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Infection and Immunity, October 2008, p. 4439-4444, Vol. 76, No. 10
0019-9567/08/$08.00+0     doi:10.1128/IAI.00740-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Differential Expression of a Putative CarD-Like Transcriptional Regulator, LtpA, in Borrelia burgdorferi

X. Frank Yang,¹ Martin S. Goldberg,² Ming He,¹ Haijun Xu,¹ Jon S. Blevins,² and Michael V. Norgard^2*

Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, Indiana 46202,¹ Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, Texas 75390²

Received 11 June 2008/ Returned for modification 11 July 2008/ Accepted 22 July 2008

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The availability of microbial genome information has provided a fruitful opportunity for studying regulatory networks in a variety of pathogenic bacteria. In an initial effort to elucidate regulatory networks potentially involved in differential gene expression by the Lyme disease pathogen Borrelia burgdorferi, we have been investigating the functions and regulation of putative transcriptional regulatory factors predicted to be encoded within the B. burgdorferi genome. Herein we report the regulation of one of the predicted transcriptional regulators, LtpA (BB0355), which is homologous to the transcriptional regulator CarD from Myxococcus xanthus. LtpA expression was assessed in response to various environmental stimuli. Immunoblot and quantitative reverse transcription-PCR analyses revealed that unlike many well-characterized differentially regulated Borrelia genes whose expression is induced by elevated temperature, the expression of LtpA was significantly downregulated when spirochetes were grown at an elevated temperature (37°C), as well as when the bacteria were cultivated in a mammalian host-adapted environment. In contrast, LtpA was induced at a lower culture temperature (23°C). Further analyses indicated that the downregulation of LtpA was not dependent on the Rrp2-RpoN-RpoS regulatory pathway, which is involved in the downregulation of OspA when B. burgdorferi is grown in a mammalian host-adapted environment. LtpA protein levels in B. burgdorferi were unaltered in response to changes in the pH in the borrelial cultures. Multiple attempts to generate an LtpA-deficient mutant were unsuccessful, which has hampered the elucidation of its role in pathogenesis. Given that LtpA is exclusively expressed during borrelial cultivation at a lower temperature, a parameter that has been widely used as a surrogate condition to mimic B. burgdorferi in unfed (flat) ticks, and because LtpA is homologous to a known transcriptional regulator, we postulate that LtpA functions as a regulator modulating the expression of genes important to B. burgdorferi's survival within its arthropod vector.

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Borrelia burgdorferi, the Lyme disease spirochete, is maintained in nature through a complex life cycle involving two diverse hosts: an arthropod tick (e.g., Ixodes scapularis) vector and a mammalian host (e.g., white-footed mice). A body of evidence now indicates that during the course of transmission, B. burgdorferi regulates the expression of many of its genes to adapt to these very diverse environments (28, 31, 34, 42, 43, 49). The best-studied example of this differential expression is the reciprocal regulation of outer surface (lipo)protein A (OspA) and OspC (for a review, see reference 44). OspA is expressed chiefly in flat ticks. During feeding, OspA tends to be downregulated concomitantly with the upregulation of OspC. This is consistent with the recent findings that OspA functions as an adhesion molecule essential for B. burgdorferi colonization and survival within ticks (36, 37, 56), whereas OspC is required for the B. burgdorferi infection of its mammalian hosts and possibly for transmission from ticks to mammals (14, 22, 38, 41, 48). Interestingly, recent data suggest that although each lipoprotein has its unique role in B. burgdorferi pathogenesis, they share a common function essential for establishing an infection in mammalian hosts (40, 51).

Although the precise environmental cues that B. burgdorferi senses to induce differential gene expression during tick feeding remain unclear, temperature has been shown to be one important factor for triggering differential gene expression, at least under the in vitro culture conditions typically examined. For example, a lower culture temperature (23°C) has been used to mimic what the spirochetes likely encounter when they are present within the unfed (flat) tick midgut (34, 42, 45, 47). In contrast, tick feeding on a mammalian host likely sends a contrasting signal of elevated temperature (35 to 37°C) as warm blood enters the tick during engorgement. In fact, OspC and many other mammalian infection-associated proteins are upregulated in response to elevated culture temperature (45, 47). Microarray analyses have revealed that more than 100 genes are induced by elevated temperature, or a combination of elevated temperature and lower pH, or in the presence of mammalian blood (34, 42, 49). In addition, more than 82 genes have been shown to be preferably expressed at lower temperature (34, 42, 49).

