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Appl Environ Microbiol. May 2007; 73(9): 2919–2930.
Published online Mar 2, 2007. doi:  10.1128/AEM.02664-06
PMCID: PMC1892873

The Alternative Sigma Factor σB and the Virulence Gene Regulator PrfA Both Regulate Transcription of Listeria monocytogenes Internalins[down-pointing small open triangle]

Abstract

Some Listeria monocytogenes internalins are recognized as contributing to invasion of mammalian tissue culture cells. While PrfA is well established as a positive regulator of L. monocytogenes virulence gene expression, the stress-responsive σB has been recognized only recently as contributing to expression of virulence genes, including some that encode internalins. To measure the relative contributions of PrfA and σB to internalin gene transcription, we used reverse transcription-PCR to quantify transcript levels for eight internalin genes (inlA, inlB, inlC, inlC2, inlD, inlE, inlF, and inlG) in L. monocytogenes 10403S and in isogenic ΔprfA, ΔsigB, and ΔsigB ΔprfA strains. Strains were grown under defined conditions to produce (i) active PrfA, (ii) active σB and active PrfA, (iii) inactive PrfA, and (iv) active σB and inactive PrfA. Under the conditions tested, σB and PrfA contributed differentially to the expression of the various internalins such that (i) both σB and PrfA contributed to inlA and inlB transcription, (ii) only PrfA contributed to inlC transcription, (iii) only σB contributed to inlC2 and inlD transcription, and (iv) neither σB nor PrfA contributed to inlF and inlG transcription. inlE transcript levels were undetectable. The important role for σB in regulating expression of L. monocytogenes internalins suggests that exposure of this organism to environmental stress conditions, such as those encountered in the gastrointestinal tract, may activate internalin transcription. Interplay between σB and PrfA also appears to be critical for regulating transcription of some virulence genes, including inlA, inlB, and prfA.

The gram-positive bacterium Listeria monocytogenes is recognized as an important human food-borne pathogen, causing manifestations ranging from mild febrile gastroenteritis to severe invasive infections. High mortality rates are associated with systemic infections, which occur predominantly among pregnant women, the immunocompromised, and the elderly (19, 24). In animals, L. monocytogenes infections can result in a variety of manifestations, including abortion, encephalitis, septicemia, and, less commonly, keratoconjunctivitis and mastitis (43).

A variety of L. monocytogenes surface proteins, including several members of the internalin family (11), are important for facilitating interactions between this pathogen and mammalian host cells. For example, internalin A (InlA) promotes invasion of human nonphagocytic cells that express the host cell receptor E-cadherin, such as the Caco-2 epithelial cell line (45). InlB mediates entry into several host cell types, including hepatocytes and several endothelial and epithelial cell lines of various human and animal origins, including HepG-2 (human hepatocyte), TIB73 (mouse hepatocyte), HUVEC (human endothelial), and Vero (African green monkey epithelial) cells (15, 29, 40, 50). InlB facilitates invasion of mammalian cells by interacting with cellular receptors, including hepatocyte growth factor receptor (Met) and other host cell components, including gC1q-R (9, 30, 31). Interactions between host-cell receptors and InlA or InlB are species specific. To illustrate, InlA recognizes human and guinea pig E-cadherin, but not mouse or rat intestinal E-cadherin, due to a single amino acid substitution in the binding site of mouse and rat E-cadherin (38). Studies with human trophoblastic cell lines suggest that InlA may contribute to L. monocytogenes targeting and crossing of the human maternofetal barrier (39); however, no clear role has been established for InlA in the crossing of the guinea pig maternofetal barrier (1). InlB also displays species specificity. Although both guinea pig and rabbit cell lines express Met and gC1q-R, neither responds to InlB. However, following transfection with the human Met gene, both guinea pig and rabbit cells support InlB-dependent entry (34). Transcription of inlC, which encodes the secreted protein InlC, is strongly induced when L. monocytogenes is in the host cell cytoplasm (18). In the absence of InlB, InlC and InlGHE appear to be required for InlA-dependent invasion of Caco-2 cells (5). The specific functions of the proteins encoded by other members of the internalin gene family (inlC2, inlD, inlE, inlF, inlG, and inlH) remain unclear.

Transcription of many confirmed and putative L. monocytogenes virulence genes is activated, at least in part, by positive regulatory factor A (PrfA) (for a recent review, see reference 37). Among the internalin genes, inlA, inlB, and inlC are at least partially regulated by PrfA (17, 18, 44). Previous reports have shown that PrfA can be present in two functional states, weakly active and highly active, which are influenced by environmental stimuli, including temperature, growth in charcoal, and the availability of readily metabolized sugars, such as cellobiose (4, 25, 28, 41, 46, 53). PrfA is highly active in intracellular L. monocytogenes when mammalian host cells are grown at 37°C (22), as well as when cells are grown in media supplemented with activated charcoal (54). On the other hand, PrfA is present in its inactive form during L. monocytogenes growth in the presence of cellobiose and other easily fermentable sugars (3, 4, 46, 53).

While PrfA is well recognized as an important activator of L. monocytogenes virulence gene expression, emerging evidence indicates that the stress-responsive alternative sigma factor, σB, encoded by sigB, also contributes to transcription of at least some L. monocytogenes virulence genes, in addition to regulating expression of a stress regulon of at least 50 genes (2, 32, 35, 59). Specifically, microarray studies using an L. monocytogenes sigB null mutant showed that transcription of the internalin genes inlA, inlB, inlC2, inlD, and inlE is at least partially σB dependent (32); σB-dependent transcription of inlA and inlB has also been confirmed by quantitative reverse transcription-PCR (qRT-PCR) (36).

The objective of this study was to measure the relative contributions of PrfA and σB to internalin gene transcription under conditions that yield (i) the active form of PrfA, (ii) the active σB and the active form of PrfA, (iii) the inactive form of PrfA, and (iv) the active σB and the inactive form of PrfA. We used qRT-PCR assays to measure mRNA transcript levels for eight internalin genes (inlA, inlB, inlC, inlC2, inlD, inlE, inlF, and inlG), two PrfA-dependent genes (prfA and plcA), two σB-dependent genes (opuCA and gadA), and two housekeeping genes (rpoB and gap) in ΔprfA and ΔsigB strains as well as in a ΔprfA ΔsigB double mutant strain grown under each of the four defined conditions. Our results, which demonstrate that internalin genes group into regulons with distinct expression patterns, provide novel insight into the regulation of L. monocytogenes internalin gene expression.

