Spirochete research: 2012 in review

Here are some of my favorite spirochete papers from 2012.  Direct access to all research articles (some behind a paywall) is provided via the DOI links.  Where present, the links above the citations lead to my blog posts about the studies.

PATHOGENESIS

Two distinct regions of the Borrelia burgdorferi BBK32 lipoprotein sequentially mediate binding to the vessel wall in vivo during escape of the spirochete from the bloodstream.

• Moriarty TJ, Shi , Lin Y-P, Ebady R, Zhou H, Odisho T, Hardy P-O, Salman-Dilgimen A, Wu J, Weening EH, Skare JT, Kubes P, Leong J, and Chaconas G (December 2012).  Vascular binding of a pathogen under shear force through mechanistically distinct sequential interactions with host macromolecules.  Molecular Microbiology 86(5):1116-1131.  DOI: 10.1111/mmi.12045

The Leptospira interrogans LigB protein protects the spirochete from complement by capturing complement regulatory proteins.

• Castiblanco-Valencia MM, Fraga TR, da Silva LB, Monaris D, Abreu PAE, Strobel S, Jozsi M, Isaac L, and Barbosa AS (March 15, 2012).  Leptospiral immunoglobulin-like proteins interact with human complement regulators factor H, FHL-1, FHR-1, and C4BP.  The Journal of Infectious Diseases 205(6):995-1004.  DOI: 10.1093/infdis/jir875
• Choy HA (July 2012).  Multiple activities of LigB potentiate virulence of Leptospira interrogans: inhibition of alternative and classical pathways of complement.  PLoS One 7(7):e41566. DOI: 10.1371/journal.pone.0041566

A B. burgdorferi lipase with hemolytic activity in vitro:

• Shaw DK, Hyde JA, and Skare JT (January 2012).  The BB0646 protein demonstrates lipase and haemolytic activity associated with Borrelia burgdorferi, the aetiological agent of Lyme disease.  Molecular Microbiology 83(2):319-334.  DOI: 10.1111/j.1365-2958.2011.07932.x

TRANSMISSION

Borrelia burgdorferi needs the alternative sigma factor RpoS to flee from the tick's midgut

• Dunham-Ems SM, Caimano MJ, Eggers CH, and Radolf JD (February 2012).  Borrelia burgdorferi requires the alternative sigma factor RpoS for Dissemination within the vector during tick-to-mammal transmission.  PLoS Pathogens 8(2):e1002532.  DOI: 10.1371/journal.ppat.1002532

MOTILITY

Video microscopy of B. burgdorferi swimming around in gelatin and mouse tissue:

• Harman MW, Dunham-Ems SM, Caimano MJ, Belperron AA, Bockenstedt LK, Fu HC, Radolf JD, and Wolgemuth CW (February 21, 2012).  The heterogeneous motility of the Lyme disease spirochete in gelatin mimics dissemination through tissue.  Proceedings of the National Academy of Sciences USA 109(8):3059-3064.  DOI: 10.1073/pnas.1114362109

L. interrogans sheath protein homologs that are not needed for flagellar sheath formation:

• Lambert A, Picardeau M, Haake DA, Sermswan RW, Srikram A, Adler B, and Murray GA (June 2012).  FlaA proteins in Leptospira interrogans are essential for motility and virulence but are not required for formation of the flagellum sheath.  Infection and Immunity 80(6):2019-2025.  DOI: 10.1128/IAI.00131-12

ULTRASTRUCTURE

A close look at the ultrastructure of Leptospira without the artifacts generated by conventional electron microscopy:

• Raddi G, Morado DR, Yan J, Haake DA, Yang XF, and Liu J (March 2012).  Three-dimensional structures of pathogenic and saprophytic Leptospira species revealed by cryo-electron tomography.  Journal of Bacteriology 194(6):1299-1306.  DOI: 10.1128/JB.06474-11

METAL TOXICITY

B. burgdorferi BicA, a protein that protects the spirochete from the toxic effects of copper and iron:

• Wang P, Lutton A, Olesik J, Vali H, and Li X (December 2012).  A novel iron- and copper-binding protein in the Lyme disease spirochaete.  Molecular Microbiology 86(6):1441-1451.  DOI: 10.1111/mmi.12068

