Did spirochetes kill off the Indians in Massachusetts before the Mayflower landed?

The coast of present-day Massachusetts was inhabited by several Native American tribes in the early 17th century.  Fishermen, traders, and explorers from the Old World encountered the Indians during their occasional travel through the area.  However by the time the Mayflower landed in Plymouth in 1620 to establish a colony, a mysterious epidemic had ravaged coastal New England, killing up to 90% of the indigenous population during the years 1616 through 1619.  Experts have yet to agree on the cause of the epidemic.  Smallpox, plague, and yellow fever, all highly lethal diseases, have been blamed.

Native American tribes of southeastern Massachusetts, approx. 1620 (Figure 1 from Marr and Cathey)

An article in the new issue of Emerging Infectious Diseases offers leptospirosis, caused by Leptospira spirochetes, as another possible agent of the 1616-1619 epidemic.  This is based not on any new information but on an examination of the lifestyle of the Native Americans of early 17th century New England.

Rats infected with Leptospira may have stowed away in the ships that sailed from Europe to the New World.  Because Leptospira lives in the kidney tubules of chronic carriers, infected rats released into the New World would have contaminated their surroundings every time they urinated.  Since Leptospira can survive in moist soil and fresh water, indigenous rodents and other animals could have become chronically infected with Leptospira, further spreading the spirochete throughout the region. The Indian lifestyle provided plenty of opportunities for exposure to Leptospira through skin abrasions and swallowing of contaminated water or food.  Their high-risk activities included the following:

• walking around barefooted
• storing food accessible to rodents
• swimming and bathing in streams and ponds
• working on moist soil to raise and harvest crops

Leptospira has little effect on the health of carrier animals yet can cause humans to fall ill.  Many escape with what may be confused with a mild case of the flu, but some end up suffering with life-threatening symptoms.  Eyewitnesses of the 1616-1619 epidemic reported that victims were afflicted with skin lesions, severe headaches, yellowing of the skin (likely jaundice), and bloody nose (possibly from lung hemorrhage), which are all symptoms of the severe form of leptospirosis.  Even today leptospirosis can be deadly with reported fatality rates of greater than 50% among those with severe lung hemorrhaging.

While the authors should be commended for even considering a disease of a spirochete that is often ignored (at least by those in the developed world), I don't think Leptospira is what killed off the Indians. One strong argument against leptospirosis being the cause of the 1616-1619 epidemic is that Leptospira is not hardy enough to survive the cold winters that Mother Nature inflicts upon New England.  Since the fatalities continued through the winter, leptospirosis is unlikely to be the culprit.

Whatever the cause, the epidemic may have been a pivotal event that facilitated English colonization of coastal Massachusetts since the surviving Indians lacked the capacity to resist the newcomers.

Featured paper

Marr, J.S., & Cathey, J.T. (2010). New Hypothesis for Cause of Epidemic among Native Americans, New England, 1616–1619 Emerging Infectious Diseases, 16 (2), 281-286 DOI: 10.3201/eid1602.090276

The Lyme disease spirochete has flagella but doesn't use them to penetrate the gut of the feeding tick

The Lyme disease agent Borrelia burgdorferi possesses flagella, which are the thin motility structures owned by many members of the bacteria world.  Flagella propel bacteria towards their destination by spinning (read this post to see how flagella function in Borrelia).  It has been assumed B. burgdorferi spin their flagella whenever they need to move from one location to another.  A recent paper in The Journal of Clinical Investigation has demonstrated otherwise, at least for B. burgdorferi in the midgut of a feeding Ixodes (blacklegged) tick.

Borrelia burgdorferi spends much of its life cycle lying dormant in the midgut of Ixodes ticks.  The spirochetes lightly pepper the inner surface of the midgut cell lining, with a few spirochetes also hiding between cells.  None live at the base of the cells at the basement membrane surrounding the midgut.  The spirochetes wake up and multiply only when the tick attaches to an animal or human and imbibes blood.  A few days into the blood meal, some spirochetes eventually breech the basement membrane and enter the hemocoel, the fluid-filled space between the tick organs where they must avoid the phagocytes patrolling the area.  From there the spirochetes invade the salivary glands, which can then release B. burgdorferi-tainted saliva into the skin of the victim.  After completing its satisfying meal of blood, the tick detaches from the skin of the victim, who may end up suffering from Lyme disease.