MATERIALS AND METHODS

Bacterial strains.

L. monocytogenes serotype 1/2a strain 10403S (6) and three isogenic in-frame null mutants, including a ΔsigB mutant (FSL A1-254 [63]), a ΔprfA strain (FSL B2-046) with a 339-bp in-frame deletion in prfA previously described by Wong and Freitag (64), and a ΔsigB ΔprfA mutant (FSL B2-068 [this study]) were used. The ΔsigB ΔprfA double mutant was generated by cloning a PCR-amplified allele with an in-frame prfA deletion from L. monocytogenes strain FSL B2-046 into the Escherichia coli-L. monocytogenes shuttle vector pKSV7. The prfA mutant allele was then introduced into the L. monocytogenes ΔsigB strain by allelic-exchange mutagenesis as previously described (12). The final double mutant was confirmed by PCR, followed by sequencing of the mutant prfA allele.

Growth conditions and cell collection for RNA isolation.

Bacterial cells for RNA collection were grown at 37°C with shaking (200 rpm) in brain heart infusion broth (BHI; Difco Laboratories, MD). For exposure to different environmental conditions, L. monocytogenes cells that had been grown in BHI to early log phase (defined as an optical density at 600 nm of 0.4) were subsequently incubated for 120 min at 37°C in (i) BHI with 0.2% charcoal (BHI-charcoal), (ii) BHI-charcoal with NaCl added (0.3 M final concentration) for the final 10 min (BHI-charcoal-NaCl), (iii) BHI with 25 mM cellobiose (BHI-cellobiose), and (iv) BHI-cellobiose with NaCl added (0.3 M final concentration) for the final 10 min (BHI-cellobiose-NaCl). Following incubation, 2 volumes of RNAprotect (QIAGEN Inc., Valencia, CA) were added to each culture. After 5 min, cells were collected for subsequent RNA isolation by centrifugation at 5,000 × g for 5 min.

Optimization of growth conditions for active or inactive PrfA.

To determine specific growth conditions that yield maximum PrfA activity in broth-grown L. monocytogenes, qRT-PCR was used to measure transcript levels for the PrfA-dependent gene, plcA, using mRNA obtained from L. monocytogenes 10403S grown in BHI as well as 10403S under conditions reported to induce (growth in 0.2% charcoal) or repress (growth in 25 mM cellobiose) PrfA activity. Specifically, early-log-phase L. monocytogenes 10403S cells were inoculated into BHI, BHI-charcoal, or BHI-cellobiose and subsequently incubated at 37°C with shaking. Normalized plcA transcript levels (averages of two independent experiments; see below) for bacteria grown in BHI-charcoal for 5 min, 30 min, 120 min, and overnight were 0.11, 0.22, 1.14, and 0.72, respectively; these plcA transcript levels were over 1 log higher than the corresponding transcript levels (averages of two independent experiments) for cells grown in BHI alone. Normalized plcA transcript levels for cells grown in BHI-cellobiose for 120 and 240 min were −2.25 and −1.37, respectively. While plcA transcript levels after growth in BHI-cellobiose for 120 min were over one-half log lower than corresponding plcA transcript levels for cells grown in BHI alone, plcA transcript levels after 240 min growth in BHI-cellobiose were similar to corresponding plcA transcript levels for cells grown in BHI alone. Based on these data, growth for 120 min at 37°C in BHI-charcoal or in BHI-cellobiose was selected as a condition that yields L. monocytogenes cells with high or low levels of PrfA activity, respectively.

Optimization of growth conditions for increased σB activity.

To define conditions that yield high σB activity in the presence of either the active or the inactive form of PrfA, qRT-PCR was used to measure transcript levels for the σB-dependent genes opuCA and gadA and the PrfA-dependent gene plcA in the presence of 0.3 M NaCl, as exposure of log-phase L. monocytogenes cells to 0.3 M NaCl in BHI for 10 min has been shown to produce high levels of σB activity (59). qRT-PCR was performed on RNA extracted from early-log-phase L. monocytogenes 10403S cells that were grown for (i) an additional 120 min in BHI with 0.3 M NaCl, (ii) an additional 120 min in BHI with 0.3 M NaCl added for the final 10 min, (iii) an additional 120 min in BHI with 0.2% charcoal and 0.3 M NaCl, (iv) an additional 120 min in BHI with 0.2% charcoal and 0.3 M NaCl added for the final 10 min, (v) an additional 120 min in BHI with 25 mM cellobiose and 0.3 M NaCl, and (vi) an additional 120 min in BHI with 25 mM cellobiose and 0.3 M NaCl added for the final 10 min. All experiments were performed on two independent RNA preparations. Transcript levels for the σB-dependent genes opuCA and gadA were similar under all six conditions, indicating that exposure to NaCl for 10 min and exposure for 120 min provided similar levels of σB activity (see Fig. S1 in the supplemental material). plcA transcript levels for cells grown in BHI-charcoal with 10 min or 120 min of exposure to NaCl were up to 1 log lower than plcA transcript levels for cells grown in BHI-charcoal without exposure to NaCl. On the other hand, plcA transcript levels for cells grown in BHI-cellobiose without exposure to NaCl were similar to transcript levels for cells grown in BHI-cellobiose with NaCl exposure for either 10 or 120 min (see Fig S2b in the supplemental material). Based on these data, we chose growth of log-phase L. monocytogenes for 120 min in BHI with 25 mM cellobiose with exposure to 0.3 M NaCl for the final 10 min as a condition that provides a high level of σB activity and minimal PrfA activity. Growth of log-phase L. monocytogenes cells for 120 min in BHI with 0.2% charcoal with exposure to 0.3 M NaCl for the final 10 min was chosen as a condition that provides high levels of σB activity and intermediate PrfA activity (as salt exposure appears to repress plcA transcription [see Fig. S2b in the supplemental material]). No in vitro conditions tested allowed maximal activity of both σB and PrfA. All subsequent experiments were performed with cells grown in the presence of charcoal or cellobiose with or without a final 10 min of exposure to 0.3 M NaCl to provide mRNA transcripts under conditions generating either active or inactive PrfA in the presence or absence of active σB and salt.

Total RNA isolation.