ANTIBIOTIC THERAPY

Inflammatory spirochete debris left behind following antibiotic treatment for Lyme disease

• Bockenstedt LK, Gonzalez DG, Haberman AM, and Belperron AA (July 2, 2012).  Spirochete antigens persist near cartilage after murine Lyme borreliosis therapy.  The Journal of Clinical Investigation 122(7):2652-2660.  DOI: 10.1172/JCI58813

A critical analysis of a study that demonstrated persistence of B. burgdorferi in infected rhesus monkeys that were treated with antibiotics:

• Wormser GP, Baker PJ, O'Connell S, Pachner AR, Schwartz I, and Shapiro ED (July 2012).  Critical analysis of treatment trials of rhesus macaques infected with Borrelia burgdorferi reveals important flaws in experimental design.  Vector-borne and Zoonotic Diseases 12(7):535-538.  DOI: 10.1089/vbz.2012.1012
• Embers ME, Barthold SW, Borda JT, Bowers L, Doyle L, Hodzic E, Jacobs MB, Hasenkampf NR, Martin DS, Narasimhan S, Phillippi-Falkenstein KM, Purcell JE, Ratterree MS, and Philipp MT (January 2012).  Persistence of Borrelia burgdorferi in rhesus macaques following antibiotic treatment of disseminated infection.  PLoS One 7(1):e29914.  DOI: 10.1371/journal.pone.0029914

A tale of two more studies: topical antibiotics applied to tick bites to prevent Lyme disease

• Wormser GP, Daniels TJ, Bittker S, Cooper D, Wang G, and Pavia CS (March 15, 2012).  Failure of topical antibiotics to prevent disseminated Borrelia burgdorferi infection following a tick bite in C3H/HeJ mice.  The Journal of Infectious Diseases 205(6):991-994.  DOI: 10.1093/infdis/jir382

DIAGNOSTICS

Not so golden?  Microscopic agglutination test for diagnosis of leptospirosis

• Limmathurotsakul D, Turner EL, Wuthiekanun V, Thaipadungpanit J, Suputtamongkol Y, Chierakul W, Smythe LD, Day NPJ, Cooper B, and Peacock SJ (August 1, 2012).  Fool’s gold: Why imperfect reference tests are undermining the evaluation of novel diagnostics: A reevaluation of 5 diagnostic tests for leptospirosis.  Clinical Infectious Diseases 55(3):322-331.  DOI: 10.1093/cid/cis403

ECOLOGY

Do nonspiral spirochetes help clean our environment?

• Caro-Quintero A, Ritalahti KM, Cusick KD, Loffler FE, and Konstandtinidis KT (May/June 2012).  The chimeric genome of Sphaerochaeta: Nonspiral spirochetes that break with the prevalent dogma in spirochete biology.  mBio 3(3):e00025-12.  DOI: 10.1128/mBio.00025-12
• Ritalahti KM, Justicia-Leon SD, Cusick KD, Ramos-Hernandez N, Rubin M, Dornbush J, and Loffler FE (January 2012).  Sphaerochaeta globosa gen. nov., sp. nov. and Sphaerochaeta pleomorpha sp. nov., free-living, spherical spirochetes.  International Journal of Systematic and Evolutionary Microbiology 62(Pt 1):210-216.  DOI: 10.1099/ijs.0.023986-0

 

BIOFILM

Biofilms of the Lyme disease spirochete

• Sapi E, Bastian SL, Mpoy CM, Scott S, Rattelle A, Pabbati N, Poruri A, Burugu D, Theophilus PAS, Pham TV, Data A, Dhaliwal NK, MacDonald A, Rossi MJ, Sinha SK, and Luecke DF (October 2012).  Characterization of biofilm formation by Borrelia burgdorferi in vitro.  PLoS One 7(10):e48277.  DOI: 10.1371/journal.pone.0048277

 

