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. 2012 Jan 13:12:7.
doi: 10.1186/1471-2180-12-7.

Role of Mycobacterium tuberculosis pknD in the pathogenesis of central nervous system tuberculosis

Nicholas A Be  1 , William R Bishai, Sanjay K Jain
Affiliations

Role of Mycobacterium tuberculosis pknD in the pathogenesis of central nervous system tuberculosis

Nicholas A Be et al. BMC Microbiol. .

Abstract

Background: Central nervous system disease is the most serious form of tuberculosis, and is associated with high mortality and severe neurological sequelae. Though recent clinical reports suggest an association of distinct Mycobacterium tuberculosis strains with central nervous system disease, the microbial virulence factors required have not been described previously.

Results: We screened 398 unique M. tuberculosis mutants in guinea pigs to identify genes required for central nervous system tuberculosis. We found M. tuberculosis pknD (Rv0931c) to be required for central nervous system disease. These findings were central nervous system tissue-specific and were not observed in lung tissues. We demonstrated that pknD is required for invasion of brain endothelia (primary components of the blood-brain barrier protecting the central nervous system), but not macrophages, lung epithelia, or other endothelia. M. tuberculosis pknD encodes a "eukaryotic-like" serine-threonine protein kinase, with a predicted intracellular kinase and an extracellular (sensor) domain. Using confocal microscopy and flow cytometry we demonstrated that the M. tuberculosis PknD sensor is sufficient to trigger invasion of brain endothelia, a process which was neutralized by specific antiserum.

Conclusions: Our findings demonstrate a novel in vivo role for M. tuberculosis pknD and represent an important mechanism for bacterial invasion and virulence in central nervous system tuberculosis, a devastating and understudied disease primarily affecting young children.

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Figures

Figure 1
Figure 1
Invasion and survival of M. tuberculosis pknD mutant in host-derived cells. A. BALB/c mice were infected with M. tuberculosis CDC1551 or pknD mutant, and sacrificed at days 1 and 49 after infection. The mutant for M. tuberculosis pknD was significantly attenuated (P = 0.004) in mouse brain, but not lung tissue, 49 days after infection. No defect was observed in the lungs at either time point. Bacterial burden is represented as log10 CFU/organ for all animal experiments. B. Invasion of host-cell monolayers by wild-type CDC1551, wild-type intergenic transposon control, pknD transposon mutant (pknD:Tn), and pknD genetic complement (pknD:Comp) was examined and normalized to the wild-type control. Invasion assays were performed in brain microvascular endothelial cells (HBMEC), epithelial A549 cells, and umbilical vein endothelia (HUVEC). No difference in invasion was observed in A549 cells (P = 0.31) or HUVEC (P = 0.41). A significant reduction in invasive capacity, however, was observed in the CNS-derived HBMEC (P = 0.02). This defect was restored by genetic complementation with the native pknD/pstS2 operon. N.S. = not significantly different. C. Intracellular survival of each of the above M. tuberculosis strains was examined in HBMEC at days 1, 3, 5, and 7 after infection. The pknD:Tn mutant demonstrated an invasion and intracellular survival defect in HBMEC relative to wild-type over the course of the seven day infection. D. Survival was also examined by infection of activated J774 macrophages. No corresponding survival defect for the pknD:Tn mutant was observed in these cells during the seven day infection. A mutant for the gene Rv0442c, known to be attenuated in the macrophage model, is included as a control. All CFU counts are represented as mean ± standard deviation.
Figure 2
Figure 2
M. tuberculosis PknD is sufficient to trigger adhesion to HBMEC. A and B. Fluorescent microspheres were coated with either PknD sensor or BSA, inoculated into HBMEC, washed, and stained for actin. Confocal microscopy demonstrated that PknD sensor-coated microspheres (panel B) adhere to brain endothelia to a greater degree than those coated with BSA (panel A). C. Confocal images were assembled into a 3D reconstruction and examined under higher magnification. PknD sensor-coated microspheres appear to be largely enveloped by actin processes (arrows) indicating that PknD-induced uptake by host cells may be an active process. D. When confocal images are examined in multiple planes, it is clear that a number of microspheres exist intracellularly. E. Wells containing endothelial cells with microspheres were analyzed for fluorescence. Quantification of fluorescence demonstrated a significant increase in the adherence of PknD-coated microspheres to the monolayer (P = 0.0002). F. Microspheres were pre-incubated with either custom anti-PknD serum or naïve serum. Incubation with anti-PknD serum (1:250 dilution) significantly reduced adherence of PknD (P = 0.0007) but not BSA-coated microspheres (P = 0.6). Moreover, no reduction in adherence was noted for PknD or BSA-coated microspheres when incubated with naïve antiserum (BSA: P = 0.4; PknD: P = 0.1; ANOVA single factor). Fluorescence readings are presented as mean ± standard deviation. *Statistically significant difference.
Figure 3
Figure 3
M. tuberculosis PknD triggers invasion of the brain endothelium. A. Brain endothelia were inoculated with either PknD sensor- or BSA-coated fluorescent microspheres, washed, and disrupted by trypsinization. Endothelia were subjected to flow cytometry and gated to remove extracellular microspheres not associated with whole cells. B. Flow cytometry analysis demonstrated that significantly more endothelial cells were positive for fluorescence when incubated with PknD sensor-coated microspheres compared to BSA-coated microspheres (7.7% vs. 0.6%; P = 0.0003). Cell counts are presented as mean ± standard deviation. C. Histograms show that discrete fluorescent-positive populations are evident in the cells inoculated with PknD sensor-coated microspheres, indicating that cell populations took up multiple quantities of microspheres. D. Microspheres were again pre-incubated with either custom anti-PknD serum or naïve serum, followed by inoculation onto endothelial cells. Pre-incubation with anti-PknD (1:250) significantly reduced the population of cells which were positive for fluorescent microspheres, compared to naïve serum, as is indicated in the figure by a horizontal bar (P = 0.001). Pre-incubation with anti-PknD (1:1250) had no effect on internalization, when compared to untreated cells (P = 0.07).
Figure 4
Figure 4
M. tuberculosis PknD sensor domain interacts with host laminin. A. M. tuberculosis WT and pknD mutant were incubated in wells coated with components of the extracellular matrix (laminin, fibronectin, and collagen). The pknD mutant demonstrated a 2-fold reduction in adhesion to the laminin matrix (P = 0.001), while not exhibiting significantly reduced adhesion to fibronectin or collagen. CFU counts are represented as mean ± standard deviation. N.S. = not significantly different. B and C. Coated microspheres were incubated with HBMEC, followed by immunostaining for laminin. Microspheres coated with PknD sensor (panel C) associated with the periphery of laminin staining more than those coated with BSA (panel B), which were evenly distributed throughout the field of view.
Figure 5
Figure 5
Invasion of brain endothelia by M. tuberculosis is reduced by anti-PknD serum. M. tuberculosis CDC1551 were pre-incubated with naïve or custom anti-PknD serum, washed, and used to infect brain endothelial cells. Following 90 minutes of infection, cells were lysed and CFU enumerated. It was observed that incubation with anti-PknD serum, but not naïve serum, significantly reduced the number of bacilli able to successfully invade HBMEC (P = 0.01). *Statistically significant difference.
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