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!

Lyrics
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.


References

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

Related posts


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.

References

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


Related posts

Sunday, September 15, 2013

Is sterilizing immunity against Leptospira possible with protein subunit vaccines?

With complete bacterial genome sequences now available, "reverse vaccinology" can be conducted to identify proteins that can function as subunit vaccines.  The "gene first" approach of reverse vaccinology relies upon computer analysis of the genome sequence to identify encoded proteins with  features common to known surface-exposed and secreted bacterial proteins.  The selected genes can then be cloned and expressed as recombinant proteins.  The proteins, which may number in the hundreds, are then purified for vaccine testing in the animal model appropriate for the bacterial pathogen.  Reverse vaccinology has been employed successfully to find protective protein antigens against Neisseria meningitidis serogroup B, Streptococcus pneumoniae, group B Streptococcus, Bacillus anthracis, Porphyromonas gingivalis, and other bacterial pathogens (reviewed in this paper).

This approach sounds straightforward but in practice may not always lead to identification of effective subunit vaccines.  An important study from Ben Adler's group down in Monash University illustrates the challenges of finding a subunit vaccine that prevents chronic Leptospira infections.  They focused on serovar Hardjo, which causes chronic infections in cattle.  They selected 263 Hardjo genes that were predicted to encode surface-exposed, secreted, or lipid-modified proteins.  Among these they successfully cloned and expressed 223 genes as 238 protein antigens in E. coli.  Some genes were expressed as two or more fragments because of their large size.  210 of the 238 (88%) aggregated into inclusion bodies during expression and had to be kept dissolved in urea during their purification.  (Strangely, the urea was not removed by dialysis prior to immunization.)  The 238 purified proteins were mixed with an aluminum hydroxide adjuvant and injected into hamsters.  169 of the 238 proteins (71%) generated an antibody response, yet none succeeded in preventing colonization of the kidneys following challenge with a Hardjo strain.

It's been hard enough to find leptospiral proteins that protect hamsters from lethal disease when tested as vaccines (see this article for a review), yet Murray and colleagues sought proteins that protected against Leptospira colonization, a more difficult endeavor that has never been achieved with subunit vaccines.  The Hardjo strain they used easily colonizes the kidneys yet fails to produce any signs of disease in hamsters.  Although several studies have demonstrated that certain versions of the LigA and LigB proteins, when administered as vaccines, protect hamsters and mice from being killed by lethal strains of Leptospira, survivors are left with infected kidneys.  A vaccine that protects against disease or death but not infection may be adequate for humans, who eventually clear the spirochetes from their kidneys even following a natural infection (assuming the disease doesn't kill them).  However, vaccinated cattle infected with Hardjo may not be able to clear the spirochetes and will continue to shed infectious Leptospira into the environment, placing the entire herd and the workers handling them at risk of infection.  Hardjo infections generally don't cause signs of disease in cattle, but they can cause fetal death and drop in milk production in cows.

The choice of adjuvant and destruction of protective conformational epitopes by urea are possible reasons for failure to find a protective antigen.  On the other hand, perhaps a different method for delivery of protein antigens into animals should been considered.  Stay tuned.

Featured paper

Murray GL, Lo M, Bulach DM, Srikram A, Seemann T, Quinsey NS, Sermswan RW, Allen A, & Adler B (2013). Evaluation of 238 antigens of Leptospira borgpetersenii serovar Hardjo for protection against kidney colonisation. Vaccine, 31 (3), 495-499 PMID: 23176980

Helpful reviews

Dellagostin, O.A., Grassmann, A.A., Hartwig, D.D., Felix, S.R., da Silva, E.F., & McBride, A.J.A. (November 2011).  Recombinant vaccines against leptospirosis.  Human Vaccines 7(11):1215-1224. DOI: 10.4161/hv.7.11.17944

Serruto, D., Serino, L., Masignani, V., & Pizza, M. (May 26, 2009).  Genome-based approaches to develop vaccines against bacterial pathogens.  Vaccine 27(25-26):3245-3250.  DOI: 10.1016/j.vaccine.2009.01.072

Wednesday, March 13, 2013

Triggering OspC production in Borrelia burgdorferi during tick feeding: Is temperature the real signal?