Total RNA was purified from bacterial cells using the RNeasy Midi kit (QIAGEN Inc.) as described previously (60). For cultures exposed to charcoal, an additional RNA clean-up step using the protocol recommended by QIAGEN was performed prior to DNase treatment. The final RNA pellet was resuspended in RNase-free water (QIAGEN). Total nucleic acid concentration and purity were estimated using absorbance readings (260/280 nm) on an ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE).

qRT-PCR.

Absolute quantification of transcript levels for eight internalin genes (inlA, inlB, inlC, inlC2, inlD, inlE, inlF, and inlG), prfA, the PrfA-dependent gene plcA, and two σB-dependent genes (gadA and opuCA) as well as two housekeeping genes (rpoB and gap) was performed using TaqMan primer and probes (see Table S1 in the supplemental material) and the ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA) essentially as previously described (14, 33). While probes and primers for prfA, plcA, gadA, opuCA, rpoB, gap, inlA, and inlB have been reported previously (32, 33, 35, 59, 60), for this study, TaqMan primer and probes for six internalin genes (inlC, inlC2, inlD, inlE, inlF, and inlG) were designed with Primer Select (Applied Biosystems) based on internalin gene sequence data for L. monocytogenes 10403S and 18 other lineage II L. monocytogenes strains sequenced in our laboratory (61; see Table S1 in the supplemental material). Primers and probes were synthesized by IDT Technologies (Coralville, IA) and Applied Biosystems, respectively. In preliminary experiments, all qRT-PCR primer/probe sets were able to reproducibly detect as few as 60 DNA copies.

Immunogold electron microscopy.

Polyclonal anti-InlB antibodies were kindly provided by Paul Leonard, Dublin City University, Ireland. Goat anti-mouse immunoglobulin G labeled with 10-nm gold particles, bovine serum albumin (BSA) background-suppressing reagent, goat normal serum, and cold-water-fish-skin gelatin manufactured by Aurion (Wageningen, The Netherlands) were supplied by Electron Microscopy Services (Hatfield, PA). All other reagents were supplied by Sigma-Aldrich Inc. (St. Louis, MO).

To prepare bacterial cells for immunogold labeling, the L. monocytogenes 10403S parent strain as well as the isogenic ΔsigB, ΔprfA, and ΔinlB strains were initially grown to early logarithmic phase (optical density at 600 nm of 0.4) in 5 ml BHI at 37°C with shaking. A 2-ml aliquot of each culture was then added to 2 ml of fresh BHI followed by incubation at 37°C for a further 2 h with addition of NaCl (at a final concentration of 0.3 M) for the final 10 min. Cells were then harvested by centrifugation and resuspended in 500 μl of phosphate-buffered saline (PBS). Immunogold labeling of whole bacterial cells was performed as described by the manufacturer (Aurion) with minor modifications. Briefly, 30 μl of suspended cells was allowed to adhere to Formvar-coated nickel grids for 45 min. The grids were washed in blocking buffer (PBS, 5% BSA, 5% normal goat serum, 0.1% cold-water-fish-skin gelatin) for 15 min, followed by two 5-min washes in incubation buffer (PBS, 0.2% BSA, 15 nM NaN3 [pH 7.4]). Grids were then transferred onto drops of a 1:10 dilution of the primary antibody, followed by incubation for 1 h and six subsequent washes with incubation buffer. Grids were then transferred to drops of a 1:20 dilution of gold conjugate reagent, followed by incubation for 1 h and six subsequent washes with incubation buffer as well as two washes with PBS. The grids were fixed for 5 min with 2% paraformaldehyde. Finally, grids were washed twice with distilled water, negatively stained with 0.5% aqueous potassium phosphotungstate (pH 6.5), and examined using a transmission electron microscope (FEI Philips TECNAI 12 BioTwin) at 120 kV.

The number of gold particles per μm2 was quantified for each strain using five separate immunogold electron microscopy images for each strain per experiment in four independent experiments conducted over four separate days. The number of gold particles in each image was recorded independently by two researchers, and particles per μm2 were calculated based on the average number of gold particles counted for each image. L. monocytogenes 10403S incubated with only the secondary gold conjugate was used as a negative control in all experiments, and no labeling was observed (data not shown).

Statistical analyses.

Absolute mRNA transcript levels for target genes were normalized prior to final statistical analyses as initially described by Vandesompele et al. (62), who demonstrated that measurement of transcript levels from a single housekeeping gene can be inadequate for normalizing quantitative transcript levels obtained from target genes under widely varying physiological conditions. Typically, housekeeping genes that are expected to maintain stable levels of expression under the test conditions are selected as targets for control reactions in quantitative gene expression studies (48); however, housekeeping gene expression can also vary under different conditions (14, 33, 60, 62). Therefore, we normalized mRNA transcript levels for the target genes to the geometric mean of the transcript levels for two housekeeping genes (gap and rpoB) as previously described (14, 33). Average log-transformed (log10) gap and rpoB transcript levels for different strains (see Fig. S3a in the supplemental material) and growth conditions (see Fig. S3b in the supplemental material) were plotted to assess whether transcript levels for the two housekeeping genes followed similar trends. While transcript levels were considerably higher for gap than for rpoB, the two genes showed similar trends in transcript levels. Thus, unless otherwise indicated, all statistical analyses were performed on the log-transformed (log10) mRNA transcript levels normalized to the geometric mean of rpoB and gap transcript levels calculated using the following formula:

equation M1

Three factors, including strain (parent strain 10403S and ΔsigB, ΔprfA, and ΔsigB ΔprfA strains), salt stress (presence or absence of NaCl), and presence of cellobiose or charcoal, were included in analysis of variance (ANOVA) models of the absolute mRNA transcript levels to determine if these factors affected transcript levels for the eight internalin genes and prfA, plcA, opuCA, and gadA (Table (Table1).1). If a significant effect of the factor “strain” was observed, a second ANOVA was conducted to determine whether sigB deletion, prfA deletion, or statistical interactions between data generated for strains with these deletions had a significant effect on transcript levels for a given gene under each of the four growth conditions (see Table S2 in the supplemental material). In cases in which mRNA transcript levels were below the qRT-PCR detection limit, a value representing the detection limit cutoff was used for statistical analyses. Initial analysis showed the mRNA transcript level data to be heteroscedastic and strongly skewed; therefore, logarithmic transformation (log10 [mRNA transcript level]) was used to correct the skewness and stabilize the variance to approximate normality. Tukey's multiple-comparison procedure was used to determine whether transcript levels for a gene differed among strains within a given condition (see Fig. Fig.111 to to4)4) or among conditions for a given strain (see Fig. S2 and S4 through S7 in the supplemental material). All statistical analyses were performed with Splus 6.2 (Insightful Corp, Seattle, WA). Standard regression diagnostics were computed for all models. Unless indicated otherwise, P values are reported as not significant (P > 0.05) or significant at P values of <0.05, <0.01, <0.001, or <0.0001.

