Unveiling the Expression Characteristics of IspC, a Cell Wall-Associated Peptidoglycan Hydrolase in Listeria monocytogenes, during Growth under Stress Conditions

Abstract

Listeria monocytogenes serotype 4b is a food-borne pathogen of public health concern, since it accounts for approximately 40% of human listeriosis cases. We have recently identified IspC, a surface-localized peptidoglycan hydrolase, as the antigen recognized by a number of monoclonal antibodies (MAbs) produced against a serotype 4b strain for diagnostic applications. To determine whether IspC, which is well conserved among various serotype 4b strains, is a useful diagnostic marker in antibody-based methods, we assessed the expression of IspC in L. monocytogenes cultured under normal and stress conditions. A functional promoter directing the transcription of the ispC gene was identified upstream of the ispC open reading frame by constructing a promoterless lacZ gene fusion with the putative ispC promoter region and by 5′ rapid amplification of cDNA ends analysis. Using both the lacZ reporter gene system and immunofluorescent staining with an IspC-specific MAb, we provide evidence that IspC is expressed on the cell surface in all growth conditions tested (temperature, osmotic stress, pH, ethanol, oxidative stress, anaerobic conditions, carbon source, and type of growth media) that allow for cellular division, although the level of ispC gene expression varies. These results demonstrated the usefulness of IspC as an excellent diagnostic marker for the serotype 4b strains and imply that IspC, in conjunction with specific MAbs, can be targeted for detection and isolation of L. monocytogenes serotype 4b strains directly from food, environmental, and clinical samples with minimal or no need for culture enrichment.

INTRODUCTION

Listeria monocytogenes is a Gram-positive pathogenic bacterium that can lead to listeriosis, a life-threatening opportunistic infection caused by the ingestion of contaminated foods. Clinical outcomes of listeriosis range from asymptomatic infection to nonspecific flu-like symptoms, gastroenteritis, septicemia, meningitis, and fetal infection followed by abortion in pregnant women (24). In the environment, L. monocytogenes is extremely hardy and actively divides between 3 and 45°C (26), in up to 10% salt (16), at a pH of between 4.4 and 9.2 (5), and under anaerobic conditions (15). The ability of L. monocytogenes to grow in extreme environments makes it a concern for the food industry, particularly in food-processing plants where ready-to-eat foods are prepared.

L. monocytogenes is divided into 13 serotypes; however, 98% of human illness is caused by serotype 1/2a, 1/2b, and 4b strains (8). Serotype 4b strains account for more cases of human listeriosis than serotype 1/2a and 1/2b isolates combined, although 1/2a and 1/2b strains are much more commonly found in foods and the environment (24, 25). This suggests that serotype 4b strains are specifically adapted to infecting human hosts (24, 25). Serotype 4b strains are also more often isolated from patients with meningoencephalitis than from patients where the infection has been limited to the bloodstream (24). Listeriosis patients also suffer a 26% mortality rate when infected with a serotype 4b strain compared to a 16% mortality rate in patients infected with a serotype 1/2a or 1/2b strain (6). These observations suggest that serotype 4b is more virulent in humans than other serotypes. Therefore, the development of a diagnostic test specific for L. monocytogenes serotype 4b strains is important.

Current gold standard methods for isolating and detecting L. monocytogenes are culture based and labor intensive and take 5 to 7 days. Molecular methods, such as PCR, have been developed to expedite the detection of L. monocytogenes in food samples but have some disadvantages. They still require the culture enrichment steps prior to detection. Inhibitory substances and background bacteria present in food samples can confound the PCR results tremendously. In addition, determining the viability of an organism, which is important in a food recall, is impossible with molecular methods. Antibody-based methods are promising to overcome these drawbacks for rapid isolation and detection of L. monocytogenes from food and environmental samples. Several monoclonal antibodies (MAbs) which react specifically with L. monocytogenes serotype 4b were developed by our laboratory (13) and have been shown to recognize a surface-localized autolysin, IspC (homologous to LMOf2365_1093) (J. Ronholm, H. van Faassen, R. MacKenzie, Z. Zhang, X. Cao, and M. Lin, unpublished data). These MAbs, together with IspC as a surface maker, have the potential for use in diagnostic tests for L. monocytogenes serotype 4b strains. The surface expression of IspC at a level allowing these specific antibodies to bind L. monocytogenes cells originating from various growth conditions is critical to success in culture-independent, antibody-based detection methods and remains to be assessed. Surface protein expression is unstable and generally dependent upon growth conditions (3, 4, 19). The variability of surface epitope expression has also been previously shown to limit the usefulness of antibodies in L. monocytogenes detection (19). Therefore, the objective of this work was to characterize the surface expression of IspC during growth under various physical and chemical stresses and to determine if IspC is a useful marker for the development of antibody-based detection methods. By analyzing the activity of the ispC promoter in response to various environmental stresses with the lacZ reporter and the surface expression of IspC with immunofluorescence microscopy, we provide evidence that in a wide range of environmental conditions that allow L. monocytogenes cells to actively divide, IspC is expressed on the surface at high enough levels for immunological detection.

