A Host-Directed Approach to the Detection of Infection in Hard-to-Heal Wounds.

Wound infection is traditionally defined primarily by visual clinical signs, and secondarily by microbiological analysis of wound samples. However, these approaches have serious limitations in determining wound infection status, particularly in early phases or complex, chronic, hard-to-heal wounds. Early or predictive patient-derived biomarkers of wound infection would enable more timely and appropriate intervention. The observation that immune activation is one of the earliest responses to pathogen activity suggests that immune markers may indicate wound infection earlier and more reliably than by investigating potential pathogens themselves. One of the earliest immune responses is that of the innate immune cells (neutrophils) that are recruited to sites of infection by signals associated with cell damage. During acute infection, the neutrophils produce oxygen radicals and enzymes that either directly or indirectly destroy invading pathogens. These granular enzymes vary with cell type but include elastase, myeloperoxidase, lysozyme, and cathepsin G. Various clinical studies have demonstrated that collectively, these enzymes, are sensitive and reliable markers of both early-onset phases and established infections. The detection of innate immune cell enzymes in hard-to-heal wounds at point of care offers a new, simple, and effective approach to determining wound infection status and may offer significant advantages over uncertainties associated with clinical judgement, and the questionable value of wound microbiology. Additionally, by facilitating the detection of early wound infection, prompt, local wound hygiene interventions will likely enhance infection resolution and wound healing, reduce the requirement for systemic antibiotic therapy, and support antimicrobial stewardship initiatives in wound care.

Mechanism of action of the uridyl peptide antibiotics: an unexpected link to a protein-protein interaction site in translocase MraY.

The pacidamycin and muraymycin uridyl peptide antibiotics show some structural resemblance to an Arg-Trp-x-x-Trp sequence motif for protein-protein interaction between bacteriophage ϕX174 protein E and E. coli translocase MraY. Members of the UPA class, and a synthetic uridine-peptide analogue, were found to show reduced levels of inhibition to F288L or E287A mutant MraY enzymes, implying that the UPAs interact at this extracellular site as part of the enzyme inhibition mechanism.

A two-step sulfation in antibiotic biosynthesis requires a type III polyketide synthase.

Caprazamycins (CPZs) belong to a group of liponucleoside antibiotics inhibiting the bacterial MraY translocase, an essential enzyme involved in peptidoglycan biosynthesis. We have recently identified analogs that are decorated with a sulfate group at the 2″-hydroxy of the aminoribosyl moiety, and we now report an unprecedented two-step sulfation mechanism during the biosynthesis of CPZs. A type III polyketide synthase (PKS) known as Cpz6 is used in the biosynthesis of a group of new triketide pyrones that are subsequently sulfated by an unusual 3'-phosphoadenosine-5'-phosphosulfate (PAPS)-dependent sulfotransferase (Cpz8) to yield phenolic sulfate esters, which serve as sulfate donors for a PAPS-independent arylsulfate sulfotransferase (Cpz4) to generate sulfated CPZs. This finding is to our knowledge the first demonstration of genuine sulfate donors for an arylsulfate sulfotransferase and the first report of a type III PKS to generate a chemical reagent in bacterial sulfate metabolism.

The biosynthesis of caprazamycins and related liponucleoside antibiotics: new insights.

The first step in the membrane cycle of reactions during peptidoglycan biosynthesis is the transfer of phospho-MurNAc-pentapeptide from UDP-MurNAc-pentapeptide to undecaprenyl phosphate, catalyzed by the integral membrane protein MraY translocase. Different MraY inhibitors are known and can be subdivided into classes depending on their structural composition. Caprazamycins belong to the liponucleoside class of antibiotics isolated from Streptomyces sp. MK730-62F2. They possess activity in vitro against Gram-positive bacteria, in particular against the genus Mycobacterium including Mycobacterium intracellulare, Mycobacterium avium and Mycobacterium tuberculosis. Caprazamycins and the structurally related liposidomycins and A-90289 share a unique composition of moieties. Their complex structure is derived from 5'-(ß-O-aminoribosyl)-glycyluridine and comprises a unique N,N'-dimethyldiazepanone ring. Recently, the corresponding biosynthetic gene clusters of caprazamycins, liposidomycins and A-90289 have been discovered and will be compared in this review. New information is also emerging regarding the biosynthesis of liponucleoside antibiotics obtained by gene disruption experiments and biochemical investigations.

A new arylsulfate sulfotransferase involved in liponucleoside antibiotic biosynthesis in streptomycetes.

Sulfotransferases are involved in a variety of physiological processes and typically use 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the sulfate donor substrate. In contrast, microbial arylsulfate sulfotransferases (ASSTs) are PAPS-independent and utilize arylsulfates as sulfate donors. Yet, their genuine acceptor substrates are unknown. In this study we demonstrate that Cpz4 from Streptomyces sp. MK730-62F2 is an ASST-type sulfotransferase responsible for the formation of sulfated liponucleoside antibiotics. Gene deletion mutants showed that cpz4 is required for the production of sulfated caprazamycin derivatives. Cloning, overproduction, and purification of Cpz4 resulted in a 58-kDa soluble protein. The enzyme catalyzed the transfer of a sulfate group from p-nitrophenol sulfate (K(m) 48.1 microM, k(cat) 0.14 s(-1)) and methyl umbelliferone sulfate (K(m) 34.5 microM, k(cat) 0.15 s(-1)) onto phenol (K(m) 25.9 and 29.7 mM, respectively). The Cpz4 reaction proceeds by a ping pong bi-bi mechanism. Several structural analogs of intermediates of the caprazamycin biosynthetic pathway were synthesized and tested as substrates of Cpz4. Des-N-methyl-acyl-caprazol was converted with highest efficiency 100 times faster than phenol. The fatty acyl side chain and the uridyl moiety seem to be important for substrate recognition by Cpz4. Liponucleosides, partially purified from various mutant strains, were readily sulfated by Cpz4 using p-nitrophenol sulfate. No product formation could be observed with PAPS as the donor substrate. Sequence homology of Cpz4 to the previously examined ASSTs is low. However, numerous orthologs are encoded in microbial genomes and represent interesting subjects for future investigations.