Tuesday, December 30, 2014

Severe Lyme arthritis: Gagging on GAGs

Janis Weis' group has been mapping genetic variants that make laboratory mice prone to severe Lyme arthritis.  One of these variants is described in a paper that appeared in The Journal of Clinical Investigation earlier this year.  The affected gene encodes the enzyme β-glucuronidase, which carries out a critical function in the lysosome. β-glucuronidase cooperates with other degradative enzymes in the lysosome to break down glycosaminoglycans (GAGs) into their individual sugar units, which are then removed from the lysosome and reused by the cells.  GAGs are long chains of specific disaccharides located on the cell surface and within the extracellular matrix.  GAGs are covalently (in proteoglycans) or noncovalently attached to proteins.  GAGs are always being degraded and resynthesized by cells. Blocking any of the enzymes involved in GAG breakdown causes accumulation of GAG fragments, which are potentially detrimental to health.  In humans, certain mutations in the β-glucuronidase gene lead to a rare condition called Sly syndrome.

Large amounts of GAGs are found in the joints, where they serve an important mechanical function.  GAGs carry a high density of negative charge due to the presence of acidic sugars such as glucuronic acid, the target of β-glucuronidase, and the sulfate groups attached to most types of GAGs.  The negative charge attracts cations, which in turn attract large numbers of water molecules.  The water within GAGs acts as a cushion that allows the joints to withstand large compressive forces.

The key to the study was having strains of mice that differed in their susceptibility to Lyme arthritis. The C3H mouse strain develops severe joint inflammation during B. burgdorferi infection. On the other hand, the B6 strain develops mild joint inflammation when infected. Weis' group had earlier narrowed the locations of the genetic variations accounting for the different susceptibilities to several distinct segments within the mouse genome.  They used the traditional techniques of mouse genetics, which involved numerous matings involving the C3H and B6 strains and their progeny (see this review for details).  The authors focused on one of the segments, and with help from mouse genome sequence data that became available, they found a nucleotide difference within the Gusb (β-glucuronidase) gene that changed a single amino acid in the enzyme.

The investigators found that β-glucuronidase activity was mildly reduced in the infected C3H strain relative to the B6 strain.  Staining tissue sections of infected mice with Alcian blue, a dye attracted to polyanions, revealed accumulation of GAGs in the joint tissues of infected C3H mice but not infected B6 mice, lending further support to the lesion in Gusb being responsible for severe inflammation.  When a functional copy of the β-glucuronidase gene was stitched into the genome of C3H mice, B. burgdorferi infection no longer caused joint inflammation.

Does the same process occur in humans with Lyme arthritis?  One hint that β-glucuronidase influences the course of Lyme arthritis is the finding from other labs that found that the concentration of the enzyme in joint fluid is higher in patients with Lyme arthritis than it is in healthy uninfected individuals, although how the high enzyme levels are mechanistically linked to arthritis remains unexplained.

So how does β-glucuronidase deficiency lead to severe Lyme arthritis?  One possibility raised by the authors is that GAG fragments worsen tissue inflammation by stimulating Toll-like receptors, as shown in other studies (see this paper for an example).

The findings may also tell us something about rheumatoid arthritis (RA).  The B6 strain ends up with a form of RA following injection with certain autoantibodies.  One of Weis' mouse crosses generated a B6 strain with its Gusb gene and flanking regions swapped for the same region of the C3H strain.  This strain developed severe arthritis when injected with the same autoantibodies and when infected with B. burgdorferi.  Therefore, the pathologic processes leading to Lyme arthritis and RA share common steps, at least in laboratory mice.  In humans, RA patients, like those with Lyme arthritis, have high levels of β-glucuronidase levels in their joint fluid.

The search for host factors affecting the development of Lyme arthritis goes on.  Weis' group continue to identify genetic variants responsible for severe Lyme arthritis.


Bramwell KK, Ma Y, Weis JH, Chen X, Zachary JF, Teuscher C, & Weis JJ (2014). Lysosomal β-glucuronidase regulates Lyme and rheumatoid arthritis severity. The Journal of Clinical Investigation, 124 (1), 311-320 PMID: 24334460

Bramwell KK, Teuscher C, & Weis JJ (2014). Forward genetic approaches for elucidation of novel regulators of Lyme arthritis severity. Frontiers in Cellular and Infection Microbiology, 4 PMID: 24926442

Pancewicz S, Popko J, Rutkowski R, Knaś M, Grygorczuk S, Guszczyn T, Bruczko M, Szajda S, Zajkowska J, Kondrusik M, Sierakowski S, & Zwierz K (2009). Activity of lysosomal exoglycosidases in serum and synovial fluid in patients with chronic Lyme and rheumatoid arthritis. Scandinavian Journal of Infectious Diseases, 41 (8), 584-589 PMID: 19513935

Jiang D, Liang J, Fan J, Yu S, Chen S, Luo Y, Prestwich GD, Mascarenhas MM, Garg HG, Quinn DA, Homer RJ, Goldstein DR, Bucala R, Lee PJ, Medzhitov R, & Noble PW (2005). Regulation of lung injury and repair by Toll-like receptors and hyaluronan. Nature Medicine, 11 (11), 1173-1179 PMID: 16244651

Monday, March 10, 2014

Video microscopy of ticks acquiring the Lyme disease spirochete from mice

The bite of an infected Ixodes hard tick transmits the Lyme disease spirochete, Borrelia burgdorferi, to humans.  Ticks acquire B. burgdorferi by feeding on reservoir hosts colonized with the spirochete.  Reservoir hosts include small mammals such as the white-footed mouse, the main reservoir of B. burgdorferi in the northeastern United States.