Dunham-Ems and colleagues wanted to follow the spirochetes in the midgut as ticks took their meal of blood.  They engineered a strain of B. burgdorferi expressing green fluorescent protein so that they could watch the spirochetes in the gut by fluorescence microscopy.  They allowed ticks with the green B. burgdorferi strain in their midguts to feed on laboratory mice.  24, 48, and 72 hours after the ticks were placed on the mice, the investigators removed the midguts and examined the organ by fluorescence microscopy to see what the spirochetes were doing.  Surprisingly, they never saw motile spirochetes in the midgut even though the spirochetes eventually found their way at 72 hours into the hemocoel, where they were highly motile.

If the spirochetes in the midgut remained nonmotile during tick feeding, how did they reach the basement membrane? The few spirochetes that initially populated the midgut multiplied exponentially and formed growing networks of spirochetes on the cell surfaces as the tick drank blood from the mice.  By 72 hours the networks eventually coalesced, encasing many gut cells in spirochetes (see the figures below).  Spirochetes at the base of the encased cells were poised to penetrate the basement membrane and invade the hemocoel.  All of this happened without B. burgdorferi ever spinning its flagella.  Only when they broke through into the hemocoel did the flagella start spinning.

Figure 4F-H from Dunham-Ems 2009.  Confocal fluorescence microscopy of a midgut from a nymph that fed on a mouse for 72 hours.  Panel F shows a network of spirochetes (green) attached to the inner surface of the midgut.  An optical section taken 24-26 µm into the lining of the midgut (panel G) reveals aggregates of spirochetes surrounding the cells. Panel H shows that some spirochetes have made it to the basement membrane, which is found 50 µm below the surface.  The midgut cell membrane is stained in red.  Scale bars = 25 µm.  Some of the gut cells are extremely large because they are differentiating as part of the digestion process.

Figure 5 A and B from Dunham-Ems 2009.  Silver stain of sections from ticks that fed for 48 hours (panel A) and 72 hours (panel B).  The edges of the epithelial cells are easier to see than in the previous figure.  Arrows point to aggregates of spirochetes (hairy bodies).  At 72 hours at least one cell is encased in spirochetes.  Scale bars = 25 µm.  Some of the cells are extremely large because they are differentiating as part of the normal digestion process of the tick (dc, differentiated cells; uc, undifferentiated cells). 

The investigators also found that something in the tick midgut inhibited the motility of B. burgdorferi.  They placed a bit of minced midgut from a tick that had been feeding on a mouse for 72 hours at the edge of a gelatin matrix containing motile fluorescent B. burgdorferi.  (Because of their helical shape, spirochetes love to move about in viscous substances such as gelatin.)  Most of the spirochetes near the tissue ceased moving and remained motionless throughout the 15 minute viewing period.  In contrast, the spirochetes continued moving when mouse blood was placed at the edge of the gelatin matrix.

Why does B. burgdorferi employ a nonmotile mode of penetration of the cell lining of the tick midgut?  Is there some advantage for the spirochete to avoid using their flagella?  As blood is known to be a powerful chemoattractant for B. burgdorferi, the authors offered the following explanation:

These results, although counterintuitive at first blush, make sense; if blood in the midgut acted as a chemoattractant, spirochetes would never disseminate during feeding.

Hence the "inhibitor" of motility released by the tick gut serves as a signal to the spirochete to not spin their flagella.

To me, this explanation isn't satisfying.  It would seem simple for B. burgdorferi to have evolved a regulatory scheme that would allow the spirochete to temporarily uncouple blood chemotaxis from flagellar motility so that they could bore through the gut lining in minutes rather than days. There must be a reason why B. burgdorferi chooses to take its time to penetrate the gut lining.

