Differentially expressed genes in PPD-stimulated PBMCs isolated from cynomolgus vaccinated controllers 8 weeks post-BCG vaccination.

<p>Fold change indicates the change in gene expression in PPD-stimulated PBMCs isolated at 8 weeks post-BCG vaccination compared with PPD-stimulated PBMCs isolated pre-vaccination when the animals were naïve (n = 6).</p

Expression of iron regulatory genes in PBMCs isolated from unvaccinated controllers and progressors at post-mortem.

<p>Gene expression of a) ferritin heavy chain b) solute carrier family 11 member A1, and c) solute carrier family 11 member A2, d) transferrin receptor 1 and e) heme oxygenase 1 in unstimulated PBMCs isolated at post-mortem. The fold change from naïve baseline levels was determined using RT-PCR. To determine statistically significant differences of relative gene expression, a two-sample t-test was performed where * and ** represent P-values of <0.05 and <0.01, respectively. The data were from 3 animals. The symbol represents the median and the error bars represent the range.</p

Comparison of gene expression profiles generated from microarray and RT-PCR analysis.

<p>RT-PCR was used to validate microarray expression data. The symbol represents the median and the error bars represent the range. Black triangles represent fold change in expression from naïve time-points as determined by microarray analysis; white triangles represent fold change in expression from naïve time-points as determined by RT-PCR analysis. * = differentially expressed (p<0.05).</p

Detectable levels of PrP^Sc on Western blots do not correlate with the levels of infectivity.

<p>10% brain homogenates from an uninfected brain (lane 2) time-course samples week 3, 6, 9, 12, 15, 18, 21 (lane 3–9), and the terminal sample (lane 10) were digested with proteinase K at 60°C for 10 minutes and assessed by Western blot. The observed signal does not correspond with the levels of infectivity found in corresponding bioassays for the week 12–21 post-exposure time-points.</p

Comparison of the cull dates for the mice challenged via the gingival margin.

<p>Panel A; Frequency distribution plots show the presence of a normally distributed population with a mean incubation period of around 250 days plus a small number of animals with significantly shorter incubations ranging from 140–188 days. Panel B; when these two groups are compared they show distinct means and distribution and are considered as distinct populations (p<0.001).</p

Singe nucleotide polymorphisms within the <i>katG</i> codon Ser³¹⁵ in populations of isoniazid-resistant <i>Mycobacterium tuberculosis</i> H37Rv.

<p>The number of singe nucleotide polymorphisms within the <i>katG</i> codon Ser³¹⁵ in populations of isoniazid-resistant <i>Mycobacterium tuberculosis</i> H37Rv colonies that were isolated from continuous cultures growing either at a slow growth rate (Culture INH69.3) or fast growth rate (Culture INH23.3) that had been exposed to isoniazid. Colonies were selected on agar containing 2 x MIC isoniazid (1 mg L^-1). One hundred colonies were picked after 6-7MGT and 12–13 MGT and were pyrosequenced at the <i>katG</i> codon Ser³¹⁵. The total number of colonies, at each time-point, for which sequence information was obtained, is shown here. Colonies picked from Culture INH69.3 and Culture INH23.3 at 12 MGT or 13 MGT, respectively, were also subjected to Sanger sequencing and 65 (shown in bold; further detail in B) out of the 77 colonies that were found to be WT for the <i>katG</i> codon Ser³¹⁵ were mutated elsewhere in the <i>katG</i> gene. Further detail on these mutations can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138253#pone.0138253.t002" target="_blank">Table 2</a>. Eleven colonies were found to have a putative duplication and partial deletion within <i>katG</i> in the fast growth culture, INH23.3, and the sequence data could not be fully interpreted.</p><p>Singe nucleotide polymorphisms within the <i>katG</i> codon Ser³¹⁵ in populations of isoniazid-resistant <i>Mycobacterium tuberculosis</i> H37Rv.</p

The mutant frequency of <i>Mycobacterium tuberculosis</i> H37Rv growing at either a fast growth rate or a slow growth rate in response to isoniazid exposure.

