Helge Christian Dorfmueller

Cell wall glycopolymers in Gram-positive bacteria are crucial for structural integrity, cell division, and stress resistance. Elucidating the structure of these glycopolymers is not only important for understanding fundamental bacterial physiology, but may facilitate vaccine development, phage therapy, antibiotic design and improvements in food fermentation safety. However, extraction protocols have so far relied on harsh chemicals that can damage these polymers, compromise their structural integrity, obscure their biological functions, and result in low yields. We present a straightforward method for isolating cell wall glycopolymers from Gram-positive bacteria that preserves essential structural features including attachment to peptidoglycan. The method was broadly applicable across phylogenetically diverse species and was not restricted to glycopolymer types. In total, we isolated 17 distinct glycopolymers from 11 species, including six with previously unreported structures, with a median yield of 49 mg per litre of culture. The simplicity and mild nature of the method, combined with the exceptional purity of the resulting products, has the potential to transform research on Gram-positive cell wall glycopolymers.

Enterococci are opportunistic pathogens classified by the World Health Organization as high-priority microorganisms. They cause a broad spectrum of infections, and their intrinsic and acquired resistance to antimicrobials makes these infections particularly difficult to treat and eradicate. In Enterococcus faecalis , the most frequently isolated enterococcal pathogen in humans, antimicrobial resistance and innate immune evasion are largely driven by the Enterococcal Polysaccharide Antigen (EPA). This surface polymer underpins key virulence traits, including resistance to host defence mechanisms, reduced susceptibility to multiple classes of antimicrobials, and susceptibility to bacteriophage infection. EPA consists of a rhamnan backbone decorated with strain-specific substituents that are essential for its biological activity. Here, we show that epaB encodes the enzyme responsible for the first committed step in assembling the EPA rhamnan chain. Using NMR spectroscopy, we demonstrate that E. faecalis lacking epaB produces an EPA polymer composed solely of decorations directly anchored to the peptidoglycan, with no detectable rhamnan backbone. The absence of this rhamnan moiety profoundly alters cell wall architecture, as revealed by atomic force microscopy of the mutant cell walls. The epaB mutation also abolishes innate immune evasion and virulence in the zebrafish infection model, while conferring resistance to bacteriophages. Collectively, these findings demonstrate that both the rhamnan backbone and its decorations are required for EPA’s full biological activity, establishing the structural and functional interdependence of these two components. IMPORTANCE: The Enterococcal Polysaccharide Antigen (EPA) is essential for normal growth and division, virulence, antimicrobial resistance, and phage infection in enterococci. This surface polymer comprises a structurally conserved rhamnan backbone substituted with strain-specific decorations. These variable decorations have been directly linked to the biological functions of EPA, whereas the rhamnan backbone has been proposed to serve primarily as a structural scaffold. Here, we use NMR spectroscopy to show that mutation of epaB , the gene responsible for the first biosynthetic step in rhamnan backbone formation, results in an EPA polymer composed exclusively of decorations anchored to the peptidoglycan. We reveal that the lack of rhamnan backbone is associated with a change in the cell wall architecture and abolishes virulence and infection by bacteriophages. Together, these findings demonstrate that assembly of the conserved rhamnan backbone is indispensable for EPA function and highlight this biosynthetic step as a promising target to combat enterococcal infections.

The Streptococcus Great Britain and Ireland (Strep GBI) conference was launched to bring together the research community studying streptococcal bacteria. The inaugural conference, Strep GBI 2026, was hosted at the Durham University from 30 to 31 March 2026 by Dr Karrera Y. Djoko, Professor Helge C. Dorfmueller and Dr Alex J. McCarthy. The conference was attended by 80 delegates from England, Scotland and Ireland with keynote lectures by Dr Angela H. Nobbs, Dr Matthew J. Sullivan and Dr Tom Parks. All other talks and flash talks were delivered by early-career researchers. Fundamental and clinical research presented at the Strep GBI meeting covered human and veterinary streptococcal pathogens, ranging from Streptococcus pyogenes , Streptococcus pneumoniae, Streptococcus agalactiae, Streptococcus dysgalactiae subsp. dysgalactiae to Streptococcus thermophilus and Streptococcus suis . Key research insights into streptococcal biology, host–pathogen biology, virulence factors, immunology, epidemiology, antimicrobial resistance, bacterial evolution and more were presented, highlighting the breadth of research on streptococcal bacteria in Great Britain and Ireland.