In recent years, a novel alternative sigma factor regulatory pathway, the Rrp2-RpoN-RpoS pathway, has been elucidated that controls the expression of ospC and other elevated temperature-induced genes in B. burgdorferi (5, 8, 16, 20, 24, 54). In this pathway, a two-component response regulator, Rrp2, in conjunction with an alternative sigma factor, RpoN (⁵⁴), controls the expression of a second alternative sigma factor, RpoS (^S), which, in turn, governs the production of OspC (24, 54) and many other mammalian infection-associated proteins, such as those encoded by bbk32, oppA5, bba64, and bba66 (12, 15, 19, 21, 29, 30, 46). The Rrp2-RpoN-RpoS pathway is activated during tick feeding and likely during mammalian infection (8) and is essential for spirochetal migration from the tick gut to the salivary glands and for B. burgdorferi to establish infection in mammalian hosts (3, 7, 16).

In contrast to rather extensive studies in B. burgdorferi of temperature-induced genes, there has been a paucity of work performed regarding the molecular mechanism(s) that may modulate gene expression at lower ambient temperatures. Of note, although it has been elegantly shown that an increased level of RpoS expression culminates in the repression of ospA and other genes when B. burgdorferi is cultivated in dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats or rabbits (6, 8), OspA is not downregulated by elevated temperature, suggesting that additional factors feed into the differential regulation of surface lipoproteins in B. burgdorferi (45, 47). Because the lower temperature condition is considered a surrogate environment to mimic that of the unfed (flat) tick, an analysis of genes that preferably are expressed at lower temperatures likely will contribute to our understanding of mechanisms underlying B. burgdorferi colonization and survival in the tick vector.

In an effort to elucidate the mechanisms of differential gene expression in B. burgdorferi, we have been focusing attention on putative transcriptional regulatory factors predicted to be encoded within the B. burgdorferi genome. One such factor is BB0355, which shares extensive sequence similarity with the transcription regulator CarD from Myxococcus xanthus (18, 32). CarD is the only reported prokaryotic protein showing structural and functional features typical of eukaryotic high-mobility group A (HMGA) transcription factors (18, 32). We found that BB0355 is preferentially expressed when spirochetes are cultivated in vitro at a lower temperature (23°C). In contrast, BB0355 was downregulated when B. burgdorferi was cultivated at an elevated temperature (37°C) in vitro or when grown in the mammalian host-adapted state. We therefore have designated BB0355 lower temperature-induced protein A (LtpA) of B. burgdorferi. Further analyses showed that unlike ospA, whose downregulation is modulated by the Rrp2-RpoN-RpoS pathway, the regulation of ltpA is not influenced by the Rrp2-RpoN-RpoS pathway. Given that LptA is homologous to the regulatory domain of the bacterial transcriptional regulator CarD, we propose that LtpA is an attractive candidate as a new Borrelia regulatory factor that is independent of the Rrp2-RpoN-RpoS pathway, and one that may be involved in regulating the expression of genes important to B. burgdorferi's survival within its arthropod vector.

