Uptake and persistence of Mycobacterium avium subsp. paratuberculosis in human monocytes

Abstract

Mycobacterium avium subsp. paratuberculosis is a bacterium sometimes found in human blood and tissue samples that may have a role in the etiology of Crohn's disease in humans. To date, however, there have been few studies examining the interactions of these bacteria with human cells. Using the THP-1 human monocytic cell line, this study shows that the uptake and trafficking of M. avium subsp. paratuberculosis in human cells are cholesterol dependent and that these bacteria localize to cholesterol-rich compartments that are slow to acidify. M. avium subsp. paratuberculosis bacteria containing phagosomes stain for the late endosomal marker Rab7, but recruitment of the Rab7-interacting lysosomal protein that regulates the fusion of bacterium-containing phagosomes with lysosomal compartments and facilitates subsequent bacterial clearance is significantly reduced. Disruption of phagosome acidification via this mechanism may contribute to M. avium subsp. paratuberculosis persistence in human cells, but there was no evidence that internalized M. avium subsp. paratuberculosis also affects the survival of bacteria taken up during a secondary phagocytic event.

Fig 1

Cholesterol aggregates at the site of M. avium subsp. paratuberculosis internalization. THP-1 cells infected with bacteria (MOI, 50:1) for 4 h were fixed, stained, and visualized for filipin-labeled cholesterol, FITC-labeled bacteria, and Texas Red-conjugated phalloidin-stained actin. The right-hand column shows an overlay image. The images, which are representative of three independent experiments, denote cells with no bacteria (A), M. bovis (B), M. avium subsp. paratuberculosis (C), and E. coli BL21 (D). Bar = 10 μm.

Fig 2

Flow cytometry assessment of internalization of labeled bacteria into cholesterol-depleted THP-1 cells. (A) A negative cholesterol gradient was created in THP-1 cells using simvastatin (SimV) and MβCD. Cells were incubated with FITC-labeled M. avium subsp. paratuberculosis (MAP) (B), M. bovis (C), or E. coli BL21 (D) (all at an MOI of 50:1) for 4 h, and then fluorescence was measured before (total associated; light bars) and after (intracellular; dark bars) the addition of trypan blue. Lowering cholesterol had a significant effect on the internalization of M. avium subsp. paratuberculosis and M. bovis (P < 0.05 and 0.01, respectively). *, **, and ***, results are statistically significantly different from those for untreated controls (P < 0.05, 0.01, and 0.001, respectively). Results are ±SEMs for three independent experiments.

Fig 3

Intracellular M. avium subsp. paratuberculosis colocalizes with cholesterol. THP-1 cells infected with FITC-labeled bacteria (MOI, 50:1) for 48 h were examined using a ×40 oil-immersion (numerical aperture, 1.3) lens and confocal microscopy (model SP5; Leica, Wetzlar, Germany). Filipin, indicated in red, was excited with 405 nm light (violet), and emission was collected from 420 to 480 nm, while FITC, shown in green, was excited with 488 nm (blue) light and fluorescence was collected from 500 to 550 nm. Bar = 5 μm.

Fig 4

Colocalization of Rab7 with M. avium subsp. paratuberculosis. Epifluorescence images of FITC-labeled live (A) and heat-killed (B) M. avium subsp. paratuberculosis, live M. bovis (C), and heat-killed E. coli BL21 (D) at 2 h postinfection of THP-1 cells (MOI, 50:1). Blue nuclei, green bacteria, and red Rab7 fluorescence are shown individually and in a merged image. Bar = 20 μm. (E) Mean ± SEM bacterial colocalization with Rab7 (yellow fluorescence) in 150 bacterium-containing phagosomes from three independent experiments.

Fig 5

Live M. avium subsp. paratuberculosis exhibits altered fusion with acidic compartments. THP-1 cells were infected with FITC-labeled M. avium subsp. paratuberculosis (MOI, 50:1) and labeled with LysoTracker Red, and using a merged image, bacterium-containing compartments were counted manually as strongly, weakly, or not stained with LysoTracker Red. Values are the means ± SEMs of live M. avium subsp. paratuberculosis colocalization with LysoTracker Red in 150 bacterium-containing compartments from three independent experiment compared to those for heat-killed M. avium subsp. paratuberculosis and live M. bovis.

Fig 6

Live M. avium subsp. paratuberculosis reduces phagosomal recruitment of RILP. At 48 h postinfection, bacterium-containing phagosomes were isolated from infected THP-1 cells by sucrose density gradient centrifugation. Phagosome preparations were dot blotted onto a PVDF membrane and probed first with an anti-RILP antibody. Bound antibody was removed with glycine-HCl, and the blot was reprobed with an antibody to Rab7. Antibody binding to RILP (A) and Rab7 (B) was visualized using an alkaline phosphatase-conjugated secondary antibody and quantified using densitometry. The ratio of RILP/Rab7 was calculated and is shown normalized (C) to that for heat-killed M. avium subsp. paratuberculosis (100%). Overall, at 48 h cells infected with live mycobacteria (both M. avium subsp. paratuberculosis and M. bovis) had significantly reduced expression of RILP relative to expression of Rab7 in phagosome preparations (P = 0.0004). ** and ***, results are significantly different from those for THP-1 cells infected with heat-killed M. avium subsp. paratuberculosis (P < 0.01 and 0.001, respectively). Results are ±SEMs for three independent experiments.

Fig 7

Secondary E. coli infection of human monocytes after primary infection with M. avium subsp. paratuberculosis. E. coli bacteria (MOI, 20:1) added to cells 2 h after infection with M. avium subsp. paratuberculosis (MOI, 50:1) were given 2 h to internalize before the addition of gentamicin to kill any remaining extracellular bacteria. Quantitative assessment of viable E. coli in THP-1 cells (A and B) and purified human monocytes (C and D) 2 and 24 h after coinfection was by serial dilution of cell lysates. Results are ±SEMs for three independent experiments.