HISTORY

Looking for the syphilis spirochete in ancient bones

• Montiel R, Solorzano E, Diaz N,  Alvarez-Sandoval BA, Gonzalez-Ruiz M, Canadas MP, Simoes N, Isidro A, and Malgosa A (May 2012).  Neonate human remains: A window of opportunity to the molecular analysis of syphilis.  PLoS One 7(5):e36371.  DOI: 10.1371/journal.pone.0036371

Presenting flawed studies directly to the public to bypass the scientific peer-review process:

• Armelagos GJ, Zuckerman MK, and Harper KN (March 2012).  The science behind pre-Columbian evidence of syphilis in Europe: research by documentary.  Evolutionary Anthropology 21(2):50-57.  DOI: 10.1002/evan.20340

REVIEWS

Here are two excellent review articles that appeared during the past year:

• Radolf JD, Caimano MJ, Stevenson B, and Hu LT (February 2012).  Of ticks, mice and men: understanding the dual-host lifestyle of Lyme disease spirochaetes.  Nature Reviews Microbiology 10(2):87-99.  DOI: 10.1038/nrmicro2714
• Charon NW, Cockburn A, Li C, Liu J, Miller KA, Miller MR, Motaleb MA, and Wolgemuth CW (2012).  The unique paradigm of spirochete motility and chemotaxis.  Annual Reviews of Microbiology 66:349-370.  DOI: 10.1146/annurev-micro-092611-150145

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

Still no solid evidence for the Old World origin of syphilis

The first recorded syphilis epidemic flared up in war-torn Naples in 1494, only two years after Columbus discovered the New World.  From there syphilis spread throughout Europe.  Ever since then, controversy has raged about the origin of syphilis.  A popular belief is that Columbus's crew got infected in the New World and brought the spirochete back to Europe, where they transmitted the disease to others while serving as mercenaries during the first Italian War.  The competing pre-Columbian hypothesis asserts that syphilis was always present in the Old World yet wasn't recognized until 1494.

Among the treponemes, Treponema pallidum subspecies pallidum, the agent of syphilis, is the only one that's sexually transmitted.  Other treponemal diseases include yaws and endemic syphilis, each caused by a genetically distinct subspecies of Treponema pallidum.  The exuberant immune response to T. pallidum during the tertiary stage of treponemal disease leaves marks on the bones.  Historical cases of treponemal disease can therefore be identified by looking for these skeletal lesions.  Diagnosing treponemal infection in this way is tricky because a number of diseases cause similar lesions.  Nevertheless, bone deformities specific to treponemal diseases do exist, most famously the worm-eaten appearance of caries sicca.  Unfortunately, skeletal lesions cannot be used to reliably distinguish syphilis from the other treponemal diseases, so I will be speaking of treponemal disease instead of syphilis when discussing the skeletal evidence.

The findings of treponemal lesions in pre-1492 skeletal remains from the Old World would upend the Columbian hypothesis.  A number of claims of pre-Columbian treponemal lesions on Old World skeletons have appeared in the scientific literature, with some garnering widespread media attention.  One example is the findings from the excavation of an English friary at Hull Magistrate's Court, which was a subject of an episode of the PBS series Secrets of the Dead.  Skeletal remains from four individuals unearthed at the site had evidence of treponemal disease.  The site where the bones were found was dated to 1300-1450.

The Syphilis Enigma, part 1 of 4


The Syphilis Enigma, part 2 of 4


The Syphilis Enigma, part 3 of 4



The Syphilis Enigma, part 4 of 4


Harper and colleagues recently took another look at the published claims of pre-Columbian treponemal disease in Old World skeletal remains.  Their work came out last month in the Yearbook of Physical Anthropology, the supplement to the Journal of Physical Anthropology  They scrutinized the data in the 54 published reports with a standardized set of criteria for diagnosing treponemal disease and radiocarbon dating the bones.  There were two parts to their analysis.  First, they looked at the description of the skeletal lesions (including photographs, when available) to make sure they were the type caused solely by treponemal diseases.  Second, they looked at method used to date the bones.  In turns out that all 54 reports were flawed in some manner.  In many reports, diagnosis of treponemal disease was based on the types of skeletal lesions that could have been induced by other diseases.  In others, the bones were dated indirectly by archeological methods instead of directly by radiocarbon dating the bones.  Or worse, details of the dating method were sometimes omitted.