Perhaps B. burgdorferi delays its journey to the salivary glands to allow the feeding tick to properly prepare the skin, which is an inhospitible environment for both tick and spirochete.  As the tick feeds, it releases a brew of anti-immune factors into the skin to protect itself from attack by the immune system.  Early arrival of B. burgdorferi to the salivary gland would release the spirochetes into the skin before the anti-immune factors have taken full effect, potentially allowing the host immune system to eliminate the spirochetes before they could establish an infection.

Reference

Dunham-Ems, S.M., Caimano, M.J., Pal, U., Wolgemuth, C.W., Eggers, C.H., Balic, A., & Radolf, J.D. (2009). Live imaging reveals a biphasic mode of dissemination of Borrelia burgdorferi within ticks. Journal of Clinical Investigation. 119(12):3652-3665. DOI: 10.1172/JCI39401

E. coli-like genes in the spirochete Brachyspira hyodysenteriae, the agent of swine dysentery

The genus Brachyspira comprises at least seven species of anaerobic spirochetes that live in the large intestines of various birds and animals (including humans).  One species, Brachyspira hyodysenteriae, causes swine dysentery, a disease that causes economic loss among pig farmers worldwide.  Afflicted pigs produce loose stools covered with mucus and blood.  In severe cases, necrotic chunks of colon lining are expelled with the stool.

An Australian group sequenced the genome of B. hyodysenteriae strain WA1 to figure out how the spirochete thrives in the complex nutritional environment of the large intestine and induces swine dysentery.  They found 2,122 protein-coding genes distributed between a 3,000,694 bp chromosome and a 35,940 bp plasmid.  A number of genes encoded degradative enzymes such as proteases, phospholipases, and hemolysins that may or may not account for the damage to the colon observed in swine dysentery cases.  Otherwise, no obvious pathogenic mechanism for the disease process could be gleaned from the genome sequence.

Remarkably, half of the proteins encoded in the B. hyodysenteriae genome were most similar in sequence to proteins of Escherichia and Clostridium, genera that are not even on the same branch on the bacterial evolutionary tree as spirochetes.  A mere 6.4% of B. hyodysenteriae proteins matched best to proteins of other spirochetes.

From Table 3 of Bellgard 2009.

Among the Escherichia- and Clostridium-like genes, those encoding proteins involved with amino acid and sugar metabolism and transport were over-represented.  Since E. coli, Clostridium species, and B. hyodysenteriae all live in the large intestine, the similarity in the proteins may simply reflect convergent evolution that enable the bacteria to metabolize the nutrients available in the colon.  The more attractive possibility is that B. hyodysenteriae acquired the genes from the other enteric bacteria by horizontal gene transfer thereby allowing the spirochete to adapt to the complex nutritional environment of the large intestine.  At least for the E. coli-like genes, examining their GC content may help distinguish between the two possibilities since the GC content of B. hyodysenteriae is only 27% versus 50% for E. coli.

How can B. hyodysenteriae acquire genes from other enteric bacteria?  A commentary in the journal Gut Pathogens raised the possibility that bacteriophage-like elements found in the B. hyodysenteriae genome could be involved, although bacteriophages generally do not transfer DNA between different species of bacteria.  Another possibility is that genes could be acquired from other bacteria by conjugation, a form of microbial mating.  Although the capacity of B. hyodysenteriae for acquiring DNA from other bacteria by conjugation is unknown, scientists have demonstrated that another spirochete could acquire DNA from E. coli by conjugation in the laboratory setting.

