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Infection and Immunity, March 2003, p. 1200-1208, Vol. 71, No. 3
0019-9567/03/$08.00+0     DOI: 10.1128/IAI.71.3.1200-1208.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Polymorphic Membrane Protein H Has Evolved in Parallel with the Three Disease-Causing Groups of Chlamydia trachomatis

The Department of Medicine, Division of Infectious Diseases, Indiana University School of Medicine, Indianapolis, Indiana 46202

Received 2 July 2002/ Returned for modification 26 September 2002/ Accepted 2 December 2002

	ABSTRACT

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Chlamydia trachomatis is a human pathogen causing trachoma, urogenital disease, and lymphogranuloma venereum (LGV). A family of nine polymorphic membrane protein genes (pmpA to pmpI), resembling autotransporter proteins, has recently been discovered in C. trachomatis. pmp genes are large and predicted to be outer membrane proteins. We hypothesized that they would contain useful nucleotide sequence variability for epidemiologic studies. Since sequence information is available only for serovars D and L2, we sought to determine the amount of diversity within an individual pmp gene among serovars. We used restriction fragment length polymorphism (RFLP) analysis as a primary screen to assess the amount of sequence divergence among the pmp genes for serovars A to L3 of C. trachomatis. RFLP analysis showed little variation for some of the genes, such as pmpA, but substantial variation in others, such as pmpI. pmpH and pmpE yielded RFLP patterns that clustered the 15 serovars into ocular, urogenital, and LGV groups, and both proteins have been localized to the outer membrane. Therefore, we chose to sequence pmpE, pmpH, and pmpI from each of the 15 serovars. Evolutionary analysis showed three distinct divergence patterns. PmpI was least variable, resulting in an ambiguous evolutionary pattern. PmpE showed a high degree of diversity in the ocular strains compared to the other strains. Finally, the evolution of PmpH shows three groups that reflect disease groups, suggesting this protein may play a role in pathogenesis.

	INTRODUCTION

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Chlamydia trachomatis is an obligate intracellular human pathogen that causes trachoma, urogenital disease, and lymphogranuloma venereum (LGV). The strains of C. trachomatis are divided into at least 18 different serovars based on immunological cross-reactivity of the major outer membrane protein (MOMP) (30, 31, 32). Additionally, the serovars of C. trachomatis are classified into two groups, the trachoma biovar and the LGV biovar, based upon differences in disease presentation and in vitro cell growth characteristics. The trachoma biovar includes serovars that cause ocular trachoma (A, B, Ba, and C) and serovars that cause urogenital disease (D through K, Da, and Ia), while the LGV biovar includes serovars L1, L2, L2a, and L3.

A superfamily of genes encoding predicted polymorphic membrane proteins (pmp) was discovered as part of the Chlamydia genome project (16, 23, 26). Several proteins encoded by these genes had previously been investigated in C. psittaci (6, 11, 12, 20, 25). Nine genes are found in C. trachomatis (pmpA to pmpI) (26). The Pmp proteins are large proteins, 90 to 187 kDa in mass, and they have no obvious homologs in nonchlamydial species (29). Transcription of all nine genes has been shown for both biovars in serovars D and L2 (19). Of the nine genes, all but pmpA have an apparent signal peptide leader sequence and are predicted outer membrane proteins (13). While expression has not been verified for all nine proteins in all serovars, expression of PmpE, G, and H, and localization of these three proteins to the outer membrane of C. trachomatis serovar L2 has been documented (21, 28).

The pmp genes are highly diverse in nucleotide sequence. None of the pmp genes are more than 52% similar to each other (13). However, the amount of sequence diversity within an individual pmp gene among the different serovars has not been investigated since sequence information is available only for serovars D and L2. Considering that the Pmp proteins are large, and thought to be expressed on the outer membrane (28, 29), we hypothesized that pmp genes might contain useful variability for epidemiologic studies. Currently, the MOMP gene serves as a molecular marker in epidemiologic studies because of its high degree of sequence variability. However, the MOMP gene for some serovars, like E and F, exhibits a high degree of conservation at the nucleotide level, decreasing its usefulness in epidemiologic studies (9, 27). Other markers, like the pmp, could improve discrimination between strains. Therefore, we examined the pmp genes as possible molecular epidemiologic markers by determining the amount of sequence diversity within the pmp genes among serovars.