Among the 11 specimens for which the diagnosis of treponemal disease was deemed to be correct by the authors, two were dated by radiocarbon methods to the pre-Columbian years.  The time of death for the two individuals were 1424-1479 for a specimen unearthed in Safed, Israel and 1426-1486 for one dug up at the Church of St. Helen-on-the-wall in York, England.  At first glance, these specimens appear to undermine the Columbian hypothesis.  However, radiocarbon dating assumes that the ratio of ¹⁴C ("new" carbon, which decays at a known rate) to ¹²C ("old" carbon) remains constant in the environment and by extension living things, which exchange carbon with the environment by eating and breathing.  Slight deviations of the ratio may throw off the calculated date by hundreds of years.  One factor that must be accounted for is the "marine reservoir effect," which could skew the isotope ratio in humans who consume seafood.  The problem is that water near the surface mixes with deeper water, which reflects the higher proportion of old carbon from the material coming off the ocean floor.  The carbon in the air does not mix quickly enough with the carbon in the water to equalize the carbon ratios.  Therefore, marine organisms do not contain the same carbon ratio as land organisms.  Humans who consume seafood will have a slightly lower proportion of new carbon than expected, making bones from seafood consumers appear older than they really are if no correction is made.  When the correction is made for the marine reservoir effect, the new dates for the specimens become 1424-1953 for the Safed specimen and 1421-1669 for the St. Helen-on-the-Walls specimen.  An additional three Old World specimens were dated to the pre-Columbian period by radiocarbon dating, corrected for the marine reservoir effect.  However, the specimens lacked lesions specific for treponemal disease.  In sum, proper dating of the skeletal remains revealed that none of the Old World skeletons bearing treponemal marks could have originated from the pre-Columbian era.  There is still no convincing evidence for the Old World origin of syphilis.

So can we now reject the pre-Columbian hypothesis?  The authors make an interesting comment about this.

The trope that absence of evidence is not the same as evidence of absence will be invoked by some. While it is true that a working hypothesis may be falsified at any time, at what point does the absence of skeletal evidence of pre-Columbian treponemal disease in the Old World become compelling? After all, the best proof of a theory is failure to disprove it, and we find that despite intense research interest, credible evidence disproving the Columbian Hypothesis is lacking.

The "intense research interest" includes past examination of tens of thousands of skeletons in continental Europe and North Africa and 50,000 in England alone, with none yielding convincing evidence of pre-Columbian treponemal disease.  The authors do point out that skeletal remains from vast regions of sub-Saharan Africa and Asia have not been well-studied.  For this reason, it may be too soon to lay the pre-Columbian hypothesis to rest.


ACKNOWLEDGEMENTS:  A big thanks to Molly Zuckerman for alerting me to her group's work and for sending me the article.

References
Harper, K.N., Zuckerman, M.K., Harper, M.L., Kingston, J.D., & Armelagos, G.J. (2011). The origin and antiquity of syphilis revisited: an appraisal of Old World pre-Columbian evidence for treponemal infection American Journal of Physical Anthropology, 146 (S53), 99-133 DOI: 10.1002/ajpa.21613

von Hunnius, T.E., Roberts, C.A., Boylston, A., & Saunders, S.R. (2006). Histological identification of syphilis in pre-Columbian England American Journal of Physical Anthropology, 129 (4), 559-566 DOI: 10.1002/ajpa.20335

Related posts by other bloggers

• Revisiting Syphilis in the Old World in Bones Don't Lie
• The French Disease, the Italian Disease, the Christian Disease--the New World Disease? in The Loom

Related posts in Spirochetes Unwound

• The origin of syphilis: a phylogenetic approach

Serologic testing for syphilis: missing the point

You may have seen several news sources touting the recent CDC finding that nearly one in five positive reactions with a newer syphilis test are wrong.  These headlines may grab the reader's attention, but the press took the finding out of context and failed to deliver the real message that the CDC was trying to convey.  Worse, the press reports may needlessly confuse and worry those who are being treated for syphilis.

