Integrity of the Actin Cytoskeleton of Host Macrophages is Necessary for Mycobacterial Entry

Abstract

Macrophages are the primary hosts for Mycobacterium tuberculosis (M. tb), an intracellular pathogen, and the causative organism of tuberculosis (TB) in humans. While M. tb has the ability to enter and survive in host macrophages, the precise mechanism of its internalization, and factors that control this essential process are poorly defined. We have previously demonstrated that perturbations in levels of cholesterol and sphingolipids in macrophages lead to significant reduction in the entry of Mycobacterium smegmatis (M. smegmatis), a surrogate model for mycobacterial internalization, signifying a role for these plasma membrane lipids in interactions at the host-pathogen interface. In this work, we investigated the role of the host actin cytoskeleton, a critical protein framework underlying the plasma membrane, in the entry of M. smegmatis into human macrophages. Our results show that cytochalasin D mediated destabilization of the actin cytoskeleton of host macrophages results in a dose-dependent reduction in the entry of mycobacteria. Notably, the internalization of Escherichia coli remained invariant upon actin destabilization of host cells, implying a specific involvement of the actin cytoskeleton in mycobacterial infection. By monitoring the F-actin content of macrophages utilizing a quantitative confocal microscopy-based technique, we observed a close correlation between the entry of mycobacteria into host macrophages with cellular F-actin content. Our results constitute the first quantitative analysis of the role of the actin cytoskeleton of human macrophages in the entry of mycobacteria, and highlight actin-mediated mycobacterial entry as a potential target for future anti-TB therapeutics.

Keywords: Actin cytoskeleton; Cytochalasin D; F-actin quantitation; Macrophages; Mycobacterium.

Fig. 1

Actin organization in THP-1 macrophages upon treatment with cytochalasin D. THP-1 macrophages were treated with increasing concentrations of CD and actin organization was evaluated using confocal microscopy. Alexa Fluor 546 phalloidin was used to stain F-actin in macrophages. Maximum intensity projections obtained by merging 11 sections from the base of the coverslip (~ 4 μm from the base into the cell) are shown. Representative projected images for control macrophages are shown in panel (A) and the corresponding images for macrophages treated with 2.5 μM, 5 μM and 10 μM CD are shown in panels (B-D), respectively. Upon treatment with increasing concentrations of CD, reduction in F-actin filaments and formation of F-actin aggregates were observed. The scale bar represents 10 μm. See Methods for more details

Fig. 2

Cytochalasin D has no effect on the viability of THP-1 macrophages. THP-1 macrophages were treated with increasing concentrations of CD and its effect on cell viability was assayed using MTT. Values of viability of CD-treated cells are expressed as percentages normalized to control cells. Data represent means ± SE from at least five independent experiments. See Methods for more details

Fig. 3

Iso-surface generation and quantification of F-actin in THP-1 macrophages. Iso-surfaces of confocal z-sections corresponding to projections shown in Fig. 1 were generated using the iso-surface tool in Imaris. Panel (A) shows iso-surfaces for control macrophages, and iso-surfaces corresponding to macrophages treated with 2.5 μM, 5 μM and 10 μM CD are shown in panels (B–D), respectively. F-actin was quantitated by normalizing the enclosed volume within the iso-surface to the projected area of cells, obtained using the Zen software provided with Zeiss LSM 880 confocal microscope. The values of F-actin content in control and CD-treated cells are shown in panel E. Data represent means ± SE of at least 15 independent measurements (* and *** correspond to significant (p < 0.05 and p < 0.001) difference in F-actin content in macrophages treated with 5 and 10 μM CD relative to control macrophages, respectively). See Methods for more details

Fig. 4

Effect of actin destabilization on the entry of M. smegmatis into THP-1 macrophages. Control and CD-treated THP-1 macrophages were exposed to M. smegmatis at a multiplicity of infection 100:1 (bacteria to macrophage) for 2 h. Following infection, macrophages were lysed and the intracellular bacilli were cultured on Middlebrook 7H10 agar upon making appropriate dilutions and the colony forming units (CFU) were counted. Data represent means ± SE of at least six independent experiments. Values are normalized to CFU counts obtained from control (untreated) macrophages (** and *** correspond to significant (p < 0.01 and p < 0.001) difference in CFU counts of CD-treated macrophages relative to control macrophages, respectively). See Methods for more details

Fig. 5

Confocal microscopic imaging confirms the requirement of intact host actin cytoskeleton for entry of M. smegmatis. Representative images showing the entry of M. smegmatis into control (untreated), and CD-treated macrophages. Macrophages were incubated with M. smegmatis stably expressing dsRed2 for 2 h and were processed for confocal microscopic imaging (as described in Methods). The panels show macrophages (DIC images) infected with M. smegmatis expressing dsRed2 (red), with the nucleus stained with DAPI (blue). Merged images are shown in the panel on the extreme right. The scale bar represents 10 μm. See Methods for more details (Color figure online)

Fig. 6

Actin destabilization has no effect on the entry of E. coli into THP-1 macrophages. Control and CD-treated THP-1 macrophages were exposed to E. coli DH5α at a multiplicity of infection 100:1 (bacteria to macrophage) for 2 h. Following infection, macrophages were lysed and the intracellular bacilli were cultured on Luria Bertani agar upon making appropriate dilutions and the colony forming units (CFU) were counted. Data represent means ± SE of at least four independent experiments. Values are normalized to CFU counts obtained from control (untreated) macrophages. See Methods for more details

Fig. 7

Entry of M. smegmatis and host F-actin content are highly correlated. Correlation of M. smegmatis CFU counts and F-actin content of host macrophages upon treatment with increasing concentrations of CD. Data for the figure are from Figs. 3 and 4. Linear regression analysis yielded a correlation coefficient (r) of ~ 0.93. All data points were bound by the 95% confidence band (plotted as red dashed lines), implying a significant dependence of mycobacterial entry on the host actin cytoskeleton. See Methods and text for further details (Color figure online)