We used restriction fragment length polymorphism (RFLP) analysis as an initial screen to assess the amount of sequence variability among the pmp genes of 15 serovars of C. trachomatis. RFLP analysis showed limited to no variation among serovars for some of the pmp genes, such as pmpA, but substantial variation was seen for other genes, such as pmpI. Interestingly, pmpH yielded RFLP patterns that clustered the 15 serovars into ocular, urogenital, and LGV disease groups. The RFLP patterns for pmpE and pmpF also showed potential segregation of serovars by disease group. However, unlike PmpE, expression of PmpF has not been demonstrated. Therefore, we chose to sequence pmpE, pmpH, and pmpI from 15 serovars of C. trachomatis. We chose pmpE and pmpH because of their provocative RFLP patterns and demonstration of their surface location and pmpI because of its apparent nucleotide diversity.

	MATERIALS AND METHODS

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Strains and isolates. Elementary bodies (EBs) were partially purified (22) by differential centrifugation followed by density gradient centrifugation in Percoll from the following laboratory strains: A/571-B/OT, B/TW-5/OT, Ba/Ap-2/OT, C/TW-3/OT, D/UW-3/Cx, E/UW-5/Cx, F/UW-6/Ur, G/UW-57/Cx, H/UW-4/Cx, I/UW-12/Ur, J/UW-36/Cx, K/UW-53/Cx, L1/440, L2/434, and L3/404.

DNA extraction. DNA was extracted from purified EBs by using previously described methods (27). The only modification was the addition of dithiothreitol (4 mM final concentration) before boiling to assist in disruption of EB outer membrane proteins. DNA from purified EBs was resuspended into 50 µl of Tris buffer (10 mM Tris-Cl [pH 8.5]).

pmp PCR amplification. The pmp genes were amplified from C. trachomatis serovars A to L3 by using PCR primers (Table 1) based on the published serovar D sequences. We were unable to amplify the pmpB gene from any serovar after trying three separate sets of primers. All PCR products were of the expected sizes based on the published serovar D genes (26).

RFLP analysis. PCR products were purified with the QIAquick PCR purification kit (QIAGEN). Products were eluted from QIAquick columns into 50 µl of elution buffer (10 mM Tris [pH 8.5]). Restriction endonuclease reactions were carried out according to the manufacturer's instructions. Reaction mixtures were 10 µl in volume and consisted of 5 µl of pmp PCR product and 1 to 5 U of enzyme in a 1x reaction buffer. Computer-generated restriction digests of the pmp genes from serovar D (Clone Manager, version 4.01; Scientific and Educational Software) were used to determine which enzymes would be appropriate for RFLP analysis and included CfoI, BamHI, HinfI, TaqI, and Tru9I (all from Roche Molecular Biochemicals). PCR products were digested with at least two different restriction endonucleases that cleaved from 3 to 19 times within each gene. The restriction products were then electrophoresed through 2 to 4% agarose gels, stained with ethidium bromide, and digitally photographed with a UV transilluminator.

DNA sequencing. PCR products for pmpE, pmpH, and pmpI were each amplified in duplicate and pooled for sequence analysis to insure sequence fidelity. All sequencing reactions were done with Big Dye terminator chemistry and an ABI Prism 310 automated DNA sequencer (Applied Biosystems, La Jolla, Calif.). To obtain full-length sequences from pmpE, pmpH, and pmpI, several internal primers were used in addition to the PCR amplification primers (Table 2). Sequences were collected from both strands in order to resolve ambiguities and ensure consistency. In addition, electropherograms were manually checked for computer miscalls.

The primers used to amplify pmpE extended from nucleotide 80 of the coding region to nucleotide 2881 based on the serovar D pmpE gene. Therefore, the first 79 bases and the last 15 bases are not included in the alignment. The sequence alignment for pmpE contained 2,811 nucleotide sites (nucleotides plus alignment gaps) and 936 amino acid sites (amino acids plus alignment gaps).

The primers used to amplify pmpH extended from nucleotide 76 of the coding region to nucleotide 3051 (terminal stop codon) based on the serovar D pmpH gene. Therefore, the first 75 bases are not included in the alignment. The sequence alignment for pmpH contained 2,991 nucleotide sites (nucleotides plus alignment gaps) and 996 amino acid sites (amino acids plus alignment gaps).