FIG. 1.
Normalized, log-transformed transcript levels of opuCA and gadA (A) and of prfA and plcA (B) for L. monocytogenes parent strain 10403S (wild type) as well as isogenic ΔsigB, ΔprfA, and ΔsigB ΔprfA strains cultured under ...
FIG. 2.
Normalized, log-transformed transcript levels of inlA and inlB for L. monocytogenes parent strain 10403S (wild type) as well as isogenic ΔsigB, ΔprfA, and ΔsigB ΔprfA strains cultured under the four different conditions ...
FIG. 3.
Normalized, log-transformed transcript levels for inlC for L. monocytogenes 10403S parent strain (wild type) as well as isogenic ΔsigB, ΔprfA, and ΔsigB ΔprfA strains cultured under the four different conditions outlined ...
FIG. 4.
Normalized, log-transformed transcript levels of inlC2 and inlD for L. monocytogenes parent strain 10403S (wild type) as well as isogenic ΔsigB, ΔprfA, and ΔsigB ΔprfA strains cultured under the four different conditions ...
TABLE 1.
Effects of different factors on transcript levels of target internalin genes and opuCA, gadA, plcA, and prfA

Statistical analysis of quantitative data for immunogold labeling used one-way ANOVA and Tukey's multiple-comparison procedure.

RESULTS

qRT-PCR was used to measure mRNA transcript levels for eight internalin genes, four control genes, and two housekeeping genes in L. monocytogenes 10403S and in otherwise isogenic ΔsigB, ΔprfA, and ΔsigB ΔprfA strains grown under defined conditions to ensure (i) the active form of PrfA, (ii) active σB and the active form of PrfA, (iii) the inactive form of PrfA, and (iv) active σB and the inactive form of PrfA.

ANOVA models with the factors “strain,” “salt stress,” and “presence of cellobiose or charcoal” were initially fitted to identify which, if any, of these factors had significant effects on transcript levels for seven internalin genes as well as for opuCA, gadA, prfA, and plcA. No detectable transcripts were found for inlE; therefore, no analyses were conducted for this gene. The factor “strain” had a significant effect on transcript levels for opuCA, gadA, prfA, and plcA as well as for five internalin genes (inlA, inlB, inlC, inlC2, and inlD) (Table (Table1),1), indicating that transcript levels for inlF and inlG were independent of both σB and PrfA. A second ANOVA model was fitted to the normalized mRNA transcript levels for the genes that were affected by the factor “strain” to investigate the main effects and interactions of the sigB and prfA deletions on transcript levels for cells grown under the four different conditions (see Table S2 in the supplemental material).

The factor “salt stress” had a significant effect on transcript levels for six internalin genes (inlA, inlC, inlC2, inlD, inlF, and inlG) as well as for opuCA, gadA, and plcA. The factor “presence of cellobiose or charcoal” had a significant effect on transcript levels of all genes, including the seven internalin genes (inlA, inlB, inlC, inlC2, inlD, inlF, and inlG), indicating that the presence of charcoal or cellobiose also affects transcript levels for internalin genes that are not PrfA dependent (i.e., inlC2, inlD, inlF, and inlG).

σB-dependent transcription of opuCA and gadA and PrfA-dependent transcription of plcA confirm that these genes are effective reporters for monitoring σB and PrfA activity.

Transcription of the compatible solute transporter protein encoded by opuCA and transcription of the glutamate dehydrogenase protein encoded by gadA have been shown to be σB dependent (21, 32, 59, 60). PrfA-dependent transcription of plcA, which encodes the phosphatidylinositol-specific phospholipase C, also has been established (8, 44). To ensure that the opuCA and gadA genes and plcA are appropriate reporter genes for σB and PrfA, respectively, the relative contributions of σB and PrfA to opuCA, gadA, and plcA transcript levels were measured under all four growth conditions. Further, as prfA has been shown to have one σB-dependent promoter (33, 52, 55), contributions of σB to prfA transcript levels were also measured.

opuCA and gadA transcript levels in the ΔsigB strain were significantly lower than those in both the isogenic parent strain and the ΔprfA strain under all four growth conditions (Fig. (Fig.1A).1A). The sigB deletion highly significantly affected gadA and opuCA transcript levels (P < 0.0001) (see Table S2 in the supplemental material). opuCA and gadA transcript levels were similar in the ΔprfA strain and the isogenic parent strain (Fig. (Fig.1A);1A); thus, the prfA deletion did not significantly affect opuCA and gadA transcript levels (see Table S2 in the supplemental material). Salt stress significantly affected opuCA and gadA transcript levels (Table (Table1);1); opuCA and gadA transcript levels in the ΔprfA strain and the isogenic parent strain exposed to NaCl were significantly higher than those in bacteria not exposed to NaCl (Fig. (Fig.1A)1A) (see Fig. S2a in the supplemental material; mRNA transcript levels Fig. S2 and S4 through S6 in the supplemental material are identical to those in Fig. Fig.11 to to4,4, but the data are rearranged to enable direct comparisons of transcript levels in the same strain under different growth conditions). The presence of cellobiose or charcoal significantly affected opuCA and gadA transcript levels (Table (Table1).1). While no clear trends of the effects of cellobiose or charcoal on gadA transcript levels are apparent (Fig. (Fig.1A;1A; see Fig. S2a in the supplemental material), opuCA transcript levels in all four strains were generally lower in cells cultured with cellobiose than in cells cultured with charcoal. This effect appears to be independent of PrfA activity, as opuCA transcript levels for bacteria grown in BHI-cellobiose were significantly lower than those for bacteria grown in BHI-charcoal, even for the ΔprfA strain.

The plcA transcript levels in the ΔprfA strain were significantly lower than these levels in either 10403S or the ΔsigB mutant background, regardless of growth condition (Fig. (Fig.1B),1B), consistent with the previously reported PrfA dependence of this gene (44). The presence of cellobiose or charcoal significantly affected plcA transcript levels (Table (Table1);1); plcA transcript levels in the isogenic parent and the ΔsigB strain grown in the presence of cellobiose were significantly lower than those of bacteria grown in the presence of charcoal (Fig. (Fig.1B;1B; see Fig. S2b in the supplemental material).