Image source

Sow with piglet, from Wikipedia

References

Bellgard, M.I., Wanchanthuek, P., La, T., Ryan, K., Moolhuijzen, P., Albertyn, Z., Shaban, B., Motro, Y., Dunn, D.S., Schibeci, D., Hunter, A., Barrero, R., Phillips, N.D., and Hampson, D.J. (2009).  Genome sequence of the pathogenic intestinal spirochete Brachyspira hyodysenteriae reveals adaptations to its lifestyle in the porcine large intestine.  PLoS ONE 4(3):e4641. DOI: 10.1371/journal.pone.0004641

Hampson, D.J. and Ahmed, N. (2009).  Spirochaetes as intestinal pathogens:  Lessons from a Brachyspira genome.  Gut Pathogens 1(1):10.  DOI: 10.1186/1757-4749-1-10

Leptospira and E. coli caught in the act

I found this web photo of E. coli mating with the spirochete Leptospira biflexa in a process called conjugation (image source, Mathieu Picardeau, Pasteur Institute).  The donor E. coli cell is transferring a copy of a plasmid bearing antibiotic resistance genes to the recipient spirochete.  The DNA is most likely pushed through a pore that forms between the mating pair where the outer membranes come together.

There are many types of plasmids, but only self-transmissible plasmids are capable of transferring copies of themselves to other bacteria by conjugation. These plasmids carry a set of at least 20 genes collectively called tra (transfer), which encode all of the proteins necessary to carry out conjugation.  The conjugational proteins assemble into several structures, including the sex pili, which bring the mating pair together, the relaxosome, which processes the DNA for transfer, and the poorly characterized pore through which the DNA traverses.  The plasmids can also harbor additional genes that have no role in conjugation, including genes encoding resistance to antibiotics.  RP4 is one example of a self-transmissible plasmid that can transfer itself to a wide range of bacteria species.  Self-transmissible plasmids have been found in many different bacteria, yet none have been discovered in spirochetes.

Transformation is the microbiologist's favorite genetic tool for delivering DNA of their choosing into bacteria. Unfortunately for those interested in leptospirosis, transformation of disease-causing species of Leptospira such as L. interrogans is difficult.  Conjugation employing a laboratory strain of E. coli as a donor provides scientists another route for delivering DNA into Leptospira.  For example, the plasmid illustrated below (Figure 1 from Picardeau, 2008) has been used to ferry the Himar1 transposon into Leptospira for random insertional mutagenesis.  Many readers may be most familiar with the F conjugational plasmid of E. coli, but the conjugational machinery found on the RP4 self-transmissible plasmid is used here since it is able to deliver DNA to a wide range of bacteria species.

Figure 1 from Picardeau, 2008.  The Himar1 transposon consists of the arrowheads and everything in between, including the kanamycin-resistance gene (Km^R).  The RP4 oriT element and the genes encoding the C9 tranposase and spectinomycin resistance (Spc^R) lie outside of the transposon.


The critical element of the plasmid is the RP4 oriT sequence where relaxase, a component of the relaxosome, nicks the DNA to initiate the transfer process.  The tra genes were removed to permit easy manipulation of the plasmid.  To perform conjugation, the plasmid was transformed into a special E. coli strain that encodes the RP4 tra genes on its chromosome.  The E. coli cells were then mixed with Leptospira and concentrated onto a filter to facilitate mating.  After allowing them to mate for 20 hours, the mating mixture was plated onto Leptospira medium agar plates containing the antibiotic kanamycin to recover Leptospira mutants with the transposon on one of its two chromosomes.  The plasmid itself is unable to replicate in Leptospira, so the transposon must hop onto a chromosome following plasmid transfer to enable growth of kanamycin-resistant Leptospira into colonies.  The donor E. coli bacteria had been genetically modified to require the nutrient diaminopimelate (DAP) to counterselect the donor on the agar plates, which were lacking DAP.

It is not feasible to screen L. interrogans insertion mutants for a desired phenotype (trait) following a single mating experiment since only a few hundred kanamycin-resistant colonies can be recovered.  Tens of thousands of mutants would be necessary to ensure coverage of (almost) all L. interrogans genes.

One application of this genetic tool is to perform multiple mating experiments to generate a library of mutants with insertions of Himar1 in different L. interrogans genes.  The sequence of the insertion site of the transposon in the chromosome can be obtained easily with today's sequencing technology.  Further experiments can be performed to examine any mutants with insertions in genes that hold the investigator's interest.  Several labs have teamed up to embark on a similar approach by delivering the Himar1 transposon into L. interrogans by transformation (see the Murray 2009 paper), which does not yield as many colonies as conjugation.