The primers used to amplify pmpI extended from nucleotide 73 of the coding region to nucleotide 2610 based on the serovar D pmpI gene. Therefore, the first 72 bases and the last 27 bases are not included in the alignment. The sequence alignment for the pmpI gene contained 2,538 nucleotides and 846 amino acids. There were no in/del events in pmpI.

Nucleotide and amino acid comparisons. After sequences were aligned, nucleotide and amino acid sequences were compared manually in a pairwise fashion for differences. Contiguous in/dels at the nucleotide and amino acid levels were counted as individual differences, i.e., a deletion of six nucleotides between two pairs was counted as six differences rather than one deletion event, and the resulting two-amino-acid deletion was counted as two deletions rather than as one deletion event.

Phylogenetic analysis. (i) Nucleotide sequences. Because the 5' and 3' terminal sequences of the pmp genes are primer defined, those regions were excluded from the phylogenetic analyses. Consequently, 2,783 of 2,811 nucleotides were compared for pmpE, 2,940 of 2,991 nucleotides were compared for pmpH, and 2,493 of 2,538 nucleotides of pmpI were compared.

All phylogenetic and evolutionary analyses were conducted with MEGA (molecular evolutionary genetics analysis), version 2.1 (17). Once aligned, the corrected nucleotide divergence of sequences was calculated in MEGA, incorporating the Kimura two-parameter correction for multiple substitutions at a single site. The branching pattern of taxa was estimated from the distance matrix by the neighbor-joining method (24) as implemented in MEGA. A cladistic reconstruction of the phylogenetic relationships among taxa also was performed by using maximum parsimony as implemented in MEGA with close-neighbor interchange. Confidence levels for the branching pattern were estimated by a bootstrap resampling of the data. Bootstrap values for the trees were obtained from a consensus tree based on 1,000 replications. Although pmp genes have been identified in C. trachomatis, Chlamydiapneumoniae, and Chlamydia psittaci, no homologues exist for C. trachomatis pmp genes. Therefore, an appropriate outgroup could not be identified, and the trees were not rooted.

(ii) Amino acid sequences. Because 5' and 3' terminal sequences of the pmp genes are primer defined, 927 of 936 amino acids of PmpE, 974 of 996 amino acids of PmpH, and 831 of 846 amino acids of PmpI were included in the analyses.

All phylogenetic and evolutionary analyses were conducted using MEGA, version 2.1 (17). Phylogenetic reconstructions based on the inferred amino acid sequences were performed by using both distance and parsimony methods as implemented in MEGA. As with the nucleotide data, bootstrap values for the trees were obtained from a consensus tree based on 1,000 randomly generated datasets and the trees were not rooted.

Nucleotide sequence accession numbers. Nucleotide sequences determined as part of this study can be found using the following GenBank accession numbers (pmpE, pmpH, and pmpI, respectively): A/571-B/OT, no. AY184140, AY184155, and AY184170; B/TW-5/OT, no. AY184141, AY184156, and AY184171; Ba/Ap-2/OT, no. AY184142, AY184157, and AY184172; C/TW-3/OT, no. AY184143, AY184158, and AY184173; D/UW-3/Cx, no. AY184144, AY184159, and AY184174; E/UW-5/Cx, no. AY184145, AY184160, and AY184175; F/UW-6/Ur, no. AY184146, AY184161, and AY184176; G/UW-57/Cx, no. AY184147, AY184162, and AY184177; H/UW-4/Cx, no. AY184148, AY184163, and AY184178; I/UW-12/Ur, no. AY184149, AY184164, and AY184179; J/UW-36/Cx, no. AY184150, AY184165, and AY184180; K/UW-53/Cx, no. AY184151, AY184166, and AY184181; L1/440, no. AY184152, AY184167, and AY184182; L2/434, no. AY184153, AY184168, and AY184183; and L3/404, no. AY184154, AY184169, and AY184184. Previously published nucleotide sequences for pmpE, pmpH, and pmpI from serovar D/UW3 can be found at the Chlamydia genome website (http://chlamydia-www.berkeley.edu:4231/).