MATERIALS AND METHODS

Bacterial strains and monoclonal antibodies.

L. monocytogenes serotype 4b LI0521, a human clinical isolate, was used in this study and was grown in either brain heart infusion (BHI) broth (BD Diagnostics) or on BHI agar plates at 37°C and supplemented with 10 μg/ml kanamycin or 50 μg/ml erythromycin as required for bacteria transformed with pTCV-PispC. L. monocytogenes cell concentrations were estimated as previously described (14). The Escherichia coli strain DH5α was used for plasmid propagation. E. coli was cultured in Luria-Bertani (LB) media supplemented with 50 μg/ml kanamycin or 150 μg/ml erythromycin as required.

The M2773 monoclonal antibody in tissue culture fluid (TCF), which was previously shown to specifically interact with the IspC protein (Ronholm et al., unpublished), was used for immunofluorescent staining.

5′ RACE analysis.

The transcription start site for the ispC gene was determined by 5′ rapid amplification of cDNA ends (RACE) analysis using total RNA derived from L. monocytogenes. All solutions, water, glassware, and utensils used for RNA extraction were treated with 0.1% diethylpyrocarbonate (DEPC) (Sigma) overnight at 37°C and autoclaved. Total RNA was extracted from 1.5 ml mid-log-phase L. monocytogenes culture. Bacterial cells were lysed after being treated with RNAprotect bacterial reagent (Qiagen) per the supplier's instructions by digestion with 13,000 U of lysozyme (Sigma) in 0.1 ml of Tris-EDTA (TE) buffer for 30 min at 37°C, followed by mechanical disruption with the FastPrep system and Lysing Matrix B (MP Biomedicals) according to the manufacturer's instructions. RNA was purified from the cell lysate with the RNeasy minikit (Qiagen) per the supplier's instructions. The integrity of the RNA sample was confirmed by agarose gel electrophoresis. 5′ RACE was carried out, to identify the transcriptional start site, according to the manufacturer's instructions (Roche) using IspC gene-specific primers (Table 1).

Table 1.

Oligonucleotide primers used in this study

Primer	Nucleotide sequence
P998 (cDNA synthesis)	5′-CTTTGTTGTAGCAACAGATACTA-3′
P995 (GSP1)	5′-GAGGCTCCATTGCAGTTACTTTA-3′
P996 (GSP2)	5′-GCGGAATTCAGCATCAATTTTAA-3′
P997 (GSP3)	5′-CCCCAACCAGATTCTAGAATTCG-3′
P920	5′-AGAGAATTCAAAATATCAAAAAGAGCATAA-3′a
P921	5′-CGCGGATCCAATTTGTTTATTGTCCTAATT-3′b
P918 (Vlac1)	5′-GTTGAATAACACTTATTCCTATC-3′
P919 (Vlac2)	5′-CTTCCACAGTAGTTCACCACC-3′

^aThe EcoRI restriction site is underlined.

^bThe BamHI restriction site is underlined.

Construction of PispC-lacZ transcriptional fusions.

The pTCV-lac plasmid vector (21), a low-copy-number and broad-host-range plasmid that contains a promoterless lacZ gene, was used to evaluate the ispC promoter activity in various growth conditions. A 454-bp DNA fragment containing the ispC promoter was amplified by PCR using primer pair P920/P921 (Table 1) from L. monocytogenes genomic DNA. After digestion with BamHI and EcoRI (New England BioLabs), the amplified promoter region was cloned into BamHI and EcoRI sites of the pTCV-lac plasmid, resulting in the recombinant plasmid pTCV-PispC. The recombinant plasmid was sequenced with the Vlac primers (P918 and P919) to verify the inserted sequence. pTCV-PispC was subsequently introduced into competent L. monocytogenes by electroporation as described previously (28).