References

Picardeau, M. (2008).  Conjugative transfer between Escherichia coli and Leptospira spp. as a new genetic tool.  Applied and Environmental Microbiology 74(1):319-322.  DOI: 10.1128/AEM.02172-07

Murray G.L., Morel, V., Cerqueira G.M., Croda, J., Srikram, A.,  Henry, R., Ko, A.I., Dellagostin, O.A., Bulach, D.M., Sermswan, R.W., Adler, B., and Picardeau, M. (2009).  Genome-wide transposon mutagenesis in pathogenic Leptospira species.  Infection and Immunity 77(2):810-816.  DOI: 10.1128/IAI.01293-08

The genetics of both host and pathogen matter in antibiotic-refractory Lyme arthritis

The arthritic form of Lyme disease was first reported in the 1970s by Allen Steere, who described the condition in a group of children (and a few adults) residing in and around the town of Lyme, Connecticut. Lyme arthritis can strike when Borrelia burgdorferi introduced into the skin by an Ixodes tick burrows into deeper tissues and ends up in the joints, usually the knee. Swelling results from an inflammatory response to B. burgdorferi residing in the joint. Lyme arthritis is treated with antibiotics, which destroy the bacteria driving inflammation. Unfortunately, arthritic symptoms endure in ~10% of treated patients despite the complete or almost complete eradication of the infection, as determined by negative PCR tests for B. burgdorferi DNA in joint fluid. Such cases are called antibiotic-refractory Lyme arthritis, which can persist for months or sometimes years. In severe cases cartilage and bone erode. Although the pathogenesis of antibiotic-refractory Lyme arthritis could involve persistence of small numbers of B. burgdorferi (or their antigens) in the joints, investigators have been seeking an autoimmune mechanism to explain the prolonged attack on joint tissue by the immune system after the spirochetes have been cleared.

Many autoimmune diseases are linked to variants of HLA (immunity) genes such as those encoding the MHC class II complex. Antibiotic-refractory Lyme arthritis is associated with MHC class II variants that are able to bind to fragments of the B. burgdorferi protein OspA (outer surface protein A) encompassing amino acid residues 165 through 173. Antigen-presenting cells whose MHC class II molecules display OspA_165-173 peptides on their surface stimulate T cells that recognize the OspA peptide. How OspA_165-173-reactive T cells cause autoimmunity has been an area of intensive research, yet a clear answer has not emerged.

One potential pathway to autoimmunity is molecular mimicry, in which a cross-reactive host protein in the joint continues to stimulate OspA_165-173-specific T cells even after the eradication of B. burgdorferi by antibiotics. Although the simplicity of the molecular mimicry model is appealing, exhaustive efforts to find a cross-reactive autoantigen that stimulates OspA_165-173-specific T cells have failed. Moreover, levels of OspA_165-173-reactive T cells decline soon after initiation of antibiotic therapy despite continuing arthritis following treatment. Thus, chronic arthritis does not seem to involve molecular mimicry driven by a cross reaction between the OspA_165-173 epitope and a self-antigen in the joint. It is possible that molecular mimicry involves another B. burgdorferi antigen that is able to bind the MHC class II variants found in genetically susceptible individuals.

Other potential routes to autoimmunity in antibiotic-refractory Lyme arthritis patients emphasize the role of the high levels of key proinflammatory cytokines and chemokines found in their joint fluid, levels even higher than those found in treatment-responsive patients prior to initiation of antibiotic therapy:

• In a model known as bystander activation, the immune response to OspA_165-173 (or another B. burgdorferi antigen) causes an excessive inflammatory response that activates other T cells that react to autoantigens in the joint.
• The immune system is unable to turn off the intense inflammatory response associated with OspA_165-173 after the spirochetes are cleared from the joint.