Based on data collected for the 10403S and ΔsigB strains (Table (Table1),1), prfA transcript levels were determined by ANOVA to be significantly affected by the sigB null mutation and by the presence of cellobiose or charcoal. Specifically, prfA transcript levels in the parent strain grown in the presence of cellobiose were higher than those of the strain grown in the presence of charcoal (Fig. (Fig.1B;1B; see Fig. S2b in the supplemental material). prfA transcript levels were only significantly lower in the ΔsigB strain than in the parent strain during growth in BHI-cellobiose with a final 10-min exposure to NaCl (Fig. (Fig.1B),1B), indicating a limited effect of σB on prfA transcription, which may be detectable only under selected growth conditions. prfA transcription could not be characterized in the ΔprfA or ΔsigB ΔprfA strain, as the prfA qRT-PCR primer and probe binding sites were deleted from these strains to create the prfA null mutations.

σB and PrfA regulate inlA and inlB transcription.

The factors “strain” and “presence of cellobiose or charcoal” affected inlA and inlB transcript levels and the factor “salt stress” affected inlA transcript levels (Table (Table1).1). inlA and inlB transcript levels were consistently and considerably lower in the ΔsigB strain than in the isogenic parent (in both the wild-type and ΔprfA backgrounds) (Fig. (Fig.2),2), indicating that σB plays an important role in inlA and inlB transcription. For example, when cells were cultured in BHI-charcoal and BHI-charcoal-NaCl, inlA and inlB transcript levels in the ΔsigB strain were up to 30-fold lower than those in the isogenic parent strain.

inlA and inlB transcript levels were not significantly different between 10403S and the ΔprfA strain under any of the four growth conditions (Fig. (Fig.2;2; see Fig. S4 in the supplemental material), suggesting that PrfA has a limited role in regulating transcription of inlA and inlB when σB is also present. The ΔsigB ΔprfA strain, however, showed consistently lower inlA transcript levels than the ΔsigB strain showed; this difference was statistically significant for cells grown in charcoal with and without NaCl (Fig. (Fig.2).2). The sigB and prfA deletions were determined to interact significantly to affect inlA transcript levels for cells grown in the presence of charcoal (see Table S2 in the supplemental material). When grown in BHI-charcoal, the ΔsigB ΔprfA strain also showed inlB transcript levels that were lower than those of the ΔsigB strain; a significant effect of prfA deletion on inlB transcript levels for cells grown in BHI-charcoal (see Table S2 in the supplemental material) was confirmed by ANOVA. Taken together, our data indicate that transcriptional regulation of inlA and inlB is affected by interplay between active PrfA and σB. The absence of a significant multiplicative effect of the sigB and prfA null mutations on inlA transcript levels for cells grown in the presence of cellobiose (Fig. (Fig.2;2; see Table S2 in the supplemental material) supports the observation that interactions between the two regulatory proteins specifically require active PrfA.

PrfA, but not σB, regulates inlC transcription.

inlC transcript levels were affected by the factors “strain,” “presence of cellobiose or charcoal,” and “salt stress” (Table (Table1).1). A significant effect of the prfA deletion on inlC transcript levels was shown by ANOVA for both cells grown in BHI-charcoal and cells grown in BHI-charcoal-NaCl (see Table S2 in the supplemental material). In both the parent strain and the ΔsigB strain, inlC transcript levels for cells grown in BHI-charcoal-NaCl were lower than those for cells grown in BHI-charcoal (see Fig. S5 in the supplemental material), indicating σB-independent down-regulation of inlC in the presence of 0.3 M NaCl. Both the ΔprfA and ΔsigB ΔprfA strains showed inlC transcript levels in BHI-charcoal that were lower than those in the 10403S and the ΔsigB strains, indicating that PrfA activates transcription of inlC under these conditions. Overall, inlC transcript levels in the wild-type strain grown in BHI-charcoal (Fig. (Fig.3)3) were low compared to transcript levels observed for inlA, inlB, inlC2, and inlD (Fig. (Fig.2).2). For cells cultured in BHI-cellobiose, inlC transcript levels were consistently below the qRT-PCR detection limit (Fig. (Fig.3).3). The very low absolute transcript levels measured for inlC likely contributed to the conclusion that differences in inlC transcript levels were not significant by analysis of individual comparisons by Tukey's multiple-comparison procedure (Fig. (Fig.33).

σB, but not PrfA, regulates transcription of inlC2 and inlD.

inlC2 and inlD transcript levels were affected by the factors “strain,” “salt stress,” and “presence of cellobiose or charcoal” (Table (Table1).1). Under all growth conditions, inlC2 and inlD transcript levels in the wild-type strain and in the ΔprfA strain were similar, but those in the ΔsigB and ΔsigB ΔprfA strains were significantly lower and were below the qRT-PCR detection limit (Fig. (Fig.4;4; see Fig. S6 in the supplemental material). A highly significant effect of the sigB deletion on inlC2 and inlD transcript levels was confirmed by ANOVA, with no effect of the prfA deletion on transcript levels (see Table S2 in the supplemental material). These data clearly indicate that, under the conditions tested, inlC2 and inlD transcription is σB dependent and PrfA independent and that the two regulators do not interact to control transcription of these two genes.

inlF and inlG transcription are not dependent on either σB or PrfA under the conditions tested.

The inlF and inlG transcript levels for 10403S and the ΔsigB, ΔprfA, and ΔsigB ΔprfA strains were similar, irrespective of growth conditions (see Fig. S7 in the supplemental material). The factor “strain” did not affect inlF or inlG transcript levels (Table (Table11).

Exposure to salt induces expression of five internalin genes and reduces expression of the PrfA-dependent inlC.