Growth conditions.

Various growth conditions were evaluated for their effects on ispC gene expression, including low temperature (4°C), high temperature (42°C), salt concentration (1 to 10%), acidity (pH 4), alkaline growth (pH 10), sublethal concentrations of ethanol (2 to 5%), oxidative media, anaerobic growth, alternative carbon sources, and various Listeria enrichment media. L. monocytogenes harboring pTCV-PispC was grown from frozen stock bacteria on a BHI agar plate and subsequently stored at 4°C. A single colony was inoculated into BHI broth and incubated for 16 to 18 h at 37°C. A 1:100 dilution of the overnight culture was subcultured into the media associated with each test condition (see below). As a control, L. monocytogenes containing the promoterless plasmid pTCV-lac was also tested under the same conditions. Each condition was examined using triplicate cultures. In addition, each condition was independently examined a minimum of 3 times, therefore a minimum of 9 measurements was recorded for each condition. Samples were taken and assayed for the β-galactosidase activity at 150 min, 350 min, and 24 h to provide a detailed look at when IspC expression occurs, except for the 4°C culture, where samples were collected after 168, 336, and 504 h of growth.

For temperature evaluation, BHI media were each equilibrated to the predetermined temperatures (4, 37, and 42°C) prior to bacterial inoculation.

The effects of various salt concentrations were examined over a 24-h period by supplementing BHI broth with 1 to 10% NaCl (wt/vol) at 1% intervals. The effects of pH were monitored by buffering BHI broth with HCl or NaOH to the desired pH (pH 2, 4, 6, 8, 10, or 12). The effect of sublethal concentrations of ethanol was assessed by adding the solvent directly to BHI broth to a final concentration of 2, 5, or 10% (vol/vol). The effects of highly oxidative conditions on IspC expression were tested by adding 7 mM cumene hydroperoxide (CHP) (Sigma) to BHI broth. Anaerobic conditions were created by autoclaving BHI in serum bottles. The bottles were sealed and the oxygen was removed by 5 cycles of vacuuming and then bubbling with a gaseous mix of 80% N₂, 10% CO₂, and 10% H₂. Resazurin was added at 0.005% (vol/vol) to verify that the medium was anaerobic. To determine if the carbon source affected ispC expression, L. monocytogenes was grown in the minimal media developed by Premaratne et al. (22) and supplemented individually with 20% (wt/vol) glucose, mannose, or fructose. Selective enrichment media have been shown to affect the antigen expression in L. monocytogenes (3 and Ronholm et al., unpublished). Therefore, the effects of various growth media on IspC expression were examined with three common enrichment culture broths: University of Vermont modified enrichment broth (UVM) (BD Diagnostics), Fraser broth (Oxiod), and Palcam broth (Oxoid).

β-Galactosidase assay.

β-Galactosidase assays were carried out essentially as described by Miller (18), with the exception that cells were collected by centrifugation for 2 min at 16,100 × g and resuspended in the assay buffer (0.06 M Na₂HPO₄, 0.04 M NaH₂PO₄, 0.01 M KCl, 0.001 M MgSO₄, and 0.05 M β-mercaptoethanol at pH 7) to remove background from the growth media prior to the assay. Enzyme activity was expressed in Miller units, defined as 1,000 × [OD₄₂₀ − (1.75 × OD₅₅₀)]/(incubation time × volume of culture × OD₆₀₀), where OD₄₂₀ is the optical density at 420 nm. As a negative control, L. monocytogenes was transformed with an empty pTCV-lac plasmid, and this was grown side by side with test samples. The number of Miller units used for the negative control was always less than 5. β-Galactosidase activity analyses were carried out in three independent experiments, each using triplicate samples, to ensure reproducibility. Results were analyzed for statistical significance with a Mann-Whitney U test, and statistical significance was defined as P < 0.001.

Immunofluorescent staining.

