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

Telomeres without telomerase in Borrelia spirochetes

You've all heard by now that the 2009 Nobel Prize in Physiology or Medicine will be awarded to Elizabeth Blackburn, Carol Greider, and Jack Szostak. They're the ones who figured out that an enzyme called telomerase combats the shortening that occurs at the ends of the linear chromosomes of eukaryotes (including ours) during each round of DNA replication. Telomerase sticks copies of a short string of nucleotides to the 3' ends of the chromosomal DNA. On the other hand, bacteria do not need telomerase because their chromosomes are circular; they do not have ends that can be shortened.

Spirochetes of the genus Borrelia, which include the agents of Lyme disease and relapsing fever, are an oddity in the bacterial world in that their chromosomes are linear. They also have a large set of linear plasmids. For example, the genome of the Lyme disease agent Borrelia burgdorferi consists of one linear chromosome and 12 linear plasmids, along with 11 circular plasmids. I will refer to the chromosomes and plasmids collectively as replicons. Despite having linear replicons, telomerase is nowhere to be found in Borrelia.

If they lack telomerase, how do Borrelia avoid having the ends of their linear replicons getting pruned during DNA replication? The key lies in the covalently closed hairpin ends of their linear replicons, something that's not found in eukaryotic telomeres. As illustrated in the figure below (figure 1 from Tourand 2003), the hairpin loops allow the replication machinery to copy the nucleotides at the very ends of the telomeres.

The replicated structure shown at the bottom of the figure illustrates that this method of replication creates a new problem. Following DNA replication, the two copies of the replicon end up fused at their ends (L'L and RR'). The fused sequences (telomere junctions) must somehow separate so that one copy of the replicon ends up in each daughter cell as the spirochete lengthens and splits in two.

Fortunately, Borrelia possesses an enzyme designated ResT, a telomere resolvase that cleaves the DNA where the two copies of the replicon are fused and reforms the hairpins at the ends of the new telomeres (see figure below). ResT was discovered by George Chaconas' laboratory in Canada. All of the work with ResT that I describe here was carried out by his group.


Chaconas' group was able to demonstrate the telomere resolvase reaction in vitro by simply mixing purified ResT with DNA containing a telomere junction. The products of the reaction were then analyzed following agarose gel electrophoresis. An example of the assay is shown below. A 4.6 kb piece of DNA containing the telomere junction formed by the left end of the B. burgdorferi linear plasmid lp17 ("L'L") is converted by ResT into the expected 2.6 and 2.0 kb products over a 2 minute time period.

Although the genome sequence of B. burgdorferi was published years ago, the sequence of the telomeres could not be determined back then because of the difficulty in cloning DNA with closed hairpin ends. The sequence of most of the telomeres finally appeared in the literature this year. The telomere sequences are aligned in the table below (figure 6 of Tourand 2009). The end of each telomere is the first nucleotide in each sequence.

The alignment shows that all of the telomeres have a "box 3" sequence. Box 3 is the recognition site for ResT binding. The evidence for this is that ResT binds to box 3 in vitro and changing the nucleotides in box 3 inhibits resolution of the telomere junction by ResT.

The telomeres were grouped based on the box 1 sequence. Type 1 telomeres carry the box 1 sequence TATAAT, and Type 2 telomeres harbor the modified box 1 sequence TATTAT. Type 3 telomeres lack the box 1 motif. When the rates of ResT resolving the different telomere junctions were measured in vitro (last column in alignment above), telomeres that lacked box 1 (Type 3) exhibited the slowest rates, with three telomeres failing to react with ResT. Since these three telomeres are obviously resolved in vivo, their resolution may require additional factors yet to be identified.

References

Tourand, Y., Deneke, J., Moriarty, T.J., & Chaconas, G. (2009). Characterization and in vitro reaction properties of 19 unique hairpin telomeres from the linear plasmids of the Lyme disease spirochete Journal of Biological Chemistry, 284 (11), 7264-7272 DOI: 10.1074/jbc.M808918200

Kobryn, K., & Chaconas, G. (2002). ResT, a telomere resolvase encoded by the Lyme disease spirochete Molecular Cell, 9 (1), 195-201 DOI: 10.1016/S1097-2765(01)00433-6

Tourand, Y., Kobryn, K., & Chaconas, G. (2003). Sequence-specific recognition but position-dependent cleavage of two distinct telomeres by the Borrelia burgdorferi telomere resolvase, ResT Molecular Microbiology, 48 (4), 901-911 DOI: 10.1046/j.1365-2958.2003.03485.x