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Bacterial strains and culture conditions. Low-passage, virulent B. burgdorferi strain 297 was described previously (23). The descriptions of the rpoN mutant, the complemented strain, and the rpoS mutant have been published previously (24). Borreliae were cultivated in vitro in Barbour-Stoenner-Kelly-H (BSK-H) medium (Sigma Chemical Co., St. Louis, MO) under various environmental conditions or in a host-adapted rat chamber model as previously described (1, 55). To adapt B. burgdorferi to culture conditions at 23°C, spirochetes first were diluted to about 1 x 10⁶ cells per ml and were incubated at 23°C for 1 week. These 23°C-adapted spirochetes then were used for subsequent inoculations into fresh BSK-H cultures that were adjusted to various pHs. HCl or NaOH was used to adjust the pH of BSK-H medium, and the medium then was filter sterilized before inoculation. Cultures were inoculated at a final concentration of 1 x 10³ spirochetes per ml and then were incubated at either 23 or 37°C. Cultures were allowed to grow to 3 x 10⁷ to 5 x 10⁷ bacteria per ml, as determined by counting spirochetes via darkfield microscopy. Escherichia coli strains XL1-Blue (Stratagene, LaJolla, CA) and TOP 10 (Invitrogen, Carlsbad, CA) were used as a cloning host, and strain BL21 was used as the host for protein purification.

Antibodies and antisera. Polyclonal antiserum against OspC and monoclonal antibody directed against FlaB (8H3-33) were described previously (52). To generate rat polyclonal antiserum directed against fusion protein LtpA, the LtpA DNA sequence first was amplified by PCR using oligonucleotide primers CAT GGG ATC CGC ATT TTT GCT AAA TCA ATC AGT AG and CGA CAA GCT TTT AAG CCT TTT TGT TAT CGA CC (letters in boldface are engineered BamHI and HindIII restriction sites, respectively). The amplicon, flanked by the two unique restriction enzyme sites, then was cloned into the appropriate polylinker region of pProEx-HTb (Qiagen, Inc.). The resulting construct encoded a 6-His tag and the LtpA protein. Fusion proteins then were purified by affinity chromatography on a nickel-nitrilotriacetic acid matrix according to protocols from the manufacturer (Qiagen, Inc.). Purified recombinant fusion proteins subsequently were used for generating rat polyclonal antisera as previously described (27).

SDS-PAGE and immunoblotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were carried out as previously described (53). Each gel lane was loaded with lysates of approximately 5 x 10⁷ spirochetes.

qRT-PCR. RNA was extracted from the wild-type, infectious clone BbAH130 of B. burgdorferi 297 cultivated at either 23 or 37°C, using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's protocol. The digestion of contaminating genomic DNA in the RNA samples was performed using RNase-free DNase I (GenHunter Technology, Nashville, TN), and the removal of DNA was confirmed by PCR amplification using primers specific for the B. burgdorferi flaB gene. cDNA was synthesized using the ThermoScript RT system (Invitrogen). Quantitative RT-PCR (qRT-PCR) was performed in triplicate on an ABI 7000 sequence detection system using Platinum SYBR green qPCR SuperMix (Invitrogen). Calculations of the relative expression of the gene of interest were normalized to 16S rRNA gene expression by using the C_T method.

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Influence of culture temperature and pH on LtpA expression. B. burgdorferi has a relatively small genome and contains few known or predicted transcriptional activators (11, 17). One of them is LtpA (BB0355), which shares extensive sequence similarity with the transcriptional regulator CarD from Myxococcus xanthus. CarD is involved in regulating light-induced carotenogenesis as well as the formation of fruiting bodies in Myxococcus xanthus. To examine whether LtpA is involved in transcriptional regulation, we first sought to determine to what extent ltpA might be differentially expressed under various environmental conditions that are known to induce differential gene expression in B. burgdorferi (10, 25, 26, 33, 45, 47, 49).

We first generated a 6x His tag recombinant fusion protein of LtpA and subsequently used it for the generation of rat antiserum. This antiserum then was used in semiquantitative immunoblots to evaluate the levels of LtpA produced by B. burgdorferi when cultivated at different temperatures. As shown previously, the production of OspA was not influenced by culture temperature, whereas OspC was markedly induced when spirochetes were cultivated at elevated temperature (Fig. 1A and B) (45, 47). In contrast, LtpA was detected within organisms cultivated at 23°C but was virtually nondetectable among spirochetes cultivated at 37°C (Fig. 1B). Therefore, the temperature-dependent expression of LtpA was neither OspA-like nor OspC-like. Because LtpA is expressed mainly at a lower culture temperature, we have designated BB0355 the Borrelia cold-inducible protein (LtpA).