Immunofluorescent staining was carried out essentially as described previously (14). Briefly, L. monocytogenes was grown in each of the tested growth conditions for 24 h (504 h for the 4°C culture) and the cells collected by centrifugation for 2 min at 16,100 × g to remove the culture supernatant. Cell pellets were resuspended in phosphate-buffered saline (PBS) containing 5% bovine serum albumin (BSA) for 1 h at room temperature to block nonspecific protein binding sites. Cells were then allowed to react for 1 h with the MAb M2773 in tissue culture fluid at a dilution of 1:50 in PBS containing 5% BSA. After washing twice with PBS, cells were incubated with a 1:2,000 dilution of Dylight 488-conjugated goat anti-mouse IgG(H+L) (Jackson ImmunoResearch) in PBS containing 5% BSA for 1 h. Cells were washed 3 times with PBS and resuspended in PBS. Cells were examined with a fluorescence microscope first under phase-contrast and then fluorescence settings. Fluorescent images were captured with a charge-coupled device (CCD) camera using the QCapture Pro software (Q Imaging) with a 3-s exposure time.

RESULTS

Identification of the ispC gene promoter and the TSS.

The sequence upstream of the ispC open reading frame (ORF) was examined for the presence of regulatory elements. It should be noted than an ORF (LMOF2365_1092) for a putative teichoic acid ABC transporter, transcribed in the same direction as ispC, ends 224 bp upstream of the ispC start codon. Sequence analysis with a prokaryotic promoter prediction algorithm (www.fruitfly.org) revealed elements of two putative promoters and corresponding predicted transcriptional start sites (TSSs) with a perfect score of 1.0 within a region of 400 bp upstream of the ispC ORF. To determine if either of these putative promoters, or an alternative promoter within this region, was active, a 454-bp DNA fragment immediately upstream of the ispC translation start codon was cloned into the pTCV-lac plasmid (21) containing a promoterless lacZ reporter gene, making pTCV-PispC. Approximately 40 Miller units of β-galactosidase activity were detected at mid-log phase (350 min) of L. monocytogenes(pTCV-PispC), which is serotype 4b strain LI0521 that had been transformed with pTCV-PispC. This indicated that a functional ispC promoter exists within the 454-bp region immediately upstream of the ispC ORF.

To define the location of the ispC promoter more precisely, we mapped the TSS using 5′ RACE to an adenine nucleotide 31 bp away from the ispC translation start codon (Fig. 1). Surprisingly, this was not one of the two TSSs predicted for two putative promoters having a perfect score of 1.0. However, within this functional promoter region another promoter was predicted with a score of 0.73, containing a predicted TSS one nucleotide away from the experimentally determined TSS. Corresponding to this experimentally determined TSS, there are −10 (TGGTAAAAT) and −35 (TTGTTA) elements spaced 19 bp apart, as predicted by a bacterial promoter program (Softberry) (Fig. 1). Examination of the sequence in this functional promoter region did not identify consensus sequence elements typical for a sigma B transcription factor (−35 GTTT and −10 GGGnAn [where n represents any nucleotide]) (10) in L. monocytogenes or for a PrfA box (TTAACAnnTGTTAA) (25), which is the binding site of the virulence factor transcriptional activator PrfA.

Fig 1.

ispC transcription start site. Using 5′ RACE, the transcription start site of IspC was identified to be an adenine residue located 31 bp upstream from the translation start codon. The approximate −10 and −35 regions, as predicted by BPROM, are also indicated in boldface italics.

Effect of temperature on ispC expression.

The ispC expression in response to growth temperature (at 37, 42, and 4°C) was investigated by assessing the β-galactosidase activity under the control of the ispC gene promoter with L. monocytogenes(pCTV-PispC) (Fig. 2). The enzyme activity showed a significant decrease at 350 min and 24 h of growth at 42°C compared to that of the culture at 37°C (P < 0.001) (Fig. 2A). Because of a long generation time at 4°C, the bacteria were allowed to grow for an extended period in order to obtain sufficient numbers of cells for the accurate measurement of the β-galactosidase activity. The enzyme activity was substantially reduced when cultured at 4°C compared to that at 37°C. These results indicate that the ispC gene expression is subjected to regulation by temperature. In spite of a low level of ispC gene expression at 24 h of growth at 42°C or at 504 h at 4°C (Fig. 2A), the IspC protein was clearly detectable on the surface by immunofluorescence microscopy with the anti-IspC MAb, M2773 (Fig. 2B to E).

Fig 2.