	RESULTS

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RFLP analysis shows differing degrees of variation within the pmp gene family. RFLP results are summarized in Table 3. Serovars that are grouped within parentheses in the table had the same RFLP pattern for the particular enzyme listed. For example, digestion of pmpA with TaqI produced two RFLP patterns, dividing the 15 serovars into two RFLP groups. One group consisted of serovars J and K while the remaining serovars had a different RFLP pattern. Interestingly, pmpA, the only pmp gene without a predicted leader peptide sequence, exhibited the least amount of variation, with two of the three enzymes demonstrating no differences among serovars. Variation in RFLP patterns was seen for the other pmp genes, but the amount of variation differed.

A very similar arrangement of serovars was seen for pmpE digests. Digestion of pmpE with HinfI produced the same serovar groupings as seen for the HinfI and CfoI digests of pmpF. Tru9I produced a similar pattern, the only difference being that LGV serovars also had the same Tru9I pattern as serovars I, J, and K.

The most interesting RFLP pattern was seen for pmpH. Two of the three enzymes used produced an arrangement of serovars for this gene that mirrors the three disease groups of C. trachomatis. Both Tru9I and CfoI produced three RFLP groups, one containing the ocular serovars, one containing the urogenital serovars, and one containing the LGV serovars. The TaqI digest showed some variation within the urogenital and LGV groups, but for all three enzymes serovars in different disease groups never shared the same RFLP pattern.

RFLP analysis of pmpI suggests that it is the most variable pmp gene, but sequence analysis shows pmpI to be highly conserved. pmpI exhibited the most variation of any of the pmp genes by RFLP analysis (Fig. 1). Three different enzymes were used to digest pmpI, and each enzyme produced a different set of RFLP patterns. Since pmpI exhibited interserovar variation, we chose to search for intraserovar variation in pmpI by using 13 serovar E clinical isolates. We selected serovar E isolates because the MOMP gene is remarkably conserved among serovar E isolates (9, 27). However, no variation was evident in the pmpI gene from the 13 clinical isolates by using either TaqI or CfoI (data not shown).

View larger version (123K): [in this window] [in a new window]   FIG. 1. RFLP analysis suggests pmpI is most variable. pmpI PCR products amplified from 15 serovars (indicated above their respective lanes) of C. trachomatis digested with TaqI endonuclease and separated on a 2% agarose gel are shown. Numbers on the left are size standards in base pairs.

View larger version (14K): [in this window] [in a new window]   FIG. 2. Evolution of pmpI shows weak segregation of serovars compatible with disease groups. Neighbor-joining trees based on the nucleotide (A) and inferred amino acid (B) sequences of pmpI are shown. Bootstrap values are shown only at the nodes separating major clades and only when they exceeded 50%. The scale bar equals a corrected sequence divergence of 0.005, which is the same as the scale used in Fig. 4 and 6. However, due to lesser divergence in pmpI, the branches and scale bar here are lengthened in order to plainly illustrate the branching patterns.

View larger version (104K): [in this window] [in a new window]   FIG. 3. PmpE digested with HinfI restriction endonuclease shows segregation of ocular, urogenital, and LGV serovars. An 8-µl aliquot of the pmpE PCR products was digested with HinfI endonuclease, and restriction fragments were separated on a 2% agarose gel. Serovars are indicated above their respective lanes. Numbers on the left are size standards in base pairs.

Neighbor-joining and parsimony analyses of the nucleotide sequences of pmpE produced the same tree (Fig. 4A). Branch lengths are proportional to nucleotide divergence, and while the scale bar represents the same amount of divergence as seen in Fig. 2, note that the actual bar is smaller due to the increased amount of divergence in pmpE compared to pmpI. The evolutionary analysis of pmpE nucleotide sequences was consistent with the RFLP patterns seen in Fig. 3 and Table 3, with serovars I, J, and K forming a distinct group separate from the rest of the urogenital serovars. The evolutionary tree obtained using the amino acid sequences of PmpE (Fig. 4B) was the same as the nucleotide tree. Most remarkable, however, is the long branch separating the ocular serovars from the remaining serovars, suggesting either an increased rate of evolution in the ocular serovars or a deceleration in the nonocular strains for this protein.

