Herein, we report the discovery and structure−activity relationships of 5-substituted-2-[(3,5-dinitrobenzyl)sulfanyl]-1,3,4-oxadiazoles and 1,3,4-thiadiazoles as a new class of antituberculosis agents. The majority of these compounds exhibited outstanding in vitro activity against Mycobacterium tuberculosis CNCTC My 331/88 and six multidrug-resistant clinically isolated strains of M. tuberculosis, with minimum inhibitory concentration values as low as 0.03 μM (0.011−0.026 μg/ mL). The investigated compounds had a highly selective antimycobacterial effect because they showed no activity against the other bacteria or fungi tested in this study. Furthermore, the investigated compounds exhibited low in vitro toxicities in four proliferating mammalian cell lines and in isolated primary human hepatocytes. Several in vitro genotoxicity assays indicated that the selected compounds have no mutagenic activity. The oxadiazole and thiadiazole derivatives with the most favorable activity/ toxicity profiles also showed potency comparable to that of rifampicin against the nonreplicating streptomycin-starved M. tuberculosis 18b-Lux strain, and therefore, these derivatives, are of particular interest.

Francisella tularensis (F. tularensis) is highly infectious for humans via aerosol route and untreated infections with the highly virulent subsp. tularensis can be fatal. Our knowledge regarding key virulence determinants has increased recently but is still somewhat limited. Surface proteins are potential virulence factors and therapeutic targets, and in this study, we decided to target three genes encoding putative membrane lipoproteins in F. tularensis LVS. One of the genes encoded a protein with high homology to the protein family of disulfide oxidoreductases DsbA. The two other genes encoded proteins with homology to the VacJ, a virulence determinant of Shigella flexneri. The gene encoding the DsbA homologue was verified to be required for survival and replication in macrophages and importantly also for in vivo virulence in the mouse infection model for tularemia. Using a combination of classical and shotgun proteome analyses, we were able to identify several proteins that accumulated in fractions enriched for membrane-associated proteins in the dsbA mutant. These proteins are substrate candidates for the DsbA disulfide oxidoreductase as well as being responsible for the virulence attenuation of the dsbA mutant.

Francisella tularensis is a Gram-negative, facultative intracellular bacterium, causing a severe disease called tularemia. It secretes unusually shaped nanotubular outer membrane vesicles (OMV) loaded with a number of virulence factors and immunoreactive proteins. In the present study, the vesicles were purified from a clinical isolate of subsp. holarctica strain FSC200. We here provide a comprehensive proteomic characterization of OMV using a novel approach in which a comparison of OMV and membrane fraction is performed in order to find proteins selectively enriched in OMV vs. membrane. Only these proteins were further considered to be really involved in the OMV function and/or their exceptional structure. OMV were also isolated from bacteria cultured under various cultivation conditions simulating the diverse environments of F. tularensis life cycle. These included conditions mimicking the milieu inside the mammalian host during inflammation: oxidative stress, low pH, and high temperature (42°C); and in contrast, low temperature (25°C). We observed several-fold increase in vesiculation rate and significant protein cargo changes for high temperature and low pH. Further proteomic characterization of stress-derived OMV gave us an insight how the bacterium responds to the hostile environment of a mammalian host through the release of differentially loaded OMV. Among the proteins preferentially and selectively packed into OMV during stressful cultivations, the previously described virulence factors connected to the unique intracellular trafficking of Francisella were detected. Considerable changes were also observed in a number of proteins involved in the biosynthesis and metabolism of the bacterial envelope components like O-antigen, lipid A, phospholipids, and fatty acids. Data are available via ProteomeXchange with identifier PXD013074.

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The facultative intracellular pathogen Francisella tularensis is the causative agent of the serious infectious disease tularemia. Despite intensive research, the virulence factors and pathogenetic mechanisms remain largely unknown. To identify novel putative virulence factors, we carried out a comparative proteome analysis of fractions enriched for membrane-associated proteins isolated from the highly virulent subspecies tularensis strain SCHU S4 and three representatives of subspecies holarctica of different virulence including the live vaccine strain. We identified six proteins uniquely expressed and four proteins expressed at significantly higher levels by SCHU S4 compared to the ssp. holarctica strains. Four other protein spots represented mass and charge variants and seven spots were charge variants of proteins occurring in the ssp. holarctica strains. The genes encoding proteins of particular interest were examined by sequencing in order to confirm and explain the findings of the proteome analysis. Our studies suggest that the subspecies tularensis-specific proteins represent novel potential virulence factors.