A Quantitative Method for the Study of HIV-1 and Mycobacterium tuberculosis Coinfection

Abstract

Mycobacterium tuberculosis and human immunodeficiency virus-1 (HIV-1) syndemic interactions are a major global health concern. Despite the clinical significance of coinfection, our understanding of the cellular pathophysiology and the therapeutic pharmacodynamic impact of coinfection is limited. Here, we use single-round infectious HIV-1 pseudotyped viral particles expressing green fluorescent protein alongside M. tuberculosis expressing mCherry to study pathogenesis and treatment. We report that HIV-1 infection inhibited intracellular replication of M. tuberculosis and demonstrate the therapeutic activity of antiviral treatment (efavirenz) and antimicrobial treatment (rifampicin). The described method could be applied for detailed mechanistic studies to inform the development of novel treatment strategies.

Keywords: Mycobacterium tuberculosis; HIV-1; coinfection; drug screening; tuberculosis.

Figure 1.

The impact of HIV-1 (HIV) infection on the growth of intracellular Mycobacterium tuberculosis over 96 hours in THP-1 macrophages. A, Live-cell confocal imaging of HIV and M. tuberculosis coinfection in THP-1 macrophage cultures singularly infected with HIV-VSV-G, infected simultaneously with HIV-VSV-G and M. tuberculosis, and infected simultaneously with HIV-BAL and M. tuberculosis. The green signal (green fluorescent protein) indicates host-cell infection by HIV. The red signal (mCherry) indicates host-cell infection by M. tuberculosis. All images were acquired 72 hours postinfection at 63× magnification. Scale bars represent 50 µm. Images are representative of 3 individual experiments. B, The impact of HIV infection on the growth of intracellular M. tuberculosis over 96 hours in THP-1 macrophages based on the detection of mCherry (587/610 nm [excitation/emission]) in AFU. Data are shown for THP-1 macrophage cultures infected with M. tuberculosis alone (closed circles; solid line), HIV-VSV-G and M. tuberculosis (closed triangle; dashed and dotted line) or HIV-BAL and M. tuberculosis (closed square; dotted line); n = 3. Error bars represent the standard deviation of the mean. Abbreviations: AFU, arbitrary fluorescent unit; HIV-1, human immunodeficiency virus-1; VSV-G, vesicular stomatitis virus glycoprotein.

Figure 2.

The impact of coinfection on the relative frequency of HIV-1 (HIV) and/or Mycobacterium tuberculosis infection in THP-1 cells. A, Cells were identified based on their size and granularity and single cells identified based on the proportionality of area and height. Dead cells were removed by staining for viability (Live/Dead) and gating on the negative population. mCherry was used to identify M. tuberculosis-infected cells and GFP was used to identify HIV–infected cells. Representative dot plots are shown for THP-1 macrophages recovered from uninfected cultures, macrophages cultured with HIV-VSV-G, macrophages cultured with M. tuberculosis, and macrophages cultured with both HIV and M. tuberculosis. B, Frequency of cells infected with HIV or M. tuberculosis for THP-1 macrophage cultures singularly infected with HIV or M. tuberculosis, or for cultures infected simultaneously with HIV-VSV-G and M. tuberculosis is shown; n = 3. C, For untreated control cultures and cultures treated with efavirenz or rifampicin, the frequency of cells singularly infected with HIV, singularly infected with M. tuberculosis, or simultaneously infected with HIV and M. tuberculosis is shown; n = 3. Statistical comparisons were made using unpaired t tests. * P < .05, ** P < .005. Abbreviations: C, control; EFV, efavirenz; FSC, forward scatter; GFP, green fluorescent protein; HIV-1, human immunodeficiency virus-1; ns, not significant; RIF, rifampicin; SSC, side scatter; VSV-G, vesicular stomatitis virus glycoprotein.