A study that just came out in The Yale Journal of Biology and Medicine is accompanied by videos of Borrelia burgdorferi being transmitted between mouse and tick.1  The authors prepared the mice by infecting them with B. burgdorferi genetically modified to express green fluorescent protein.  After waiting two weeks to allow the spirochetes to disseminate, they placed one hungry Ixodes scapularis tick onto an ear of each infected mouse.

Figure 1 from Bockenstedt et al., 20141.  A feeding tick (arrow) attached to the ear of a mouse.  The tick is engorged with blood.

Nymphal ticks feed for an average of 2.5 to 8 days.  The meal starts with the tick inserting its barbed feeding apparatus into the skin.  (For a close-up view of this process, head over to the blog Phenomena: Not Exactly Rocket Science.)  The tick releases saliva through the feeding canal into the skin.  Tick saliva contains substances that damage host tissue surrounding the feeding apparatus and pharmacologic agents that inhibit clotting and engage the immune system. Intervals of salivation alternate with ingestion of blood, tissue fluid, and lymph that pool at the feeding site.2

The authors examined the feeding site by two photon intravital microscopy to observe what was happening to the spirochetes. They saw spirochetes in the dermis moving towards the feeding apparatus and disappearing as they presumably got sucked into the feeding canal.  One such spirochete is digitally colored in red in the video below, which was shot 48 hours into the blood meal.  (One hour of video footage was compressed into 30 seconds.)  The feeding apparatus is the green structure near the top of the viewing field.  Previous studies have suggested that ticks acquire B. burgdorferi from skin, not from blood.3  The videos from this study provide support for this notion, according to the authors.


Are the spirochetes mere passengers that get caught in the flow of fluid being drawn into the feeding canal, or are they active participants?  The authors argue for an active role for the spirochetes:
Spirochete movement is unlikely to be due simply to the mechanical flux of tissue fluid as the tick feeds because close examination of individual spirochetes that move toward the hypostome reveals both the oscillating movements that we observe in the absence of tick feeding as well as directional translocation.


1.  Bockenstedt LK, Gonzalez D, Mao J, Li M, Belperron AA, & Haberman A (2014). What ticks do under your skin: two-photon intravital imaging of Ixodes scapularis feeding in the presence of the Lyme disease spirochete. The Yale Journal of Biology and Medicine, 87 (1), 3-13 PMID: 24600332

2.   Anderson JF, & Magnarelli LA (2008). Biology of ticks. Infectious Disease Clinics of North America, 22 (2) PMID: 18452797

3.  Nakayama Y, & Spielman A (1989). Ingestion of Lyme disease spirochetes by ticks feeding on infected hosts. The Journal of infectious diseases, 160 (1), 166-7 PMID: 2732513

Monday, December 16, 2013

"...and a dog with lepto in its pee."

I saw this video over at the Worms & Germs blog.  It's a new take on a popular Christmas carol.  Enjoy!

On the twelfth day of Christmas my true love gave to me,
Twelve tubs of Purell,
Eleven raccoon roundworms,
Ten cats-a-scratching
Nine hungry hookworms,
Eight dogs-a-biting,
Seven cats with ringworm,
Six big fat dog ticks,
Five cats with fleas.
Four rats with cowpox.
Three tapeworms,
Two toxic turtles,
And a dog with lepto in its pee.

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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Tuesday, October 15, 2013

Towards sterilizing immunity against Leptospira with a DNA vaccine

In my previous post, I described the failure of researchers to come up with a conventional protein-based subunit vaccine that confers sterilizing immunity against leptospirosis.  What I mean by "conventional" subunit vaccine is a mixture of purified recombinant Leptospira protein with an adjuvant (either aluminum hydroxide or Freund's).  Although an antibody response was detected against most proteins tested, immunization failed to prevent kidney colonization in every case, including those animals that survived challenge with lethal strains of Leptosipra.  It's becoming clear that the leptospirosis vaccine field must move beyond simple formulations of protein plus adjuvant if sterilizing immunity is desired.

Several labs have explored more modern approaches to delivering leptospirosis vaccines.  Odir Dellagostin's group down in Brazil tested the efficacy of DNA vaccines in protecting hamsters against leptospirosis, as described in this paper in Clinical and Vaccine Immunology.  Forster and colleagues targeted the Leptospira interrogans LigA and LigB proteins, which are surface proteins that disrupt (or exploit) multiple host functions.