The Ixodes tick, the vector of the Lyme disease spirochete, goes months without a meal.  During this time, the Borrelia burgdorferi spirochetes living in its midgut live quiet lives, sipping on the tick's antifreeze to sustain themselves. When the tick finally takes a blood meal from a warm-blooded victim, B. burgdorferi responds by producing a number of new proteins, some of which are needed for transmission to and infection of the mammalian host.  Among these proteins is the outer surface lipoprotein OspC, whose function involves capture of tick (see this post) and mammalian host proteins.  How does B. burgdorferi know when to start making these critical proteins?  The favored model has been that the the warmth of the blood entering the tick triggers B. burgdorferi to make these proteins.  It's been known for almost two decades that B. burgdorferi growing in culture medium produces miniscule amounts of OspC at low temperatures (23º-24ºC) and larger amounts at higher temperatures (32º-37ºC), as shown in the figure below from the classic 1995 report by Tom Schwan and colleagues.

Figure 4 from Schwan et al., 1995B. burgdorferi incubated at 24ºC (lanes 2 and 6), transferred from 24ºC to 37ºC (lanes 3 and 7), incubated at 37ºC (lanes 4 and 8), or transferred from 37ºC to 24ºC (lanes 5 and 9).  Panel A, SDS-PAGE gel stained for total proteins with Coomassie  brilliant blue.  Arrow marks location of OspC.  Panel B, Western blot with flagellin antibody (Fla) and OspC antibody.
As reasonable as this model sounds, findings from a recent paper from Brian Stevenson's group (Jutras et al., 2012) challenge the model.  Although not emphasized in earlier papers, the authors noted that B. burgdorferi multiplies much more quickly at higher temperatures.  In their hands, B. burgdorferi proliferated with a doubling time of 32 hours at 23ºC and 12 hours at 34ºC.  As expected, their Western blots showed that more OspC was produced by the spirochetes growing at the higher temperature.  Members of the Erp family of surface proteins, whose levels also rise during tick feeding, were produced at higher levels at the higher temperature as well, as shown in earlier studies.  The investigators devised an experiment to test whether B. burgdorferi could tie OspC and Erp expression to its growth rate instead of temperature.

The standard culture medium for Borrelia is BSK-II with 6% rabbit serum, a complex nutrient-rich concoction.  They made two new formulations of the culture medium to slow the growth rate:  (1) quarter strength BSK-II with the rabbit serum concentration remaining at 6%; (2) full-strength BSK-II with the rabbit serum concentration reduced to 1.2%.  Medium #1 slowed the doubling time at 34ºC to 40 hours, and medium #2 reduced it to 32 hours.  Western blots of the spirochetes harvested from both cultures revealed low levels of the OspC and Erp proteins.  When these spirochetes were inoculated into the standard culture medium (BSKII/6% rabbit serum) and incubated at 34ºC, high levels of the proteins were again detected.  Therefore, B. burgdorferi is capable of adjusting OspC and Erp expression by monitoring its growth rate, even if the surrounding temperature does not change.

The final experiment from the study demonstrates that not even growth rate is the direct signal.  The authors froze B. burgdorferi at -80ºC for at least a month and then inoculated the bacteria into standard culture medium for incubation at 23ºC.  As a control, bacteria being maintained at 34ºC were also transferred to standard culture for incubation at 23ºC.  Both cultures grew with the same doubling time.  Nevertheless, the spirochetes that were revived from the frozen state produced more OspC and Erp proteins that those that were initially maintained at 34ºC.

So what's the real cue?  Going back to the natural life cycle of B. burgdorferi, the spirochetes living in the unfed tick's midgut do not really grow or divide.  The metabolism of B. burgdorferi is slowed by the nutrient-poor conditions in the tick's midgut.  When the tick finally takes a blood meal, the surge of nutrients entering the tick signals B. burgdorferi to rev up its metabolism, triggering production of OspC.  This model would explain why the frozen spirochetes, whose metabolism was undoubtedly slowed, were able to produce large amounts of OspC and Erp proteins when inoculated into standard culture medium at 23ºC, the temperature usually associated with diminished production of the proteins.  The challenge will be to figure out how B. burgdorferi is sensing its metabolic state at the molecular level.

References

Jutras, B.L., Chenail, A.M., & Stevenson, B. (2012). Changes in bacterial growth rate govern expression of the Borrelia burgdorferi OspC and Erp infection-associated surface proteins. Journal of Bacteriology, 195 (4), 757-764 DOI: 10.1128/JB.01956-12

Schwan, T.G., Piesman, J., Golde, W.T., Dolan, M.C., & Rosa, P.A. (1995). Induction of an outer surface protein on Borrelia burgdorferi during tick feeding. Proceedings of the National Academy of Sciences, 92 (7), 2909-2913 DOI: 10.1073/pnas.92.7.2909

Related posts

Tuesday, February 19, 2013

Is the major outer membrane lipoprotein LipL32 really exposed on the surface of Leptospira?

