Looking for the syphilis spirochete in ancient bones

PCR is a powerful tool that has been used to detect microbial DNA in human remains unearthed by archaeologists.  This approach has helped reveal when and where infectious diseases such as tuberculosis and the plague have afflicted human populations in the past.  With the controversy raging over the question of whether the syphilis spirochete was present in Europe before Columbus sailed to America, one would think that scientists would have tried PCR to detect Treponema pallidum DNA in skeletal remains.  Well, they have, but in almost every case they failed to detect T. pallidum DNA, even in bones bearing the lesions of syphilis.  The problem is that adults who die in the later stages of syphilis do not have many T. pallidum spirochetes in their bones.

On the other hand, spirochetes are relatively abundant in the bones of infants afflicted with congenital syphilis.  Therefore the skeletal remains of the very young may be a better source for detection of T. pallidum DNA by PCR.  As reported in their recent PLoS One article, Montiel and colleagues looked for T. pallidum DNA in skeletal remains gathered from a 16th-17th century crypt in Spain.  The investigators found four infant bones with lesions that were consistent with congenital syphilis.  Since there were two left humeri (specimens ELS551 and ELS558 in the image below), the bones must have belonged to at least two newborns.

Figure 1 from Montiel et al., 2012.  Source.

The PCR reactions were conducted on specimens from both newborns in each of three different laboratories.  Two segments along the T. pallidum chromosome were targeted.  One lab targeted the arp gene, the second lab targeted the 5' UTR of the 15 kDa lipoprotein gene, and the third targeted both sequences.  All attempts but one led to the generation of PCR products.  To confirm that they derived from T. pallidum sequences, the PCR products were either analyzed by restriction digestion or cloned and sequenced.  The 5' UTR of the lipoprotein gene was critical to this effort because its sequence can be used to distinguish the syphilis spirochete from the other disease-causing treponemes.  The PCR products from both newborns turned out to have the Eco47III restriction site that is unique to the syphilis spirochete among modern treponemes.  The molecular analysis therefore supported the diagnosis of congenital syphilis in the two long-deceased infants.

The investigators took special precautions to minimize the risk of contamination, which is always a concern of paleomicrobiologists running PCR reactions.  For example, the experiments were done in laboratories in which Treponema-containing samples had never been handled.  The investigators even excluded positive controls from their PCR reactions.

Scientists are still not certain whether congenital syphilis can be correctly diagnosed by examining bone pathology alone (see pp. 102-103 of this paper for a nice discussion of this issue).  The PCR method will therefore aid scientists wishing to identify skeletal remains afflicted with congenital syphilis.  It also gives paleomicrobiologists hope that PCR methods will help answer the centuries-old question about the origin of syphilis.

References

Montiel R, Solórzano E, Díaz N, Álvarez-Sandoval BA, González-Ruiz M, Cañadas MP, Simões N, Isidro A, & Malgosa A (2012). Neonate human remains: a window of opportunity to the molecular study of ancient syphilis. PloS one, 7 (5) PMID: 22567153

Bouwman, AS, & Brown, TA (2005). The limits of biomolecular palaeopahology: ancient DNA cannot be used to study venereal syphilis Journal of Archaeological Science, 32, 703-713 DOI: 10.1016/j.jas.2004.11.014

Related posts

• Still no solid evidence for the Old World origin of syphilis
• The origin of syphilis: a phylogenetic approach

BAM! A rare outer membrane protein of the stealth pathogen Treponema pallidum hidden in plain sight

Our immune system is capable of generating antibody against bacterial proteins located inside and outside of the bacterial cell.  However only those antibodies targeting surface proteins (or other surface components) have the potential to eliminate the bacteria.

Unlike typical Gram negative bacteria, the external surface of the Treponema pallidum outer membrane is bare except for a tiny number of proteins protruding from the membrane.  Most of the proteins targeted by the host antibody response are safely tucked away beneath the surface, inaccessible to the antibodies that recognize them.  T. pallidum even lacks LPS, which is a major target found on typical Gram negative bacteria.  The barren surface of T. pallidum is therefore one factor that may allow the "stealth pathogen" to persist in the host despite a strong antibody response.

