Friday, December 13, 2013

Escape of the Lyme disease spirochete from the bloodstream involves multiple adhesins and receptors

Microbial pathogens that cause systemic infections often travel within the circulatory system to spread throughout the host.  Eventually, these pathogens exit from the bloodstream to get to their target organ.  The first step of exit, adherence to the inner surface of the vessel wall, is probably the most challenging one because the rapidly flowing blood shoots the microbes through the capillaries.  A recent review described the process akin to "a spider trying to gain a foothold on the wall of a garden hose with the tap turned on full."  Most in vitro studies of adherence involve placement of microbe-mammalian cell cocultures in a stationary incubator.   These static conditions poorly reflect what microbes experience as they are carried throughout the circulatory system.  A better way to study vascular adhesion is to watch the process occur within a live animal.

A Canadian group has been doing just that.  As I explained in this post, they used intravital microscopy to shoot videos of the Lyme disease spirochete, Borrelia burgdorferi, escaping from skin capillaries of living mice (see the earlier post to watch a few of the videos).  The bacteria were genetically engineered to express green florescent protein so that they could be visualized with a fluorescence microscope.

From analyzing the videos, the investigators concluded that escape occurs in a series of steps.  The spirochete first "tethers" itself to the wall.  Within a second (assuming it doesn't let go), the spirochete starts crawling ("dragging") along the inner wall of the capillary.  The crawling spirochete eventually squeezes between the cells in the vessel wall (endothelial cells) to complete its escape.

In a follow-up study (see this post) they showed that expression of bbk32, encoding a B. burgdorferi surface protein, restored the ability of a highly-passaged, nonadherent B. burgdorferi strain to tether and drag along the vessel wall.  BBK32 clings to fibronectin and glycosaminoglycans (GAGs) in vitro.  The Canadian group found that fibronectin, which circulates in the bloodstream, and GAGs, which line the inner surface of capillaries, were necessary for B. burgdorferi to interact with the microvasculature.  Fibronectin, by its ability to anchor itself to GAGs, may serve as a lifeline that allows bacteria with fibronectin-binding proteins to tether themselves to the vessel wall.

Moriarty and colleagues conducted a third study to gain a better molecular understanding of BBK32's role in vascular adhesion.  The study was published in Molecular Microbiology over a year ago (here's the link to the study), but I still think it's worth going over today because of the significance of their findings, not only for Lyme disease but also for other diseases involving bloodstream dissemination of microbial pathogens.

Since separate surfaces of the BBK32 protein are responsible for fibronectin and GAG binding, the investigators wanted to see if BBK32's binding activities were deployed sequentially to carry out tethering and dragging.  Therefore, BBK32 variants defective in binding one or the other host protein were constructed by deleting small segments in each binding region.  The mutant genes encoding the variants were then introduced on plasmids into the highly-passaged, nonadhesive B. burgdorferi strain that they used in their earlier study.  The transformants expressing the BBK32 variants were injected into the bloodstream of mice, and skin capillaries were examined by intravital microscopy so that the investigators could count the tethering and dragging interactions like they did in their two earlier studies.

B. burgdorferi expressing the BBK32 variant defective in fibronectin binding (Δ158-182 in panel A below) underwent fewer tethering interactions than spirochetes expressing full-length BBK32 (BBK32 FL).  On the other hand, the BBK32 variant that bound GAG poorly (Δ45-68) mediated as many tethering interactions as wild-type BBK32.  These results indicate that the first step of adherence to the microvasculature, tethering, involves BBK32 interaction with fibronectin.  The BBK32 variant defective in GAG binding was impaired in promoting the second step of vascular adherence, dragging (panel B).

Figure 5 from Moriarty et al., 2012.  BBK32 Δ45-68 binds GAG poorly; BBK32 Δ158-182 binds fibornectin poorly.
Therefore, the exit pathway involving BBK32 goes as follows.  B. burgdorferi first uses BBK32 to tether to fibronectin, which is anchored to the vessel wall.  For the second step, BBK32 uses a different surface to bind to GAGs, allowing the spirochete to make more extensive contacts with the wall, leading to dragging interactions along the inner surface of the capillary.  Subsequent steps involving sequential contact of other bacterial factors with other host factors results in penetration of the spirochete through the vessel wall.

BBK32 is clearly capable of mediating vascular adhesion of an otherwise nonadherent B. burgdorferi mutant lacking most of its other adhesins.  However, to determine whether BBK32 really has a role in vascular adhesion of infectious B. burgdorferi requires a loss-of-function analysis, as opposed to the gain-of-function experiment just described.  Therefore, the investigators started with an infectious B. burgdorferi strain and knocked out the bbk32 gene.  They injected the bbk32 mutant and wild-type strains into the bloodstream of mice and again counted the number of tethering and dragging interactions in skin capillaries.  Here's where the results get interesting.  In comparison with the wild-type strain, they found that knocking out bbk32 reduced the number of vascular interactions by only 20%, and this reduction wasn't even statistically significant.  This result indicates that the contribution of BBK32 to vascular adhesion in skin capillaries is minor, at best.  Another B. burgdorferi adhesin (or adhesins) is responsible for the majority of vascular interactions in this tissue.

Figure 2B from Moriarty et al., 2012.  "Interactions" is the sum of tethering and dragging interactions counted in skin capillaries.

So where in the host does BBK32 function as a major adhesin?  Since B. burgdorferi is known to colonize joint tissue, the investigators decided to train their microscope on the capillaries supplying blood to the joints.  They saw that the spirochetes underwent the same tethering and dragging interactions that they observed in the skin. When they compared the bbk32 mutant against the infectious strain, they found that BBK32 accounts for roughly half of the tethering and dragging interactions (infectious vs. bbk32 KO in the graph below).  Whatever adhesin or adhesins are responsible for the other 50% do not interact with GAGs since coninjection of large amounts of a fibronectin peptide that binds GAGs failed to reduce the number of early vascular interactions of the bbk32 mutant (bbk32 KO vs. bbk32 KO + FN-C/H II).

Figure 2C from Moriarty et al., 2012.  "Interactions" is the sum of tethering and dragging interactions counted in joint capillaries.

Here's the bottom line:
  • BBK32 is capable of mediating the first two steps of B. burgdorferi adhesion to the vasculature.  These steps involve distinct surfaces of BBK32 undgoing sequential contacts with fibronectin and GAGs.
  • Whether BBK32 is actually involved in vascular adhesion depends on where the spirochete is located within the host.  For example, BBK32 has a major role in vascular adhesion in the joint, whereas it has little or no role in the skin.
  • Clearly, there are other (currently undiscovered) bacterial adhesins and host receptors that promote vascular adhesion and escape.


Moriarty TJ, Shi M, Lin YP, Ebady R, Zhou H, Odisho T, Hardy PO, Salman-Dilgimen A, Wu J, Weening EH, Skare JT, Kubes P, Leong J, & Chaconas G (2012). Vascular binding of a pathogen under shear force through mechanistically distinct sequential interactions with host macromolecules. Molecular Microbiology, 86 (5), 1116-31 PMID: 23095033

Coburn J, Leong J, & Chaconas G (2013). Illuminating the roles of the Borrelia burgdorferi adhesins. Trends in Microbiology, 21 (8), 372-9 PMID: 23876218

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