Here's a study that may come as a surprise to those in the leptospirosis field.  The outer membrane lipoprotein LipL32 is believed to be the dominant protein on the cell surface of pathogenic species of Leptospira.  However, according to a new PLoS One article written by Pinne and Haake at UCLA, LipL32 may not be present on the surface at all.  This is an important issue to get right because the function proposed for LipL32, attachment to the extracellular matrix during infection, assumes that the lipoprotein is exposed on the surface of the spirochete.  More importantly, a number of research groups have already committed a lot of time and resources towards generating LipL32-based vaccines, which in current formulations confer (at best) weak protection against leptospirosis in rodent models (see this review for a critical analysis of the vaccine studies).

Pinne and Haake assessed surface exposure of LipL32 by two methods.  The first involved adding proteinase K to suspensions of Leptospira to digest proteins exposed on the surface of the spirochete.  When they did this, they saw that the known surface-exposed proteins OmpL37 and OmpL47 were degraded.  On the other hand, LipL32 didn't break down at all unless the spirochetes were first lysed by boiling them in a detergent (see Western blot below).

Figure 1B from Pinne and Haake (2013).  Increasing concentrations of proteinase K (up to 150 ug/ml) were added to suspensions (first five lanes) or lysates (last five lanes) of L. interrogans.  Following incubation, LipL32 was examined in a Western blot.  Source.

They next added LipL32 antibodies to Leptospira to see if they bound to the surface of the spirochete.  They did not, providing additional evidence that LipL32 was not exposed on the cell surface.  The authors tested LipL32 antibodies from different sources in an attempt to rule out the  possibility that failure of antibody binding was due to the surface-exposed portion of LipL32 not being antigenic.  LipL32 antiserum raised in rabbits, monoclonal LipL32 antibodies raised in mice, and LipL32 antibodies purified from the sera of leptospirosis patients all failed to bind the surface of Leptospira unless the outer membrane was chemically (with methanol or EDTA) or physically disrupted.  Note that antibodies raised against OmpL54, a known suface-exposed protein, reacted strongly with intact Leptospira (last pair of images below).

Figure 3A from Pinne and Haake (2013).  Bound antibody was detected with a secondary fluorescent antibody (green).  The spirochetes were also stained with DAPI, a penetrating dye that stains DNA (blue).  Left column, intact Leptospira, right column, methanol-treated Leptospira. Source.

In light of these results, the authors took another look at the 2005 study by Cullen and coauthors, who claimed LipL32 was surface exposed.  In contrast to Pinne and Haake, Cullen and colleagues detected binding of LipL32-specific antibodies to intact Leptospira in three different assays.  However, Pinne and Haake point out that antibody binding in their assays was extremely weak when  the abundance of LipL32 is considered.  The most striking example was the immunoelectron microscopy image of Leptospira treated with gold-labeled LipL32 antibody (see next image).  Yes, the surface ended up labeled, with a mean of 10.8 gold particles per spirochete cell.  However, we now know that there are 38,000 copies of LipL32 in each bacterial cell, making LipL32 the most abundant protein of L. interrogans (see this blog post about the Leptospira protein census).  If LipL32 were really surface exposed, the surface of the spirochete should have been packed with gold particles.

Figure 5 from Cullen et al. (2005).

Cullen and colleagues also mixed suspensions of Leptospira with a biotin probe that reacts with primary amines (mostly on lysine side chains).  The probe should have reacted solely with surface-exposed proteins since it's unable to penetrate the outer lipid bilayer and is assumed to be too large to diffuse through outer membrane porins.  Biotinylated proteins were separated by two-dimensional electrophoresis and identified by mass spectrometry (see next figure).  Although LipL32 was one of the few proteins labeled with biotin, it's hard to make a firm conclusion about its surface exposure because proteins known to be located underneath the outer membrane, FlaB1 (a flagellar protein) and GroEL (a cytoplasmic heat shock protein), were also labeled with biotin. It's possible that LipL32 was labeled despite being located underneath the surface because the membrane was damaged while the spirochetes were being harvested for the experiment.

Modified from Figure 2 of Cullen et al., 2005.  Biotinylated proteins were separated by two-dimnesional electrophoresis.  Spots were removed and analyzed by mass spectrometry to identify proteins.  Multiple spots for each protein in the result of members of the population of each protein reacting with different numbers of biotin molecules.  "LipL32.16" was generated by proteolysis of LipL32.