None of the rare T. pallidum outer membrane proteins (Omps) have been identified with certainty, until now.  Scientists at the University of Connecticut have finally confirmed that TP0326 is one of these rare Omps.¹  Desrosiers and colleagues showed that the protein TP0326 was digested when proteinase K was added to live T. pallidum.  Proteins known to be located in the periplasm were left untouched by the protease, indicating that the fragile outer membrane remained intact while the spirochetes were being harvested for the experiment.  These results indicated that TP0326 was exposed on the surface of T. pallidum.

TP0326 (also called "Tp92") was first identified as a candidate Omp over a decade ago when rabbit antibodies against the protein were shown to promote opsonophagocytosis (engulfment) of T. pallidum.²   Antibodies must physically link bacteria to phagocytes for opsonophagocytosis to proceed.  Opsonophagocytosis therefore occurs only when antibodies against surface-exposed proteins are present.  The amino acid sequence of TP0326 also gave clues to its location.  TP0326 was identified as a homolog of the Omp85 family,² a set of proteins known to reside in the outer membrane of other Gram negative bacteria.  Omp85 was later renamed BamA when it was shown to be the core component of the outer membrane protein complex "BAM" that inserts newly expressed Omps into the outer membrane.³

A number of computer programs predicted that TP0326 spanned the outer membrane as a β-barrel structure, which I described in an earlier post.⁴  The gallery of E. coli transmembrane Omps displayed below shows that the loops on one side of the barrel are exposed on the surface of the bacterium.  The transmembrane portion of BamA is depicted as a box because its structure has yet to be determined experimentally, but it is also likely to have a β-barrel structure.

Figure 1 from Burgess 2008.  The outer membrane is colored gray.  The numbers indicate the number of β strands that cross the membrane.  The periplasm is located below the outer membrane.

The amino terminus of BamA consists of five repeating structures called POTRA, which are thought to guide the β strands of new transmembrane Omps into the outer membrane.³  Note that the POTRA domains protrude into the periplasmic space.  The amino terminus of TP0326 is thought to possess the POTRA domains as well.

Given the prediction that TP0326 structurally resembles BamA, it wasn't too surprising that TP0326 was exposed on the surface of T. pallidum.  What was surprising was how TP0326 was targeted by the immune system in syphilis patients.  Among the six patients examined by Desrosiers et al., three lacked any antibody whatsover against TP0326.  Although the other three syphilis patients managed to generate antibody against TP0326, the antibodies reacted weakly (1 patient) or not at all (2 patients) with the β-barrel domain, which contained the surface-exposed loops.  Instead, the antibodies targeting TP0326 reacted strongly with the subsurface POTRA domains in these three patients.  Assuming that the results with these six patients can be extrapolated to other syphilis patients, humans infected with T. pallidum are incapable of generating a strong antibody response against the surface-exposed loops of TP0326.

Since the exposed regions of TP0326 appear to be an Achilles heel of T. pallidum, TP0326 may have evolved to avoid generation of antibodies targeting its vulnerable segments.  Experimentally infected rabbits, which are not a natural host of T. pallidum, succeeded in generating antibody against the TP0326 β-barrel domain. Unfortunately the authors didn't present any data indicating whether the surface-exposed loops were recognized by the rabbit antibodies.  However, as I mentioned above, earlier work showed that infected rabbits produce opsonic antibody against TP0326.²  The same paper showed that TP0326 was somewhat effective as a subunit vaccine in the rabbit model of syphilis.²  Both of these observations suggest that rabbits are able to produce antibody that reacts with the surface-exposed regions of TP0326.  The rabbit model could be used to figure out conclusively whether the effectiveness of TP0326 as a vaccine relies upon generation of antibodies against the surface-exposed loops of the protein.  If so, an approach for developing a syphilis vaccine would be to coax the human immune system into generating antibodies that target the surface-exposed loops of TP0326.


