Gish, C. | bioRxiv Science

bioRxiv2025

Methicillin-Resistant Staphylococcus aureus has Phenotypic Variation in mecA Expression that Alters Antibiotic Sensitivity

Methicillin resistant Staphylococcus aureus (MRSA) sepsis has a high rate of morbidity and mortality. Multiple clinical studies have demonstrated improved outcomes when MRSA sepsis is treated with dual antibiotic therapy that includes a {beta}-lactam antibiotic such as cefazolin. This is a paradox as MRSA should be inherently resistant to this class of antibiotics. We report a serendipitous observation revealed a phenotype where MRSA became sensitive to cefazolin when cultured in a physiologic relevant media of fetal bovine serum as well as in synovial fluid. This could be observed across multiple clinical isolates. Expected resistance was maintained when cultured in Muller Hinton Broth (MHB). MRSA {beta}-lactam antibiotic resistance is mediated by PBP2a, a penicillin-binding protein encoded by mecA. We hypothesized that this phenotype of antibiotic sensitivity in physiologic medium was based, in part, on levels of PBP2a expression and post-translational modifications of peptidoglycan wall teichoic acid (WTA). We therefore conducted quantitative RT-PCR analysis and Western blotting which demonstrated limited mecA expression in the mRNA level and limited PBP2a protein level when cultured in FBS or synovial fluid as compared to the clinical microbiology standard MHB, respectively. Whole genome sequencing of loss of function mutants generated through serial passaging in FBS revealed that the clp family of proteins and rpo genes were involved in {beta}-lactam resistance. Cell wall peptidoglycan analysis suggested that WTA glycosylation was altered between {beta}-lactam resistant and sensitive MRSA phenotypes. Together, this suggests pathways for clpP, rpoB, and WTA glycosylation can be new potential targets for MRSA treatment. IMPORTANCEThis study demonstrates that MRSA has phenotypic variation in mecA expression, PBP2a protein levels, and patterns of wall teichoic acid glycosylation based on environmental conditions which can result in a phenotype of {beta}-lactam antibiotic sensitivity. The presence of an antibiotic resistance gene does not necessarily result in antibiotic resistance. This provides a possible explanation for the clinical observations of potential therapeutic benefit of {beta}-lactam antibiotics in dual therapy treatment of MRSA sepsis and suggests new therapeutic strategies to combat multidrug resistant bacteria. More importantly, identifying genes potentially responsible for this phenotype offers new potential therapeutic targets for the treatment of multidrug resistant infection.

microbiology↗

bioRxiv2024

Visual circuitry for distance estimation in Drosophila

Animals must infer the three-dimensional structure of their environment from two-dimensional images on their retinas. In particular, visual cues like motion parallax and binocular disparity can be used to judge distances to objects. Studies across several animal models have found neural signals that correlate with visual distance, but the causal role of these neurons in distance estimation as well as the range of possible neural properties that can inform distance estimation have remained poorly understood. Here, we developed a novel high-throughput behavioral assay to identify neurons in the Drosophila visual system that are involved in distance estimation during free locomotion. We found that silencing the primary motion detectors in the fly visual system eliminated their ability to perceive distance, consistent with a reliance on motion parallax to judge distance. Through a targeted silencing screen of visual neurons during behavior and through in vivo two-photon microscopy, we identified a visual projection neuron that encodes the parallax signal in the relative motion of foreground and background. Interestingly, it differs from previously identified parallax-tuned neurons in its lack of direction selectivity both to moving bars and to moving backgrounds. This non-canonical tuning is interpretable in the context of parallax signals that the fly would likely encounter during naturalistic walking behavior. Our results demonstrate how both direction selective and non-direction selective feature-detecting neurons can contribute to distance estimation using parallax cues, providing a framework for considering broader classes of parallax-encoding neurons in distance estimation across visual systems.

neuroscience↗