Based on the intense labeling of LipL32 with biotin, Cullen and coauthors declared LipL32 the most abundant protein on the cell surface.  They speculated that LipL32 is poorly accessible to large molecules such as antibodies and proteases (which in their study failed to digest any protein when added to intact Leptospira) because the LPS side chains act as a "rainforest canopy" that can be penetrated only by smaller molecules such as biotin. This is a reasonable supposition because LipL32 is up to 60Å in length, whereas the distance between the outer membrane and the surface of the LPS layer is 92Å, according to a cryoelectron microscopy study of L. interrogans.  On the other hand, Pinne and Haake concluded that LipL32 is entirely or almost entirely subsurface since their assays failed to detect even a hint of the lipoprotein on the surface of Leptospira.  They maintain that the reactivity of surface probes with LipL32 observed by Cullen and colleagues was an artifact generated by the presence of damaged spirochetes in their assays and the massive copy number of LipL32. 

The results from Pinne and Haake's study do not rule out the "rainforest canopy" model since they did not test smaller surface probes that could penetrate into the LPS side chain layer.  Additional studies are needed to pin down the location of LipL32 relative to the surface of Leptospira.

References

Pinne, M., & Haake, D.A. (2013). LipL32 is a subsurface lipoprotein of Leptospira interrogans: Presentation of new data and reevaluation of previous studies. PLoS ONE, 8 (1) DOI: 10.1371/journal.pone.0051025

Cullen, P.A., Xu, X., Matsunaga, J., Sanchez, Y., Ko, A.I., Haake, D.A., & Adler, B. (2005). Surfaceome of Leptospira spp. Infection and Immunity, 73 (8), 4853-4863 DOI: 10.1128/IAI.73.8.4853-4863.2005

Related posts

Monday, February 4, 2013

An autoantigen targeted during Lyme arthritis

Infection by the spirochete Borrelia burgdorferi, if left untreated, can lead to a form of Lyme disease called Lyme arthritis.  About 10% of Lyme arthritis patients end up with a chronic form that doesn't go away with antibiotic treatment.  Allen Steere's group has long suspected that the antibiotic-refractory form of Lyme arthritis involves an autoimmune process.  This notion seems reasonable since those with antibiotic-refractory Lyme arthritis tend to have certain forms of the HLA-DR gene that are also common among those afflicted with the autoimmune disease rheumatoid arthritis.

Several groups have been searching for autoantigens (self antigens) that could drive the joint inflammation seen in Lyme arthritis patients.  Several candidate protein autoantigens were identified based on their short sequence similarities (molecular mimicry) to a T-cell or antibody epitopes in the B. burgdorferi OspA protein, which is targeted by the immune system in many Lyme arthritis patients, especially those with the antibiotic-refractory form.  However, further studies demonstrated that none of these autoantigens were likely to stimulate a sufficiently robust T-cell or antibody response that could account for the prolonged joint swelling experienced by patients with antibiotic-refractory Lyme disease (see this excellent review article for the complete story).  Therefore, an additional approach is needed to identify additional autoantigen candidates, an approach that does not assume that molecular mimicry underlies antibiotic-refractory Lyme arthritis.

An unbiased approach for finding autoantigens is to gather all of the different self-peptides being displayed by the HLA-DR molecules in the synovial tissue of the swollen joint and then figure out which of these peptides are capable of stimulating T cells.  At one time this approach wasn't possible since the individual peptides presented by HLA-DR molecules are found in such tiny amounts in human tissues, but the sensitivity of today's liquid chromatography/tandem mass spectrometry systems have improved to the point where many of the peptides can now be sorted and sequenced.

Steere's new study, which appeared in January's print issue of Arthritis and Rheumatism, was conducted in collaboration with Catherine Costello's group in Boston University.  The study was a follow-up to an earlier one published two years ago.  The authors extracted the inflamed synovial tissue from the swollen knee of a 12 year old boy suffering from antibiotic-refractory Lyme arthritis (see picture below).  He had gone through three months of antibiotic therapy a year prior to the procedure.  The tissue was culture and PCR negative.  When the patient's HLA-DR genes were examined, he turned out to have a copy of the DRB1*0101 allele, one of the HLA-DRB gene variants that places individuals at a higher risk for antibiotic-refractory Lyme arthritis.

Figure 1A from Drouin et al., 2013

The boy's synovial tissue was ground up, and HLA-DR-specific antibodies were used to capture the HLA-DR molecules with their bound peptides.  The peptides were then analyzed by liquid chromatography/tandem mass spectrometry.  The authors identified 120 different self-peptides from this analysis.  When each peptide was chemically synthesized and mixed with the boy's blood mononuclear cells, one peptide turned out to stimulate proliferation of his T cells.  This peptide came from a human protein called endothelial cell growth factor, or ECGF.

What's the function of ECGF?  The protein stimulates angiogenesis, the sprouting of new blood vessels from pre-existing ones.  Angiogenesis is a general feature of inflammatory arthritis, including Lyme arthritis and rheumatoid arthritis.