View larger version (19K): [in this window] [in a new window]   FIG. 4. Sequence analysis of pmpE shows an extraordinary amount of evolution in ocular serovars. Phylogenetic trees based on the nucleotide (A) and inferred amino acid (B) sequences of pmpE. Bootstrap values are shown only at the nodes separating major clades and only when they exceeded 50%. The scale bar equals a corrected sequence divergence of 0.005, which is the same as the scale used in Fig. 2 and 6. However, because there is significantly more divergence in pmpE and pmpH than in pmpI, the actual bars here and in Fig. 6 are similar in length compared to the elongated bar used in Fig. 2.

View larger version (131K): [in this window] [in a new window]   FIG. 5. RFLP analysis of pmpH yields three groups consistent with the three disease groups of C. trachomatis. An 8-µl aliquot of the pmpH PCR products was digested with CfoI endonuclease, and restriction fragments were separated on a 4% Metaphor agarose gel. In the LGV strains, the loss of the 54-bp fragment is difficult to see because it comigrates with the 58-bp fragment in the other serovars, and the new 15-bp band in the LGV strains is not visible. However, the new 39-bp fragment in the LGV strains is faintly visible below the 58-bp fragment. Serovars are indicated above their respective lanes. Numbers on the left are size standards in base pairs.

View larger version (14K): [in this window] [in a new window]   FIG. 6. Evolution of pmpH indicates strong divergence of serovars compatible with disease groups. Phylogenetic trees based on the nucleotide (A) and inferred amino acid (B) sequences of pmpH. Bootstrap values are shown only at the nodes separating major clades and only when they exceeded 50%. Based on pmpH, the 15 serovars clearly evolve into three distinct disease groups, with strong bootstrap support (100%) for the three clades. The scale bar equals a corrected sequence divergence of 0.005, which is the same as the scale used in Fig. 2 and 4. However, because there is significantly more divergence in pmpE and pmpH than in pmpI, the actual bars here and in Fig. 4 are similar in length compared to the elongated bar used in Fig. 2.

	DISCUSSION

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The pmp genes are large, ranging from 2,600 to 5,300 bp. An exhaustive sequencing survey of nucleotide diversity within these large genes for the 15 serovars of C. trachomatis would result in about 500,000 bp of nucleotide sequence data. Therefore, we chose instead to first screen for diversity by using RFLP analysis and to then further investigate three of the potentially interesting genes with sequence analysis.

The original goal of our study was to investigate the pmp genes for use as molecular epidemiologic markers. We obtained RFLP analysis of eight genes and sequenced three that looked the most interesting by RFLP analysis. Based on our results, however, it is unlikely that there is significant variation among strains of the same serovar. pmpI was highly conserved, and a recent report suggests that pmpG is also highly conserved (W. J. Jackson, R. B. Taylor, J. H. Tian, K. Johnson, X. Ding, N. Chang, and H. H. Yang, Abstr. 102nd Gen. Meet. Am. Soc. Microbiol., abstr. E-53, 2002). pmpE exhibited up to 6.5% nucleotide and amino acid dissimilarity among serovars in different disease groups, but between serovars within a disease group, pmpE differed by less than 0.5%. A similar result was obtained for pmpH, which showed 8% nucleotide dissimilarity among serovars in different disease groups, but nearly identical sequence for serovars within a disease group. Thus, it is unlikely there will be substantial intraserovar variation in any of the pmp genes.

Evolutionary analysis of pmpE, pmpH, and pmpI produced trees that are significantly different from the published MOMP/omp1 tree (9, 27). In the MOMP evolutionary tree, ocular, urogenital, and LGV serovars are intermixed and form three clades that are not congruent with disease groups: the B serogroup (B, Ba, D, L1, and L2), the C serogroup (A, C, H, I, J, K, and L3), and the intermediate group (serovars F and G). Analysis of pmpE, pmpH, and pmpI indicated that these genes have evolved such that the serovars of C. trachomatis diverge into clades that are more consistent with disease groups. However, the evolutionary pattern of each pmp gene is somewhat unique.

We were surprised to find that pmpI is highly conserved. The paucity of genetic variability resulted in an ambiguous phylogenetic pattern (Fig. 2). In evolutionary terms, highly conserved genes either code for important proteins for which primary amino acid sequence as well as tertiary structure must be conserved or are only recently evolved and have not had time to accumulate mutational variation (18). The high degree of sequence conservation suggests three possibilities for PmpI: (i) PmpI may play a general role in pathogenesis, (ii) PmpI may not be expressed, or (iii) PmpI may not be exposed on the cell surface and thus not under selective pressure by host defenses. Consistent with the latter two suggestions, PmpI has not been demonstrated yet to be in the outer membrane of C. trachomatis (28).

