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  • 1
    Publication Date: 2018-06-22
    Description: Borrelia burgdorferi , the agent of Lyme disease (LD), uses host-derived signals to modulate gene expression during the vector and mammalian phases of infection. Microarray analysis of mutants lacking the B orrelia host ad aptation r egulator (BadR) revealed the downregulation of genes encoding enzymes whose role in the pathophysiology of B. burgdorferi is unknown. Immunoblot analysis of the badR mutants confirmed reduced levels of these enzymes, and one of these enzymes, encoded by bb0086 , shares homology to prokaryotic magnesium chelatase and Lon-type proteases. The BB0086 levels in B. burgdorferi were higher under conditions mimicking those in fed ticks. Mutants lacking bb0086 had no apparent in vitro growth defect but were incapable of colonizing immunocompetent C3H/HeN or immunodeficient SCID mice. Immunoblot analysis revealed reduced levels of proteins critical for the adaptation of B. burgdorferi to the mammalian host, such as OspC, DbpA, and BBK32. Both RpoS and BosR, key regulators of gene expression in B. burgdorferi , were downregulated in the bb0086 mutants. Therefore, we designated BB0086 the B orrelia host ad aptation p rotein (BadP). Unlike badP mutants, the control strains established infection in C3H/HeN mice at 4 days postinfection, indicating an early colonization defect in mutants due to reduced levels of the lipoproteins/regulators critical for initial stages of infection. However, badP mutants survived within dialysis membrane chambers (DMCs) implanted within the rat peritoneal cavity but, unlike the control strains, did not display complete switching of OspA to OspC, suggesting incomplete adaptation to the mammalian phase of infection. These findings have opened a novel regulatory mechanism which impacts the virulence potential of B . burgdorferi .
    Print ISSN: 0019-9567
    Electronic ISSN: 1098-5522
    Topics: Medicine
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  • 2
    Publication Date: 2018-06-27
    Description: Due to the rise of drug-resistant forms of tuberculosis, there is an urgent need for novel antibiotics to effectively combat these cases and shorten treatment regimens. Recently, drug screens using whole-cell analyses have been shown to be successful. However, current high-throughput screens focus mostly on stricto sensu life/death screening that give little qualitative information. In doing so, promising compound scaffolds or nonoptimized compounds that fail to reach inhibitory concentrations are missed. To accelerate early tuberculosis (TB) drug discovery, we performed RNA sequencing on Mycobacterium tuberculosis and Mycobacterium marinum to map the stress responses that follow upon exposure to subinhibitory concentrations of antibiotics with known targets, ciprofloxacin, ethambutol, isoniazid, streptomycin, and rifampin. The resulting data set comprises the first overview of transcriptional stress responses of mycobacteria to different antibiotics. We show that antibiotics can be distinguished based on their specific transcriptional stress fingerprint. Notably, this fingerprint was more distinctive in M. marinum . We decided to use this to our advantage and continue with this model organism. A selection of diverse antibiotic stress genes was used to construct stress reporters. In total, three functional reporters were constructed to respond to DNA damage, cell wall damage, and ribosomal inhibition. Subsequently, these reporter strains were used to screen a small anti-TB compound library to predict the mode of action. In doing so, we identified the putative modes of action for three novel compounds, which confirms the utility of our approach.
    Print ISSN: 0066-4804
    Electronic ISSN: 1098-6596
    Topics: Biology , Medicine
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  • 3
    Publication Date: 2018-05-26
    Description: In Toxoplasma gondii , calcium-dependent protein kinase 1 (CDPK1) is an essential protein kinase required for invasion of host cells. We have developed several hundred CDPK1 inhibitors, many of which block invasion. Inhibitors with similar 50% inhibitory concentrations (IC 50 s) were tested in thermal shift assays for their ability to stabilize CDPK1 in cell lysates, in intact cells, or in purified form. Compounds that inhibited parasite growth stabilized CDPK1 in all assays. In contrast, two compounds that showed poor growth inhibition stabilized CDPK1 in lysates but not in cells. Thus, cellular exclusion could explain exceptions in the correlation between the action on the target and cellular activity. We used thermal shift assays to examine CDPK1 in two clones that were independently selected by growth in the CDPK1 inhibitor RM-1-132 and that had increased 50% effective concentrations (EC 50 s) for the compound. The A and C clones had distinct point mutations in the CDPK1 kinase domain, H201Q and L96P, respectively, residues that lie near one another in the inactive isoform. Purified mutant proteins showed RM-1-132 IC 50 s and thermal shifts similar to those shown by wild-type CDPK1. Reduced inhibitor stabilization (and a presumed reduced interaction) was observed only in cellular thermal shift assays. This highlights the utility of cellular thermal shift assays in demonstrating that resistance involves reduced on-target engagement (even if biochemical assays suggest otherwise). Indeed, similar EC 50 s were observed upon overexpression of the mutant proteins, as in the corresponding drug-selected parasites, although high levels of CDPK1(H201Q) only modestly increased resistance compared to that achieved with high levels of wild-type enzyme.
    Print ISSN: 0066-4804
    Electronic ISSN: 1098-6596
    Topics: Biology , Medicine
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