Tourand, Y., Lee, L., & Chaconas, G. (2007). Telomere resolution by Borrelia burgdorferi ResT through the collaborative efforts of tethered DNA binding domains Molecular Microbiology, 64 (3), 580-590 DOI: 10.1111/j.1365-2958.2007.05691.x

Baby steps towards unraveling transcriptional regulation in the unculturable syphilis spirochete

I would never select Treponema pallidum as my experimental model if I had to study gene regulation in a spirochete. The main problem is that no one has figured out how to grow T. pallidum in any type of culture medium. T. pallidum can be propagated only by growing the spirochete in the testes of rabbits. Consequently, investigators have not even begun to develop the genetic tools (e.g., gene knock outs, shuttle plasmids) necessary to unravel the regulatory mechanisms that control T. pallidum gene expression.

Despite the limitations imposed by T. pallidum upon those who wish to study gene regulation, a group of syphilis researchers at the University of Washington in Seattle have started to dissect the regulation of several members of the 12-gene tpr (Treponema pallidum repeat) family. No one has figured out what the Tpr proteins do, but syphilis researchers are interested in them in part because they show how the immune response battles T. pallidum infections. For example, antibodies generated against TprK during infection bind to TprK exposed on the surface of T. pallidum and mark them for destruction by macrophages. More recent studies suggest that TprK undergoes antigenic variation (a topic of a future post), which may allow T. pallidum to persist in the host.

The Seattle group's studies on gene regulation have focused on the Subfamily II tpr genes tprE, tprG, and tprJ, as reported in the journal Molecular Microbiology. The sequences upstream of their transcription start sites contain a sequence that closely matches the consensus binding sequence for the E. coli global transcriptional regulator CRP (cAMP regulatory protein), also known as CAP (catabolite activator protein). The T. pallidum genome encodes a CRP homolog designated TP0262. In E. coli and a few other Gram negatives, CRP is an integral component of the complex network of transporter, regulatory, and enzymatic proteins that allow bacteria to selectively metabolize the preferred sugar, usually glucose, from those available in the environment. When glucose is absent, the enzyme adenylate cyclase is activated and synthesizes the second messenger cAMP (cyclic AMP), which turns on CRP by allosteric activation. (Here's a nice description of the allosteric activation of CRP.) The cAMP-CRP complex then binds upstream of various promoters and activates transcription by recruiting RNA polymerase to the promoter. Additional layers of regulation ensure that the genes are transcribed only when the sugar that is to be broken down by the gene products is present.

Because it's not possible to examine gene regulation in T. pallidum, the Seattle group transferred the tpr genes to E. coli, a genetically pliable bacterium. They fused each tpr gene, including the upstream sequences containing the proposed CRP binding site and the promoter, to a gene whose product is easily measurable, green fluorescent protein (gfp). They then introduced the plasmid carrying the gene fusion into an E. coli strain missing its crp gene so that they could measure tpr-driven GFP levels in the presence and absence of a second plasmid expressing TP0262. They found that TP0262 increased tprE'-gfp and tprJ'-gfp fusion expression while decreasing trpG'-gfp expression. The ability of TP0262 to control tpr'-gfp expression was lost when the CRP binding site was removed from the fusion constructions. They also showed that control of the tprJ'-gfp fusion by TP0262 was lost when the adenylate cyclase gene in E. coli was removed, indicating that cAMP was needed to activate TP0262 (data for tprE and tprG were not presented). Their in vitro experiments demonstrated binding of purified recombinant TP0262 to the proposed CRP binding site upstream of the three tpr genes by DNase I protection and gel shift assays.

What was missing from the study, as acknowledged by the authors, were experiments to demonstrate that TP0262 does the same thing in T. pallidum. For future studies, they plan to show that TP0262 is bound upstream of the Subfamily II tpr genes in T. pallidum by chromatin immunoprecipitation, which entails determining the sequence of the segment of DNA that is bound when TP0262 is immunoprecipitated from a T. pallidum extract. Such experiments would not require genetic manipulation or the ability to cultivate T. pallidum. It would only require harvesting a large number of T. pallidum spirochetes from infected rabbits.