Featured paper

1. Desrosiers DC, Anand A, Luthra A, Dunham-Ems SM, LeDoyt M, Cummings MAD, Eshghi A, Cameron CE, Cruz AR, Salazar JC, Caimano MJ, and Radolf JD (June 2011).  TP0326, a Treponema pallidum β-barrel assembly machinery A (BamA) orthologue and rare outer membrane protein.  Molecular Microbiology 80(6):1496-1515.  DOI: 10.1111/j.1365-2958.2011.07662.x

Related papers

2. Cameron CE, Lukehart SA, Castro C, Molini, B, Godornes C, and Van Voorhis WC (April 2000).  Opsonic potential, protective capacity, and sequence conservation of the Treponema pallidum subspecies pallidum Tp92.  The Journal of Infectious Diseases 181:1401-1413.  DOI: 10.1086/315399


3. Knowles TJ, Scott-Tucker A, Overduin M, and Henderson IR (March 2009).  Membrane protein architects: the role of the BAM complex in outer membrane protein assembly.  Nature Reviews Microbiology 7(3):206-214.  DOI: 10.1038/nrmicro2069

4. Cox DL, Luthra A, Dunham-Ems S, Desrosiers DC, Salazar JC, Caimano MJ , and Radolf JD (December 2010).  Surface immunolabeling and consensus computational  framework to identify candidate rare outer membrane proteins of Treponema pallidum.  Infection and Immunity 78(12):5178-5194.  DOI: 10.1128/IAI.00834-10

Image source

Burgess NK, Dao TP, Stanley AM, and Fleming KG (September 26, 2008).  β-barrel proteins that reside in the Escherichia coli outer membrane in vivo demonstrate varied folding behavior in vitro.  Journal of Biological Chemistry 283(39):26748-26758.  DOI: 10.1074/jbc.M80275420

Related posts

• The quest for outer membrane proteins of the stealth pathogen Treponema pallidum:  the cliffhanger episode

The quest for outer membrane proteins of the stealth pathogen Treponema pallidum: the cliffhanger episode

Syphilis patients are able to generate antibodies against the spirochete Treponema pallidum.  However, if you were to mix sera from these patients with T. pallidum in a test tube, very few of the antibodies in the sera would bind to the spirochetes.  The reason is that the strange outer membrane architecture of T. pallidum makes the spirochete invisible to the antibodies.  Most of the proteins and lipid molecules targeted by the antibodies lie beneath the outer membrane.

The outer membrane of T. pallidum differs considerably from that of a typical Gram-negative bacterium.  The most glaring difference is that T. pallidum lacks lipopolysaccharide (LPS), a favorite target of the immune response.  The outer membrane is also bare of other potential surface antigens except for a very small number of transmembrane outer membrane proteins (Omps) and (possibly) surface lipoproteins.  The poor surface antigencity may help the so-called "stealth pathogen" persist in the body despite a robust immune response to the infection.

The few Omps displayed on the surface of T. pallidum must be doing something really important if the spirochete is willing to risk exposing them to attack by antibodies.  For this reason, scientists have been seeking the identity of these Omps to figure out what they do.  These rare Omps could also be fashioned into a long-desired syphilis vaccine.

Unfortunately, the scarcity of Omps and the delicate nature of the outer membrane of T. pallidum have stymied efforts to identify Omps.  The routine centrifugation and washing steps used to prepare other bacteria for analysis easily damage the outer membrane of T. pallidum, causing the loss of Omps and exposing the abundant periplasmic and inner membrane proteins.  Consequently, probes used to identify exposed proteins may react with the periplasmic and inner membrane proteins, which are normally shielded by the outer membrane.  Without the proper controls, this would lead one to conclude wrongly that a non-Omp that reacts with the probe (such as antibodies raised against the protein of interest) is surface exposed.  In addition, when the outer membrane is purified with the intention to identifying Omps, it is hard to distinguish the tiny amounts of Omps from proteins from other bacterial compartments contaminanting the outer membrane preparation.