Although much attention has been focused on the role of host genetics, a recent study indicates that the genetics of the pathogen could also influence the course of Lyme arthritis. In the July 2009 issue of Arthritis and Rheumatism, Allen Steere and his collaborators showed that antibiotic-refractory Lyme arthritis is associated with different strains of B. burgdorferi. The strains were typed from joint fluid samples collected before or during antibiotic treatment. Among the methods available to group B. burgdorferi isolates, they used the 16S-23S ribosomal RNA intergenic spacer type (RST), of which there are three. Antibiotic-refractory arthritis was defined as joint swelling lasting for at least 3 months after the start of antibiotic treatment. Antibiotic treatment consisted of 8 weeks of oral antibiotics or up to 4 weeks of antibiotics administered intravenously. Joint fluid from all 17 patients in the study tested positive by PCR for B. burgdorferi DNA prior to or during antibiotic treatment.

The authors found that all 7 Lyme arthritis patients infected with RST1 strains had the antibiotic-refractory form. Joint fluid was obtained after antibiotic treatment from 5 of the 7 patients; all 5 samples tested negative for B. burgdorferi DNA by PCR. In contrast, 2 of 6 and 3 of 4 infected with RST2 and RST3 strains, respectively, were successfully treated with antibiotics (see the table below from the Jones et al. 2009 article). A larger number of samples is needed to demonstrate that the difference observed between RST1 and RST2 strains is statistically significant, but there is a clear trend towards RST1 infections having the greatest association with antibiotic treatment failure and RST3 having the least, with RST2 having an intermediate effect. The duration of arthritis also depended on the infecting RST strain.


How do RST1 strains cause arthritis to persist even after the apparent eradication of the spirochetes by the recommended course of antibiotics? The investigators proposed that RST1 strains provoke a stronger inflammatory response in the joint than RST2 or RST3 strains. Coupled with an immune response to OspA_165-173 in genetically susceptible patients, this could cause inflammation to continue at high levels even after elimination of the spirochetes from the joints. RST1 strains may be more likely than the other genotypes to spark intense joint inflammation even in patients who are not genetically prone to antibiotic-refractory arthritis.

In future studies, it would be interesting to see if proinflammatory cytokine levels are related to the RST type that infects the joint. Ultimately, researchers need to identify the B. burgdorferi gene or genes whose variation among the RSTs causes the different treatment outcomes of Lyme arthritis.

Featured paper

Jones, K.L., McHugh, G.A., Glickstein, L.J., & Steere, A.C. (2009). Analysis of Borrelia burgdorferi genotypes in patients with Lyme arthritis: High frequency of ribosomal RNA intergenic spacer type 1 strains in antibiotic-refractory arthritis
Arthritis & Rheumatism, 60 (7), 2174-2182 DOI: 10.1002/art.24812

Other references

Drouin E.E., Glickstein, L., Kwok, W.W., Nepom, G.T., and Steere, A.C. (2008). Human homologues of a Borrelia T cell epitope associated with antibiotic-refractory Lyme arthritis. Molecular Immunology 45(1):180-189. DOI: 10.1016/j.molimm.2007.04.017

Kannian, P., Drouin, E.E., Glickstein, L., Kwok, W.W., Nepom, G.T., and Steere A.C. (2007). Decline in the frequencies of Borrelia burgdorferi OspA_161-175-specific T cells after antibiotic therapy in HLA-DRB1*0401-positive patients with antibiotic-responsive or antibiotic-refractory Lyme arthritis. The Journal of Immunology 179(9):6336-6342.

Shin J.J., Glickstein, L.J., and Steere, A.C. (2007). High levels of inflammatory chemokines and cytokines in joint fluid and synovial tissue throughout the course of antibiotic-refractory Lyme arthritis. Arthritis & Rheumatism 56(4):1325-1335. DOI: 10.1002/art.2241

Steere, A.C., Klitz, W., Drouin, E.E., Falk, B.A., Kwok, W.W., Nepom, G.T., and Baxter-Lowe, L.A. (2006). Antibiotic-refractory Lyme arthritis is associated with HLA-DR molecules that bind a Borrelia burgdorferi peptide. The Journal of Experimental Medicine 203(4):961-971. DOI: 10.1084/jem.20052471