Among the six internalin genes significantly affected by exposure to NaCl, five (inlA, inlC2, inlD, inlF, and inlG) showed generally higher transcript levels for 10403S cells exposed to salt, while one (inlC) showed generally lower mRNA transcript levels after salt exposure (Fig. (Fig.22 to to4;4; see Fig. S4 through S7 in the supplemental material).

inlA transcript levels in both 10403S and the ΔprfA strain grown with cellobiose with a 10-min final exposure to NaCl were higher than those in cells grown in cellobiose without exposure to NaCl (Fig. (Fig.2;2; see Fig. S4 in the supplemental material). However, in ΔsigB and ΔsigB ΔprfA cells, inlA transcript levels for cells exposed to NaCl were not higher than those of cells not exposed to NaCl; rather, in the presence of charcoal, inlA transcript levels were generally lower for cells exposed to NaCl. These data indicate that increased inlA mRNA transcript levels in cells grown with NaCl reflect σB-dependent induction of inlA transcription. No clear pattern of higher transcript levels in cells exposed to NaCl was observed for inlB, which is downstream of inlA and shows lower transcript levels than inlA under our experimental conditions (Fig. (Fig.2;2; see Fig. S4 in the supplemental material). Lower inlB transcript levels may indicate limited NaCl-based induction of inlB transcription, possibly due to low levels of read-through transcription from inlA. It is also possible that more than 10 min of post-NaCl exposure is needed to yield a detectable increase in inlB transcription.

Trends for inlC2 and inlD transcript levels were similar to those of inlA; for cells with an intact sigB, transcript levels for both inlC2 and inlD were consistently higher for cells exposed to NaCl than for cells not exposed to NaCl, particularly in the presence of cellobiose (Fig. (Fig.4;4; see Fig. S6 in the supplemental material). As inlC2 and inlD transcript levels in the ΔsigB background were below the qRT-PCR detection limit, it is not possible to determine whether higher inlC2 and inlD transcript levels in cells exposed to salt are solely σB dependent or also include a σB-independent component.

inlF and inlG transcript levels in cells exposed to NaCl were also generally higher than the transcript levels in cells not exposed to NaCl, with a relative increase for inlG transcripts larger than that for inlF transcripts. The presence of higher inlF and inlG transcript levels in cells exposed to NaCl was independent of strain (see Fig. S7 in the supplemental material), indicating that induction of inlF and inlG transcription in the presence of 0.3 M NaCl is independent of both σB and PrfA.

inlC was the only internalin gene that showed transcript levels in cells exposed to NaCl that were significantly lower than the transcript levels in cells not exposed to NaCl (Fig. (Fig.3;3; see Fig. S5 in the supplemental material). These observations parallel the finding that transcript levels for the PrfA-dependent plcA in strains with an intact prfA (i.e., 10403S and the ΔsigB strains) that were cultured in BHI-charcoal were also higher than those in such strains in BHI-charcoal-NaCl (Fig. (Fig.1B;1B; see Fig. S2b in the supplemental material). As inlC transcript levels were below the qRT-PCR detection limit for cells grown in the presence of cellobiose (Fig. (Fig.3;3; see Fig. S5 in the supplemental material), comparisons of inlC transcript levels were possible only between cells grown in BHI-charcoal or BHI-charcoal-NaCl. We hypothesize that (i) NaCl exposure down-regulates PrfA activity (rather than prfA transcription), as prfA transcript levels appear unaffected by NaCl exposure (Fig. (Fig.1B;1B; see Fig. S2b in the supplemental material) and (ii) reduced PrfA activity in NaCl-exposed cells results in reduced transcription of PrfA-dependent genes such as plcA and inlC. The hypothesis of NaCl-mediated down-regulation of PrfA activity is supported by the observation that transcript levels for the PrfA-dependent plcA were not affected by NaCl exposure in the ΔprfA and ΔprfA ΔsigB strains (Fig. (Fig.1B;1B; see Fig. S2b in the supplemental material).

inlA, inlB, inlC, inlC2, inlD, inlF, and inlG show lower transcript levels when grown in BHI-cellobiose than when grown in BHI-charcoal.

All seven internalin genes with detectable transcript levels (i.e., inlA, inlB, inlC, inlC2, inlD, inlF, and inlG) were significantly affected by the presence of cellobiose or charcoal (Table (Table1).1). The transcript levels for all of these genes in cells grown in BHI-cellobiose were lower than those in cells grown in BHI-charcoal (Fig. (Fig.22 to to4;4; see Fig. S4 through S7 in the supplemental material). Similarly, plcA transcript levels in cells of the 10403S and ΔsigB strain cells grown in BHI-cellobiose were also lower than those in cells grown in BHI-charcoal (Fig. (Fig.1B;1B; see Fig. S2b in the supplemental material). In 10403S, prfA transcript levels in cells grown in BHI-cellobiose were significantly higher than those in cells grown in BHI-charcoal (Fig. (Fig.1B;1B; see Fig. S2b in the supplemental material); this effect was not apparent in the ΔsigB strain. This may indicate that active PrfA (produced in the presence of charcoal) down-regulates its own transcription, possibly in a σB-dependent manner. Overall, these data also indicate that cellobiose and charcoal differentially affect transcript levels of different virulence and stress response genes.

When cells were cultured in BHI-cellobiose (with or without NaCl), inlA and inlB transcript levels were lower than the levels in cells grown in BHI-charcoal (with or without NaCl); these differences were often but not always statistically significant (Fig. (Fig.2;2; see Fig. S4 in the supplemental material). While lower transcript levels in the presence of cellobiose were observed for both inlA and inlB, differences in inlB transcript levels were smaller and generally not statistically significant. For inlA, the transcript levels in the presence of cellobiose that were lower than those in the presence of charcoal were generally less pronounced when growth conditions included a final 10-min exposure to NaCl. Interestingly, the effect of cellobiose on inlA and inlB transcript levels appears to be mediated through mechanisms that are both PrfA dependent and PrfA independent. Specifically, even in the ΔprfA strain, inlA and inlB transcript levels of cells grown in the presence of cellobiose were lower than those of cells grown in the presence of charcoal (Fig. (Fig.2;2; see Fig. S4 in the supplemental material), suggesting that a cellobiose-dependent mechanism that is at least partially PrfA independent down-regulates inlA and inlB expression.

inlC transcript levels, while detectable for all four strains grown in BHI-charcoal (with or without NaCl), were below the qRT-PCR detection limit for all four strains grown in the presence of cellobiose (Fig. (Fig.3;3; see Fig. S5 in the supplemental material), clearly indicating that inlC transcription is down-regulated during growth in the presence of cellobiose. Lower inlC transcript levels in the presence of cellobiose were also observed for ΔprfA cells, providing evidence that down-regulation of inlC in the presence of cellobiose is at least partially PrfA independent.

inlC2, inlD, inlF, and inlG transcript levels of cells grown in the presence of cellobiose generally were lower than those of cells grown in the presence of charcoal (Fig. (Fig.4;4; see Fig. S6 and S7 in the supplemental material). Lower transcript levels for these genes in the presence of cellobiose were observed in both the wild-type and ΔprfA backgrounds, consistent with the observation that transcription of these genes is PrfA independent. These data suggest that a PrfA-independent mechanism is responsible for differential transcript levels between cells grown in the presence of either charcoal or cellobiose for inlC2, inlD, inlF, and inlG.

