Methicillin-resistant Staphylococcus aureus (MRSA) infections represent a significant clinical challenge, with high rates of β-lactam resistance and reinfection. Here, we report a combination therapy combining methicillin with a Toll-like receptor 9 (TLR9) agonist (CpG-ODN M362) delivered via a subcutaneously injectable hyaluronic acid - methylcellulose (HAMC) hydrogel for the treatment of MRSA kidney infections. In a murine renal infection model, combination therapy achieved logarithmic reductions in renal bacterial burden compared to either monotherapy, while also preserving protective immune memory that methicillin alone failed to generate. Formulation within a thermosensitive, shear-thinning HAMC hydrogel enabled sustained codelivery of both agents from a single subcutaneous injection, enhancing bacterial clearance and humoral immune responses compared to daily bolus administration. This work demonstrates that hydrogel-mediated immunostimulatory combination therapy represents a promising single-dose strategy for treating MRSA infection with otherwise ineffective antibiotics while simultaneously guarding against reinfection.
Abstract The cell envelope of bacteria is coated with an array of glycans that are poised to engage carbohydrate-binding proteins, including antibodies and lectins, on host cells. The interactions between bacterial glycans and host cell receptors ultimately underpin downstream responses, tipping the balance toward symbiosis or disease. Bacterial glycans are challenging to study due to their complex structures and the transient nature of their interactions with carbohydrate-binding proteins. However, an array of chemical biology tools has been developed to ease the study of bacterial glycans and their binding to proteins on host cells. This review highlights chemical approaches that can be used to better understand the engagement of bacterial glycans with cognate receptors, as well as burgeoning strategies to delineate the interactions of mammalian glycans that have the potential to be translated for the study of bacterial glycans. Ultimately, these tools will help shed light on the full landscape of cell–cell communication mediated by bacterial glycans.
Abstract Rapid detection of choline-containing streptococcal pathogens is important for timely diagnosis and antimicrobial treatment. Here, we developed gCBMLytA, a GFP-fused choline-binding module derived from the pneumococcal autolysin LytA, as a fluorescence-based biosensor. gCBMLytA specifically labeled all 14 tested Streptococcus pneumoniae strains and also recognized Streptococcus oralis, while showing no detectable binding to nontarget ESKAPE pathogens or Haemophilus influenzae. These results indicate that gCBMLytA recognizes choline within the structural context of streptococcal teichoic acids rather than choline-containing structures in unrelated bacteria. In mouse pneumonia models, intravenously administered gCBMLytA selectively accumulated in the lungs of S. pneumoniae-infected mice, but not in healthy controls or Staphylococcus aureus-infected mice. Together, these findings demonstrate that gCBMLytA is a selective fluorescence-based probe for choline-containing streptococcal pathogens and may serve as a versatile recognition module for diagnostic platforms, including fluorescence imaging, lateral flow assays, ELISA, and flow-cytometry-based detection systems.
Gepotidacin, the first-in-class triazaacenaphthylene antibacterial, kills bacteria by stabilizing DNA cleavage complexes and inhibiting the catalytic activities of the type II topoisomerases, gyrase and topoisomerase IV. Mycobacterial species encode gyrase as their sole type II topoisomerase, meaning that inhibition of this enzyme blocks several essential cellular functions through a single target. Unlike fluoroquinolones and spiropyrimidinetriones, which inhibit gyrase by inserting two drug molecules, one at each of the cleaved scissile bonds, gepotidacin intercalates a single molecule into the intact double helix between the two scissile bonds within the enzyme active site. To determine how the distinct binding mechanism of gepotidacin affects its inhibitory profile, we examined the ability of the drug to inhibit decatenation, supercoiling of relaxed DNA, and relaxation of positive supercoils catalyzed by Mycobacterium tuberculosis and Mycobacteroides abscessus gyrase. Gepotidacin inhibited decatenation and supercoiling at comparable concentrations but required higher concentrations to inhibit relaxation of positively supercoiled DNA, a pattern distinct from fluoroquinolones and spiropyrimidetriones. Similar results were observed with GSK000, a Mycobacterium tuberculosis gyrase inhibitor that also intercalates into intact DNA in the gyrase active site. Using mutant gyrase enzymes capable of relaxing both positively and negatively supercoiled DNA, we found that gepotidacin and GSK000 inhibited relaxation of negative supercoils at substantially lower concentrations (similar to 13-fold on average) than relaxation of positive supercoils. This sensitivity to supercoil handedness was markedly higher than observed for nonintercalative drugs (moxifloxacin, ciprofloxacin, zoliflodacin, and novobiocin). These findings suggest that the intercalative binding mechanism of gepotidacin confers heightened sensitivity to DNA supercoil handedness.
Reverse transcriptases from bacterial group II introns (GII RTs) demonstrate superior thermostability and processivity compared with retroviral counterparts. While GII RTs have proven advantageous for RNA-seq, their performance in other applications, such as reverse transcription loop-mediated isothermal amplification (RT-LAMP), remains poorly studied. RT-LAMP is a widely used method suitable for point-of-care diagnostics of RNA viruses and other pathogens. In this study, we compared the performance in RT-LAMP of GII RT from the thermophilic bacterium (Afl RT) with that of the retroviral RNase H-deficient reverse transcriptase M-MuLV. A duplex RT-LAMP targeting SARS-CoV-2 and MS2 phage genomic RNA (internal control) served as a model. Preheating at 95 degrees C and 1.5 M betaine increased the efficiency of RT-LAMP with both reverse transcriptases. Under these conditions, the optimal temperature range and time of reverse transcription were similar for both enzymes, 55-60 degrees C and 15 min, respectively. However, high concentrations of Afl RT (>50 U/reaction) inhibited RT-LAMP. Notably, RT-LAMP with Afl RT demonstrated higher sensitivity for detecting both in vitro-transcribed RNA fragments and SARS-CoV-2-positive samples. In validation against RT-qPCR using nasal swabs (200 SARS-CoV-2-positive and 200 negative samples) from patients with respiratory symptoms, the observed sensitivity of RT-LAMP with Afl RT was 100% (200/200; 95% CI, 98.17-100%), whereas with M-MuLV RT, sensitivity was 97.0% (194/200; 95% CI, 93.58-98.89%). Specificity was 100% for both RTs (200/200; 95% CI, 98.17-100%). These observations demonstrate that Afl RT is suitable for RT-LAMP and may increase the sensitivity of LAMP-based diagnostics.