Intraspecies interactions shape microbial community structure and evolution, yet the mechanisms determining competitive outcomes among closely related strains remain unclear. The soil bacterium Bacillus subtilis is a model for microbial social interactions, where quorum-sensing systems regulate cooperation and antagonism. Here, we take a multifaceted approach to dissect the role of quorum-sensing regulation in competitive fitness. Isolate NCIB 3610 carries a signal unresponsive RapP-PhrP module that alters quorum-sensing control and promotes faster growth. Modelling and mutant analysis demonstrate that the small differences in growth rate conferred by RapP-PhrP ³⁶¹⁰ are sufficient to drive competitive exclusion. The importance of quorum sensing control is further exemplified by experimental evolution of distinct wild isolates, which revealed recurrent mutations in the sensor kinase comP , which phenocopy complete comP or comA deletions and confer a growth-linked competitive advantage. Key quorum sensing mechanisms are abandoned even in structured microbial communities, where it might be expected that communal traits are favoured. Furthermore, a phylogenomic survey of 370 B. subtilis genomes identified disruptive comP mutations in ~16% of isolates. However, growth rate alone does not explain all interaction outcomes as even isogenic strains with equivalent doubling times differ in competitiveness. Transcriptomic profiling and validation experiments implicated a type VII secretion system toxin as an additional effector. These findings reveal that disruption of quorum-sensing pathways, whether naturally or through selection, provides a rapid route to competitive advantage, highlighting a fundamental trade-off between communal signalling and individual fitness in microbial populations.

Intraspecies interactions shapes microbial community structure and evolution, yet the mechanisms determining competitive outcomes among closely related strains remain unclear. The soil bacterium Bacillus subtilis is a model for microbial social interactions, where quorum-sensing systems regulate cooperation and antagonism. Here, we take a multifaceted approach to dissect the role of quorum-sensing regulation in competitive fitness. Isolate NCIB 3610 carries a signal unresponsive RapP-PhrP module that alters quorum-sensing control and promotes faster growth. Modelling and mutant analysis demonstrate that the small differences in growth rate conferred by RapP-PhrP ³⁶¹⁰ are sufficient to drive competitive exclusion. The importance of quorum sensing control is further exemplified by experimental evolution of distinct wild isolates, which revealed recurrent mutations in the sensor kinase comP , which phenocopy complete comP or comA deletions and confer a growth-linked competitive advantage. Key quorum sensing mechanisms are abandoned even in structured microbial communities, where it might be expected that communal traits are favoured. Furthermore, a phylogenomic survey of 370 B. subtilis genomes identified disruptive comP mutations in ∼16% of isolates. However, growth rate alone does not explain all interaction outcomes as even isogenic strains with equivalent doubling times differ in competitiveness. Transcriptomic profiling and validation experiments implicated a type VII secretion system toxin as an additional effector. These findings reveal that disruption of quorum-sensing pathways, whether naturally or through selection, provides a rapid route to competitive advantage, highlighting a fundamental trade-off between communal signalling and individual fitness in microbial populations. Importance: Microbial competition and cooperation are key in shaping the structure and evolution of microbial communities. Our study on Bacillus subtilis , a model for microbial social interactions, reveals how alterations in cell-cell communication can enhance competitive fitness. We show, through a combination of modelling, mutational analysis, and experimental evolution, that certain strains of B. subtilis gain competitive advantage by disrupting their quorum sensing pathways, which leads to faster growth and enhanced competitiveness. Such mutations are prevalent in ∼16% of analysed genomes, underscoring their widespread evolutionary benefit under competitive conditions. Our research highlights the delicate balance between individual success and community cooperation in microbes, providing insights into microbial behaviour and potential applications in modifying microbial ecosystems.