The authors went on to examine the T- and B-cell responses to ECGF in other Lyme arthritis patients.  The T-cell response was determined by measuring the amount of interferon-γ secreted by the patients' blood mononuclear cells upon exposure to ECGF in vitro.  In antibiotic-refractory patients, the T-cell response was observed in 38% (14/37) subjects against 30% (8/27) among Lyme arthritis patients who responded to antibiotics. The difference between the two groups was not statistically significant.  The B-cell (antibody) response was examined by ELISA in a larger group of patients.  17% (19/109) of antibiotic-refractory patients and 8% (6/77) of antibiotic-responsive patients had an IgG antibody response against ECGF that was higher than among healthy controls, yet the difference between the antibiotic-refractory and -responsive groups again was not statistically significant (P = 0.09).  So a link between an autoimmune response to ECGF and antibiotic-refractory arthritis was not clear-cut.  However, in support of a link, the authors mentioned that almost all of the Lyme arthritis patients with a T-cell response to ECGF (20/21, 98%) had one of the HLA-DR alleles known to be a risk factor for antibiotic-refractory arthritis.

The authors also looked at the levels of ECGF in the swollen joints of Lyme arthritis patients.  Those with antibiotic-refractory Lyme arthritis had much higher levels of ECGF in their joint fluid (mean 448 ng/ml, 37 subjects) than those whose arthritis responded to antibiotic treatment (mean 154 ng/ml, 19 subjects, P < 0.0001)

Further evidence for a link between an immune response to ECGF and chronic Lyme arthritis came from a group of untreated Lyme disease patients who were followed in the late 1970s, before the cause of Lyme disease was known.  Sera from sequential bleeds were still available from many of these patients.  If an autoimmune process involving ECGF was responsible for the disease, then the immune response to the autoantigen should have appeared before the disease symptoms.  This turned out to be the case.  Six of the seven Lyme arthritis patients who had antibodies against ECGF developed the antibody response before their joints swelled up.  The duration of the arthritis attack was longer in Lyme arthritis patients with an immune response to ECGF, lasting a median of 67 weeks in the seven patients with an ECGF antibody response and only 17 weeks in the 20 Lyme arthritis lacking the response (P = 0.004).

Steere's paper proposes that the immune response to ECGF leads to a persisting, autoimmune form of arthritis in those who have a high level of ECGF in their joint fluid.  In those patients, T cells that recognize ECGF would be activated more easily because of the high levels of ECGF available for phagocytes to engulf, process, and display to the T cells.  These events would lead to a chronic form of arthritis that would persist even when the spirochetes were cleared from the joints by the immune system or antibiotics.  These patients also have a lot of ECGF in their synovial tissue.  Antibody against ECGF could bind to the tissue and trigger attack by complement, contributing to the tissue damage.

Molecular mimicry doesn't appear to be involved in triggering an immune response to ECGF.  The authors were unable to identify any B. burgdorferi proteins that could cross-react with ECGF.

The immune response to ECGF can't be the whole story since most patients with antibiotic-refractory Lyme arthritis don't generate a T-cell or antibody response to the protein.  An autoimmune process in these other patients may involve other self-antigens waiting to be discovered.  Other host and spirochete factors also influence the course of Lyme arthritis (see this post, which gives the spirochete's point of view).


References

Drouin, E.E., Seward, R.J., Strle, K., McHugh, G., Katchar, K., Londoño, D., Yao, C., Costello, C.E., & Steere, A.C. (2013). A novel human autoantigen, endothelial cell growth factor, is a target of T and B cell responses in patients with Lyme disease. Arthritis & Rheumatism, 65 (1), 186-196 DOI: 10.1002/art.37732

Seward, R.J., Drouin, E.E., Steere, A.C., & Costello, C.E. (2010). Peptides presented by HLA-DR molecules in synovia of patients with rheumatoid arthritis or antibiotic-refractory Lyme arthritis. Molecular & Cellular Proteomics, 10 (3) DOI: 10.1074/mcp.M110.002477

A helpful review

Steere, A.C., Drouin, E.E., & Glickstein, L.J. (2011). Relationship between immunity to Borrelia burgdorferi Outer-surface protein A (OspA) and Lyme arthritis. Clinical Infectious Diseases, 52 (Supplement 3) DOI: 10.1093/cid/ciq117

Related post

Monday, January 14, 2013

LigB of Leptospira interrogans: Avoiding or exploiting complement?

LigB has turned out to be a versatile surface protein for Leptospira interrogans.  The protein is one of several that the spirochete uses to stick to the extracellular matrix, a critical step in colonizing host tissues.  In addition, LigB's ability to bind fibrinogen may help L. interrogans spread within the host by slowing clot formation.  According to separate studies from the U.S. and Brazil published last year, LigB also helps L. interrogans fend off attack by the host complement system.