InlB immunogold microscopy confirms that a sigB deletion has a greater effect on inlB expression than a prfA deletion has.

Surface localization of L. monocytogenes InlB by immunoelectron microscopy on intact cells of the 10403S parent strain as well as the ΔprfA, ΔsigB, and ΔinlB strains clearly showed reduced InlB on the surface of the ΔsigB strain (Fig. (Fig.5)5) relative to the wild-type and ΔprfA strains. The ΔinlB mutant, which was used as a negative control, showed virtually no labeling. Average counts of gold particles per μm2 were 126 ± 38 for the wild type, 91 ± 16 for the ΔprfA strain, 32 ± 12 for the ΔsigB strain, and 2 ± 5 for the ΔinlB strain. The ΔsigB and ΔinlB strains both showed significantly (P < 0.05; Tukey's multiple-comparison procedure) lower gold particle counts than the 10403S and ΔprfA strains. The lower gold particle counts associated with the ΔsigB strain relative to the ΔprfA strain confirm that σB contributes more than PrfA to InlB expression under the conditions examined in these experiments.

FIG. 5.
Surface localization of L. monocytogenes InlB by immunoelectron microscopy using whole cells of L. monocytogenes 10403S (A) and the ΔprfA (B), ΔsigB (C), and ΔinlB (D) strains. Arrows indicate the bacterial cell surface and point ...

DISCUSSION

Bacterial cells sense changes in their external environment and respond rapidly by altering protein expression via complex interactions among signal transduction pathways and global gene regulators. In L. monocytogenes, induction of gene expression under conditions of environmental stress is controlled, at least in part, by the stress-responsive alternative sigma factor σB (13, 20, 32, 60, 63). Regulation of many genes involved in host-pathogen interactions is coordinated by PrfA (8, 48, 56, 58, 64). In this report, we show that L. monocytogenes internalin genes can be grouped into distinct regulons controlled by σB, PrfA, both regulators, or neither. Our data also show that transcript levels for opuCA, gadA, inlA, inlC, inlC2, inlD, inlF, and inlG are significantly affected by the presence of NaCl. Transcript levels were lower for inlC, but higher for all others, in the presence of NaCl. Transcript levels for opuCA, gadA, plcA, inlA, inlB, inlC, inlC2, inlD, inlF, and inlG were also significantly affected by the presence of cellobiose or charcoal; for all of these genes, transcript levels were higher in the presence of charcoal.

Internalin genes can be grouped into distinct regulons controlled by σB, PrfA, both regulators, or neither.

σB and PrfA contribute differentially to the transcriptional regulation of different internalins under the conditions tested in these experiments such that (i) both σB and PrfA contribute to transcription of inlA and inlB, (ii) only σB contributes to transcription of inlC2 and inlD; (iii) only PrfA contributes to transcription of inlC, and (iv) neither σB nor PrfA contributes to transcription of inlF and inlG. L. monocytogenes internalin genes thus group into four distinct regulons, which suggests that specific internalins are expressed under different environmental conditions.

PrfA is considered to be a key regulator of L. monocytogenes virulence gene expression and has been shown to directly regulate 12 genes (48). The observation that σB and PrfA contribute to transcription of at least four and three internalin genes, respectively, illustrates that both proteins are important for regulation of known and putative L. monocytogenes virulence genes. This hypothesis is further supported by the fact that other L. monocytogenes genes that contribute to intrahost survival and infection are also regulated by σB or by σB and PrfA (e.g., bsh, opuCA, hfq [32, 33]). While previous studies have shown that the prfAp2 promoter region includes a functional σB-dependent promoter (49, 52, 55), our data indicate that transcription of inlA and inlB is predominantly and directly dependent on σB, as σB-dependent transcription of these genes was also observed in a ΔprfA background. Further, our observations of the relative importance of σB contributions to inlA transcription provide mechanistic support for the previous observation that σB is critical for L. monocytogenes gastrointestinal invasion in guinea pigs, which requires InlA (23). Combined with previous reports, our data support an emerging model in which σB is critical for expression of virulence and stress response genes important in the environment external to the host and for intestinal stages of infection, while PrfA predominantly up-regulates genes important for intracellular stages of infection.

Our data clearly show that inlC2 and inlD expression is σB dependent and independent of PrfA under the conditions studied. These findings extend results from previous microarray experiments with a ΔsigB strain, which provided initial evidence of σB-dependent expression of inlC2 and inlD, along with identification of a σB consensus promoter sequence upstream of the inlC2D operon (32). The PrfA-dependent expression of inlC reported here, which is also consistent with previous findings (18, 42, 48), has been further confirmed through a comparison of inlC transcript levels in isogenic prfA* (which expresses a constitutively active PrfA [57]) and ΔprfA strains (P. McGann, M. Wiedmann, and K. Boor, unpublished data). One previous study (5) reported that InlC plays a supportive role for InlA-mediated invasion in Caco-2 cells. In addition, inlC is strongly transcribed in the cytoplasm of phagocytic J774 cells (18), suggesting that it also may play a postinvasion role in L. monocytogenes infection. Although intracellular replication of a ΔinlC strain in Caco-2 and J774 cells appeared comparable to that of the wild-type strain, the strain showed reduced virulence in an intravenous mouse model (18). Our expression data, which indicate that inlC groups into a regulon with plcA and other PrfA-dependent genes (with transcription patterns that are both PrfA dependent and repressed by salt), while being distinct from inlA in its transcriptional regulation, support a possible role for inlC in systemic spread, which also requires plcA and other PrfA-dependent genes. The low inlE transcript levels reported here (i.e., transcript levels below the qRT-PCR detection limit) are in agreement with the observations of Dramsi et al. (16), who showed, using Western blot analysis, that InlE was not expressed during growth in bacterial medium.

Exposure to 0.3 M NaCl affected transcript levels of six internalin genes, with all but one (inlC) showing higher transcript levels under salt stress.

