Coenzyme M biosynthesis in bacteria involves phosphate elimination by a functionally distinct member of the aspartase/fumarase superfamily.

For nearly 30 years, coenzyme M (CoM) was assumed to be present solely in methanogenic archaea. In the late 1990s, CoM was reported to play a role in bacterial propene metabolism, but no biosynthetic pathway for CoM has yet been identified in bacteria. Here, using bioinformatics and proteomic approaches in the metabolically versatile bacterium Xanthobacter autotrophicus Py2, we identified four putative CoM biosynthetic enzymes encoded by the xcbB1, C1, D1, and E1 genes. Only XcbB1 was homologous to a known CoM biosynthetic enzyme (ComA), indicating that CoM biosynthesis in bacteria involves enzymes different from those in archaea. We verified that the ComA homolog produces phosphosulfolactate from phosphoenolpyruvate (PEP), demonstrating that bacterial CoM biosynthesis is initiated similarly as the phosphoenolpyruvate-dependent methanogenic archaeal pathway. The bioinformatics analysis revealed that XcbC1 and D1 are members of the aspartase/fumarase superfamily (AFS) and that XcbE1 is a pyridoxal 5'-phosphate-containing enzyme with homology to d-cysteine desulfhydrases. Known AFS members catalyze ß-elimination reactions of succinyl-containing substrates, yielding fumarate as the common unsaturated elimination product. Unexpectedly, we found that XcbC1 catalyzes ß-elimination on phosphosulfolactate, yielding inorganic phosphate and a novel metabolite, sulfoacrylic acid. Phosphate-releasing ß-elimination reactions are unprecedented among the AFS, indicating that XcbC1 is an unusual phosphatase. Direct demonstration of phosphosulfolactate synthase activity for XcbB1 and phosphate ß-elimination activity for XcbC1 strengthened their hypothetical assignment to a CoM biosynthetic pathway and suggested functions also for XcbD1 and E1. Our results represent a critical first step toward elucidating the CoM pathway in bacteria.

Hydrogels for Local and Sustained Delivery of Bacteriophages to Treat Wound Infections.

Bacteriophages (phages) are viruses that specifically target and kill bacteria, serving as a promising therapeutic to combat multidrug-resistant (MDR) pathogens such as Pseudomonas aeruginosa (Pa). However, delivering adequate concentrations of active phages directly to the infection site over sufficient times to eradicate infections remains an outstanding challenge to phage therapy (PT). Here we present "HydroPhage", a biocompatible hydrogel system for the sustained release of high-titre phages to effectively treat infections caused by MDR pathogens. We develop injectable hydrogels comprised of hyaluronic acid (HA) and polyethylene glycol (PEG) crosslinked through static covalent thioether bonds and hemithioacetal-based dynamic covalent crosslinks (DCC), which encapsulate phages at concentration up to 1011 PFU per mL gel, and achieve sustained release over a week with more than 60% total phage recovery. In a preclinical mouse model of extended wound infection, we demonstrate enhanced bacterial clearance compared to intravenous treatment. Thus, using hydrogels for local and sustained delivery of phage may represent an effective approach to eradicating MDR infections broadly.

Regulatory T cells use heparanase to access IL-2 bound to extracellular matrix in inflamed tissue.

Although FOXP3+ regulatory T cells (Treg) depend on IL-2 produced by other cells for their survival and function, the levels of IL-2 in inflamed tissue are low, making it unclear how Treg access this critical resource. Here, we show that Treg use heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) of inflamed central nervous system tissue. HPSE expression distinguishes human and murine Treg from conventional T cells and is regulated by the availability of IL-2. HPSE-/- Treg have impaired stability and function in vivo, including in the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Conversely, endowing monoclonal antibody-directed chimeric antigen receptor (mAbCAR) Treg with HPSE enhances their ability to access HS-sequestered IL-2 and their ability to suppress neuroinflammation in vivo. Together, these data identify a role for HPSE and the ECM in immune tolerance, providing new avenues for improving Treg-based therapy of autoimmunity.