What's the evidence that LigB protects Leptospira from complement?  L. interrogans, like many pathogens, survives even when complement is present.  Its resistance to complement is reflected by its ability to survive in human serum, which contains all the complement components necessary to assemble the deadly membrane attack complex within exposed microbial membranes.  On the other hand, the nonpathogen Leptospira biflexa, which lacks the ligB gene, is rapidly killed by human serum.  The U.S. study demonstrated that transformation of L. biflexa with a plasmid expressing LigB allowed the nonpathogen to survive in human serum diluted to 5%, a concentration that easily killed L. biflexa harboring a similar plasmid lacking ligB.

How exactly does LigB protect Leptospira from the onslaught of complement?  A defensive strategy deployed by many pathogens is to seize host complement regulators, which are present to prevent complement activation on host cells.  The Brazilian study showed that LigB grabs several complement regulators that diminish the levels of the key complement proteins C3b and C4b on the bacterial surface.  Two of these regulators are the factor H protein and the C4-binding protein (C4BP).  Both complement regulators break apart the C3 convertases, the enzyme complexes that generate C3b from C3.  Factor H and C4BP also serve as cofactors for the protease factor I, which cleaves C3b and C4b into smaller pieces to prevent their assembly into the C5 convertase.  The C5 convertase is what triggers assembly of the membrane attack complex.  As expected, the investigators found that the breakdown of C3b and C4b by factor I in the presence of its complement regulators was accelerated when LigB was present.

LigB is not the only Leptospira protein that captures complement regulators.  The proteins LenA (originally called LfhA) and LcpA bind factor H and C4BP, respectively.  The importance of factor H in protecting L. interrogans can be seen in the bar graph below.  Survival of L. interrogans was poor in serum lacking factor H.  Addition of factor H to the serum up to the concentration found in blood (500 μg/ml) enhanced survival of the spirochete.

Figure 1 from Castiblanco-Valencia et al. (2012).  Survival of L. interrogans in factor H-depleted serum with 500 μg/ml factor H (FH) is set to 100%.

Surprisingly, the U.S. study revealed that a small segment of LigB grabbed C3b and C4b.  Why would L. interrogans risk capturing complement proteins while simultaneously collecting complement regulators that inactivate those same proteins?  Other pathogens do just fine capturing complement regulators without actively grabbing complement components.  It's possible that C3b and C4b are inactivated more effectively by the complement regulators when all components are bound to LigB.

There's another possibility that should be considered.  What went unmentioned in both papers is that C3b and its cleavage products, which may remain attached to the bacterial surface, are opsonins recognized by phagocytes aiming to grab and engulf microbial intruders.  However, several intracellular pathogens use complement receptors as an entry point to invade macrophages.  L. interrogans has been shown to survive within cultured macrophages and even remained intact within macrophages in a zebrafish model, as I've explained in another post.  Is it possible that L. interrogans uses C3b to grab and invade macrophages?

Featured papers

Castiblanco-Valencia MM, Fraga TR, da Silva LB, Monaris D, Abreu PAE, Strobel S, Jozsi M, Isaac L, & Barbosa AS (2012). Leptospiral immunoglobulin-like proteins interact with human complement regulators factor H, FHL-1, FHR-1, and C4BP. Journal of Infectious Diseases, 205 (6), 995-1004 DOI: 10.1093/infdis/jir875

Choy H (2012). Multiple activities of LigB potentiate virulence of Leptospira interrogans: Inhibition of alternative and classical pathways of complement. PLoS ONE, 7 (7) DOI: 10.1371/journal.pone.0041566

Other helpful papers

Verma A, Hellwage J, Artiushin S, Zipfel PF, Kraiczy P, Timoney JF, and Stevenson B (March 2006).  LfhA, a novel factor H-binding protein of Leptospira interrogansInfection and Immunity 74(5):2659-2666.  Link

Barbosa AS, Monaris D, Silva LB, Morais ZM,  Vasconcellos SA,  Cianciarullo AM, Isaac L, and Abreu PAE (July 2010).  Functional characterization of LcpA, a surface-exposed protein of Leptospira spp. that binds the human complement regulator C4BP.  Infection and Immunity 78(7):3207-3216.  DOI: 10.1128/IAI.00279-1010.1128/IAI.00279-10

Related posts

Thursday, January 3, 2013

Spirochete research: 2012 in review

Here are some of my favorite spirochete papers from 2012.  Direct access to all research articles (some behind a paywall) is provided via the DOI links.  Where present, the links above the citations lead to my blog posts about the studies.