The investigators cloned various fragments of the lengthy ligA and ligB genes downstream of the human cytomegalovirus promoter of the commercial expression plasmid pTARGET.  They mixed the plasmid DNA with aluminum hydroxide adjuvant and injected the material into the muscle of the hind leg of hamsters.  The animals were given a booster with the same material 21 days later.  An IgG immune response was detected against four of the the five Lig protein fragments being tested (see figure below).

Figure 2 from Forster et al., 2013.  Sera were drawn before immunization and after the first and second immunizations with pTARGET-based lig plasmid DNA.  Purified recombinant protein encoded by each plasmid was used as antigen in ELISAs.  Left, middle, and right bar for each DNA: before immunization, after first immunization, and after second immunization, respectively.

21 days after the boost, the animals were challenged with a lethal strain of L. interrogans.  The survival curves are shown below.  Note that animals immunized with the vector alone (small filled circles) were all dead by day 11.

Figure 3 from Forster et al., 2013.

Among the lig gene fragments, the one expressing the "LigBrep" fragment was effective, protecting five of the eight animals in the group (62.5%) from death.  LigBrep comprises amino acid residues 1 through 628 of LigB, whose total length is 1891 residues.  Although the survival rate is nothing to get excited over, what distinguishes the LigBrep DNA vaccine from the conventional subunit vaccines tested in earlier studies is that the kidneys from 4 of the 5 survivors were culture negative, indicating that sterilizing immunity was achieved in 80% of the animals that survived infection.

Another notable outcome of the study was that protection was achieved even though the challenge strain and the vaccine's lig gene originated from different Leptospira serovars.  One of the problems with killed whole-cell vaccines is that they only protect against Leptospira serovars present in the vaccine formulation because they target LPS, whose structure varies among different serovars.  Leptospira proteins tend to be similar in amino acid sequence across different species and are therefore more attractive as vaccines.  (The "killed-whole leptospires" control plotted in the graph above was generated from the challenge strain).

So how does DNA vaccination induce sterilizing immunity against Leptospira?  As always, more studies are needed to explore this issue, but I will go ahead and speculate. DNA vaccines that are administered by standard injection stimulate a Th1-biased immune response.  Studies with cattle have suggested that vaccines must stimulate Th1 immunity to minimize kidney colonization by Leptospira (see this study, for example).  Moreover, an earlier study by Dellagostin's group demonstrated sterilizing immunity against L. interrogans in some animals immunized with a Mycobacterium bovis BCG strain that was engineered to express LipL32, the major outer membrane protein of L. interrogans.  BCG also stimulates Th1 immunity.

Why would a Th1 response be necessary for sterilizing immunity against Leptospira?  Th1 cytokines help steer B cells into producing an IgG isotype that is strongly recognized by Fc receptor on phagocytes.  Consequently, bacteria bound by these IgG molecules are engulfed by opsonophagocytosis.  During Leptospira infections, opsonophagocytosis clears spirochetes from the circulation during the antibody response, raising the possibility that opsonophagocytosis also leads to sterilizing immunity by vaccines that induce production of the "right" IgG.

Th1 cells are also necessary for cellular immunity, which enhances the killing functions of macrophages so that they can rid themselves of intracellular pathogens.  Leptospira is considered to be an extracellular pathogen.  Nevertheless, there may be a transient intracellular phase that is critical during infection.  Although intracellular Leptospira has not been observed in vivo, L. interrogans is known to survive and replicate in cultured macrophages.


Forster KM, Hartwig DD, Seixas FK, Bacelo KL, Amaral M, Hartleben CP, & Dellagostin OA (2013). A conserved region of leptospiral immunoglobulin-like A and B proteins as a DNA vaccine elicits a prophylactic immune response against leptospirosis. Clinical and Vaccine Immunology : CVI, 20 (5), 725-731 PMID: 23486420

Zuerner RL, Alt DP, Palmer MV, Thacker TC, & Olsen SC (2011). A Leptospira borgpetersenii serovar Hardjo vaccine induces a Th1 response, activates NK cells, and reduces renal colonization. Clinical and Vaccine Immunology : CVI, 18 (4), 684-91 PMID: 21288995

Seixas FK, da Silva EF, Hartwig DD, Cerqueira GM, Amaral M, Fagundes MQ, Dossa RG, & Dellagostin OA (2007). Recombinant Mycobacterium bovis BCG expressing the LipL32 antigen of Leptospira interrogans protects hamsters from challenge. Vaccine, 26 (1), 88-95 PMID: 18063449

Toma C, Okura N, Takayama C, & Suzuki T (2011). Characteristic features of intracellular pathogenic Leptospira in infected murine macrophages. Cellular microbiology, 13 (11), 1783-1192 PMID: 21819516

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