Temperature-dependent regulation of ispC expression. The ispC promoter activity was measured in L. monocytogenes(pTCV-PispC) using a β-galactosidase assay. (A) After 150 min of growth at 42°C, there was no change in promoter activity compared to growth at 37°C at the same time point. However, after 350 min and 24 h of growth at 42°C, IspC expression was significantly less (P < 0.001) than that in cells grown at 37°C. IspC expression in cells grown at 4°C was shown to be very low. Statistically significant changes, in this and other figures, of promoter activity are indicated by two asterisks. In Fig. 2 to 8, the bars indicate the median value of all observations (n = 9), and the error bars are used to indicate the interquartile deviation. Cells grown for 24 h at 42°C were visualized using phase contrast microscopy (B) and were detectable by immunofluorescence microscopy with an anti-IspC MAb (C). Cells grown for 504 h at 4°C were visualized using phase contrast microscopy (D) and were also detectable by immunofluorescence microscopy (E).

Osmotic stress and ispC expression.

The expression of ispC in response to growth in high salt conditions (1 to 10% NaCl) was similarly investigated using the L. monocytogenes(pCTV-PispC) cells. After 150 min of growth, cells grown in 4 and 5% (wt/vol) sodium chloride had statistically significant reductions in enzyme activity (P < 0.001) compared to cells grown in BHI broth without additional salt. Cultures grown in the presence of 6 to 10% (wt/vol) NaCl did not show detectable levels of β-galactosidase activity at the 150-min time point (Fig. 3A). Cells grown for 350 min in 5 to 10% (wt/vol) NaCl had significant ispC expression (P < 0.001). After 24 h of growth, cultures supplemented with 3 to 10% (wt/vol) NaCl had significantly reduced enzyme activity (P < 0.001) (Fig. 3A). This indicated that the ispC gene expression is subjected to regulation by osmotic stress. Despite significantly decreased ispC promoter activity in osmotically stressed cells, the IspC protein was detectable on the surface of cells after 24 h of growth in BHI containing various salt concentrations by immunofluorescence microscopy with M2773, although the fluorescence signal decreased in a NaCl concentration-dependent manner (Fig. 3B to I). Cell morphological changes, such as chaining, started to occur in bacteria grown at 5% (wt/vol) NaCl and became more prominent as the salt concentration increased, although chained cells were still able to express the surface IspC, which was detectable by immunofluorescence microscopy with M2773.

Fig 3.

Sodium chloride downregulation of ispC expression. The ispC promoter activity was measured in Miller units at 1 to 10% NaCl. Concentration-dependent activity suppression was observed at all time points. (A) Statistically significant decreases in ispC gene expression (P < 0.001) were found for several conditions compared to cells at the same time point grown in BHI without additional salt (positive control). Although cellular growth and division were also suppressed by osmotic stress, the IspC protein was detectable on the cell surface by immunofluorescence in 1% (C), 6% (E), 7% (G), and 10% (I) NaCl. Panels B, D, F, and H show phase contrast images to demonstrate the presence and abundance of L. monocytogenes cells. Significant cellular chaining was apparent in the presence of >5% NaCl.

Minimal effect of culture pH on ispC expression.

L. monocytogenes has been reported to grow in the pH range of 4.4 to 9.2 (5); however, our preliminary experiments indicated that cellular division does not occur below pH 6 or above pH 10, and the activity of β-galactosidase was investigated in L. monocytogenes(pTCV-PispC) only within the pH range of 6 to 10. At the 150-min time point, the activity of the enzyme expressed under the control of the ispC promoter was not affected by the culture medium pH. However, cultures at pH 6 and 10 had significantly lower enzyme activity than the positive control (pH 7.6) (P < 0.001) at the 350-min time point (Fig. 4A). At 24 h there was no difference in the enzyme activity between cultures at different pHs (Fig. 4A). Consistent with these results is that after 24 h of growth, surface expression of IspC was detected by immunofluorescence microscopy in cells cultured under each condition, although qualitative observations indicated that growth in alkaline conditions makes cells easier to detect with the anti-IspC MAb (Fig. 4B to G).

Fig 4.

pH has a minimal effect on ispC expression. Cultures with a pH outside the range 6 to 10 could not actively divide within the time periods for this experiment. (A) Within the pH 6 to 10 range, ispC expression was relatively constant, with statistically different (P < 0.001) expression only occurring at 350 min postinoculation at pH 6 and 10. At 24 h of growth there was no significant difference in ispC expression between any of the cultures. IspC was also detectable on the cell surface by immunofluorescence at pH 6 (C), pH 8 (E), and pH 10 (G). Corresponding phase contrast images are shown in panels B, D, and F for comparison.

Effect of sublethal ethanol concentrations on ispC expression.