Despite these technical challenges, several Omp candidates have been proposed, but those proteins are either mired in controversy (TprK, for example) or await experimental confirmation of their surface exposure.  To date, no protein that has been demonstrated unambiguously to be displayed on the exterior of T. pallidum.

The past decade has seen the development of several computer programs that can be used to predict whether a given gene encodes a transmembrane Omp.  The algorithms differ, but all of these programs attempt to identify amino acid sequences that fold into a β-barrel, which forms the core of transmembrane Omps whose 3D structures are known.  The β-barrel forms when an anti-parallel β-sheet rolls into the shape of a barrel.  Most of the β-barrel is embedded in the outer membrane so that the loops connecting the β-strands stick out from the two surfaces of the membrane.  The loops displayed on the external face of the outer membrane would be accessible to antibodies.  The size and composition of the loops vary among different Omps, but all β-strands tend to have alternating hydrophobic amino acids with their nonpolar side chains acid protruding out from the barrel into the hydrophobic interior of the lipid bilayer.  Each β-strand consists of 9-11 amino acid residues and is tilted up to 45° out of the transmembrane axis.  Different β-barrels have as few as 8 and as many as 22 transmembrane β-strands.

The ribbon representation of OmpA, an 8-stranded transmembrane Omps from E. coli, is shown below as one example.

From Figure 1b of Smith et al., 2007.  The N- and C-terminal β-strands are colored brown and blue, respectively.  The side chains of the "aromatic girdle" are shown.

Below, OmpA is unfurled to show the topology of the protein:

From Figure 1a of Smith et al., 2007.  β-strand amino acid residues are depicted as diamonds, and loop residues are depicted as circles.  Alternating hydrophobic amino acid residues within the  β-strand are colored red (aromatic residues of the girdle) and yellow.

Assuming that the rare transmembrane Omps of T. pallidum share the β-barrel structure, the obvious computational approach to finding these Omps would be to run all of the proteins encoded by the T. pallidum genome through one of these programs.  One problem with these programs is that they will pick up a few proteins that are not truly transmembrane Omps.  To minimize this problem, Justin Radolf's group, as reported in the December 2010 issue of Infection and Immunity, ran the 1038 protein-coding sequences of T. pallidum through seven different Omp-predicting programs.  They found that two proteins were predicted by all seven programs to have the β-barrel structure; another four candidates were identified by six programs.

One of the proteins at the top of the list, identified by all seven programs, was TP0326, a BamA homolog encoded by the genomes of many Gram-negative bacteria.  Experiments with other bacteria have shown that BamA is a member of an outer membrane protein complex that assembles other transmembrane Omps into the outer membrane, so it would make sense for T. pallidum to possess such a protein.  BamA itself is thought to be a transmembrane Omp. 

This wasn't the first time that a syphilis researcher has encountered TP0326.  In a study published 11 years ago, before the function of BamA was known, Caroline Cameron and colleagues demonstrated that antibodies raised against TP0326 (also called "Tp92" in their paper) stimulated macrophages to engulf T. pallidum in a process called opsonophagocytosis.  In addition, TP0326 was somewhat effective as a vaccine in the rabbit model of syphilis:  rabbits that had been immunized with TP0326 experienced milder skin lesions than unimmunized rabbits following inoculation of T. pallidum into the skin.  These observations indirectly supported the localization of TP0326 to the outer membrane since opsonophagocytosis and effective vaccination require a target that is accessible on the surface of the spirochete.  However, this earlier work lacked a more direct test such as the indirect immunofluorescence assay to confirm that TP0326 was exposed on the surface.

The problem with the standard two-step indirect immunofluorescence assay is that it is not sensitive enough to detect the rare Omps of T. pallidum.  Therefore, as described in the Infection and Immunity paper, Radolf's group tinkered with the assay and managed to amplify the output signal by adding a third step to the procedure.  To minimize damage to the outer membrane during the centrifugation and washing steps, the spirochetes were encased in gel microdroplets, which protected the delicate outer membrane while allowing antibodies to permeate to probe the T. pallidum surface.