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FIG. 1. Effect of spirochete culture temperature on the expression of LtpA. Low-passage B. burgdorferi strain 297 was cultivated at either 23°C (23) or 37°C (37) in BSK-H medium (pH 7.5). Spirochetes were harvested at a cell density of 3 x 107 per ml, and cell lysates were subjected to SDS-PAGE and immunoblotting. Each gel lane for SDS-PAGE was loaded with lysates of approximately 5 x 107 spirochetes. Numbers at the left of the figure denote molecular mass markers, in kilodaltons. (A) Coomassie-stained SDS-PAGE gel. The bands corresponding to OspA and OspC are labeled on the right. (B) Immunoblot analyses performed by using antisera directed against LtpA, OspC, or FlaB (loading control) (positions are denoted by arrowheads).

To examine whether the influence of temperature on LtpA expression correlated at the mRNA level, real-time RT-PCR assays were performed. Equal amounts of RNA from spirochetes cultivated at 23 or 37°C were used, and the data were normalized using the 23°C RNA level. As shown in Fig. 2, both ospC and rpoS transcripts were upregulated at 37°C, as previously described (7, 24, 42, 52). Consistently with the immunoblot results, the ltpA mRNA level was fivefold higher within spirochetes cultivated at 23°C than in those that were cultivated at 37°C. These results indicate that the temperature regulation of ltpA (and the subsequent production of LtpA) occurs chiefly at the transcriptional level.

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FIG. 2. Regulation of the expression of LtpA gene is primarily at the transcriptional level. RNA was extracted from the wild-type, infectious clone BbAH130 of B. burgdorferi 297 cultivated at either 23 or 37°C, and qRT-PCR was performed for the ltpA, flaB, ospC, and rpoS genes. The calculation of the relative expression of the gene of interest was normalized to the level of 16S rRNA.

Because pH is another major parameter influencing Borrelia gene expression (9, 10, 52), we further sought to determine whether pH affected ltpA expression. Wild-type B. burgdorferi cells were cultivated at 23°C under various pH culture conditions (pH 6.8, 7.5, or 8.0). As shown in Fig. 3, the level of LtpA was not influenced by the culture pH.

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FIG. 3. Culture pH does not influence LtpA expression. Low-passage B. burgdorferi strain 297 was cultivated under various pHs (6.8, 7.5, or 8.0; labeled on top) at 23°C in BSK-H medium. Spirochetes were harvested at a cell density of 3 x 107 per ml, and then the cell lysates were subjected to immunoblotting using antisera directed against LtpA and FlaB (loading control) (positions are denoted by arrowheads). Each gel lane for SDS-PAGE was loaded with lysates of approximately 5 x 107 spirochetes. Note that the pH 8.0 lane was inadvertently underloaded for FlaB.

Expression of LtpA by mammalian host-adapted spirochetes. B. burgdorferi cultivated within DMCs implanted into the peritoneal cavities of small mammals mimics, at least in part, the mammalian host-adapted state (1, 4). We thus sought to examine the expression of LtpA among mammalian host-adapted B. burgdorferi. As previously reported, B. burgdorferi cultivated for 7 to 10 days within DMCs caused a dramatic downregulation of OspA concomitantly with the upregulation of OspC (1) (Fig. 4A). In contrast to spirochetes cultivated in vitro at 23°C, spirochetes grown within DMCs did not produce quantities of LtpA that were detectable by immunoblotting (Fig. 4B); this regulatory pattern is akin to that observed for OspA (1, 4).

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FIG. 4. Mammalian host-adapted spirochetes do not express appreciable levels of LtpA. Low-passage B. burgdorferi strain 297 was cultivated at 23°C (23) or in rat host-adapted DMCs (HA). Spirochetes were harvested at a cell density of 3 x 107 per ml, and the lysates then were subjected to SDS-PAGE and immunoblotting. Each gel lane for SDS-PAGE was loaded with lysates of approximately 5 x 107 spirochetes. (A) Coomassie-stained SDS-PAGE gel. The bands corresponding to OspA and OspC are labeled on the left. (B) Immunoblot analyses performed by using antisera directed against LtpA and FlaB (loading control) (positions are denoted by arrowheads).

