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CD154 as a potential early molecular biomarker for rapid quantification analysis of active Staphylococcus enterotoxin A

Reuven Rasooly, Bradley J. Hernlem
DOI: http://dx.doi.org/10.1111/j.1574-695X.2011.00874.x 169-174 First published online: 1 March 2012

Abstract

Staphylococcus aureus is a major bacterial pathogen producing a group of 21 enterotoxins (SEs). These enterotoxins have two separate but related biological activities. They cause gastroenteritis, and they function as superantigens that activate large numbers of T cells. In the current study, we demonstrate that short-term ex vivo exposure of primary naïve CD4+ T-cells to SEA induces differential expression of the T cell surface receptor CD154 in a time- and dose-dependent manner. In addition, we show that SEA induces higher CD154 protein expression and higher splenocyte cell proliferation compared with SEB. We also demonstrate that expression of CD154 can be used for rapid detection of active SEA in milk.

Keywords
  • enterotoxin
  • CD154 expression
  • cell proliferation

Introduction

Staphylococcus aureus is one of the major bacterial pathogens causing clinical infection and foodborne illness (Dinges et al., 2000). This bacterium produces a group of 21 known enterotoxins (SEs) that have two separate biological activities: they act on the gastrointestinal tract, and as a superantigen on the immune system. These two activities (emetic and superantigenic) are related (Hui et al., 2008; Hu et al., 2009). When superantigenic activity is abolished by mutation, enterotoxic activity is also lost (Harris et al., 1993). Comparison of amino acid sequence homology among different types of SEs shows that SEA, SEE and SED belong to one group and SEB and SEC to another group, with 31% homology between SEA and SEB (Balaban & Rasooly, 2000).

SEB is recognized as a potential bioweapon (Henghold, 2004), and SEA is associated with 75% of staphylococcal outbreaks. Recent contamination of powdered skim milk with SEA caused an extensive outbreak with 13 420 cases in Japan (Asao et al., 2003; Ikeda et al., 2005). This outbreak emphasizes the need to develop better methods to detect active SEs. In our previous study (Rasooly & Do, 2009), we developed a non-radioactive splenocyte cell-based assay that can detect active SEA in food after incubation of the spiked food with splenocyte for 48 h. This cell-based assay is much more sensitive than the in vivo monkey or kitten bioassays. The purpose of the present study is to further improve the assay by developing a more rapid method (< 6 h of incubation) for detection of active SEA in food. It was demonstrated that γδ T cells are capable of expressing MHC class II molecules, and function as antigen presenting cells (APC) (Cheng et al., 2008). Our previous work has shown that a subtype of mouse naïve CD4+ T cells expresses MHC class II on their cell surface and that these CD4+ T cells can perform the role of both APC and T cells, able to present SEA to itself or neighboring CD4+ T cells via MHC class II, thus inducing CD4+ T-cell proliferation in the absence of macrophages, dendritic cells or B lymphocytes (Rasooly et al., 2011). In this work, we identified genes that were altered in CD4+ T-cells following short-term ex vivo exposure to SEA. The results show that CD154 (CD40 ligand) that is expressed on CD4+ T cell, providing costimulatory signals to B cells and APC is differentially expressed, and that its protein expression could be used for early SEA detection in milk and potentially other food products.

Material and methods

Chemicals and reagents

SEA and SEB were obtained from Toxin Technology (Sarasota, FL). Nonfat dry milk was obtained from Nestle Carnation. Bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU) was obtained from Calbiochem (San Diego, CA). Anti-CD4 antibody labeled with APC was obtained from eBioscience (San Diego, CA). CD154 detection cocktail contains a CD40 blocking antibody that prevents the downregulation of CD154 expression, CD28 antibody, and anti CD154 antibody labeled with PE were obtained from Miltenyi Biotec (Auburn, CA). CD4+ T-cell positive isolation kits were obtained from Invitrogen (Carlsbad, CA).