PmpE showed a remarkable and quite unexpected evolutionary pattern. The amount of diversity in PmpE in the ocular strains compared to the STD and LGV strains was three times the amount of diversity between STD and LGV strains, and the majority of nucleotide substitutions were nonsynonymous. The amount of genetic change taking place in PmpE within the ocular group suggests that PmpE may be under different selective pressure in the ocular strains versus the STD and LGV strains. Two possible hypotheses regarding the source of this difference are (i) pressure for variability in the ocular strains, for instance, due to selection at the cell surface and (ii) pressure towards conservation in the nonocular strains, for instance, due to constraints on the amino acid structure of the protein. However, until the function of PmpE is known, it is difficult to speculate on the source of the difference. Furthermore, while one study has demonstrated PmpE to be surface exposed in serovar L2 (28), a different study failed to find this protein on the surface of serovar L2 (21). Therefore, further study of PmpE is necessary.

Although we did not sequence pmpF for this study, the RFLP patterns for this gene are very similar to those seen for pmpE, suggesting a possible divergence of serovars consistent with disease group. In all three digests of pmpF, serovars in different disease groups do not share the same RFLP pattern. In addition, pmpE and pmpF are separated by only 3 bp, and they are located in the same cluster of pmp genes as pmpG and pmpH (26). Therefore, sequencing pmpF may demonstrate that evolution of this gene is comparable to patterns seen in pmpE and/or pmpH.

PmpH exhibited up to 8% nucleotide and amino acid dissimilarity and several in/del events among serovars when comparing sequences among disease groups. However, within a disease group, the serovars were nearly identical in sequence (Table 6). Phylogenetic analysis of pmpH nucleotide and inferred amino acid sequences (Fig. 6) indicates that, based on this gene, the serovars of C. trachomatis diverge distinctly into three clades that are compatible with disease groups: an ocular-trachoma clade (serovars A, B, Ba, and C), a urogenital-STD clade (serovars D, E, F, G, H, I, J, and K), and an LGV clade (serovars L1, L2, and L3). No other C. trachomatis gene studied to date shows a pattern of evolution that parallels disease. The bootstrap support for this pattern was very strong for both the nucleotide and amino acid trees, with values hovering around 100%. In addition, two studies have shown PmpH to be one of the proteins located in the outer membrane of C. trachomatis serovar L2 (21, 28). Therefore, based on its location on the cell surface and its evolutionary pattern consistent with disease groups, we hypothesize that PmpH may be essentially involved in the mechanisms of pathogenesis, perhaps as a ligand on the cell surface involved in attachment or invasion.

The three pmp genes investigated here each show a different pattern of evolution. The evolution of PmpH is most provocative, however, especially with recent evidence that the Chlamydia spp. pmp family closely resembles the autotransporter or type V secretion family of proteins (3, 7, 14). Proteins within this family are known toxins, adhesins, and mediators of intracellular motility, including Bordetella pertactin, FHA, and TcfA; E. coli AIDA-I and TibA; Haemophilusinfluenzae Hap, Hia, and Hsf; Rickettsia spp. rOmpA and rOmpB; Moraxella catarrhalis UspA1 and UspA2 h; and Shigella flexneri IcsA (reviewed in reference 15). Therefore, we hypothesize that C. trachomatis PmpH may play an important role in pathogenesis based on its evolutionary pattern, together with its outer membrane location and similarity to type V secretion proteins. It is important to further investigate the function of PmpH and those of the other pmp proteins to determine if they play a role in pathogenesis. It is possible that PmpH may be a ligand responsible for attachment or invasion and would thus be an attractive vaccine candidate.

	FOOTNOTES

 
* Corresponding author. Mailing address: Department of Medicine, Division of Infectious Diseases, Indiana University School of Medicine, 435 Emerson Hall, 545 Barnhill Dr., Indianapolis, IN 46202. Phone: (317) 278-1791. Fax: (317) 274-1587. E-mail: dstothar{at}iupui.edu.

Editor: J. T. Barbieri

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