Biology subjects

Reglinski, M.

Publications and source records attributed to Reglinski, M..

8 recordsLinked to original sources

bioRxiv2026

Mild extraction preserves labile structural features of Gram-positive bacterial cell wall glycopolymers

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

microbiology↗

bioRxiv2026

M1 protein expression determines niche-specific spread of Streptococcus pyogenes

Invasive infections with Streptococcus pyogenes account for significant mortality. Cell surface M protein is believed to be essential for S. pyogenes virulence, with critical roles in dissemination and immune evasion. In contrast, the role of secreted M protein in infection is less clear. Here, we assessed the role of M protein in streptococcal colonisation, persistence, and invasion in respiratory and soft tissue murine infection models, and report that while cell-surface M protein is required to establish nasopharyngeal infection, it does not drive invasive infection. Cell-surface M protein was important for binding of major host matrix proteins and growth in immune human blood, but soluble M protein was the predominant determinant of lymphatic survival, tissue spread, and systemic infection in mice. A mutant emm1 strain which only expresses secreted M protein exhibited enhanced survival in the lymph node niche and overall competitive advantage over the wildtype parent strain in co-infections. Absence of M protein at the bacterial cell surface led to enhanced hyaluronan binding, providing explanation for homing to the draining lymph node. Together, these findings highlight potential nuance in the role of M protein that may impact vaccine approaches targeting M protein. Author SummaryS. pyogenes is a significant cause of illness, both mild and lethal, worldwide. Despite this, there are currently no approved vaccines. Many vaccine candidates are based on the M protein, a surface expressed anti-phagocytic factor that is thought to be critical for the ability of S. pyogenes to evade the host immune system. The unexpected selection of an M1 protein mutant strain following in vivo soft tissue infection raised many questions for us. If M protein is meant to be the critical factor in avoiding immune killing, how was this strain able to persist? One explanation could be that, although the strain was negative for M1 protein on the bacterial surface, it still secreted a truncated version of the protein with potential inflammatory effects. Importantly, the strain was selected in the draining lymph node pointing to a niche-specific advantage. We showed that cell surface-associated M1 protein, although required to establish nasopharyngeal infection, was not required in soft tissue infection, and that expression of a soluble, truncated form of M1 protein gave a survival advantage over the wild-type strain expressing cell wall-associated M1 protein. Furthermore, the absence of cell surface M protein led to enhanced hyaluronan binding providing explanation for lymphatic niche homing.

microbiology↗

bioRxiv2024

A widely-occurring family of pore-forming effectors broadens the impact of the Serratia Type VI secretion system

The ability to compete with diverse competitors is essential for bacteria to succeed in microbial communities. A widespread strategy for inter-bacterial competition is the delivery of antibacterial toxins, or effector proteins, directly into rival cells using the Type VI secretion system (T6SS). Whilst a large number of broad-spectrum enzymatic T6SS effectors have been described, relatively few which form pores in target cell membranes have been reported. Here, we describe a widely-occurring new family of T6SS-dependent pore-forming effectors, exemplified by Ssp4 of Serratia marcescens Db10. We show in vitro that Ssp4 forms regulated pores that have higher selectivity for cations and use molecular dynamics simulations to support a high resolution structural model of a tetrameric membrane pore formed by Ssp4. Notably, Ssp4 displays a distinct ion selectivity, phylogenetic distribution and impact on intoxicated cells compared with Ssp6, the other cation-selective pore-forming toxin delivered by the same T6SS. Ssp4 is also active against a wider range of target species than Ssp6, highlighting that T6SS effectors are not always broad-spectrum. Finally, use of Tn-seq to identify Ssp4-resistant mutants reveals that a mucA mutant of Pseudomonas fluorescens, which overproduces extracellular polysaccharide, provides resistance to T6SS attacks. We conclude that possession of two distinct T6SS-dependent pore-forming toxins may be a common strategy to ensure effective de-energisation of closely- and distantly-related competitors.

microbiology↗

bioRxiv2024

Rhamnose Polysaccharide-Decorated Outer Membrane Vesicles as a Vaccine Candidate Targeting Group A Streptococcus from Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis

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.

microbiology↗

bioRxiv2024

A platform for the recombinant production of Group A Streptococcus glycoconjugate vaccines

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.