With the modified immunofluorescence assay, the investigators were able to detect surface proteins with syphilitic antibodies for the first time, although only in a small minority of the spirochetes in the field of view lit up with the red color (see figure below).  Presumably, the other spirochetes failed to react with the antibodies because they didn't quite have enough Omp antigens being expressed on their surface (although a more interesting explanation would be that the nonreactive spirochetes had down-regulated their surface Omps).  Regardless of the true explanation, these results indicated that at least some of the antibodies generated by syphilis patients were directed against surface components of T. pallidum.  When the investigators treated the spirochetes with the detergent Triton X100 to intentionally damage the outer membrane, all of the spirochetes glowed, indicating that most of the antibodies targeted proteins beneath the surface of T. pallidum.  As a negative control, they demonstrated that sera from healthy patients failed to react with intact spirochetes.

To keep track of how many spirochetes were damaged by the procedure, the investigators added antibody raised against the periplasmic flagella along with the patient antibodies.  The flagellar antibodies would bind to the spirochetes only if the integrity of the outer membrane was compromised by handling the spirochetes.  The assay was designed so that bound flagellar antibodies would glow green.

From Figure 4 of Cox et al., 2010.   (A) All spirochetes, whether or not they fluoresced, could be seen with darkfield optics (DF).  Spirochetes that bound to antibodies from syphilis patients (HSS) glowed red.   Spirochetes with a disrupted outer membrane reacted with the flagellar antibody (anti-FlaA) and glowed green.  (B) 5.8% of the spirochetes observed were undamaged and reacted with patient antibodies (glowed red but not green).  Another 5.1% were damaged (glowed red and green).  89.0% of the spirochetes failed react with the patient antibodies.  100% of the spirochetes fluoresced when treated with the detergent Triton X100 before adding the antibodies.

With an improved immunofluorescence assay, the investigators were poised to test the proteins at the top of the list for surface exposure.  As I was nearing the end of the paper, I was expecting the authors to describe their test of the BamA homolog TP0326 for surface exposure.  Surprisingly, they ended the paper without testing any of the proteins near the top of the list.

I can only assume that the authors are planning to submit a separate manuscript in the future describing the successful detection of TP0326 or another protein near the top of the list.  But the problem with ending the paper without demonstrating surface localization of even a single protein is that one can question whether even the 3-step immunofluorescence assay is sensitive enough to detect an Omp exposed on the T. pallidum surface.  They did test two proteins lower down on the list that other labs believe are surface exposed (TprK and a fibronectin-binding lipoprotein)  but neither protein was detected on the outer membrane surface by the modified immunofluorescence assay.  So we are left with an assay that certainly has more sensitivity, but is it sensitive enough?

To be continued...(?)

Featured paper

Cox, D.L., Luthra, A., Dunham-Ems, S., Desrosiers, D.C., Salazar, J.C., Caimano, M.J.., and Radolf, J.D. (December 2010).  Surface immunolabeling and consensus computational framework to identify candidate rare outer membrane proteins of Treponema pallidum.  Infection and Immunity 78(12):5178-5194.  DOI: 10.1128/IAI.00834-10

Other references

Radolf, J.D. (June 1995).  Treponema pallidum and the quest for outer membrane proteins.  Molecular Microbiology 16(6):1067-1073.

Cameron, C.E., Lukehart, S.A., Castro, C., Molini, B., Godornes, C., and Van Voorhis, W.C. (April 2000).  Opsonic potential, protective capacity, and sequence conservation of the Treponema pallidum subspecies pallidum Tp92.  Journal of Infectious Diseases 181(4):1401-1413.  DOI: 10.1086/315399

Cox, D.L., Akins, D.R., Porcella, S.F., Norgard, M.V., and Radolf, J.D. (1995).  Treponema pallidum in gel microdroplets:  a novel strategy for investigation of treponemal molecular architecture.  Molecular Microbiology 15(6):1151-1164.