β-Galactosidase activity was assessed in L. monocytogenes (pTCV-PispC) cultivated in BHI broth containing sublethal ethanol concentrations (2, 5, and 10%, vol/vol). Ethanol at a concentration of 10% (vol/vol) was inhibitory to cell division, therefore this condition was eliminated from the study. Although generation time was increased for bacterial cells in the presence of ethanol, the level of ispC expression was not significantly affected (P > 0.001) by the ethanol concentration used at any of the time points (Fig. 5A). Therefore, expression of the ispC gene is not regulated by exposure to sublethal ethanol, and the presence of the IspC protein in cells grown in 2 and 5% ethanol was easily detected by immunofluorescence microscopy using the anti-IspC MAb M2773 (Fig. 5B and E).

Fig 5.

Sublethal dose of ethanol does not affect ispC expression. (A) Growth in the presence of 2 or 5% ethanol did not significantly (P < 0.001) affect ispC expression compared to the culture in the presence of 0% ethanol. IspC is readily detectable on the cell surface of L. monocytogenes cells grown in 2% (C) and 5% (E) ethanol. Phase contrast images are shown in panels B and D for comparison.

Effect of oxidative and anaerobic growth conditions on ispC expression.

L. monocytogenes(pTCV-PispC) was grown in CHP to provide highly oxidative conditions and also under anaerobic conditions before being analyzed for β-galactosidase activity (Fig. 6). Previous studies have shown that L. monocytogenes survives in 13.8 mM CHP (2); however, our preliminary experiments indicated that L. monocytogenes cannot actively divide in CHP concentrations as low as 7 mM; therefore, this condition was eliminated from the study. Similar levels of enzyme activity were observed at 150- and 350-min time points for cell cultures under both aerobic and anaerobic conditions, revealing no effect of these conditions on the expression of ispC. However, after 24 h of growth, the ispC promoter showed more activity in aerobically cultured cells than in anaerobic conditions (P < 0.001) (Fig. 6A). Bacterial cells grown in both aerobic and anaerobic conditions express the IspC protein and are easily detectable by using immunofluorescence microscopy with the M2773 antibody (Fig. 6B to E).

Fig 6.

ispC is expressed in anaerobic conditions. (A) ispC expression was measured from L. monocytogenes(pTCV-PispC) grown in an anaerobic culture and compared to cells grown in an aerobic culture. At 350 min after inoculation, there were no differences in ispC expression. Although ispC was also expressed at 24 h after inoculation, levels of expression were significantly (P < 0.001) lower in the anaerobic culture than in the aerobic culture. However, IspC was detectable on the cell surface 24 h after inoculation of both aerobic (C) and anaerobic (E) growth media. The same field of view is shown in panels B and D, respectively, in phase contrast to allow for comparison.

Carbon source does not regulate ispC expression.

The expression of the ispC gene was examined in response to three carbon sources (glucose, fructose, and mannose). Carbon source has been cited as having a major effect on the expression of various proteins in L. monocytogenes (17, 22). L. monocytogenes(pTCV-PispC) showed similar growth (data not shown) and β-galactosidase activity in the minimal media supplemented with each carbon source (Fig. 7A). Therefore, the ispC gene expression is not regulated by carbon source. Cells grown with each carbon source were also easily detected by the presence of the IspC protein by immunofluorescence microscopy (Fig. 7B to G).

Fig 7.

Carbon source regulation of ispC expression. (A) ispC was expressed at the same level in minimal media when supplemented individually with a glucose, fructose, or mannose carbon source. L. monocytogenes cells are also detectable by an anti-IspC antibody when grown in glucose (C), fructose (E), or mannose (G). In addition, phase contrast images of cells grown in glucose (B), fructose (D), or mannose (F) are shown for comparison.

Effect of various L. monocytogenes enrichment media on ispC expression.

Several types of media are routinely used for the selective enrichment of L. monocytogenes in most Listeria methods prior to bacterial isolation and detection. To expand the potential of MAbs which recognize IspC beyond direct culture-independent detection into postenrichment detection, the expression of the ispC gene, as assessed by the activity of β-galactosidase transcribed by the ispC promoter in L. monocytogenes(pTCV-PispC), was examined over the span of the growth curve in four media, including BHI broth (Fig. 8A), Fraser broth (Fig. 8B), Palcam broth (Fig. 8C), and UVM (Fig. 8D). Although the levels of β-galactosidase activity varied between media, it was detectable in all of the media used. Expression of the ispC gene was significantly higher (P < 0.001) at all time points when cells were grown in BHI broth than when they were grown in UVM. ispC gene expression is the same in cells grown in Fraser and Palcam broths (P > 0.001) except for at the 750-min time point, when the ispC gene is expressed more in Fraser broth (P < 0.001). The activity of β-galactosidase under the control of the ispC promoter is generally higher in cells grown in BHI broth than in cells grown in Fraser and Palcam broths, although significance varies between time points. IspC was present on the cell surface at high enough levels to allow cells to be easily detected by immunofluorescence microscopy after 24 h of growth regardless of growth media (Fig. 8E to J).