PATHOGENESIS

Two distinct regions of the Borrelia burgdorferi BBK32 lipoprotein sequentially mediate binding to the vessel wall in vivo during escape of the spirochete from the bloodstream.
  • Moriarty TJ, Shi , Lin Y-P, Ebady R, Zhou H, Odisho T, Hardy P-O, Salman-Dilgimen A, Wu J, Weening EH, Skare JT, Kubes P, Leong J, and Chaconas G (December 2012).  Vascular binding of a pathogen under shear force through mechanistically distinct sequential interactions with host macromolecules.  Molecular Microbiology 86(5):1116-1131.  DOI: 10.1111/mmi.12045

The Leptospira interrogans LigB protein protects the spirochete from complement by capturing complement regulatory proteins.
  • Castiblanco-Valencia MM, Fraga TR, da Silva LB, Monaris D, Abreu PAE, Strobel S, Jozsi M, Isaac L, and Barbosa AS (March 15, 2012).  Leptospiral immunoglobulin-like proteins interact with human complement regulators factor H, FHL-1, FHR-1, and C4BP.  The Journal of Infectious Diseases 205(6):995-1004.  DOI: 10.1093/infdis/jir875
  • Choy HA (July 2012).  Multiple activities of LigB potentiate virulence of Leptospira interrogans: inhibition of alternative and classical pathways of complement.  PLoS One 7(7):e41566. DOI: 10.1371/journal.pone.0041566

A B. burgdorferi lipase with hemolytic activity in vitro:
  • Shaw DK, Hyde JA, and Skare JT (January 2012).  The BB0646 protein demonstrates lipase and haemolytic activity associated with Borrelia burgdorferi, the aetiological agent of Lyme disease.  Molecular Microbiology 83(2):319-334.  DOI: 10.1111/j.1365-2958.2011.07932.x

TRANSMISSION

Borrelia burgdorferi needs the alternative sigma factor RpoS to flee from the tick's midgut
  • Dunham-Ems SM, Caimano MJ, Eggers CH, and Radolf JD (February 2012).  Borrelia burgdorferi requires the alternative sigma factor RpoS for Dissemination within the vector during tick-to-mammal transmission.  PLoS Pathogens 8(2):e1002532.  DOI: 10.1371/journal.ppat.1002532

MOTILITY

Video microscopy of B. burgdorferi swimming around in gelatin and mouse tissue:
  • Harman MW, Dunham-Ems SM, Caimano MJ, Belperron AA, Bockenstedt LK, Fu HC, Radolf JD, and Wolgemuth CW (February 21, 2012).  The heterogeneous motility of the Lyme disease spirochete in gelatin mimics dissemination through tissue.  Proceedings of the National Academy of Sciences USA 109(8):3059-3064.  DOI: 10.1073/pnas.1114362109

L. interrogans sheath protein homologs that are not needed for flagellar sheath formation:
  • Lambert A, Picardeau M, Haake DA, Sermswan RW, Srikram A, Adler B, and Murray GA (June 2012).  FlaA proteins in Leptospira interrogans are essential for motility and virulence but are not required for formation of the flagellum sheath.  Infection and Immunity 80(6):2019-2025.  DOI: 10.1128/IAI.00131-12

ULTRASTRUCTURE

A close look at the ultrastructure of Leptospira without the artifacts generated by conventional electron microscopy:
  • Raddi G, Morado DR, Yan J, Haake DA, Yang XF, and Liu J (March 2012).  Three-dimensional structures of pathogenic and saprophytic Leptospira species revealed by cryo-electron tomography.  Journal of Bacteriology 194(6):1299-1306.  DOI: 10.1128/JB.06474-11

METAL TOXICITY

B. burgdorferi BicA, a protein that protects the spirochete from the toxic effects of copper and iron:
  • Wang P, Lutton A, Olesik J, Vali H, and Li X (December 2012).  A novel iron- and copper-binding protein in the Lyme disease spirochaete.  Molecular Microbiology 86(6):1441-1451.  DOI: 10.1111/mmi.12068

ANTIBIOTIC THERAPY

Inflammatory spirochete debris left behind following antibiotic treatment for Lyme disease
  • Bockenstedt LK, Gonzalez DG, Haberman AM, and Belperron AA (July 2, 2012).  Spirochete antigens persist near cartilage after murine Lyme borreliosis therapy.  The Journal of Clinical Investigation 122(7):2652-2660.  DOI: 10.1172/JCI58813