<p>The mutant frequency of <i>Mycobacterium tuberculosis</i> H37Rv growing at either a fast growth rate (Panel A, solid lines; circles: Culture INH23.2 & triangles: Culture INH23.3) or a slow growth rate (Panel B solid lines; circles: Culture INH69.2 & triangles: Culture INH69.3) for at least 7 MGT in response to isoniazid (0.5 mg L^-1) added at 0h and continuously throughout culture. Each line represents the mutant frequency from an individual chemostat culture. The dotted lines in each case are control cultures without isoniazid addition</p

Genes that were more highly expressed during fast growth compared with slow growth prior to isoniazid addition.

<p>Genes that were more highly expressed by at least two-fold during fast growth compared with slow growth prior to isoniazid addition. AS and S indicate whether the higher levels of transcript were detected in the sense or antisense strand respectively.</p><p>Genes that were more highly expressed during fast growth compared with slow growth prior to isoniazid addition.</p

<i>Mycobacterium tuberculosis</i> Is Resistant to Isoniazid at a Slow Growth Rate by Single Nucleotide Polymorphisms in <i>katG</i> Codon Ser³¹⁵

<div><p>An important aim for improving TB treatment is to shorten the period of antibiotic therapy without increasing relapse rates or encouraging the development of antibiotic-resistant strains. In any <i>M</i>. <i>tuberculosis</i> population there is a proportion of bacteria that are drug-tolerant; this might be because of pre-existing populations of slow growing/non replicating bacteria that are protected from antibiotic action due to the expression of a phenotype that limits drug activity. We addressed this question by observing populations of either slow growing (constant 69.3h mean generation time) or fast growing bacilli (constant 23.1h mean generation time) in their response to the effects of isoniazid exposure, using controlled and defined growth in chemostats. Phenotypic differences were detected between the populations at the two growth rates including expression of efflux mechanisms and the involvement of antisense RNA/small RNA in the regulation of a drug-tolerant phenotype, which has not been explored previously for <i>M</i>. <i>tuberculosis</i>. Genotypic analyses showed that slow growing bacilli develop resistance to isoniazid through mutations specifically in <i>katG</i> codon Ser³¹⁵ which are present in approximately 50–90% of all isoniazid-resistant clinical isolates. The fast growing bacilli persisted as a mixed population with <i>katG</i> mutations distributed throughout the gene. Mutations in <i>katG</i> codon Ser³¹⁵ appear to have a fitness cost <i>in vitro</i> and particularly in fast growing cultures. Our results suggest a requirement for functional <i>katG</i>-encoded catalase-peroxide in the slow growers but not the fast-growing bacteria, which may explain why <i>katG</i> codon Ser³¹⁵ mutations are favoured in the slow growing cultures.</p></div

Mutations identified in <i>katG</i> under fast or slow growth rates including mutations in <i>katG</i> codon Ser³¹⁵.

<p>Colonies picked from slow growth (Culture INH69.3) and fast growth culture (Culture INH23.3) after 12–13 MGT were subjected to Sanger sequencing and found to contain <i>katG</i> mutations other than those found in <i>katG</i> codon Ser³¹⁵. These mutants included 65 out of the 77 colonies that were previously found to be WT for the <i>katG</i> codon Ser³¹⁵ but were mutated elsewhere in the <i>katG</i> gene. The notation used for each mutation is as follows: original nucleotide followed by the nucleotide number and finally the new nucleotide. The corresponding amino acid changes or effects at each position are also indicated. In cases where an amino acid alteration is indicated by an asterisk there was a truncation of the gene. The square brackets indicate that the mutation was found in both growth rates, curly brackets indicate a mutation found only during slow growth and no brackets indicate a mutation only found during fast growth.</p><p>Mutations identified in <i>katG</i> under fast or slow growth rates including mutations in <i>katG</i> codon Ser³¹⁵.</p