Tuesday, February 15, 2011

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 pallidumInfection 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

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