Image source

Smith, S.G.J, Mahon, V., Lambert, M.A., and Fagan, R.P. (August 2007).  A molecular Swiss army knife:  OmpA structure, function and expression.  FEMS Microbiology Letters 273(1):1-11.  DOI: 10.1111/j.1574-6968.2007.00778.x

Congenital syphilis, upward trend (again) in the United States

Syphilis can be deadly if passed from an infected mother to her unborn child. The most recent CDC data show that 6.5% of U.S. infants with congenital syphilis (CS) in 2008 were stillborn or died within 30 days of birth.¹

Newborns with CS who are destined to live begin to show signs of disease within the first few weeks of life.  The main features of CS in early infancy include fever, skin lesions, enlarged liver and spleen, and a chronic runny nose ("snuffles"), which may be tinged with blood.  Bone lesions may lead to Parrot's pseudoparalysis, a condition so painful that the infant will refuse to move the affected extremities.  Ongoing damage to bony tissue may later lead to childhood deformities including saddle nose, sabre shins, and Hutchinson incisors (notched central incisors).  Other late signs of CS include inflammation of the cornea and sudden hearing loss.

The lesions and deformities associated with congenital syphilis are sparked by Treponema pallidum, a spirochete that can cross the placenta from the mother's bloodstream.  The probability of transmission to the fetus depends on how long the mother has been infected with T. pallidum.  The risk of transmission is lower in mothers at later stages of syphilis.  After crossing the placenta, the spirochete invades the fetal organs.  The continuing immune response to persistent T. pallidum infection causes the damage seen in CS.  Early treatment of the mother with penicillin, at least 30 days before delivery, is essential to stop the disease.

The rate of congenital syphilis in the United States has started to creep back up after plummeting over two decades.¹  The incidence of congenital syphilis has gone up from 8.2 cases per 100,000 live births in 2005 to 10.1 in 2008 with most of the increase having occurred in the South.  CS rates in infants born to black mothers have gone up from 26.6 in 2005 to 34.6 per 100,000 live births in 2008 and now account for half of all CS cases.  Since CS is transmitted from mothers with syphilis, CS rates have historically tracked the combined primary and secondary syphilis rate seen in women, which has also started to climb (see figure below).  What factors account for the increased incidence of syphilis?  In one Alabama county, increased syphilis rates in black women were linked to crack cocaine use and the exchange of sex for money or drugs.²  Although more studies are needed to determine whether the same factors are linked to syphilis throughout the South, it should be pointed out that the same factors were associated with the previous syphilis epidemic that peaked in the early 1990s, when there were several thousand yearly cases of CS as opposed to the several hundred seen today.³

Figure from CDC¹

Now that the upward trend in the CS rate has been recognized, public health authorities in partnership with community-based groups must allocate some of their scarce resources to reverse the trend.  With prenatal care and prompt treatment, congenital syphilis can be prevented.

1. Centers for Disease Control and Prevention (CDC) (April 16, 2010). Congenital syphilis - United States, 2003-2008. MMWR Morbidity and Mortality Weekly Report 59(14):413-417.  link

2. Centers for Disease Control and Prevention (CDC) (May 8, 2009).  Primary and secondary syphilis - Jefferson County, Alabama, 2002-2007.  MMWR Morbidity and Mortality Weekly Report 58(17):463-467.  link

3. Nakashima, A.K., Rolfs, R.T., Flock, M.L., Kilmarx, P., and Greenspan, J.R. (Jan-Feb 1996). Epidemiology of syphilis in the United States, 1941-1993.  Sexually Transmitted Diseases 23(1):16-23.  PMID: 8801638

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

Does male circumcision protect against syphilis?