What signal does TP0262 respond to? Does it respond to the glucose found in the host? The insightful Commentary by Radolf and Desrosiers sheds some light on the question. They note that T. pallidum is missing the special transporter genes that in E. coli encode the components necessary to link sugar availability to cAMP and CRP. They surmise that TP0262 has thus been freed to regulate genes not related to sugar metabolism, such as the tpr genes. Since CRP is a global transcriptional regulator in other bacteria, it is likely to regulate expression of not only the Subfamily II tpr genes but also additional genes in T. pallidum.

Near the end of their commentary, Radolf and Desrosiers made one comment that stood out:

One of the most important outcomes of the present study is that it will help put to rest the pregenomic view of the syphilis spirochaete as a transcriptionally invariant organism.

Maybe I'm too young to appreciate their point, but I can't believe that there ever was a time when syphilis researchers believed that T. pallidum genes were not regulated!

Featured articles

Giacani, L., Godornes, C., Puray-Chavez, M., Guerra-Giraldez, C., Tompa, M., Lukehart, S.A., & Centurion-Lara, A. (2009). TP0262 is a modulator of promoter activity of tpr Subfamily II genes of Treponema pallidum ssp. pallidum
Molecular Microbiology, 72 (5), 1087-1099 DOI: 10.1111/j.1365-2958.2009.06712.x

Radolf, J.D., & Desrosiers, D.C. (2009). Treponema pallidum, the stealth pathogen, changes, but how?
Molecular Microbiology, 72 (5), 1081-1086 DOI: 10.1111/j.1365-2958.2009.06711.x

Protein census of Leptospira interrogans

A census of proteins in a bacterial cell was conducted for the first time ever. By "census," I don't mean merely identifying all cellular proteins (which can be accomplished by shotgun tandem mass spectrometry). What I mean is counting the number of copies of every protein. The bacterium targeted for the census was the spirochete Leptospira interrogans. Like the census conducted here in the U.S. every ten years, some proteins were missed. The strategy developed by Malmström and colleagues, as described in the August 6 issue of Nature, allowed them to determine the abundance of 1,864 (or 83%) of the 2,221 proteins that were detectable by tandem mass spectrometry (MS) in Leptospira interrogans that had been grown in standard Leptospira culture medium.

The results of the protein census are compiled in the bar graph below. Proteins with related biological functions were grouped together and are color coded. The "Proteome" bar tabulates the number of different proteins in each group.
The next bar, "Copies per cell (control)," gives you an idea of how much of the protein expression machinery in L. interrogans is directed towards the synthesis of proteins in each functional category. The percentage reflects the amounts and size of the proteins in each category. For example, proteins of unknown function (hypothetical proteins) represent only 12.7% (blue) of the total protein synthesis capacity even though they constitute 30% of the identified proteins and over 40% of all genes in L. interrogans. I would surmise that these hypothetical proteins would account for a more sizable fraction of total protein synthesis under some other condition that L. interrogans would encounter during its life cycle (e.g., during infection).

The other observation noted by the authors is that L. interrogans gears 15% of its protein synthesis effort to make a small number of proteins deemed to be components of the "external encapsulating structure" (green), which is a fancy Gene Ontology term encompassing abundant Leptospira proteins that have been demonstrated to be in the inner or outer membrane. Most of the 15% is accounted for by five outer membrane proteins: LipL32, Loa22, LipL41, LipL21, and LipL36, the functions of which are not entirely clear. The five proteins are among the 10 most abundant proteins in L. interrogans.

The last bar shows the effect of the antibiotic ciprofloxacin ("cipro") on global protein levels in L. interrogans. The most striking change is the massive increase in 15 proteins of unknown function (light blue) leading them to constitute ~20% of the total protein content. As ciprofloxacin is an inhibitor of DNA gyrase, transcription of the genes encoding the 15 proteins may be extremely sensitive to DNA topology.

Did the enormous increase in the copy number of the 15 proteins following ciprofloxacin treatment increase the total number of protein molecules in L. interrogans? The authors found little change in the total cellular protein content:

Interestingly, this large redistribution of the proteome did not significantly change the total cellular protein concentration. Therefore, the large increase in the abundance of [the 15 proteins of unknown function] after ciprofloxacin exposure was compensated by a slight reduction of other high abundant protein classes.... This indicates that in L. interrogans, the cells strive to maintain a certain total number of protein components, that is, a constant cellular proteome concentration.

Featured paper

Malmström, J., Beck M., Schmidt, A., Lange, V., Deutsch, E.W., and Aebersold, R. (2009). Proteome-wide cellular protein concentrations of the human pathogen Leptospira interrogans. Nature 460(7256):762-765. DOI: 10.1038/nature08184