microbiology↗

bioRxiv2021

Non-conventional serine protease activity of the CXC chemokine-cleaving streptococcal enzyme, SpyCEP

The Streptococcus pyogenes cell envelope protease (SpyCEP) is vital to streptococcal pathogenesis and disease progression. Despite its strong association with invasive disease, little is known about enzymatic function beyond the ELR+ CXC chemokine substrate range. As a serine protease, SpyCEP has a catalytic triad consisting of aspartate (D151), histidine (H279), and serine (S617) residues which are all thought to be mandatory for full activity. We utilised a range of SpyCEP constructs to investigate the protein domains and catalytic residues necessary for enzyme function. We designed a high-throughput mass spectrometry assay to measure CXCL8 cleavage and applied this for the first time to study the enzyme kinetics of SpyCEP. Results revealed a remarkably low Michaelis-Menton constant (KM) of 82 nM and a turnover of 1.65 molecules per second. We found that an N-terminally-truncated SpyCEP C-terminal construct containing just the catalytic dyad of H279 and S617 was capable of cleaving CXCL8 with a similar KM of 55 nM, albeit with a reduced substrate turnover of 2.7 molecules per hour, representing a 2,200- fold reduction in activity. We conclude that the SpyCEP C-terminus plays a key role in high affinity substrate recognition and binding, but that the N-terminus is required for full catalytic activity.

microbiology↗

bioRxiv2021

Recombinant Group A Carbohydrate backbone embedded into Outer Membrane Vesicles is a potent vaccine candidate targeting Group A Streptococcus from Streptococcus pyogenes and Streptococcus dysgalactiae subsp. equisimilis

BackgroundGroup A Streptococcus (GAS) are responsible for a wide range of human-exclusive infections, annually killing more than 500,000 people. Antibiotic resistance incidence of invasive GAS tripled in the past decade and emphasises the need to develop a universal GAS vaccine. We have produced, for the first time, a recombinant polyrhamnose backbone (pRha), a validated universal GAS vaccine candidate. E. coli outer membrane vesicles (OMVs) carrying pRha were investigated for their immunogenicity and efficacy in an animal model. MethodsOMVs decorated with pRha were administered to C57BL/6J mouse and rabbit models. Flow cytometry, ELISA, Luminex, immunofluorescence microscopy and serum bactericidal assay assays were conducted to investigate the ability of pRha-specific antibodies to recognise and kill clinical (hypervirulent) GAS strains. ResultsOur results suggest that pRha-OMVs induce specific antibodies which recognise Group A Carbohydrate (GAC) from S pyogenes and S. dysgalactiae subsp. equisimilis. Increased IgG levels correlate with increased bactericidal killing of the hypervirulent GAS M89 strain. Elevated IL-17a from pRha-OMV-immunised splenocytes indicates possible stimulation of long-term memory immune cells. ConclusionWe are the first to report efficacy and potency of this unique, exogenously produced polysaccharide, pRha, in the induction of humoral-mediated immune responses to GAS. TopicStreptococcus pyogenes, immunoglobulins, polysaccharides, opsonophagocytosis, acute rheumatic fever, M protein, invasive Group A Streptococcus, hyaluronic acid

microbiology↗

bioRxiv2020

Vaccine-induced, but not natural immunity, against the Streptococcal Inhibitor of Complement protects against invasive disease

Highly pathogenic emm1 Streptococcus pyogenes strains secrete the multidomain Streptococcal inhibitor of complement (SIC) that binds and inactivates components of the innate immune response. We aimed to determine if naturally occurring or vaccine-induced antibodies to SIC are protective against invasive S. pyogenes infection. Immunisation with full length SIC protected mice against systemic bacterial dissemination following intranasal or intramuscular infection with emm1 S. pyogenes. Vaccine-induced rabbit anti-SIC antibodies, but not naturally occurring human anti-SIC antibodies, enhanced bacterial clearance in an ex vivo whole blood assay. SIC vaccination of both mice and rabbits resulted in antibody recognition of all domains of SIC, whereas naturally occurring human anti-SIC antibodies recognised the proline-rich region of SIC only. We therefore propose a model whereby natural infection with S. pyogenes generates non-protective antibodies against the proline-rich region of SIC, while vaccination with full length SIC permits development of protective antibodies against all SIC domains.

microbiology↗