Real-time bottom-up characterization of protein mixtures enabled by online microdroplet-assisted enzymatic digestion (MAED).

Enzymatic digestion remains one of the "rate-determining steps" in the bottom-up analysis of proteins. However, by performing digestion in microdroplets generated from electrosonic spray, the reaction could be accelerated to a timescale lower than milliseconds. Here, we describe a simple and rapid online digestion platform named online microdroplet-assisted enzymatic digestion (MAED). It involves the integration of intact protein separation with enzymatic digestion in microdroplets. Via online MAED, various protein standards, including an antibody standard, were characterized in a bottom-up manner without prior digestion, and high sequence coverages were obtained. We further extended the application of online MAED to a more complex sample, mouse brain extract, where protein identifications were successfully yielded. Compared with the conventional bottom-up approach, a more comprehensive characterization could be obtained particularly for low molecular weight proteins. In short, we provide a rapid and alternative bottom-up analysis in a top-down fashion as well as a new possibility for microdroplet chemistry.

Roles of SmeYZ, SbiAB, and SmeDEF Efflux Systems in Iron Homeostasis of Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia, a nonfermenting Gram-negative rod, is frequently isolated from the environment and is emerging as a multidrug-resistant global opportunistic pathogen. S. maltophilia harbors eight RND-type efflux pumps that contribute to multidrug resistance and physiological functions. Among the eight efflux pumps, SmeYZ pump is constitutively highly expressed. In our previous study, we demonstrated that loss-of-function of the SmeYZ pump results in pleiotropic phenotypes, including abolished swimming motility, decreased secreted protease activity, and compromised tolerance to oxidative stress and antibiotics. In this study, we attempted to elucidate the underlying mechanisms responsible for ΔsmeYZ-mediated pleiotropic phenotypes. RNA-seq transcriptome analysis and subsequent confirmation with qRT-PCR revealed that smeYZ mutant experienced an iron starvation response because the genes involved in the synthesis and uptake of stenobactin, the sole siderophore of S. maltophilia, were significantly upregulated. We further verified that smeYZ mutant had low intracellular iron levels via inductively coupled plasma mass spectrometry (ICP-MS). Also, KJΔYZ was more sensitive to 2,2'-dipyridyl (DIP), a ferrous iron chelator, in comparison with the wild type. The contribution of SmeYZ, SmeDEF, and SbiAB pumps to stenobactin secretion was suggested by qRT-PCR and further verified by Chrome Azurol S (CAS) activity, iron source utilization, and cell viability assays. We also demonstrated that loss-of-function of SmeYZ led to the compensatory upregulation of SbiAB and SmeDEF pumps for stenobactin secretion. The overexpression of the SbiAB pump resulted in a reduction in intracellular iron levels, which may be the key factor responsible for the ΔsmeYZ-mediated pleiotropic phenotypes, except for antibiotic extrusion. IMPORTANCE Efflux pumps display high efficiency of drug extrusion, which underlies their roles in multidrug resistance. In addition, efflux pumps have physiological functions, and their expression is tightly regulated by various environmental and physiological signals. Functional redundancy of efflux pumps is commonly observed, and mutual regulation occurs among these functionally redundant pumps in a bacterium. Stenotrophomonas maltophilia is an opportunistic pathogen that shows intrinsic multi-drug resistance. In this study, we demonstrated that SmeYZ, SbiAB, and SmeDEF efflux pumps of S. maltophilia display functional redundancy in siderophore secretion. Inactivation of smeYZ led to the upregulation of smeDEF and sbiAB. Unexpectedly, sbiAB overexpression resulted in the reduction of intracellular iron levels, which led to pleiotropic defects in smeYZ mutant. This study demonstrates a previously unidentified connection between efflux pumps, siderophore secretion, and intracellular iron levels in S. maltophilia.