A critical analysis of a study that demonstrated persistence of B. burgdorferi in infected rhesus monkeys that were treated with antibiotics:
  • Wormser GP, Baker PJ, O'Connell S, Pachner AR, Schwartz I, and Shapiro ED (July 2012).  Critical analysis of treatment trials of rhesus macaques infected with Borrelia burgdorferi reveals important flaws in experimental design.  Vector-borne and Zoonotic Diseases 12(7):535-538.  DOI: 10.1089/vbz.2012.1012
  • Embers ME, Barthold SW, Borda JT, Bowers L, Doyle L, Hodzic E, Jacobs MB, Hasenkampf NR, Martin DS, Narasimhan S, Phillippi-Falkenstein KM, Purcell JE, Ratterree MS, and Philipp MT (January 2012).  Persistence of Borrelia burgdorferi in rhesus macaques following antibiotic treatment of disseminated infection.  PLoS One 7(1):e29914.  DOI: 10.1371/journal.pone.0029914

A tale of two more studies: topical antibiotics applied to tick bites to prevent Lyme disease
  • Wormser GP, Daniels TJ, Bittker S, Cooper D, Wang G, and Pavia CS (March 15, 2012).  Failure of topical antibiotics to prevent disseminated Borrelia burgdorferi infection following a tick bite in C3H/HeJ mice.  The Journal of Infectious Diseases 205(6):991-994.  DOI: 10.1093/infdis/jir382

DIAGNOSTICS

Not so golden?  Microscopic agglutination test for diagnosis of leptospirosis
  • Limmathurotsakul D, Turner EL, Wuthiekanun V, Thaipadungpanit J, Suputtamongkol Y, Chierakul W, Smythe LD, Day NPJ, Cooper B, and Peacock SJ (August 1, 2012).  Fool’s gold: Why imperfect reference tests are undermining the evaluation of novel diagnostics: A reevaluation of 5 diagnostic tests for leptospirosis.  Clinical Infectious Diseases 55(3):322-331.  DOI: 10.1093/cid/cis403

ECOLOGY

Do nonspiral spirochetes help clean our environment?
  • Caro-Quintero A, Ritalahti KM, Cusick KD, Loffler FE, and Konstandtinidis KT (May/June 2012).  The chimeric genome of Sphaerochaeta: Nonspiral spirochetes that break with the prevalent dogma in spirochete biology.  mBio 3(3):e00025-12.  DOI: 10.1128/mBio.00025-12
  • Ritalahti KM, Justicia-Leon SD, Cusick KD, Ramos-Hernandez N, Rubin M, Dornbush J, and Loffler FE (January 2012).  Sphaerochaeta globosa gen. nov., sp. nov. and Sphaerochaeta pleomorpha sp. nov., free-living, spherical spirochetes.  International Journal of Systematic and Evolutionary Microbiology 62(Pt 1):210-216.  DOI: 10.1099/ijs.0.023986-0

 

BIOFILM

Biofilms of the Lyme disease spirochete
  • Sapi E, Bastian SL, Mpoy CM, Scott S, Rattelle A, Pabbati N, Poruri A, Burugu D, Theophilus PAS, Pham TV, Data A, Dhaliwal NK, MacDonald A, Rossi MJ, Sinha SK, and Luecke DF (October 2012).  Characterization of biofilm formation by Borrelia burgdorferi in vitroPLoS One 7(10):e48277.  DOI: 10.1371/journal.pone.0048277

 

HISTORY

Looking for the syphilis spirochete in ancient bones
  • Montiel R, Solorzano E, Diaz N,  Alvarez-Sandoval BA, Gonzalez-Ruiz M, Canadas MP, Simoes N, Isidro A, and Malgosa A (May 2012).  Neonate human remains: A window of opportunity to the molecular analysis of syphilis.  PLoS One 7(5):e36371.  DOI: 10.1371/journal.pone.0036371

Presenting flawed studies directly to the public to bypass the scientific peer-review process:
  • Armelagos GJ, Zuckerman MK, and Harper KN (March 2012).  The science behind pre-Columbian evidence of syphilis in Europe: research by documentary.  Evolutionary Anthropology 21(2):50-57.  DOI: 10.1002/evan.20340

REVIEWS

Here are two excellent review articles that appeared during the past year:
  • Radolf JD, Caimano MJ, Stevenson B, and Hu LT (February 2012).  Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes.  Nature Reviews Microbiology 10(2):87-99.  DOI: 10.1038/nrmicro2714
  • Charon NW, Cockburn A, Li C, Liu J, Miller KA, Miller MR, Motaleb MA, and Wolgemuth CW (2012).  The unique paradigm of spirochete motility and chemotaxis.  Annual Reviews of Microbiology 66:349-370.  DOI: 10.1146/annurev-micro-092611-150145