Serological tests for syphilis are grouped into two categories.  Nontreponemal tests such as the VDRL and RPR are based on antibody generated against the lipid cardiolipin.  Presumably cardiolipin is released from damaged tissue in syphilis patients and gets incorporated into the membrane of Treponema pallidum.  The reason that these tests are "nontreponemal" is that antibodies to cardiolipin accompany many other conditions.  On the other hand, treponemal tests use T. pallidum proteins or even the entire spirochete as antigen to detect antibodies against the spirochete.  Although the classic treponemal tests such as the FTA-ABS (fluorescent treponemal antibody-absorption) and TP-PA (Treponema pallidum particle agglutination) are still used, the newer automated EIA (enzyme immunoassay) and CIA (immunochemiluminescence) treponemal tests enable clinical laboratories to rapidly screen a large number of sera.

The traditional approach to syphilis testing is to first screen the patient's serum with a nontreponemal test.  Since nontreponemal tests can give false positive reactions, reactive sera are retested with one of the treponemal tests.  However, the low cost of executing the automated treponemal tests have led some high-volume clinical laboratories to reverse the order of the assays:  they screen with the EIA/CIA treponemal test and confirm positive results with a nontreponemal test.  The CDC report in the Morbidity and Mortality Weekly Report deals with this so-called "reverse sequence" testing.

So where did the "nearly one in five" figure come from?  From 2006 to 2010, five large clinical laboratories screened 140,176 sera specimens with the reverse sequence procedure.  Of the 4,834 reactive with the EIA/CIA treponemal test, 2,743 gave negative results with the nontreponemal RPR test.  When the samples that gave discrepant results were tested further with one of the classic treponemal tests, 866 of the 2,743 samples were negative.  Overall, among the 4,834 samples that were reactive with the newer treponemal test, 866 or 18% were nonreactive with two subsequent tests.  These 866 were assumed to be false positives.

The news media pounced on the 18% figure and declared that hundreds may have been given antibiotics to treat a disease that they didn't have.  But they ignored the fact that doctors don't diagnose syphilis on the basis of a single lab test.  It is standard practice to perform a second test when the first comes back positive and to do even a third one if warranted.  Doctors also take into account the physical exam and the sexual and medical history of the patient before making the decision to treat with antibiotics.

Here's how the CDC responded to the assertion that those among the 18% may have been falsely diagnosed and treated unnecessarily with antibiotics:

There are two problems with this assertion. First, the current report does not document whether or not treatment was provided. Second, in those cases where treatment was provided, it may have been justified based on sexual risk and findings on clinical evaluation. It is also important to note that syphilis is not diagnosed on the basis of a single blood test. Many labs routinely will do additional testing when the first test is positive, without notifying the patient. Doctors diagnose syphilis after considering at least two syphilis tests, the patient's history, the physical exam, and a review of past syphilis test results. The MMMR analysis, while important, does not allow us to conclude that the newer tests led to inaccurate syphilis diagnosis or inappropriate treatment.

So what was the message that the CDC was trying to communicate to readers of the MMMR report?  Their intention was to provide guidance in the management of cases for which the reverse sequence screening is performed instead of the traditional sequence, which is still recommended by the CDC.  Specifically, when conflicting results occur (positive with the treponemal test, negative with the nontreponemal test), a third test should be done with the TP-PA.  (The CDC does not recommend the FTA-ABS because it is less specific and probably less sensitive.)  A positive reaction with the TP-PA indicates past or present syphilis; a negative reaction indicates that syphilis is unlikely.  As always, the clinical observations and medical history of the patient should also be considered in making an informed treatment decision.


Reference

Centers for Disease Control and Prevention (February 11, 2011).  Discordant results from reverse sequence syphilis screening -- five laboratories, United States, 2006-2010.  MMMR. Morbidity and Mortality Weekly Report 60(5):133-137.  link

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.