Exposure of L. monocytogenes to 0.3 M NaCl for 10 min induces high levels of σB activity (60); therefore, these parameters were selected for conditions requiring σB activity. Interestingly, in addition to inducing σB activity and, thus, higher transcript levels for most σB-dependent internalin genes, exposure to 0.3 M NaCl also induced higher transcript levels for the σB-independent internalin genes, inlF and inlG. These results suggest that salt or osmotic stress may be an important environmental stimulus for up-regulation of at least some L. monocytogenes virulence genes. Since 0.3 M NaCl mimics osmotic stress conditions that could be encountered by L. monocytogenes in the lumen of the human intestine (60), it is tempting to speculate that salt or osmotic stress conditions might represent a stimulus that signals to L. monocytogenes that it is present in an intestinal tract environment. If this hypothesis is true, then the specific internalin genes that are induced by salt stress might be important during gastrointestinal infection. In support of the importance of salt and osmotic stress as a stimulus that induces virulence gene expression, transcriptome analysis of Yersinia pestis identified a number of virulence genes that are up-regulated in response to salt and/or hyperosmotic stress conditions (26). Further, Heusipp et al. reported that transcription of Yersinia enterocolitica rpoE, which encodes the alternative sigma factor σE, is induced in vivo in infected mice and by osmotic stress, further supporting a link between salt stress and virulence, particularly as σE has been shown to contribute to virulence for a number of bacteria (27).

Interestingly, transcript levels for inlC, the only internalin gene that showed only PrfA-dependent transcription, were lower for cells exposed to NaCl than for unexposed cells. It is thus tempting to speculate that InlC contributes to L. monocytogenes virulence and/or survival during systemic spread in a host or during intracellular infection, with limited contributions outside of the host cell. Transcript levels for the PrfA-dependent gene plcA were also reduced in the presence of NaCl. These data suggest that osmotic or salt stress down-regulates all or some PrfA-dependent genes. Since plcA and inlC transcript levels, but not prfA transcript levels, were lower in the presence of NaCl, exposure to salt stress appears to mediate a decrease in PrfA activity, by acting directly either on the PrfA protein itself or on some other PrfA-regulating element (e.g., PrfA-activating factor [7]).

Both PrfA-dependent and -independent mechanisms mediate lower transcript levels for the seven internalin genes with detectable transcripts when cells are grown in BHI-cellobiose than when cells are grown in BHI-charcoal.

A wider range of L. monocytogenes genes beyond those that are PrfA dependent, including the σB-dependent opuCA, as well as all seven internalins with detectable transcripts examined in this study, showed reduced transcript levels when cells were grown in cellobiose. Specifically, transcription of the PrfA-dependent plcA and inlC for cells grown in BHI-cellobiose was lower than that for cells grown in BHI-charcoal, consistent with previous data, which showed that cellobiose mediates repression of PrfA activity (46). Down-regulation of PrfA-independent internalin gene transcription (i.e., inlC2, inlD, inlF, and inlG) in BHI-cellobiose, as well as that of PrfA-dependent genes in a ΔprfA null mutant background, indicates that differential regulation of internalin gene transcription in the presence of cellobiose or charcoal occurs through both PrfA-dependent and -independent mechanisms. These findings indicate that cellobiose and/or other easily metabolized sugars may serve as important environmental signals for L. monocytogenes to down-regulate transcription of virulence genes, including a number of internalin genes. In support of this hypothesis, previous studies have shown that repression of L. monocytogenes virulence gene expression by the presence of sugars is not confined to the presence of the β-glucoside cellobiose but is also associated with other sugars, including arbutin (10, 25, 46, 51). PrfA-dependent gene repression by sugars appears to be regulated by at least two separate mechanisms, one involving the catabolite repression pathway and another that specifically responds to β-glucosides (10). Although Milenbachs Lukowiak et al. (47) proposed that both repression pathways may act through PrfA, our data suggest that at least one pathway also acts through a PrfA-independent mechanism. Further studies will be needed to better understand the global effects of cellobiose, as well as other easily metabolized sugars, on gene expression in L. monocytogenes, particularly since initial transcriptome analyses by Milohanic et al. (48) also indicated broad and complex effects of cellobiose on L. monocytogenes gene expression. To illustrate, while a prfA deletion in strain 10403S did not affect opuCA transcript levels in our study, opuCA transcript levels were consistently lower in bacteria grown in the presence of cellobiose. In contrast, for a different L. monocytogenes strain (EGD), Milohanic and coworkers (48) classified opuCA as a class III gene, which was found to be up-regulated by PrfA, even in the presence of cellobiose. PrfA-dependent and -independent mechanisms of gene regulation under different environmental conditions, including the presence of easily fermentable sugars, appear complex. A better understanding of transcriptional regulation under these conditions is likely to provide insight into how different L. monocytogenes strains respond to different environments encountered during transmission.

Transcription of L. monocytogenes internalin genes is regulated by a diversity of mechanisms that may provide clues into the roles of specific internalin proteins in different environments and host-associated niches.

Taking into consideration the ubiquitous nature of L. monocytogenes and its remarkable ability to invade a wide variety of cell types, the existence of multiple and complex regulatory systems for controlling expression of cell surface molecules is not surprising. Our data demonstrate that expression of the L. monocytogenes internalin gene family is subject to a diverse set of regulatory mechanisms. Specifically, our results suggest that σB and PrfA function in an interactive network, as illustrated by the fact that L. monocytogenes grown in the presence of charcoal, a condition that activates PrfA (54), also down-regulates prfA transcription in 10403S but not in the ΔsigB strain, indicating a σB-dependent negative-feedback loop for prfA transcription and possibly providing a mechanistic explanation for early reports of the ability of PrfA to negatively regulate its own transcription (22). In summary, both PrfA and σB contribute to transcription of internalin genes and other genes involved in virulence. Our results indicate that interactions between PrfA and σB are complex, suggesting the existence of an intricate network that allows L. monocytogenes to regulate gene expression in response to changing environmental cues.

Supplementary Material

[Supplemental material]

Acknowledgments

We thank B. Bowen for creation of mutant strain FSL B2-068 and James Booth, Department of Biological Statistics and Computational Biology, Cornell University, for assistance with statistical analyses.

This work was supported in part by National Institutes of Health award no. RO1-AI052151-01A1 (to K.J.B.).

Footnotes

[down-pointing small open triangle]Published ahead of print on 2 March 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

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