Influence of the Rrp2-RpoN-RpoS regulatory pathway on the expression of LtpA. Because the Rrp2-RpoN-RpoS pathway is a central regulatory pathway that modulates the differential expression of many genes in B. burgdorferi, we sought to determine the influence of this pathway on the production of LtpA. To facilitate this, we exploited rpoN and rpoS mutants of B. burgdorferi that have been described previously (24). As shown in Fig. 5, the expression of LtpA was influenced mainly by the culture temperature and not by the presence or absence of either RpoN or RpoS. These data indicate that the temperature-dependent expression of LtpA is independent of the Rrp2-RpoN-rpoS regulatory pathway.

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FIG. 5. Expression of LtpA is not influenced by the Rrp2-RpoN-RpoS regulatory pathway. Wild-type (wt), RpoN–, RpoS–, or RpoN–/RpoN+ (RpoN–/+) strains of low-passage B. burgdorferi were cultivated at either 23°C (23) or 37°C (37) in BSK-H medium (pH 7.5) (labeled on top). Spirochetes were harvested at a cell density of 3 x 107 per ml, and the lysates were subjected to immunoblotting using antisera directed against LtpA and FlaB (loading control) (positions are denoted by arrowheads).

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Our recent efforts to elucidate regulatory networks involved in differential gene expression by B. burgdorferi led to the discovery of a regulatory pathway, the Rrp2-RpoN-RpoS regulatory cascade, which now has emerged as a central pathway modulating differential gene expression during the infectious cycle of B. burgdorferi (5, 7, 8, 16, 20, 24, 54). In an effort to identify additional regulators in B. burgdorferi, we report herein our initial study of a predicted transcriptional regulator, LtpA, present in B. burgdorferi. The purpose of our investigation was to determine the differential regulation of LptA and investigate a possible relationship between LtpA and the Rrp2-RpoN-RpoS regulatory network. Our data showed that LtpA is differentially expressed at both the mRNA and protein levels by an alteration in culture temperature. LtpA was preferentially expressed, however, at 23°C as opposed to 37°C. In addition, LtpA was not expressed to any appreciable degree by spirochetes that were mammalian host adapted. This result was unanticipated, because recent microarray analyses have shown that a number of genes are induced at lower temperatures, ranging from 19 to 85 genes among the various studies (34, 42, 49), but none of the microarray analyses identified the gene encoding LtpA (BB0355) as a low-temperature-induced gene (34, 42, 49). Prior failure to recognize ltpA as a low-temperature-induced gene likely is due to the intrinsic technical limitations of microarrays.

BLAST analysis has revealed that LtpA belongs to a CarD_TRCF superfamily (Pfam02559; expect = 1.2e-32). Of note, Pfam02559 originally was denoted the CarD family. Upon subsequent findings that LtpA and many other proteins have homology only to the N-terminal domain of CarD, a region that shares homology to transcription repair coupling factor (TRCF) (see below), we communicated with the Sanger group (http://www.sanger.ac.uk), and subsequently, Pfam02559 was renamed the CarD_TRCF superfamily. CarD is the only reported prokaryotic protein showing structural and functional features typical of eukaryotic HMGA transcription factors (2). Eukaryotic HMGA proteins contain multiple repeats of a conserved RGRP motif (the AT hook motif) that specifically binds to the minor groove of DNA with AT-rich sequences (35) and serve as DNA architectural factors that remodel chromatin to aid in the assembly of specific nucleoprotein complexes essential for transcription, replication, recombination, or repair (50). Like HMGA, CarD is a global regulator involved in many cellular processes in M. xanthus, including light-induced carotenogenesis, starvation-induced multicellular development, and vegetative gene expression. CarD contains two functional domains (35) (Fig. 6). The C-terminal domain (HMGA domain) has homology to HMGA with four repeats of the AT hook motif, along with a conserved acidic region. The N-terminal domain (TRCF domain) of CarD, however, is not found in eukaryotic HMGA proteins. This domain contains a region homologous to the RNA polymerase-binding module of the TRCF (13), along with a leucine zipper motif. TRCF displaces RNA polymerase stalled at a lesion, binds to the damage recognition protein UvrA, and increases the template strand repair rate during transcription (13).