Hyaluronan in the pathogenesis of acute and post-acute COVID-19 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) recently emerged as the cause of a global pandemic. Infection with SARS-CoV-2 can result in COVID-19 with both acute and chronic disease manifestations that continue to impact many patients long after the resolution of viral replication. There is therefore great interest in understanding the host factors that contribute to COVID-19 pathogenesis. In this review, we address the role of hyaluronan (HA), an extracellular matrix polymer with roles in inflammation and cellular metabolism, in COVID-19 and critically evaluate the hypothesis that HA promotes COVID-19 pathogenesis. We first provide a brief overview of COVID-19 infection. Then we briefly summarize the known roles of HA in airway inflammation and immunity. We then address what is known about HA and the pathogenesis of COVID-19 acute respiratory distress syndrome (COVID-19 ARDS). Next, we examine potential roles for HA in post-acute SARS-CoV-2 infection (PASC), also known as "long COVID" as well as in COVID-associated fibrosis. Finally, we discuss the potential therapeutics that target HA as a means to treat COVID-19, including the repurposed drug hymecromone (4-methylumbelliferone). We conclude that HA is a promising potential therapeutic target for the treatment of COVID-19.

FOXP3+ regulatory T cells use heparanase to access IL-2 bound to ECM in inflamed tissues.

FOXP3+ regulatory T cells (Treg) depend on exogenous IL-2 for their survival and function, but circulating levels of IL-2 are low, making it unclear how Treg access this critical resource in vivo. Here, we show that Treg use heparanase (HPSE) to access IL-2 sequestered by heparan sulfate (HS) within the extracellular matrix (ECM) of inflamed central nervous system tissue. HPSE expression distinguishes human and murine Treg from conventional T cells and is regulated by the availability of IL-2. HPSE-/- Treg have impaired stability and function in vivo, including the experimental autoimmune encephalomyelitis (EAE) mouse model of multiple sclerosis. Conversely, endowing Treg with HPSE enhances their ability to access HS-sequestered IL-2 and their tolerogenic function in vivo. Together, these data identify novel roles for HPSE and the ECM in immune tolerance, providing new avenues for improving Treg-based therapy of autoimmunity.

Hyaluronan synthesis inhibition impairs antigen presentation and delays transplantation rejection.

A coat of pericellular hyaluronan surrounds mature dendritic cells (DC) and contributes to cell-cell interactions. We asked whether 4-methylumbelliferone (4MU), an oral inhibitor of HA synthesis, could inhibit antigen presentation. We find that 4MU treatment reduces pericellular hyaluronan, destabilizes interactions between DC and T-cells, and prevents T-cell proliferation in vitro and in vivo. These effects were observed only when 4MU was added prior to initial antigen presentation but not later, consistent with 4MU-mediated inhibition of de novo antigenic responses. Building on these findings, we find that 4MU delays rejection of allogeneic pancreatic islet transplant and allogeneic cardiac transplants in mice and suppresses allogeneic T-cell activation in human mixed lymphocyte reactions. We conclude that 4MU, an approved drug, may have benefit as an adjunctive agent to delay transplantation rejection.

Low-affinity CD4+ T cells are major responders in the primary immune response.

A robust primary immune response has been correlated with the precursor number of antigen-specific T cells, as identified using peptide MHCII tetramers. However, these tetramers identify only the highest-affinity T cells. Here we show the entire CD4+ T-cell repertoire, inclusive of low-affinity T cells missed by tetramers, using a T-cell receptor (TCR) signalling reporter and micropipette assay to quantify naive precursors and expanded populations. In vivo limiting dilution assays reveal hundreds more precursor T cells than previously thought, with higher-affinity tetramer-positive T cells, comprising only 5-30% of the total antigen-specific naive repertoire. Lower-affinity T cells maintain their predominance as the primary immune response progresses, with no enhancement of survival of T cells with high-affinity TCRs. These findings demonstrate that affinity for antigen does not control CD4+ T-cell entry into the primary immune response, as a diverse range in affinity is maintained from precursor through peak of T-cell expansion.