Biophysical Interaction Landscape of Mycobacterial Mycolic Acids and Phenolic Glycolipids with Host Macrophage Membranes

Abstract

Lipidic adjuvant formulations consisting of immunomodulatory mycobacterial cell wall lipids interact with host cells following administration. The impact of this cross-talk on the host membrane's structure and function is rarely given enough consideration but is imperative to rule out nonspecific perturbation underlying the adjuvant. In this work, we investigated changes in the plasma membranes of live mammalian cells after exposure to mycobacterial mycolic acid (MA) and phenolic glycolipids, two strong candidates for lipidic adjuvant therapy. We found that phenolic glycolipid 1 softened the plasma membrane, lowering membrane tension and stiffness, but MA did not significantly change the membrane characteristics. Further, phenolic glycolipid 1 had a fluidizing impact on the host plasma membrane, increasing the fluidity and the abundance of fluid-ordered-disordered coexisting lipid domains. Notably, lipid diffusion was not impacted. Overall, MA and, to a lesser extent, phenolic glycolipid 1, due to minor disruption of host cell membranes, may serve as appropriate lipids in adjuvant formulations.

Keywords: fluidity; infectious diseases; lateral membrane organization; mycobacterial lipids; plasma membrane; vaccine adjuvants.

Figure 1. Chemical structures of (A) alpha, keto, and methoxy MAs and (B) phenolic glycolipid, PGL1.

Red color in PGL1 structure depicts the saccharide units, and blue is the phenyl motif; absence of these generates PDIM. (C) Quantitative mass analysis of alpha, keto, and methoxy MAs (in mol %) in the extracted and purified fraction of Mycobacterium bovis. Lower panels depict the mol % abundance of constituting acyl chains in increasing order of carbon chain lengths within each class of MA.

Figure 2. Effect of mycobacterial lipids on the THP-1 membrane properties. Representative force−indentation curves of control and mycobacterial lipid-treated cells.

First, 0.5 μm of the force curves were fitted with Hertz equation to obtain estimates of cortical stiffness. (B) Elastic modulus distribution of THP-1 cells with and without mycobacterial lipid treatment, MA and PGL1 (10 μg/mL) for 4 h (n = 70 cells per condition) fitted with a single log−normal distribution. Median elastic modulus is shown in the insets. (C) Relative frequency of the membrane tether forces in cells with (n = 855 elements) and without (n = 633 elements) treatment with mycobacterial lipids fitted with a log−normal distribution. The median values are provided in the inset. For data in B,C, P < 0.05, Mann−Whitney test was used. (D) Changes in the tether number with the median values in the inset and (E) changes in tether length of cells in the presence and absence of mycobacterial lipids (10 μg/mL, 4 h). (***P < 0.005 unpaired student t-test.)

Figure 3

Fluorescence lifetime images of the THP-1 cell plasma membrane in the absence and presence of indicated mycobacterial lipids along with associated histograms (no of cells = 42(control), 35 (MA) and 32 (PGL-1), N = 2.) Scale bar 10 μm.

Figure 4. Mycobacterial PGL1 regulates the host cell membrane order and hydration.

(A) Representative GP images of THP-1 cells in the absence and presence of the indicated lipids (4 h, 10 μg/mL) (B) Segmented images derived from (A) showing only the plasma membrane regions. (C) GP distribution associated with the stack of GP images (n = 45, N = 2) fitted to Gaussian function. It shows peak centroids for each identified population. The surface coverage of each of these populations (area under the curve) is indicated as shown. Scale bar: 10 μm, 60× oil objective.