Group A Streptococcus (Strep A) is a human-exclusive bacterial pathogen killing annually more than 500,000 patients, and no current licensed vaccine exists. Strep A bacteria are highly diverse, but all produce an essential, abundant, and conserved surface carbohydrate, the Group A Carbohydrate, which contains a rhamnose polysaccharide (RhaPS) backbone. RhaPS is a validated universal vaccine candidate in a glycoconjugate prepared by chemical conjugation of the native carbohydrate to a carrier protein. We engineered the Group A Carbohydrate biosynthesis pathway to enable recombinant production using the industry standard route to couple RhaPS to selected carrier proteins within Escherichia coli cells. The structural integrity of the produced recombinant glycoconjugate vaccines was confirmed by Nuclear Magnetic Resonance (NMR) spectroscopy and mass spectrometry. Purified RhaPS glycoconjugates elicited carbohydrate-specific antibodies in mice and rabbits and bound to the surface of multiple Strep A strains of diverse M-types, confirming the recombinantly produced RhaPS glycoconjugates as valuable vaccine candidates.

Group A Streptococcus (Strep A) cause a wide range of human-exclusive infections, annually killing more than 500,000 people. Antibiotic resistance incidence of invasive Strep A tripled in the past decade and emphasises the need to develop a universal Strep A vaccine. In this study, we developed recombinant rhamnose polysaccharides (RhaPS), a validated universal Strep A vaccine candidate, presented on E. coli outer membrane vesicles (OMVs). We investigated OMV-RhaPS for their immunogenicity in the mouse and rabbit models. Through flow cytometry, ELISA, Luminex assays, and immunofluorescence microscopy, we demonstrated that RhaPS-specific antibodies recognise Strep A strains via the Group A Carbohydrate (GAC) in S. pyogenes and the newly emerged S. dysgalactiae subsp. equisimilis . Elevated IL-17a levels from RhaPS-OMV-immunised splenocytes indicate the RhaPS-specific stimulation of long-term memory immune cells. We are the first to report the efficacy and potency of recombinant produced RhaPS inducing humoral-mediated immune responses and triggering antibodies that recognise Strep A bacteria.

Strep A is a human-exclusive bacterial pathogen killing annually more than 500,000 patients, and no current licensed vaccine exists. Strep A bacteria are highly diverse, but all produce an essential, abundant, and conserved surface carbohydrate, the Group A Carbohydrate, which contains a rhamnose polysaccharide (RhaPS) backbone. RhaPS is a validated universal vaccine candidate in a glycoconjugate prepared by chemical conjugation of the native carbohydrate to a carrier protein. We engineered the Group A Carbohydratte biosynthesis pathway to enable recombinant production using the industry standard route to couple RhaPS to selected carrier proteins within E. coli cells. The structural integrity of the produced recombinant glycoconjugate vaccines was confirmed by NMR spectroscopy and mass spectrometry. Purified RhaPS glycoconjugates elicited carbohydrate-specific antibodies in mice and rabbits and bound to the surface of multiple Strep A strains of diverse M-types, confirming the recombinantly produced RhaPS glycoconjugates as valuable vaccine candidates.

The Bacillus subtilis extracellular biofilm matrix includes an exopolysaccharide (EPS) that is critical for the architecture and function of the community. To date, our understanding of the biosynthetic machinery and the molecular composition of the EPS of B. subtilis remains unclear and incomplete. This report presents synergistic biochemical and genetic studies built from a foundation of comparative sequence analyses targeted at elucidating the activities of the first two membrane-committed steps in the EPS biosynthetic pathway. By taking this approach, we determined the nucleotide sugar donor and lipid-linked acceptor substrates for the first two enzymes in the B. subtilis biofilm EPS biosynthetic pathway. EpsL catalyzes the first phosphoglycosyl transferase step using uridine diphosphate (UDP)-di- N -acetyl bacillosamine as phospho-sugar donor. EpsD is a predicted GT-B fold (GT4 family) retaining glycosyl transferase that catalyzes the second step in the pathway that utilizes the product of EpsL as an acceptor substrate and UDP- N -acetyl glucosamine as the sugar donor. Thus, the study defines the first two monosaccharides at the reducing end of the growing EPS unit. In doing so, we provide the first evidence of the presence of bacillosamine in an EPS synthesized by a Gram-positive bacterium. IMPORTANCE: Biofilms are the communal way of life that microbes adopt to increase survival. Key to our ability to systematically promote or ablate biofilm formation is a detailed understanding of the biofilm matrix macromolecules. Here, we identify the first two essential steps in the Bacillus subtilis biofilm matrix exopolysaccharide (EPS) synthesis pathway. Together, our studies and approaches provide the foundation for the sequential characterization of the steps in EPS biosynthesis, using prior steps to enable chemoenzymatic synthesis of the undecaprenyl diphosphate-linked glycan substrates.