Sunday, September 6, 2015

Do Lyme disease spirochetes produce a toxin?

According to the current view of Lyme disease pathogenesis, tissue damage is caused by the inflammatory response to the spirochetes.  Borrelia species do not produce toxins that injure the host directly.  A new study published in BMC Microbiology may force us to modify our view.

The study shows that some Borrelia strains carry a set of genes with the potential to generate a peptide resembling streptolysin S (SLS), a potent toxin produced by the pathogen Streptococcus pyogenes.  The enzymes that produce SLS in S. pyogenes are expressed from a cluster of genes surrounding sagA, a tiny gene encoding the SLS precursor.  The peptide produced from sagA is nontoxic; it has to undergo several alterations to its structure to become toxic.  A critical modification is carried out by the SagBCD protein complex, which converts the side chains of cysteine, serine, and threonine into ring structures.
Figure 2 from Molloy et al., 2011

Other genes surrounding sagA encode a peptidase that is thought to trim the leader peptide from the amino terminus of the SLS precursor and an ABC transporter that may be responsible for expelling SLS from the cytoplasm.

Figure 1A from Molloy et al., 2011

SLS targets neutrophils and possibly other immune cells during S. pyogenes infection.  SLS-like toxins are also produced by other Gram-positive pathogens, including Staphylococcus aureus, Listeria monocytogenes and Clostridium botulinum.

The investigators mined the genomes of other bacteria in search for genes encoding the machinery that generates SLS-like toxins.  They found SLS-like gene clusters in various Firmicutes and Actinobacteria, both Gram-positive groups of bacteria.

The researchers also found the gene cluster in the genomes of Borrelia afzelii strain PKo, Borrelia valaisiana strain VS116, and Borrelia spielmanii strain A14S.  B. afzelii is a major cause of Lyme disease in Europe and Asia.  B. valaisiana and B. spielmanii are responsible for occasional cases of Lyme disease.

Figure 4 from Molloy et al., 2015.  Top: organization of SLS-like gene cluster in S. pyogenes and three Borrelia strains. Bottom: sequence of the SLS precursor (SagA) and the borrelial SLS-like precursors.

They also used PCR to screen the DNA of 140 patient and tick isolates of Lyme Borrelia for the genes encoding the SLS-like biosynthetic machinery.  Most of the isolates were obtained from Europe and the U.S., with a few coming from Asia.  Design of the PCR primers was based on the sequence of the B. valaisiana bvalB, bvalC, and bvalD genes, which encode homologs of the S. pyogenes sagB, sagC, and sagD gene products.  Most of the B. garinii, B. afzelii, B. valaisiana, B. spielmanii, and B. lusitaniae isolates that were examined tested positive.  On the other hand, none of the 22 isolates of B. burgdorferi or 13 isolates of B. bavariensis were PCR positive.  These results indicate that SLS-like sequences are widespread among Lyme disease spirochetes (though not in B. burgdorferi).

The next step was to show that the SLS-like borrelial gene actually encoded a peptide that damages mammalian cells.  A simple assay based on the ability of many toxins to rupture (hemolyze) red blood cell in vitro is available.  Hemolysis is measured easily by mixing the toxin with sheep red blood cells.  Hemoglobin released from the ruptured cells is quantified with a spectrophotometer.

They decided to test the SLS-like peptide encoded by B. valaisiana, BvalA, for hemolytic activity.  The researchers succeeded in expressing and purifying a recombinant form of BvalA.  Not surprisingly,  BvalA was not hemolytic because its amino acid side chains had to be converted into ring structures necessary for the peptide to injure red blood cells.  They wanted to mix BvalA with the BvalBCD protein complex so that the peptide would be modified, but they could not generate the protein complex.  Instead, they used the SagBCD complex from S. pyogenes to modify the BvalA peptide.  When they did this, they finally observed hemolytic activity.

Red blood cells are unlikely to be a major target of borrelial SLS-like peptides during infection.  So what is the real target?  More studies are needed to answer this question, but we should consider the possibility that the toxin has nothing to do with Lyme disease.  Instead, it may help the spirochete to survive during its residence within the tick vector.  A number of nonpathogenic bacteria carry gene clusters distantly related to the ones that produce SLS.  Several peptide toxins produced by these bacteria are known to kill competing microbes.  Like us humans, ticks have a microbiome inhabiting their gut.  Some Lyme spirochetes may need to secrete the toxin to ward off their microbial neighbors.


Molloy EM, Casjens SR, Cox CL, Maxson T, Ethridge NA, Margos G, Fingerle V, & Mitchell DA (2015). Identification of the minimal cytolytic unit for streptolysin S and an expansion of the toxin family. BMC Microbiology, 15 PMID: 26204951

Molloy EM, Cotter PD, Hill C, Mitchell DA, & Ross RP (2011). Streptolysin S-like virulence factors: the continuing sagA. Nature Reviews Microbiology, 9 (9), 670-81 PMID: 21822292

Monday, August 17, 2015

A biosignature of early Lyme disease

Laboratory testing for Lyme disease involves two-tier antibody testing with sera from patients suspected of having the disease.  The first step is usually an ELISA with a cell lysate of Borrelia burgdorferi as antigen.  If the ELISA results are positive or borderline, a Western blot is done to confirm that the patient has Lyme disease.  Direct detection of Borrelia burgdorferi by culture would be the preferred laboratory test, but it takes too long for the spirochete to grow.  Culture of patient specimens is done only for research studies.

