Browsing by Author "Song, Xinhao"

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    Developing Microdroplet Emulsions as a System for the Study of Microbial Social Interactions
    (2023-04-18) Song, Xinhao; Shamoo, Yousif
    The evolution and maintenance of social behaviors, especially cooperative interactions, is an intensively-studied field in ecology. Social microbes that interact through the secretion of diffusible chemical signals are ideal model organisms for both fundamental and translational studies of this fascinating process. In this thesis, I investigate the social interactions among soil-dwelling Gram-positive bacteria Streptomyces spp. Streptomyces species have contributed to more than two-thirds of current antibiotics and have a remarkable capability for the synthesis of secondary metabolites. Moreover, in their natural soil habitats, neighboring Streptomyces species can form complex social interaction networks that are mediated by diffusible secondary metabolites, further adding to their qualities as model organisms for the study of microbial social interactions. One of the key factors for the evolution and maintenance of social interactions among microbes is spatially structured environments. Such environments limit the diffusion of “public good” molecules such as beneficial metabolites and chemical signals and may favor social traits with indirect fitness benefits. In this thesis, I used microdroplet emulsions to construct artificial and configurable spatially segregated environments for the investigation of nascent social interactions. Two comprehensive case studies of inhibition and cooperative Streptomyces pairs were conducted. First, a collection of Streptomyces environmental isolates was obtained and characterized both phenotypically and genomically. From this collection, isolates T4-11 and AMS-5 were identified as inhibitive and cooperative partner strains for the model strain S. venezuelae. Next, using both plasmid-based and genome integration approaches, various RFP and GFP fluorescence reporter strains were constructed to facilitate the investigation of social interactions. Subsequently, interactions between T4-11/S. venezuelae and AMS-5/S. venezuelae pairs were thoroughly investigated using conditioned medium and co-culture assays in both liquid suspension and microdroplet environments. The inhibition of T4-11 on S. venezuelae was potent and reproducible across various conditions, whereas the interactions between AMS-5 and S. venezuelae varied significantly in different environments. To expand further the set of tools available to study social interactions, a volatile gas-based S. venezuelae reporter strain was constructed and characterized. It is well known that fluorescence reporters suffer from limitations when used in microdroplets, such as inaccurate readings due to strong background or limited sensitivity during early growth stages. To overcome such limitations, I showed that volatile gas produced by S. venezuelae gas reporters could be quantified by GC-MS in situ and could serve as an alternative non-optical detection approach for microbes encapsulated microdroplets. In addition, a 100-1000 fold improvement in sensitivity was observed for the gas reporter when compared to an RFP fluorescence reporter.
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    Enolpyruvate transferase MurAAA149E, identified during adaptation of Enterococcus faecium to daptomycin, increases stability of MurAA–MurG interaction
    (Elsevier, 2023) Zhou, Yue; Utama, Budi; Pratap, Shivendra; Supandy, Adeline; Song, Xinhao; Tran, Truc T.; Mehta, Heer H.; Arias, Cesar A.; Shamoo, Yousif
    Daptomycin (DAP) is an antibiotic frequently used as a drug of last resort against vancomycin-resistant enterococci. One of the major challenges when using DAP against vancomycin-resistant enterococci is the emergence of resistance, which is mediated by the cell-envelope stress system LiaFSR. Indeed, inhibition of LiaFSR signaling has been suggested as a strategy to “resensitize” enterococci to DAP. In the absence of LiaFSR, alternative pathways mediating DAP resistance have been identified, including adaptive mutations in the enolpyruvate transferase MurAA (MurAAA149E), which catalyzes the first committed step in peptidoglycan biosynthesis; however, how these mutations confer resistance is unclear. Here, we investigated the biochemical basis for MurAAA149E-mediated adaptation to DAP to determine whether such an alternative pathway would undermine the potential efficacy of therapies that target the LiaFSR pathway. We found cells expressing MurAAA149E had increased susceptibility to glycoside hydrolases, consistent with decreased cell wall integrity. Furthermore, structure–function studies of MurAA and MurAAA149E using X-ray crystallography and biochemical analyses indicated only a modest decrease in MurAAA149E activity, but a 16-fold increase in affinity for MurG, which performs the last intracellular step of peptidoglycan synthesis. Exposure to DAP leads to mislocalization of cell division proteins including MurG. In Bacillus subtilis, MurAA and MurG colocalize at division septa and, thus, we propose MurAAA149E may contribute to DAP nonsusceptibility by increasing the stability of MurAA–MurG interactions to reduce DAP-induced mislocalization of these essential protein complexes.
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    Intracellular Experimental Evolution of Francisella tularensis Subsp. holarctica Live Vaccine Strain (LVS) to Antimicrobial Resistance
    (American Chemical Society, 2023) Mehta, Heer H.; Song, Xinhao; Shamoo, Yousif
    In vitro experimental evolution has complemented clinical studies as an excellent tool to identify genetic changes responsible for the de novo evolution of antimicrobial resistance. However, the in vivo context for adaptation contributes to the success of particular evolutionary trajectories, especially in intracellular niches where the adaptive landscape of virulence and resistance are strongly coupled. In this work, we designed an ex vivo evolution approach to identify evolutionary trajectories responsible for antibiotic resistance in the Live Vaccine Strain (LVS) of Francisella tularensis subsp. holarctica while being passaged to increasing ciprofloxacin (CIP) and doxycycline (DOX) concentrations within macrophages. Overall, adaptation within macrophages advanced much slower when compared to previous in vitro evolution studies reflecting a limiting capacity for the expansion of adaptive mutations within the macrophage. Longitudinal genomic analysis identified resistance conferring gyrase mutations outside the Quinolone Resistance Determining Region. Strikingly, FupA/B mutations that are uniquely associated with in vitro CIP resistance in Francisella were not observed ex vivo, reflecting the coupling of intracellular survival and resistance during intracellular adaptation. To our knowledge, this is the first experimental study demonstrating the ability to conduct experimental evolution to antimicrobial resistance within macrophages. The results provide evidence of differences in mutational profiles of populations adapted to the same antibiotic in different environments/cellular compartments and underscore the significance of host mediated stress during resistance evolution.