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FIG. 6. Domain structure of LtpA and CarD in M. xanthus. CarD can be divided into two domains. The C-terminal domain (HMGA domain) has homology to eukaryotic HMGA transcription factors that contain four repeats of the AT hook motif for binding to the minor groove of DNA with AT-rich sequences, as well as a conserved acidic region. The N-terminal domain (TRCF domain) of CarD is absent from HMGA proteins. It contains a region homologous to the RNA polymerase-binding (RPB) module of the TRCF, along with a leucine zipper motif. LtpA is homologous to CarD but only has an N-terminal TRCF domain, suggesting that unlike CarD, LtpA influences gene expression.

LtpA is homologous to CarD, but only at the N-terminal TRCF domain; CarD does not possess a C-terminal HMGA-like DNA binding domain (Fig. 6). Thus, it is unlikely that LtpA is a DNA-binding protein. The function of the N-terminal domain of CarD is not fully understood, but it was shown to be involved in dimerization (39). Its homology to TRCF suggests that it also interacts with RNA polymerase. However, coimmunoprecipitation and two-hybrid analyses to demonstrate such interactions have yielded negative results (39). One factor that specifically interacts with the N-terminal domain of CarD recently has been identified in M. xanthus: CarG, a zinc-binding H/C-rich metallopeptidase. carG is colocalized and cotranscribed with carD and, like carD, is essential for regulating multiple processes in M. xanthus. It was shown that CarG does not affect the DNA-binding activity of CarD, and it has been postulated that CarG functions similarly as zinc-associated eukaryotic transcriptional adaptors to recruit other unknown transcription factors to the CarD-CarG complex.

Upon searching the Borrelia genome, no CarG homologue has been identified. Thus, identifying the factors that LtpA may interact with in B. burgdorferi would provide insights into the actual function of LtpA, as well as functions of the TRCF domain-containing proteins in general. Alternatively, generating an isogenic ltpA mutant would reveal the role of LtpA in gene regulation and in the enzootic life cycle of B. burgdorferi. In this regard, multiple attempts have been carried out unsuccessfully to inactivate ltpA. Thus far, this limitation has prevented us from implementing a genetic approach to study the function of LtpA. Despite the lack of direct evidence, it still is conceivable, if not plausible, based on its differential expression and homology to a known transcriptional regulator that LtpA is a new Borrelia regulator that is independent of the Rrp2-RpoN-RpoS pathway. It also remains tempting to speculate that LtpA plays a role in facilitating Borrelia adaptation to its tick vector.

 
Funding for this work was provided partially by an American Heart Association scientist development grant (to X.F.Y.), Indiana INGEN and METACyt grants of Indiana University, funded by the Lilly Endowment, Inc. (to X.F.Y.), and NIH grant AI-59062 from the National Institute of Allergy and Infectious Diseases (to M.V.N.). J.S.B. was supported by National Institutes of Health training grant T32-AI07520 and by Ruth L. Kirschstein National Research service award F32-AI058487 from the National Institutes of Health. This investigation was conducted partially in a facility constructed with support from research facilities improvement program grant number C06 RR015481-01 from the National Center for Research Resources, NIH.

 
* Corresponding author. Mailing address: Department of Microbiology, U.T. Southwestern Medical Center, 6000 Harry Hines Blvd., Dallas, TX 75390-9048. Phone: (214) 648-5900. Fax: (214) 648-5905. E-mail: michael.norgard{at}utsouthwestern.edu

Published ahead of print on 28 July 2008.

Editor: A. J. Bäumler

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Infection and Immunity, October 2008, p. 4439-4444, Vol. 76, No. 10
0019-9567/08/$08.00+0     doi:10.1128/IAI.00740-08
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