Circumcision has been shown to reduce the risk of men contracting several sexually transmitted infections (STIs). Three randomized controlled trials (RCTs) published over the last few years have demonstrated that removing the foreskin of adult men diminished the risk of HIV infection by at least 50%. An article by Tobian and colleagues in last week's issue of the New England Journal of Medicine revealed a weak protective effect of circumcision against two other infections, herpes simplex virus 2 (HSV-2) and human papilloma virus (HPV), which cause genital herpes and penile warts, respectively. The same study showed no effect of circumcision on acquisition of Treponema pallidum, the agent of syphilis. You can find a nice critical analysis of the study here. Because I am interested in diseases caused by spirochetes, I will focus on the syphilis data.

Tobian et al. conducted two RCTs with similar designs. When the data were combined, they found that 50 of 2083 (2.4%) male adolescents and adults in Uganda who underwent circumcision became infected with T. pallidum over the following 24 month period. Similarly, 45 of 2143 (2.1%) control subjects became infected within the same time period, suggesting that circumcision had no effect on contracting T. pallidum. An editorial in the same journal issue points out that the study may have been underpowered to detect a protective effect (i.e., not enough subjects in the study).

Since the Tobian et al. study failed to give a simple answer to the question, I thought it would be illuminating to look at the older observational studies that examined the effects of male circumcision on syphilis transmission. Fortunately, I found a meta-analysis that compiled data from 14 research papers, most of which described cross-sectional studies.

The meta-analysis presented by Weiss and colleagues revealed a slight protective effect of male circumcision. The relative risks (RRs) along with the 95% confidence intervals (CI) are plotted in the graph. The RR in 11 of the 14 studies were adjusted for potential confounding factors such as age. The summary statistics listed at the bottom of the graph indicate a small protective effect (summary RR, 0.67; 95% CI, 0.54-0.83).

Ideally, all studies included in a meta-analysis would have similar RRs. However, if you look carefully at the graph, you will notice a wide variation in the RRs with some of the 95% confidence intervals failing to overlap. Using standard statistical calculations, the investigators determined that it was unlikely that the variation of the RR among the studies was due to chance (P = 0.01). In other words, differences in how the studies were designed and conducted led to significant variation in the outcomes. Consequently, the authors declared that there was significant heterogeneity among the studies and warned that the summary RR "should be interpreted cautiously."

The authors described one potential source of the heterogeneity. Looking at the plots again, you will note that the Cook and Parker studies demonstrated the largest statistically significant protective effect of male circumcision. Those two studies were conducted in the United States and Australia, respectively, where males are circumcised as infants. In contrast, the two largest studies, authored by Gray and Urassa, showed no effect of circumcision on the risk of becoming infected with T. pallidum. Those studies were conducted in Uganda and Tanzania, respectively, where males are not circumcised until they are adolescents or young adults. Many of the circumcised males examined in the two African studies, which were cross-sectional and case-control studies, could have contracted syphilis before being circumcised. Weiss et al. excluded subjects who were circumcised after their first sexual intercourse or after age eleven, but this information was not available for all studies. This would lead to an underestimate of the protective effects of circumcision.

You may look at the large protective effects of infant circumcision observed in the U.S. and Australian studies (RR = 0.25 and 0.19, respectively) and conclude that mass infant circumcision would be beneficial (at least for protection against syphilis). However, both studies involved men visiting STD clinics, and the results may not apply to the general population in those countries.

References

Tobian, A.A.R., Serwadda, D., Quinn, T.C., Kigozi, G., Gravitt, P.E., Laeyendecker, O., Charvat, B., Ssempijja, V., Riedesel, M., Oliver, A.E., Nowak, R.G., Moulton, L.H., Chen M.Z., Reynolds, S.J., Wawer, M.J., Gray, R.H. (2009). Male circumcision for the prevention of HSV-2 and HPV infections and syphilis. The New England Journal of Medicine 360(13):1298-1309.

Golden M.R. and Wasserheit, J.N. (2009) Prevention of viral sexually transmitted infections--foreskin at the forefront (Editorial). The New England Journal of Medicine 360(13):1349-1350.

