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