Fig 8.

ispC expression in various enrichment media. The β-galactosidase activity under the control of the IspC promoter was measured in Miller units in L. monocytogenes cells grown from inoculation to lag phase in BHI broth (A), Fraser broth (B), Palcam broth (C), and UVM (D). A clear peak in promoter activity during mid-log phase could be seen only in BHI broth. The presence of the protein IspC on the surface of cells grown in Fraser broth (F), Palcam broth (H), and UVM broth (J) was detectable after 24 h of growth by immunofluorescence microscopy. The phase-contrast images of the same field of view are shown in panels E, G, and I, respectively, for comparison.

DISCUSSION

In this study, we characterized the expression characteristics of IspC, a surface-associated peptidoglycan hydrolase, through the use of the lacZ reporter gene system and immunofluorescence microscopy in order to assess the value of IspC as a diagnostic marker for L. monocytogenes serotype 4b. A functional IspC promoter was active in L. monocytogenes and was identified immediately upstream of the ispC translation start site. This promoter remained active in ispC gene transcription in all environmental conditions tested which are conducive to bacterial cell division; however, the levels of its activity varied. We have further demonstrated that L. monocytogenes serotype 4b cells are capable of displaying IspC on the cell surface in each of the tested conditions, making cells detectable by immunofluorescence microscopy using an anti-IspC MAb. Even in an extreme stress condition, where there was only weak expression of IspC, there was still a sufficient amount of IspC localized on the cell surface to allow detection using the anti-IspC MAb. We recently have demonstrated that IspC is highly conserved among L. monocytogenes serotype 4b isolates but not in other serotypes (Ronholm et al., unpublished). Since IspC is unique to serotype 4b isolates and its expression is detectable under normal or stress growth conditions, it can serve as an excellent diagnostic marker, with the aid of anti-IspC antibodies, for detection or isolation of this important serotype of L. monocytogenes.

Transcription of IspC started from a TSS 31 bp upstream of the translation start site under the direction of its own promoter (Fig. 1). Expression of ispC appears to peak, when bacterial cells are grown in BHI broth, during mid-log phase (350 min) (Fig. 8A). This finding correlated well with the result of a previous study which showed by reverse transcription-PCR (RT-PCR) that the IspC gene was transcribed at the highest levels during early log phase (27). The IspC protein was detectable in all growth conditions examined in this study; however, particularly low levels of expression were observed during growth in BHI broth supplemented with 10% (vol/wt) NaCl (Fig. 3) or during growth at 4°C (Fig. 2).

An anti-IspC MAb was able to detect the target protein on the cell surface by immunofluorescence microscopy after 24 h of growth (504 h of culture at 4°C) regardless of growth conditions. These findings were an important addition to the ispC promoter activity analysis, which suggested that IspC can serve as a good diagnostic marker because gene expression is always observed in L. monocytogenes. In contrast, other studies conducted to determine which L. monocytogenes growth conditions allow for expression of the antigens targeted by various antibodies did not use cells grown entirely under a stress condition (3, 7). In these studies, cells were grown either in a nonstressed condition followed by subculturing for a limited time in the stress condition (7) or in a stress environment and then rescued in an enrichment broth (3). The problem with growing cells for a limited time in the stress condition followed by transfer to enrichment broth is that residual protein from the original culture does not get degraded quickly enough to disappear after the short incubation time and therefore is still detected (Ronholm et al., unpublished). Our findings that IspC was detectable with this specific MAb in all growth conditions tested makes this protein antigen a novel potential diagnostic marker for diagnostics for L. monocytogenes serotype 4b, since studies which have used a methodology similar to the one presented here have found that their antigens were not expressed at detectable levels under all tested conditions (4, 11, 19, 20). Since the primary objective of this study was to validate IspC as a marker for diagnostic use in pre-enrichment bacterial detection, possibly in a biosensor in a microfludics-based flow cytometer, our methodology, where cells were grown entirely in the stress condition and detected by immunofluorescence signal, allowed us to examine IspC expression in cells from selected environments under detection conditions simulating those that would be present in a biosensor applied to the detection of L. monocytogenes directly from food or environmental samples.

