ABSTRACT
Bacteria are involved in numerous interactions during infection and among host-associated microbial populations. Salmonella enterica serovar Typhimurium is a foodborne pathogen of great importance as well as a model organism to study interactions within a microbial community. In this study, we found that S. Typhimurium becomes tolerant to azithromycin when co-cultured with lactobacilli strains. Similarly, acidified media, from cell-free supernatant of lactobacilli cultures for instance, also induced the tolerance of S. Typhimurium to azithromycin. The addition of membrane disruptors restored the normal sensitivity to azithromycin in acidified media, but not when lactobacilli were present. These results suggested that the acidification of the media led to modification in envelope homeostasis, but that a different mechanism promoted the tolerance to azithromycin in the presence of lactobacilli strains. To further understand how lactobacilli strains modify the sensitivity of S. Typhimurium to azithromycin, a high-throughput assay was performed using the single-gene deletion collection of the S. Typhimurium (1) in co-culture with Lacticaseibacillus rhamnosus and (2) in sterile acidic conditions (pH 5.5 media only). As expected, both screens identified genes involved in envelope homeostasis and membrane permeability. Our results also suggest that changes in the metabolism of S. Typhimurium induce the tolerance observed in the presence of L. rhamnosus. Our results thus highlight two different mechanisms by which lactobacilli induce the tolerance of S. Typhimurium to azithromycin.IMPORTANCEThis study provides valuable insights into the intricate interactions between bacteria during infections and within host-associated microbial communities. Specifically, it sheds light on the significant role of lactobacilli in inducing antibiotic tolerance in Salmonella enterica serovar Typhimurium, a critical foodborne pathogen and model organism for microbial community studies. The findings not only uncover the mechanisms underlying this antibiotic tolerance but also reveal two distinct pathways through which strains of lactobacilli might influence Salmonella's response to antibiotics. Understanding these mechanisms has the potential to enhance our knowledge of bacterial infections and may have implications for the development of strategies to combat antibiotic resistance in pathogens, such as Salmonella. Furthermore, our results underscore the necessity to explore beyond the direct antimicrobial effects of antibiotics, emphasizing the broader microbial community context.
ABSTRACT
The lutropin receptor consists of an extracellular N-terminal half and a membrane-associated C-terminal half. hCG initially binds the exodomain with a high affinity and the resulting complex is thought to interact with the endodomain through a secondary contact generating a hormonal signal. Therefore, the exodomain and endodomain are likely to associate directly or indirectly with each other, but lack of fruitful materials and technology has hampered knowledge about their physical relationship and contact sites. In this work, we engineered a double-recombinant (separate exodomain and endodomain) baculovirus system successfully expressing on the surface of insect cells high levels of split LH receptor, binding the hormone with high affinity and inducing cAMP synthesis. In contrast, the exodomain and endodomain expressed separately were mostly trapped in cells. Our data indicate that the exodomain and endodomain are disulfide linked in the split receptor. When the disulfide links were reduced, the split receptor still induced cAMP up to 60%, which raises the intriguing possibility of a residual induction activity of the endodomain in the absence of high-affinity ligand binding. Our results also underscore that the targeting and transport of the LH receptor to plasma membrane require both domains, whereas each domain is independently sufficient for folding. The expression level of functional lutropin receptors is the highest ever reported. Our system may also be useful for future studies requiring a high amount of soluble secreted exodomain.
