50 research outputs found
Overexpression of <i>M</i>. <i>bovis</i> BCG lipoproteins increases HIV susceptibility of <i>M</i>. <i>smegmatis</i>-infected human CD4+ T cells.
<p>A. Levels of expression of <i>M</i>. <i>bovis</i> BCG lipoprotein genes in various <i>M</i>. <i>smegmatis</i> knock-in strains. Cycle threshold values were first normalized to the housekeeping gene, <i>sigA</i>, and expressed as fold increase relative to wild-type <i>M</i>. <i>bovis</i> BCG. Each cycle difference was assumed to represent a 2-fold difference in gene expression. The data are representative of three independent experiments. B. The percentage of X4-tropic, GFP-expressing pseudovirus-infected CD4+ cells without any stimulation (Unstim) or after infection with <i>M</i>. <i>bovis</i> BCG Copenhagen (BCG), <i>M</i>. <i>smegmatis</i> MC<sup>2</sup>155 containing empty vector (M. smeg), or <i>M</i>. <i>smegmatis</i> strains overexpressing BCG lipoproteins PhoS1, LprQ, LprD, LprI, LprH, LprF, LppX, LprP, or MPT83. C. The TLR2 agonist zymosan (Zym), TLR2 and TLR4 antagonist OxPAPC, or the TLR4-specific antagonist CLI-095 was added to PBMC prior to HIV infection. Results represent combined flow cytometric analysis of CD4+ cells after infection with an X4-tropic GFP+ pseudovirus, from three independent experiments. * p< 0.05; ** p< 0.005.</p
DataSheet6.xls
<p>The development of antibiotic tolerance is believed to be a major factor in the lengthy duration of current tuberculosis therapies. In the current study, we have modeled antibiotic tolerance in vitro by exposing Mycobacterium tuberculosis to two distinct stress conditions: progressive hypoxia and nutrient starvation [phosphate-buffered saline (PBS)]. We then studied the bacterial transcriptional response using RNA-seq and employed a bioinformatics approach to identify important transcriptional regulators, which was facilitated by a novel Regulon Enrichment Test (RET). A total of 17 transcription factor (TF) regulons were enriched in the hypoxia gene set and 16 regulons were enriched in the nutrient starvation, with 12 regulons enriched in both conditions. Using the same approach to analyze previously published gene expression datasets, we found that three M. tuberculosis regulons (Rv0023, SigH, and Crp) were commonly induced in both stress conditions and were also among the regulons enriched in our data. These regulators are worthy of further study to determine their potential role in the development and maintenance of antibiotic tolerance in M. tuberculosis following stress exposure.</p
Deficiency of LspA partially reverses <i>M</i>. <i>bovis</i> BCG-mediated induction of HIV infectivity of CD4+ T cells.
<p>The percentage of GFP+ CD4+ T cells following no stimulation (Unstim), or stimulation of PBMC with <i>M</i>. <i>bovis</i> BCG Copenhagen (BCG), <i>M</i>. <i>smegmatis</i> MC<sup>2</sup>155 containing empty vector (M. smeg), or a <i>M</i>. <i>bovis</i> BCG strain lacking lipoprotein signal peptidase A (BCGΔ<i>lspA</i>), which fails to produce mature lipoproteins. Results represent combined flow cytometric analysis of CD4+ cells after infection with an X4-tropic GFP+ pseudovirus, from 3 independent experiments. * p< 0.05</p
DataSheet1.xls
<p>The development of antibiotic tolerance is believed to be a major factor in the lengthy duration of current tuberculosis therapies. In the current study, we have modeled antibiotic tolerance in vitro by exposing Mycobacterium tuberculosis to two distinct stress conditions: progressive hypoxia and nutrient starvation [phosphate-buffered saline (PBS)]. We then studied the bacterial transcriptional response using RNA-seq and employed a bioinformatics approach to identify important transcriptional regulators, which was facilitated by a novel Regulon Enrichment Test (RET). A total of 17 transcription factor (TF) regulons were enriched in the hypoxia gene set and 16 regulons were enriched in the nutrient starvation, with 12 regulons enriched in both conditions. Using the same approach to analyze previously published gene expression datasets, we found that three M. tuberculosis regulons (Rv0023, SigH, and Crp) were commonly induced in both stress conditions and were also among the regulons enriched in our data. These regulators are worthy of further study to determine their potential role in the development and maintenance of antibiotic tolerance in M. tuberculosis following stress exposure.</p
DataSheet2.XLS
<p>The development of antibiotic tolerance is believed to be a major factor in the lengthy duration of current tuberculosis therapies. In the current study, we have modeled antibiotic tolerance in vitro by exposing Mycobacterium tuberculosis to two distinct stress conditions: progressive hypoxia and nutrient starvation [phosphate-buffered saline (PBS)]. We then studied the bacterial transcriptional response using RNA-seq and employed a bioinformatics approach to identify important transcriptional regulators, which was facilitated by a novel Regulon Enrichment Test (RET). A total of 17 transcription factor (TF) regulons were enriched in the hypoxia gene set and 16 regulons were enriched in the nutrient starvation, with 12 regulons enriched in both conditions. Using the same approach to analyze previously published gene expression datasets, we found that three M. tuberculosis regulons (Rv0023, SigH, and Crp) were commonly induced in both stress conditions and were also among the regulons enriched in our data. These regulators are worthy of further study to determine their potential role in the development and maintenance of antibiotic tolerance in M. tuberculosis following stress exposure.</p
DataSheet5.XLS