Source: CDC
In general, the problem with antibody testing for infectious diseases is that it takes time for the immune system to generate antibody against the pathogen.  Therefore, patients in the early stages of infection may test negative.  False-negative tests may delay appropriate treatment until the illness worsens.  For these reasons, scientists have been trying to come up with better laboratory tests for infections whose diagnosis relies on detecting antibody against the infectious agent.

One new approach being developed for a few pathogens involves measuring the amounts of each of the thousands of small molecules found in the sera of infected patients.  This is done by liquid chromatography/mass spectrometry (LC-MS), which accurately and precisely measures the size of small molecules, even in complex substances like serum.  The assumption is that the composition of small molecules (the so-called "metabolome") starts to change in a predictable manner as soon as someone is infected.  The metabolome changes because tissues react to the pathogen by generating inflammatory molecules that leak into the bloodstream.  Another critical assumption is that the changes that occur in the metabolome are unique to each pathogen.  Analysis of the patient's metabolome may therefore allow clinicians to quick diagnose any infectious disease whose metabolome has been characterized.

A recent CDC study revealed the metabolome of early Lyme disease.  The investigators obtained sera from 89 patients who had early-stage Lyme disease.  All had erythema migrans (EM), the rash characteristic of Lyme disease.  Most were also culture or PCR positive for Borrelia burgdorferi.  The patient sera were compared with sera from 50 healthy individuals by LC-MS.

After two runs of LC-MS with each sample, the researchers identified a set of 95 small molecules whose levels consistently differed between patients with early Lyme disease and healthy individuals.  Statistical modeling of the data allowed the investigators to refine the biosignature to a set of 44 molecules that identified Lyme disease in the 139 (89 + 50) subjects with the highest sensitivity and specificity.

To better gauge the performance of the biosignature in identifying those with early Lyme disease, the investigators conducted LC-MS on sera from another group of 91 patients shown to have early Lyme disease by the same criteria as the first set of patients.  Control sera came from 108 healthy individuals.  Another set of control sera was obtained from 101 patients with other diseases that could be confused with Lyme disease clinically, serologically, or microbiologically: syphilis, severe periodontitis, infectious mononucleosis, and fibromyalgia.  All patient and control sera were also tested by the standard two-tier antibody test.

The sensitivity of LC-MS testing turned out to be much higher than that of two-tier testing: 88% vs. 44%.  The specificity of LC-MS was 94% with healthy sera and 95% when sera from patients with other diseases were tested.  These values were not significantly different from the specificities of 100% and 95% achieved with two-tier testing.

These results show the promise of using the metabolic biosignature to help diagnose early Lyme disease.  However, note that all patient sera used to uncover the biosignature and assess its performance came from individuals with EM.  In practice, a clinical diagnosis involving the classic bulls-eye EM with a patient history suggestive of Lyme disease does not require confirmation by laboratory testing.  Patients without EM are more likely to need laboratory testing.  According to the CDC, 20-40% of Lyme disease patients do not have EM.

To get an idea of how well the biosignature performs on patients without EM, the investigators obtained sera drawn from 22 cases with early Lyme disease who tested positive with the C6 ELISA, a newer antibody test.  The antigen for the C6 ELISA is a highly-conserved peptide from the B. burgdorferi surface protein VlsE.  Eight of the 22 patients did not have EM.  The EM status was unknown in another eight patients.  The remaining six patients had EM.  Unfortunately, the results for each subgroup were not presented by the authors, so we don't have a firm answer about the performance of LC-MS testing on patients without EM.  What we can say is that even though more than a third of the 22 patients did not have EM, the sensitivity of LC-MS testing remained high at 86%.  In contrast, the sensitivity of two-tier testing with this group was only 41%, even though the investigators stacked the deck by using the C6 ELISA as the first tier with this group.  Future testing of the biosignature should include a larger number of sera from EM-negative patients in the early stages of Lyme disease.

As discussed in the paper, sera from patients with skin conditions that could be confused with EM (e.g., STARI, cellulitis) should be examined in future studies to make sure that the early Lyme biosignature can be used to rule out those conditions.  The authors recommend that sera from patients with neurologic, cardiac, and arthritic forms of Lyme disease also be examined to see if biosignatures specific for these more serious forms of Lyme disease could be identified.


Molins CR, Ashton LV, Wormser GP, Hess AM, Delorey MJ, Mahapatra S, Schriefer ME, & Belisle JT (2015). Development of a metabolic biosignature for detection of early Lyme disease. Clinical Infectious Diseases, 60 (12), 1767-1775 PMID: 25761869

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.