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> MC²155 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

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> MC²155 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

Differential susceptibility to HIV infection is not dependent on increased expression of the immune activation markers CD38 and HLA DR.

<p>Cells stimulated with different antigens are activated to a similar extent as evidenced by expression of immune activation marker CD38 (A). Dot plots from unstimulated CD4+ cells and CD4+ cells stimulated with PHA (150 g/ml), <i>M. bovis</i> BCG (Copenhagen) and <i>M. tuberculosis</i> (CDC1551) depicting the expression of immune activation marker CD38 (B). Cells stimulated with <i>M. bovis</i> BCG (Copenhagen) and <i>M. tuberculosis</i> (CDC1551) show lower expression of MHC II molecule HLA DR when compared to cells stimulated with <i>M. smegmatis</i> MC²155 (C) *p<0.05.</p

<em>Mycobacterium tuberculosis</em> Complex Enhances Susceptibility of CD4 T Cells to HIV through a TLR2-Mediated Pathway

<div><p>Among HIV-infected individuals, co-infection with <em>Mycobacterium tuberculosis</em> is associated with faster progression to AIDS. We investigated the hypothesis that <em>M. bovis</em> BCG and <em>M. tuberculosis</em> (Mtb complex) could enhance susceptibility of CD4+ cells to HIV infection. Peripheral blood mononuclear cells (PBMCs) collected from healthy donors were stimulated with <em>M. bovis</em> BCG, <em>M. tuberculosis</em> CDC1551 and <em>M. smegmatis</em> MC²155, and stimulated CD4+ cells were infected with R5-and X4-tropic single replication-competent pseudovirus. CD4+ cells stimulated with Mtb complex showed enhanced infection with R5- and X4-tropic HIV, compared to unstimulated cells or cells stimulated with <em>M. smegmatis</em> (p<0.01). Treatment with TLR2 siRNA reversed the increased susceptibility of CD4+ cells with R5- and X4-tropic virus induced by Mtb complex. These findings suggest that TB infection and/or BCG vaccination may be a risk factor for HIV acquisition.</p> </div

Silencing of TLR2 expression attenuates the increased susceptibility to HIV.

<p>Expression of TLR2 increases at the transcription level in cells stimulated with <i>M. bovis</i> BCG when compared to cells stimulated with <i>M. smegmatis</i> (A). The expression levels of TLR4 and TLR9 do not show a significant difference in cells stimulated with different strains of mycobacteria as evidenced by RT-PCR. Comparison of HIV infectivity of CD4+ cells transfected with scrambled siRNA and subsequently stimulated with <i>M. bovis</i> BCG to CD4+ cells transfected with TLR2 siRNA and subsequently stimulated with <i>M. bovis</i> BCG (B) **p<0.005. TLR2 expression on CD4+ cells after stimulation with <i>M. bovis</i> BCG, <i>M. tuberculosis</i>, and <i>M. smegmatis</i> compared to TLR2 expression on CD4+ cells transfected with TLR2 siRNA and subsequently stimulated with <i>M. bovis</i> BCG (C). The expression showed similar trends for the four samples tested. Dot plots comparing the percentage of surface expression of TLR2 on unstimulated CD4+ cells and CD4+ cells stimulated with <i>M. bovis</i> BCG, <i>M. tuberculosis</i> (CDC1551), <i>M. smegmatis</i> (MC²155), and CD4+ cells transfected with TLR2 siRNA and subsequently stimulated with <i>M. bovis</i> BCG (D).</p

<i>M. tuberculosis</i> complex increases susceptibility to HIV infectivity.

<p>The percentage of unstimulated CD4+ cells and CD4+ cells stimulated with PHA (50 µg/ml), <i>M. bovis</i> BCG (Copehnhagen), <i>M. tuberculosis</i> (CDC1551), <i>M. smegmatis</i> (MC²155) from individual subjects infected with X4-tropic pseudovirus (A) or R5-tropic pseudovirus (B). Results from individual donors are color-coded. Statistical analysis was performed using student T test (** p<0.005). Flow cytometry analysis of the representative CD4+ cells after infection with GFP-expressing pseudovirus (C).</p