<p>The development of antibiotic tolerance is believed to be a major factor in the lengthy duration of current tuberculosis therapies. In the current study, we have modeled antibiotic tolerance in vitro by exposing Mycobacterium tuberculosis to two distinct stress conditions: progressive hypoxia and nutrient starvation [phosphate-buffered saline (PBS)]. We then studied the bacterial transcriptional response using RNA-seq and employed a bioinformatics approach to identify important transcriptional regulators, which was facilitated by a novel Regulon Enrichment Test (RET). A total of 17 transcription factor (TF) regulons were enriched in the hypoxia gene set and 16 regulons were enriched in the nutrient starvation, with 12 regulons enriched in both conditions. Using the same approach to analyze previously published gene expression datasets, we found that three M. tuberculosis regulons (Rv0023, SigH, and Crp) were commonly induced in both stress conditions and were also among the regulons enriched in our data. These regulators are worthy of further study to determine their potential role in the development and maintenance of antibiotic tolerance in M. tuberculosis following stress exposure.</p
DataSheet3.XLS
<p>The development of antibiotic tolerance is believed to be a major factor in the lengthy duration of current tuberculosis therapies. In the current study, we have modeled antibiotic tolerance in vitro by exposing Mycobacterium tuberculosis to two distinct stress conditions: progressive hypoxia and nutrient starvation [phosphate-buffered saline (PBS)]. We then studied the bacterial transcriptional response using RNA-seq and employed a bioinformatics approach to identify important transcriptional regulators, which was facilitated by a novel Regulon Enrichment Test (RET). A total of 17 transcription factor (TF) regulons were enriched in the hypoxia gene set and 16 regulons were enriched in the nutrient starvation, with 12 regulons enriched in both conditions. Using the same approach to analyze previously published gene expression datasets, we found that three M. tuberculosis regulons (Rv0023, SigH, and Crp) were commonly induced in both stress conditions and were also among the regulons enriched in our data. These regulators are worthy of further study to determine their potential role in the development and maintenance of antibiotic tolerance in M. tuberculosis following stress exposure.</p
Recombinant MT0516 exhibits concentration- and time-dependent exopolyphosphatase activity.
<p>A. Polyacrylamide gel electrophoresis showing the fractions of recombinant His-tagged MT0516 protein (36.6 KDa), expressed in <i>E.coli</i> Arctic Express (DE3) BL 21. Lane 1: Molecular weight markers; Lanes 2-3: Supernatant and pellet, respectively, prior to Ni<sup>++</sup>NTA column binding; Lanes 4-5: Elution fraction 1 and 2, respectively, eluted sequentially from Ni<sup>++</sup>NTA column. B. Western blot using Penta-His antibody. Lane 1: Molecular weight markers. Lane 2: Elution fraction 2 after native purification demonstrating the expected size band (36.6 KDa). C. Exopolyphosphatase activity of recombinant MT0516 expressed as % hydrolysis of substrate (65-mer poly P) as a function of protein concentration (μg/ml). **p <0.001. D. Exopolyphosphatase activity of recombinant MT0516 expressed as % hydrolysis of substrate (65-mer poly P) as a function of time (hours). E =  elution fraction 2; AP =  alkaline phosphatase (positive control); NC =  negative control comprising supernatant obtained following IPTG induction of Arctic <i>E coli</i> transformed with empty vector. In each reaction, 1 μg/ml poly P was used as substrate. Data for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028076#pone-0028076-g005" target="_blank">Fig. 5C and 5D</a> are derived from individual experiments, each of which was performed three times with separate samples yielding similar results.</p
MT0516 is required for full virulence of Mtb in guinea pigs.
<p>A. Growth and survival of the mutant following low-dose aerosol infection in guinea pig lungs relative to wild-type and complement strains (*p≤0.01; **p≤0.001 for differences in bacillary counts between mutant- and wild-type-infected lungs). B. Gross pathology of Mtb-infected guinea pig lungs at Day 84 after aerosol infection. C. Microscopic examination of Mtb-infected lungs at Day 84 (H&E stain, 2× magnification. Insets: Ziehl-Neelsen staining, 50x magnification. CDC1551 =  wild-type strain; MT0516::Tn =  MT0516-deficent mutant; MT0516::Tn Comp =  MT0516::Tn complement strain.</p
Complementation of transposon mutant MT0516::Tn.
<p>A. Diagram of expected recombination between integrating plasmid and genomic DNA. B. PCR analysis of genomic DNA. A list of relevant primers is included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0028076#pone-0028076-t001" target="_blank">Table 1</a>. Lane 1: Molecular weight markers. Lanes 2-4: Amplification of the MT0516 gene from wild-type, MT0516::Tn, and MT0516::Tn Comp, respectively. The expected 1035-bp PCR product is present in Lanes 2 and 4 but absent in Lane 3. Lane 5: Molecular weight markers. Lanes 6-8: Amplification of the kanamycin resistance cassette from wild type, MT0516::Tn and MT0516::Tn Comp, respectively, yielding the expected 226-bp product only in Lanes 7 and 8. Lane 9: Molecular weight markers. Lanes 10-11: Amplification of the hygromycin resistance cassette from MT0516::Tn and MT0516::Tn Comp, respectively, yielding the expected 319-bp product only in Lane 11. C. Diagram of expected sizes of <i>EcoRI</i>-digested genomic fragments expected to hybridize to probe recognizing region of MT0516 coding sequence by Southern blot (D). Note that MT0516::Tn Comp is expected to have two different size fragments hybridizing to the MT0516 probe, including that found in MT0516::Tn (1.8 Kb) and a distinct fragment unique to the attB integration site (1.7 Kb). D. Southern blot detecting the presence of DNA fragments bound to the MT0516 hybridization probe. Lane 1: Molecular weight markers. Lane 2: Blank. Lanes 3-5: Wild type, MT0516::Tn, and MT:0516::Tn Comp, respectively.</p