strains that confirmed the loss of multiple LPxTG-containing virulence factors, including ZmpB, NanA, and IgA1 protease, consistent with an essential role for SrtA in surface protein anchoring. To enable mechanistic studies, we established a biochemical framework to produce monomeric Streptococcus pneumoniae SrtA by refolding and developed a gel-based assay using recombinant substrates to monitor catalytic activity. The refolded monomer, but not the swapped dimer, catalyzed cleavage and transpeptidation of a canonical LPxTG substrate in a metal-independent manner under the conditions examined. We further report high-resolution NMR backbone assignments for the active monomer and identify substrate-induced chemical shift perturbations that localize to the active site. Together, these findings provide an integrated proteomic, biochemical, and NMR characterization of monomeric, catalytically active Streptococcus pneumoniae SrtA and reveal residue-specific interactions with a canonical LPNTG recognition peptide.
Eunjeong Lee, Blaine H. Gordon, J. Redzic et al.· Biomolecules· 0 citations
Streptococcus mutans is a major cariogenic pathogen whose proliferation, acidogenesis, and pathogenicity are strongly impacted by dietary sugars, resulting in dental biofilm-associated illnesses. This study evaluated the effects of sucrose, glucose, and alternative sweeteners (allulose, erythritol, stevia, sucralose, and xylitol) on S. mutans biofilm formation, adhesion, autoaggregation, acidogenicity, metabolic activity, and virulence-related gene expression across different dental surfaces. Sucrose significantly enhanced acid production and biofilm formation, with 3% sucrose resulting in the highest biofilm biomass on both hydroxyapatite (HA) and stainless steel (SS) surfaces. In contrast, alternative sweeteners exhibited variable antibiofilm activity, with sucralose demonstrating the most potent and consistent inhibitory effects across all tested concentrations. Sucralose effectively suppressed sucrose-induced S. mutans biofilm formation, exhibiting dose-dependent antibiofilm activity. Moreover, sucralose significantly reduced bacterial adhesion, the viability of established biofilms, autoaggregation, acid production, intracellular ATP levels, and biofilm structural integrity, as confirmed by confocal laser scanning microscopy (CLSM) and field emission scanning electron microscopy (FE-SEM). Phenol-sulfuric acid analysis further demonstrated a significant reduction in extracellular polymeric substances (EPS) carbohydrate production, while Fourier-transform infrared (FTIR) study demonstrated that sucralose influences EPS composition, namely by lowering polysaccharide-associated components connected to glucan structures. Furthermore, sucralose markedly downregulated key virulence genes involved in biofilm architecture, quorum sensing (QS), and pathogenicity (vicR, gtfC, gbpC, and comX) in a concentration-dependent manner. Notably, this study shows that sucralose disrupts both EPS structural components and virulence gene regulatory networks in S. mutans, offering novel mechanistic insights into its antibiofilm efficacy against cariogenic biofilms.
Syeda Roufun Nesa, Nigar Sultana Meghla, Hyobin Lee et al.· Microbial Pathogenesis· 0 citations
ABSTRACT Listeria monocytogenes (Lm) is a major foodborne pathogen capable of persisting on fresh produce and in food-processing environments. Although exopolysaccharides (EPSs) are recognized as central components of resilient bacterial biofilms, Lm biofilms have long been considered to be EPS-less or EPS-poor. We previously identified a listerial EPS, Pss, whose biosynthesis is encoded by the highly conserved pss operon and is stimulated by elevated c-di-GMP levels. However, the prevalence of Pss in wild-type strains remained unclear, in part, due to the lack of a specific detection probe. Here, we engineered a genetically encoded fluorescent probe, PssZ*-mS, based on a catalytically inactive variant (E72Q) of the Pss hydrolase PssZ. Structural modeling predicted that the E72Q substitution abolishes catalysis without disrupting Pss binding. PssZ*-mS bound purified Pss with high affinity (Kd, 50 nM) and showed no detectable interaction with other tested EPSs, including cellulose, pectin, maltodextrin, or alginate. Using PssZ*-mS, we demonstrate that the reference wild-type strain EGDe produces substantial Pss under standard conditions in liquid minimal glucose-containing medium and on diverse abiotic surfaces relevant to food environments. A survey of Lm isolates revealed that Pss production is common, though not universal, among Lm strains and is also present in non-monocytogenes Listeria species. Notably, three genetically distinct strains from the infamous 2011 whole-cantaloupe listeriosis outbreak synthesize Pss. These findings challenge the prevailing view of listerial biofilms and identify Pss as a widespread component of Lm biofilms, underscoring the need for targeted anti-Pss strategies to enhance food safety. IMPORTANCE Listeria monocytogenes persists on fresh produce and in food-processing environments despite rigorous sanitation, yet the molecular basis of this resilience remains poorly understood. We developed a specific fluorescent probe for the conserved Pss exopolysaccharide (EPS) and show that Pss is a common component of listerial biofilms. These findings challenge the long-standing view that Listeria forms primarily EPS-less or EPS-poor biofilms and identify Pss as an important biofilm component. By enabling direct visualization of Pss, this work provides a new tool for studying listerial biofilms and opens avenues for targeted antibiofilm strategies to improve food safety and public health. Listeria monocytogenes persists on fresh produce and in food-processing environments despite rigorous sanitation, yet the molecular basis of this resilience remains poorly understood. We developed a specific fluorescent probe for the conserved Pss exopolysaccharide (EPS) and show that Pss is a common component of listerial biofilms. These findings challenge the long-standing view that Listeria forms primarily EPS-less or EPS-poor biofilms and identify Pss as an important biofilm component. By enabling direct visualization of Pss, this work provides a new tool for studying listerial biofilms and opens avenues for targeted antibiofilm strategies to improve food safety and public health.
Aili Wang, Ahmed M. Elbakush, Oliver Trunschke et al.· Journal of Bacteriology· 0 citations
The WT and Δ Flo11 strains grew similarly under alkaline and physiological conditions, while growth of the Δ Flo11 mutant was significantly impaired, and the importance of Flo11 to static biofilm formation and adherence under alkaline conditions in vitro was tested.
