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. 2021 Feb 16;22(4):1945.
doi: 10.3390/ijms22041945.

Generation of Liposomes to Study the Effect of Mycobacterium Tuberculosis Lipids on HIV-1 cis- and trans-Infections

Marion Pouget  1   2 , Anna K Coussens  3   4 , Alessandra Ruggiero  1   5 , Anastasia Koch  3 , Jordan Thomas  1 , Gurdyal S Besra  6 , Robert J Wilkinson  3   7   8 , Apoorva Bhatt  6 , Georgios Pollakis  1 , William A Paxton  1
Affiliations

Generation of Liposomes to Study the Effect of Mycobacterium Tuberculosis Lipids on HIV-1 cis- and trans-Infections

Marion Pouget et al. Int J Mol Sci. .

Abstract

Tuberculosis (TB) is the leading cause of death among HIV-1-infected individuals and Mycobacterium tuberculosis (Mtb) co-infection is an early precipitate to AIDS. We aimed to determine whether Mtb strains differentially modulate cellular susceptibility to HIV-1 infection (cis- and trans-infection), via surface receptor interaction by their cell envelope lipids. Total lipids from pathogenic (lineage 4 Mtb H37Rv, CDC1551 and lineage 2 Mtb HN878, EU127) and non-pathogenic (Mycobacterium bovis BCG and Mycobacterium smegmatis) Mycobacterium strains were integrated into liposomes mimicking the lipid distribution and antigen accessibility of the mycobacterial cell wall. The resulting liposomes were tested for modulating in vitro HIV-1 cis- and trans-infection of TZM-bl cells using single-cycle infectious virus particles. Mtb glycolipids did not affect HIV-1 direct infection however, trans-infection of both R5 and X4 tropic HIV-1 strains were impaired in the presence of glycolipids from M. bovis, Mtb H37Rv and Mtb EU127 strains when using Raji-DC-SIGN cells or immature and mature dendritic cells (DCs) to capture virus. SL1, PDIM and TDM lipids were identified to be involved in DC-SIGN recognition and impairment of HIV-1 trans-infection. These findings indicate that variant strains of Mtb have differential effect on HIV-1 trans-infection with the potential to influence HIV-1 disease course in co-infected individuals.

