doi: 10.1038/srep35835.
A dual-targeting approach to inhibit Brucella abortus replication in human cells
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- PMID: 27767061
- PMCID: PMC5073326
- DOI: 10.1038/srep35835
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A dual-targeting approach to inhibit Brucella abortus replication in human cells
Daniel M Czyż et al.
Sci Rep.
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Abstract
Brucella abortus is an intracellular bacterial pathogen and an etiological agent of the zoonotic disease known as brucellosis. Brucellosis can be challenging to treat with conventional antibiotic therapies and, in some cases, may develop into a debilitating and life-threatening chronic illness. We used multiple independent assays of in vitro metabolism and intracellular replication to screen a library of 480 known bioactive compounds for novel B. abortus anti-infectives. Eighteen non-cytotoxic compounds specifically inhibited B. abortus replication in the intracellular niche, which suggests these molecules function by targeting host cell processes. Twenty-six compounds inhibited B. abortus metabolism in axenic culture, thirteen of which are non-cytotoxic to human host cells and attenuate B. abortus replication in the intracellular niche. The most potent non-cytotoxic inhibitors of intracellular replication reduce B. abortus metabolism in axenic culture and perturb features of mammalian cellular biology including mitochondrial function and receptor tyrosine kinase signaling. The efficacy of these molecules as inhibitors of B. abortus replication in the intracellular niche suggests "dual-target" compounds that coordinately perturb host and pathogen are promising candidates for development of improved therapeutics for intracellular infections.
Figures
Compounds from the ICCB bioactives library were arrayed into plates containing THP-1 macrophage-like cells infected with wild-type B. abortus constitutively expressing mCherry. ICCB compounds are plotted sequentially along the X-axis; relative mCherry B. abortus fluorescence is plotted on the Y-axis in all graphs. Dotted line delineates 20% below the mean normalized fluorescence across all conditions; compounds that fall below this line were considered hits (orange). (A) Graph of B. abortus fluorescence (72 hours post-infection) in the presence of each ICCB compound in cells treated pre-infection (circles) or post-infection (squares) as described in Methods. (B) Twenty-one non-cytotoxic compounds that inhibit intracellular growth of B. abortus presumably by targeting the host. Orange-filled circles represent compounds that inhibited B. abortus infection by 20% or more. Green-filled circle represents a single compound that in our screen was found to be effective only when applied pre-infection.
(A) A panel of six 96-well plates arrayed with 480 small molecules from the ICCB bioactives library. B. abortus growth/metabolism was assessed colorimetrically in test and control wells containing a minimal-defined base medium and tetrazolium dye as a metabolic indicator (purple color, monitored at 630 nm) as described in Methods. (B) B. abortus metabolism in minimal-defined medium in the presence of 480 ICCB compounds, as assessed by quantifying tetrazolium formazan absorbance at 630 nm. Orange circles represent twenty-six compounds that inhibited Brucella metabolism by ≥1 standard deviation from the mean (dotted horizontal line represents 1 standard deviation below the mean). ICCB compounds are plotted sequentially along the x-axis. The twenty-six hit compounds are listed in Table 1.
Cytotoxicity of the 26 Brucella-specific (white bars) and 18 host-specific compounds (shaded bars) was assessed by quantifying the average number of nuclei in a viewing area per each treatment. The results are expressed as an average number of nuclei calculated from 6–24 images per treatment in a single experiment. Error bars represent standard deviation. Toxic compounds were also identified based on a qualitative image analysis. Compounds that had a significantly lower average number of nuclei than control (p < 0.05) were deemed toxic. While compounds that are labeled as moderately-toxic had a lower average number of nuclei, the image analysis revealed adherent cells that had normal morphology (Supplementary Fig. S1). Compounds that had no apparent affect on morphology and had a high average number of nuclei were deemed non-toxic. *Three overlapping host- and Brucella-specific compounds.
(A) Quantification of inhibitory efficacy of compounds, measuring the average ratio of bacteria-to-nucleus in THP-1 cells treated with each of the 13 non-cytotoxic pathogen-targeting compounds in a single experiment. Statistical significance was evaluated from ≈7 images per treatment using one-way ANOVA followed by Dunnett’s test (*p < 0.05, ***p < 0.001, ****p < 0.0001). Error bars represent +/− standard deviation. (B) Sample fluorescence images of nuclei (blue) and intracellular B. abortus (pseudocolor green) assessing the efficacy of each of the 5 final pathogen-targeting compounds that significantly inhibited infection by B. abortus: tyrphostin 9, FCCP, AG-879, manumycin A, and mitomycin C. Doxycycline is a positive control and TPCK represents a non-hit compound that did not have any effect on infection. Scale bar = 50 μm. (C) B. abortus CFU counts generated from infected THP-1 treated with 13 identified non-toxic compounds that inhibit B. abortus metabolism in axenic culture. Data represent an average from 3–7 biological replicates. Error bars represent +/− SEM.
(A) Quantification of inhibitory efficacy of compounds, measuring the average ratio of bacteria-to-nucleus in THP-1 cells treated with each of the 18 non-cytotoxic host-specific compounds in a single experiment. Statistical significance was evaluated from ≈7 images per treatment using one-way ANOVA followed by Dunnett’s test (*p < 0.05, ****p < 0.0001). Error bars represent +/− standard deviation. (B) Sample fluorescence images of nuclei (blue) and intracellular B. abortus (pseudocolor green) assessing efficacy of each of the 9 final host-specific compounds that significantly inhibited infection by B. abortus. Doxycycline is a positive control and AG-213 represents a non-hit compound that did not have any effect on infection. Scale bar = 50 μm. (C) B. abortus CFU counts generated from infected THP-1 treated with 18 identified non-toxic compounds that presumably target host cells to inhibit B. abortus infection. Data represent an average from 3–7 biological replicates. Error bars represent +/− SEM.
Images of B. abortus (green pseudocolor) and Hoechst-stained THP-1 nuclei (blue) of cells treated with compunds at their respective minimum inhibitory concentrations (MICs). The images were taken 72 hours post-infection. Control column represents mock treated controls matching each treatment in the Compound column. Magnified inset regions (white box) appear to the right of each image. Scale bars = 50 μm.
References
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