Weiss, H.A., Thomas, S.L., Munabi, S.K., and Hayes, R.J. (2006). Male circumcision and risk of syphilis, chancroid, and genital herpes: a systematic review and meta-analysis. Sexually Transmitted Infections 82(2):101-109.

The origin of syphilis: a phylogenetic approach

K.N. Harper, P.S. Ocampo, B.M. Steiner, R.W. George, M.S. Silverman, S. Bolotin, A. Pillay, N.J. Saunders, and G.J. Armelagos. (2008). On the origin of the treponematoses: A phylogenetic approach. PLoS Neglected Tropical Diseases 2(1):e148.

The first recorded outbreak of syphilis occurred in Europe in 1495, a few years after Columbus sailed the ocean blue. Was syphilis a New World disease newly introduced into Europe by Columbus and his crew, or was it an Old World disease that simply was not noticed until 1495? A study from Harper and colleagues published last January described a molecular genetic analysis that may have yielded important clues hidden within the genetic material of Treponema pallidum.

The authors first examined the evolutionary relationships among Treponema pallidum strains from subspecies pertenue, endemicum, and pallidum, which are responsible for the diseases yaws, bejel, and syphilis, respectively. The small collection of strains or their DNA was obtained from different patients throughout the past century. The tree illustrated below (Figure 3 of Harper et al.) was constructed from the alignment of 70 SNPs (single nucleotide polymorphisms) and 12 indels (insertions/deletions). The branching pattern indicates that pertenue emerged the earliest. Subspecies endemicum later emerged from pertenue, and pallidum, the agent of syphilis, arose most recently.
The authors also obtained scrapings from yaws skin lesions on two aboriginal children living deep in the rainforests of Guyana. Since these children were members of a population that had been living for generations with minimal contact with the rest of the world, these pertenue strains may be closely related to those present in the Americas before the European explorers arrived. Unfortunately, the samples collected by the authors had degraded extensively by the time the DNA was extracted for analysis. Consequently, the two Guyanan strains could not be included in the phylogenetic analysis shown above; only regions encompassing 17 of the 70 SNPs could be sequenced from the degraded DNA. Nevertheless, they went ahead and aligned the 17 nucleotides with those from the strains used to construct the phylogenetic tree. The alignment revealed that among the nonveneral strains (pertenue and endemicum), only the Guyanan strains had as many as 4 nucleotides that were identical to those of the pallidum strains.

The world map illustrated below (Figure 4 of Harper et al.) depicts the path of sequence changes in the 4 SNPs among the Treponema strains. The dots mark the geographic source of each strain used in the analysis. The red and green colors demark areas of endemicity of the nonvenereal diseases yaws and bejel, respectively, around the year 1900. The map shows that T. pallidum first appeared as pertenue in the Old World and gave rise to the endemicum subspecies, which migrated with humans to the Middle East and Europe. The Old World pertenue or endemicum strain then eventually gave rise to the New World pertenue strain as humans crossed the Bering Land Bridge and spread throughout the Americas. The sequence identity of the Guyanan strains with the pallidum strains at all 4 positions is consistent with the New World strain being introduced back into the Old World as a progenitor of today's syphilis-causing pallidum strains, which are now found worldwide. Clinical evidence also supports the New World model: the nonvenereal skin lesions in the Guyanan yaws patients resembled syphilis chancres rather than the typical skin lesions found with yaws.I do not believe that the results presented in the paper support the New World origin of syphilis. As Harper et al. state in the Discussion of the paper, the close evolutionary relationship of the South American pertenue strains with the pallidum (syphilis) strains is based on a mere four nucleotides. Still, the authors concluded that pallidum arose from a descendant of the New World pertenue that was brought to Europe by Columbus. However, the results do not rule out the possibility that pallidum and New World pertenue strains evolved independently from a common ancestor, such as the "unknown" strain illustrated in the map. The New World strains would need to be included in the phylogenetic tree to distinguish the two possibilities. It was unfortunate that the entire set of 70 SNPs and 12 indels could not be examined in the Guyanan strains.