Keywords: BCG; CDC1551; DC-SIGN; EU127; H37Rv; HIV-1; HN878; M. smegmatis; Mycobacterium tuberculosis; PDIM; SL1; TB; TDM; in vitro; liposomes; trans-infection.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Liposome-size analysis by NanoSight particle tracking. (A) Liposomes generated with the Extruder system at 100 nm size, (B) Liposomes generated with the Extruder system at 200 nm size (C) 0.8PC:0.2Ch, (D) BCG batch n° 1, (E) BCG batch n° 2 and (F) H37Rv liposomes made by sonication without the Extruder step. Liposome suspensions were diluted 1:1000–1:2000 in PBS, and three 60 s videos were recorded. The data shown are from one experiment. On the right are graphs showing the results of particle concentration and their size measurement; and on the left are liposomes observed at the screen shot from NTA video.
Figure 2
Figure 2
Thin-layer chromatography (TLC) analyses of 0.8PC:0.2Ch, BCG and H37Rv liposomes. 10 µL of liposomes solution made in water were spotted and dried on a silica gel 60 F254 plate. Separation occurred in 60:16:2 CHCl3:MeOH:H2O solvent and was visualised by staining with molybdophosphoric acid and charring. The arrows show the presence of lipids from mycobacterial origin. The data are from one representative experiment.
Figure 3
Figure 3
Influence of Mycobacterium liposomes on HIV-1 cis-infection. Cells were infected with 8 ng CA-p24 of (A,B) pSG3-LAI (HIV-1 X4), (C,D) pSG3-BAL (HIV-1 R5) and pSG3Δenv (ΔpSG3) where (A,C) virus input with 100 ng of liposomes at the same time or (B,D) 100 ng of liposomes added to TZM-bl cells 30 min prior to adding virus. 48 h after infection cells were lysed to measure luciferase activity (relative light unit, RLU). The RLUs produced for each experiment were normalised to the average value of the negative control 0.8PC:0.2Ch liposomes. 0.8PC:0.2Ch is used here as a negative control and reference. For the data shown, n = 3. Mann–Whitney unpaired t-test was performed for (A–D).
Figure 4
Figure 4
Influence of the presence of Mycobacterium liposomes on HIV-1 trans-infection via Raji-DC-SIGN cells. (A) 12.5 ng CA-p24 pSG3-LAI (HIV-1 X4) or (B) 20 ng CA-p24 pSG3-BAL (HIV-1 R5). After capture the cells were washed and co-cultured with TZM-bl cells. The luciferase activity (RLU) was read after 48 h. The RLUs produced for each experiment were normalised to the average value of the negative control 0.8PC:0.2Ch liposomes. The data shown are a pool of at least two independent experiments where n ≥ 3 in total. Mann–Whitney unpaired t-test was performed for (A,B) and p- value represented with * for p-value < 0.05, ** for p-value < 0.01, *** for p-value < 0.001, **** for p-value < 0.0001.
Figure 5
Figure 5
Comparison of the influence different H37Rv on HIV-1 trans-infection via Raji-DC-SIGN cells. (A) 12.5 ng CA-p24 pSG3-LAI (HIV-1 X4) or (B) 20 ng CA-p24 pSG3-BAL (HIV-1 R5). After capture the cells were washed and co-cultured with TZM-bl cells. The luciferase activity was read after 48 h. The RLUs produced for each experiment were normalised to the average value of the negative control 0.8PC:0.2Ch liposomes. The data shown are a pool of at least two independent experiments where n ≥ 6 in total. Mann–Whitney unpaired t-test was performed for (A,B) and p-value represented with * for p-value < 0.05, ** for p-value < 0.01, *** for p-value < 0.001, **** for p-value < 0.0001.
Figure 6
Figure 6
Role of H37Rv glycolipids in HIV-1 trans-infection via DC-SIGN. (A) H37Rv fractions 1 to 8 have been integrated into liposomes following the ratio 0.6PC:0.2Ch:0.2fraction and were tested on HIV-1 trans-infection via DC-SIGN in parallel of 0.8PC:0.2Ch and H37Rv liposomes. The data shown are from one experiment, n = 3. (B) H37Rv papA1Δ and H37Rv papA1Δ + SL1 were tested in parallel of 0.8PC:0.2Ch and H37Rv liposomes on HIV-1 trans-infection. The data shown are a pool of at least two independent experiments where n ≥ 6 in total. For (A,B) 0.5 × 106 Raji DC-SIGN were pre-incubated during 30 min with 100 μg liposomes or 50 µL of media. The cells were then incubated for 2 h with HIV-1 pseudo-typed virus (1) 12.5 ng CA-p24 pSG3-LAI (HIV-1 X4) or (2) 20 ng CA-p24 pSG3-BAL (HIV-1 R5). After capture the cells were washed and co-cultured with TZM-bl cells. The luciferase activity was read after 48 h. The RLUs produced for each experiment were normalised to the average value of the negative control 0.8PC:0.2Ch liposomes. Mann–Whitney unpaired t-test was performed for (A,B) and p-value represented with * for p-value < 0.05, ** for p-value < 0.01, *** for p-value < 0.001, **** for p-value < 0.0001.
Figure 7
Figure 7
Influence of Mycobacterium liposomes on HIV-1 trans-infection via iDCs or mDCs. (A) and (B) iDCS, (C,D) mDCs, was pre-incubation for 30 min with 100 μg of liposomes or 50 µL of media. The cells were then incubated 2h with HIV-1 pseudo-typed virus: 12.5 ng CA-p24 pSG3-LAI (HIV-1 X4, panels (A,C)) or 20 ng CA-p24 pSG3-BAL (HIV-1 R5, panels (B,D)). For the data shown, the experiment has been performed using cells isolated from on at least two different donors where n ≥ 3 in total. The RLUs produced for each experiment were normalised to the average value of the negative control 0.8PC:0.2Ch liposomes. Mann–Whitney unpaired t-test was performed for (A–D) and p-value represented with * for p-value < 0.05, ** for p-value < 0.01, *** for p-value < 0.001, **** for p-value < 0.0001.
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