RT-PCR analysis of the putative co-operonic pairs <i>PE5-PPE4</i> and <i>PE15-PPE20</i>.

<p>Schematic representation of the genomic organization of the <i>PE5</i> (A) and <i>PE15</i> (C) loci in the <i>M.tb</i> genome showing the positions of the primers used in the study. (B) RT-PCR amplification products of the <i>PE5-PPE4</i> gene pair: Lane 1- (5F+5R), Lane 2- (5JF+4JR), Lane 3 - (4F+4R), Lanes 5, 6 and 7 correspond to -RT controls for these respective primer pairs, Lane 4–100 bp DNA ladder (D) RT-PCR amplification products of the <i>PE15-PPE20</i> gene pair: Lane 1- (15F+15R), Lane 2- (15JF+20JR), Lane 3 - (20F+20R); Lanes 5, 6 and 7 correspond to -RT controls for these respective primer pairs. Lane 4–100 bp DNA ladder.</p

Sub-cellular localization and surface accessibility.

<p>(A) Schematic representation of the c-myc fusion constructs of PE5/PE15 generated in the episomal plasmid pJEX55 under the control of the constitutive <i>hsp60</i> promoter. (B) Immuno-detection of PE5 and PE15 in sub-cellular fractions of Proteinase K treated recombinant <i>M.smegmatis</i> strains expressing PE5-myc and PE15-myc. All proteins were detected using an anti c-myc mAb. CW - cell wall fraction, CM - cell membrane fraction, CY - cytoplasmic fraction.</p

Physiology of recombinant <i>M.smegmatis</i> strains expressing <i>PE5</i> and <i>PE15</i> in resting J774.1 macrophages.

<p>(A) CFU counts of <i>M.smegmatis</i> expressing empty vector (pMV261), <i>PE5</i> (PE5) and <i>PE15</i> (PE15) 24, 48 and 72 h post infection. (B) Input and T₀ (post-infection) CFU counts of infecting bacilli (± SEM). (C and D) Transcript levels of pro- and anti-inflammatory cytokines and <i>iNOS</i>, 24, 48 and 72 h post infection, p<0.05 for all data points. Error bars represent ± SEM from three biological replicates. **p<0.005, *p<0.05.</p

MAP kinase signalling and its correlation to IL-10 levels in infected THP-1 macrophages.

<p>Phosphorylation levels of p38 (A) and ERK1/2 (B) and their densitometric quantitation from resting THP-1 cells infected with <i>M.smegmatis</i> expressing pMV261, <i>PE5</i> and <i>PE15</i> 24 h post infection. Error bars represent ± SEM of three biological replicates. **p<0.005, *p<0.05. The western blots shown are representative of at least two biological replicates.</p

Inhibition of MAP kinase signalling in infected THP-1 macrophages.

<p>Phosphorylation levels of p38 (C) and ERK1/2 (D) and their densitometric quantitation from resting THP-1 cells infected with <i>M.smegmatis</i> expressing pMV261, PE5 and PE15 24 h post infection in the presence of p38 and ERK1/2 inhibitors. Error bars represent ± SEM of three biological replicates. **p<0.005, *p<0.05. The western blots shown are representative of at least two biological replicates.</p

Viability of <i>M.smegmatis</i> strains expressing <i>PE5</i> & <i>PE15</i> in THP-1 macrophages.

<p>CFU counts of <i>M.smegmatis</i> expressing empty vector (pMV261), <i>PE5</i> (PE5) and <i>PE15</i> (PE15) in resting (A) and activated THP-1 macrophages (B) 24, 48 and 72 h post infection. (C) Input and T₀ (post-infection) CFU counts of infecting bacilli (± SEM). Error bars represent ± SEM from three biological replicates. **p<0.005, * p<0.05.</p

The <em>Mycobacterium tuberculosis</em> PE Proteins Rv0285 and Rv1386 Modulate Innate Immunity and Mediate Bacillary Survival in Macrophages

<div><p>The unique PE/PPE multigene family of proteins occupies almost 10% of the coding sequence of <em>Mycobacterium tuberculosis</em> (<em>M.tb</em>), the causative agent of human tuberculosis. Although some members of this family have been shown to be involved in pathways essential to <em>M.tb</em> pathogenesis, their precise physiological functions remain largely undefined. Here, we investigate the roles of the conserved members of the ‘PE only’ subfamily Rv0285 (PE5) and Rv1386 (PE15) in mediating host-pathogen interactions. Recombinant <em>Mycobacterium smegmatis</em> strains expressing PE5 and PE15 showed enhanced survival <em>vs</em> controls in J774.1 and THP-1 macrophages - this increase in viable counts was correlated with a reduction in transcript levels of inducible nitric oxide synthase. An up-regulation of anti- and down-regulation of pro-inflammatory cytokine levels was also observed in infected macrophages implying an immuno-modulatory function for these proteins. Induction of IL-10 production upon infection of THP-1 macrophages was associated with increased phosphorylation of the MAP Kinases p38 and ERK1/2, which was abolished in the presence of the pharmacological inhibitors SB203580 and PD98059. The <em>PE5-PPE4</em> and <em>PE15-PPE20</em> gene pairs were observed to be co-operonic in <em>M.tb</em>, hinting at an additional level of complexity in the functioning of these proteins. We conclude that <em>M.tb</em> exploits the PE proteins to evade the host immune response by altering the Th1 and Th2 type balance thereby favouring <em>in vivo</em> bacillary survival.</p> </div

Sequence conservation of <i>M.tb</i> PE5 and PE15.

<p>(A) Multiple sequence alignment of the 6 proteins in the indicated cluster in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051686#pone-0051686-g001" target="_blank">Figure 1</a>. Multiple sequence alignment of protein sequences of PE5 (B) and PE15 (C) from members of the <i>M.tb</i> complex.</p

<i>In vivo</i> effects of THP-1 macrophage infection with <i>M.smegmatis</i> strains expressing <i>PE5</i> and <i>PE15</i>.

<p>(A) and (B) Transcript levels of IL-10, IL-12 and <i>iNOS,</i> in resting macrophages infected with <i>M.smegmatis</i> expressing empty vector (pMV261), <i>PE5</i> (PE5) and <i>PE15</i> (PE15) 24, 48 and 72 h post infection, p<0.05 for all data points. IL-10 (C) and IL-12 (D) levels in the culture supernatants of infected cells 24, 48 and 72 h post infection, p<0.005 for all data points. Error bars represent ± SEM from three biological replicates.</p

Gene expression analysis and growth of recombinant <i>M.smegmatis</i> expressing <i>PE5</i> and <i>PE15</i>.

<p><i>In vitro growth</i> profiles of <i>M.smegmatis</i> expressing pMV261, PE5 and PE15 generated by measuring OD (A) and enumeration of CFU counts (B). Real time RT-PCR quantitation of PE5 (C) and PE15 (D) transcripts as a function of growth. Transcript levels of are represented relative to mRNA levels of the cognate gene at 4 h which is assigned a value of 1. Error bars represent ± SEM.</p