RESUMO
The role of commensal anaerobic bacteria in chronic respiratory infections is unclear, yet they can exist in abundances comparable to canonical pathogens in vivo . Their contributions to the metabolic landscape of the host environment may influence pathogen behavior by competing for nutrients and creating inhospitable conditions via toxic metabolites. Here, we reveal a mechanism by which the anaerobe-derived short chain fatty acids (SCFAs) propionate and butyrate negatively affect Staphylococcus aureus physiology by disrupting branched chain fatty acid (BCFA) metabolism. In turn, BCFA impairment results in impaired growth, diminished expression of the agr quorum sensing system, as well as increased sensitivity to membrane-targeting antimicrobials. Altered BCFA metabolism also reduces S. aureus fitness in competition with Pseudomonas aeruginosa , suggesting that airway microbiome composition and the metabolites they produce and exchange directly impact pathogen succession over time. The pleiotropic effects of these SCFAs on S. aureus fitness and their ubiquity as metabolites in animals also suggests that they may be effective as sensitizers to traditional antimicrobial agents when used in combination.
RESUMO
Daptomycin is a membrane-targeting last-resort antimicrobial therapeutic for the treatment of infections caused by methicillin- and/or vancomycin-resistant Staphylococcus aureus. In the rare event of failed daptomycin therapy, the source of resistance is often attributable to mutations directly within the membrane phospholipid biosynthetic pathway of S. aureus or in the regulatory systems that control cell envelope response and membrane homeostasis. Here we describe the structural changes to the cell envelope in a daptomycin-resistant isolate of S. aureus strain N315 that has acquired mutations in the genes most commonly reported associated with daptomycin resistance: mprF, yycG, and pgsA. In addition to the decreased phosphatidylglycerol (PG) levels that are the hallmark of daptomycin resistance, the mutant with high-level daptomycin resistance had increased branched-chain fatty acids (BCFAs) in its membrane lipids, increased membrane fluidity, and increased cell wall thickness. However, the successful utilization of isotope-labeled straight-chain fatty acids (SCFAs) in lipid synthesis suggested that the aberrant BCFA:SCFA ratio arose from upstream alteration in fatty acid synthesis rather than a structural preference in PgsA. Transcriptomics studies revealed that expression of pyruvate dehydrogenase (pdhB) was suppressed in the daptomycin-resistant isolate, which is known to increase BCFA levels. While complementation with an additional copy of pdhB had no effect, complementation of the pgsA mutation resulted in increased PG formation, reduction in cell wall thickness, restoration of normal BCFA levels, and increased daptomycin susceptibility. Collectively, these results demonstrate that pgsA contributes to daptomycin resistance through its influence on membrane fluidity and cell wall thickness, in addition to phosphatidylglycerol levels. IMPORTANCE: The cationic lipopeptide antimicrobial daptomycin has become an essential tool for combating infections with Staphylococcus aureus that display reduced susceptibility to ß-lactams or vancomycin. Since daptomycin's activity is based on interaction with the negatively charged membrane of S. aureus, routes to daptomycin-resistance occur through mutations in the lipid biosynthetic pathway surrounding phosphatidylglycerols and the regulatory systems that control cell envelope homeostasis. Therefore, there are many avenues to achieve daptomycin resistance and several different, and sometimes contradictory, phenotypes of daptomycin-resistant S. aureus, including both increased and decreased cell wall thickness and membrane fluidity. This study is significant because it demonstrates the unexpected influence of a lipid biosynthesis gene, pgsA, on membrane fluidity and cell wall thickness in S. aureus with high-level daptomycin resistance.
Assuntos
Antibacterianos , Parede Celular , Daptomicina , Farmacorresistência Bacteriana , Fluidez de Membrana , Testes de Sensibilidade Microbiana , Staphylococcus aureus , Daptomicina/farmacologia , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Fluidez de Membrana/efeitos dos fármacos , Antibacterianos/farmacologia , Farmacorresistência Bacteriana/genética , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Mutação , Fosfatidilgliceróis/metabolismoRESUMO
Daptomycin is a membrane-targeting last-resort antimicrobial therapeutic for the treatment of infections caused by methicillin- and/or vancomycin-resistant Staphylococcus aureus. In the rare event of failed daptomycin therapy, the source of resistance is often attributable to mutations directly within the membrane phospholipid biosynthetic pathway of S. aureus or in the regulatory systems that control cell envelope response and membrane homeostasis. Here we describe the structural changes to the cell envelope in a daptomycin-resistant isolate of S. aureus strain N315 that has acquired mutations in the genes most commonly reported associated with daptomycin-resistance: mprF, yycG, and pgsA. In addition to the decreased phosphatidylglycerol (PG) levels that are the hallmark of daptomycin-resistance, the mutant with high-level daptomycin resistance had increased branched-chain fatty acids (BCFAs) in its membrane lipids, increased membrane fluidity, and increased cell wall thickness. However, the successful utilization of isotope-labeled straight-chain fatty acids (SCFAs) in lipid synthesis suggested that the aberrant BCFA:SCFA ratio arose from upstream alteration in fatty acid synthesis rather than a structural preference in PgsA. RT-qPCR studies revealed that expression of pyruvate dehydrogenase (pdhB) was suppressed in the daptomycin-resistant isolate, which is known to increase BCFA levels. While complementation with an additional copy of pdhB had no effect, complementation of the pgsA mutation resulted in increased PG formation, reduction in cell wall thickness, restoration of normal BCFA levels, and increased daptomycin susceptibility. Collectively, these results demonstrate that pgsA contributes to daptomycin resistance through its influence on membrane fluidity and cell wall thickness, in addition to phosphatidylglycerol levels.
RESUMO
Purpose: Medicine has yet to increase the representation of historically excluded persons in medicine to reflect the general population. The lack of support and guidance in the medical training of these individuals is a significant contributor to this disparity. The Engage, Mentor, Prepare, Advocate for, Cultivate, and Teach (EMPACT) Mentoring program was created to address this problem by providing support for learners who are historically underrepresented in medicine (URiM) as they progress through medical school. Methods: The EMPACT Pilot Program was formed and conducted during the 2019-2020 academic year. A total of 19 EMPACT mentorship groups were created, each consisting of two mentors and four medical student mentees. Additionally, four professional development workshops were held along with a final Wrap-up and Awards event. Pre and post pilot program surveys along with surveys after each workshop and focus groups were conducted with a random selection of program participants. Results: When compared to data from before and after the implementation of the EMPACT program, there were statistically significant differences (p < 0.05) in EMPACT mentees reporting they agree or strongly agree they felt ready to handle their clinical rotations (28% to 65%), felt the need to have an advocate (85% to 47%), possessed insight on day-to-day activities of an attending (26% to 56%) and felt a sense of community (79% to 94%). Mentors revealed an increase in their awareness of the concepts of microaggressions and imposter phenomenon. Finally, both groups felt an increase in their support system and sense of community at the school of medicine. Conclusion: Despite COVID-19 limitations, the EMPACT program met its goals. We effectively supported URiM medical students through mentorship, networking, and community.