L. monocytogenes was shown to have chained morphology when subjected to extreme osmotic stress (Fig. 3). This finding was in agreement with other studies (4, 9). Cellular chaining indicates that the autolysins involved in cell division either are not expressed or are not active under these conditions.

An anti-IspC MAb was able to detect L. monocytogenes cells grown in each of the selective enrichment broths, although there was variation in the activity of the ispC promoter between broths (Fig. 8A to D). These experiments were carried out to assess if IspC can be detected by anti-IspC antibodies on the surface of L. monocytogenes after enrichment. Selective enrichment broths have been shown to affect the expression of several surface antigens (3, 11, 19, 20). Similar studies, using different MAbs (EM-7G1 and C11E9) which also recognize an Listeria autolysin (lmo2691) in L. monocytogenes serotype 1/2a, found that growth in UVM entirely inhibits the expression of the antigen, rendering these antibodies useless for detection when enrichment takes place in this medium (3, 19, 20). UVM was also found to reduce the expression of ActA, making detection impossible using an anti-ActA antibody after UVM enrichment (11). In agreement with these findings, we also showed that expression of IspC was more reduced in UVM than in any other media. However, despite weak activity of the ispC promoter, cells grown in UVM were still detectable by immunofluorescence microscopy with an anti-IspC MAb. This is likely because IspC is a very stable protein (Ronholm et al., unpublished) (27), and even minor expression during any growth phase leads to surface protein accumulation. The anti-IspC MAb was also able to detect cells grown in Fraser broth almost as well as cells grown in BHI broth. The EM-7G1 and C11E9 antibodies were unable to detect cells grown in Fraser broth (3, 19).

IspC is a peptidoglycan hydrolase with N-acetylglucosaminidase activity (23). It is involved in virulence, as evidenced by the attenuation seen in the ΔispC deletion mutant (28). Elucidating the mechanisms behind regulation of virulence-associated autolysins is an important step in determining their role in pathogenesis, which appears to be separate from their roles in cellular division or growth (12). We are very interested in the biological significance of the IspC autolysin, which appears to be serotype 4b specific and is expressed over a broad range of environmental conditions. The ispC promoter appears to be most active during early log phase. In addition, we found that ispC is always expressed in cells which are able to divide. These two observations suggest that IspC has a role in cell division. However, previous work indicates that IspC is not directly involved in cellular division, since ispC knockout mutants do not have the characteristic chaining phenotype that is seen in cells where essential division molecules have been knocked out (28). One possibility is that IspC has a role in cell division; however, a phenotype is not observed in the deletion mutant because another autolysin with an overlapping function is able to functionally compensate for the ispC knockout. IspC expression is also highly upregulated during rabbit infection (30), and based on the rate of cell division proportionate to its upregulation, it is likely that there are requirements for IspC during infection besides those related to growth and division. The role of IspC in infection is not currently understood but is of interest.

Food-borne bacteria encounter a variety of environmental stresses which alter surface protein expression, and this affects our ability to detect bacteria using antibody-based methods (19). Several studies have demonstrated that the ability of polyclonal and monoclonal antibodies to detect Listeria is highly dependent on culture conditions (3, 4, 7, 11, 19, 20, 29). Ideally, next-generation rapid detection techniques will allow for specific pathogen detection without the need for enrichment. Developing technologies, such as flow cytometry, immunomagnetic separation, and biosensors, have the potential to eliminate the need for cultural enrichment (1). However, a major hurdle to overcome for such technologies is the need to identify surface antigen targets that are consistently expressed regardless of sample matrix or growth phase.

Our laboratory has produced MAbs which are specific to L. monocytogenes serotype 4b and bind to the cell wall-associated IspC with high affinity (13 and Ronholm et al., unpublished). However, before these antibodies are used in diagnostics, it is crucial to evaluate the influence of stress on expression of the target protein and determine how this affects antibody reactions (7). We have shown that IspC is expressed in each of our tested conditions and that a MAb to IspC is able to detect bacterial cells in every tested growth environment, including extreme stress conditions and enrichment media, supporting the claim that IspC, together with its specific MAbs, has value in antibody-based diagnostics for L. monocytogenes serotype 4b.