Splenocyte isolation

Spleens from C57BL/6 female mice were aseptically removed and disrupted using a syringe and needle in Russ-10 cell culture medium (made by combining 450 mL of RPMI 1640 medium without glutamine (Gibco), 50 mL fetal bovine serum (Hyclone, Logan, UT), 5 mL 200 mM glutamine (Gibco), 5 mL antibiotic-antimycotic (containing penicillin, streptomycin, and fungizone; Gibco), 5 mL nonessential amino acid mix (Gibco), 5 mL sodium pyruvate (Gibco), and 0.25 mL of 100 mM beta mercaptoethanol (Sigma). Cells were centrifuged at 200 g at 4 °C for 10 min. Red blood cells were then lysed by adding red cell lysis buffer (0.15 M NH4Cl, 10 mM KHCO3, 0.1 mM Na2EDTA). Cells were again centrifuged and resuspended in Russ-10 medium, and viable cells were counted using trypan blue and a hemocytometer.

SEA or SEB induced naive T-cell proliferation assay

Cells were placed in 96-well plates (1 × 106 cells mL−1, 0.2 mL) in Russ-10 medium and treated with various concentrations of SEA or SEB ranging from 5 pg mL−1 to 5 µg mL−1 and then incubated at 37 °C in a 5% CO2 incubator. After 48 h, cell proliferation was measured by adding bromodeoxyuridine (5-bromo-2-deoxyuridine, BrdU), which was incorporated into the DNA of dividing cells, 4 h before fixation as described by manufacturer instructions (Calbiochem). Spectrophotometric absorbance was measured at 620 and 450 nm.

Positive isolation of murine CD4+ T-cells

Murine CD4+ T-cells were isolated using a positive isolation method (Dynabeads Mouse CD4 L3T4 Isolation Kit), according to the manufacturer's instructions. Briefly, splenocytes were resuspended in isolation buffer (PBS supplemented with 0.1% BSA and 2 mM EDTA) at a concentration of 1 × 107 cells mL−1, and incubated with washed Dynabeads (25 µL of Dynabeads per 107 cells) for 20 min on ice with gentle rotation. After incubation, the cells and Dynabeads were placed on a magnet for 2 min. The supernatant was removed and the bead-bound cells were washed with isolation buffer three times. The bead-bound cells were resuspended in Russ-10 media (107 cells per 100 µL of media) and DETACHaBEAD mouse CD4 was added (10 µL per 107 cells) and incubated for 45 min with gentle rotation at room temperature. The detached cells were washed three times and resuspended in media.

Quantitative RT-PCR analysis

Quantitative RT-PCR (qRT-PCR) analysis to confirm the microarray result was carried out as described previously (Rasooly et al., 2007). The forward primer used for CD154 was 5′-ACGTTGTAAGCGAAGCCAAC-3′, and the reverse primer used was 5′-TATCCTTTCTTGGCCCACTG-3′. The housekeeping gene hypoxanthine-guanine phosphoribosyl transferase (Hprt) forward primer was 5′-GTTGGATACAGGCCAGACTTTGTTG-3′ and the reverse primer used was 5′-GAGGGTAGGCTGGCCTATA GGCT-3′.

Sample preparation of food

Milk was prepared with 5% non-fat dry milk dissolved in water and spiked with 200 or 500 ng mL−1 of SEA and then incubated with cells.

Flow cytometry

T cells (0.5 × 106 cells) were cleaned of clumps and large debris by passing through a cell strainer on a 5-mL polystyrene tube (BD Falcon Cat# 352235). As suggested by manufacturer's instruction, 4 µL of PE conjugated CD154 detection cocktail and 0.125 mg APC-conjugated anti-CD4 antibody were added to 200 µL of cell suspension followed by incubation for 40 min at 4 °C. Cells were washed twice in 200 mL of PBS and then resuspended in 0.5 mL of PBS. Flow cytometric analysis was performed using a FACS Vantage SE (BD Biosciences, San Jose, CA) fitted with a Cobolt Calypso™ 491 nm, 100 mW laser (Cobolt AB, Sweden) and a Cube™ 640 nm, 40 mW laser (Coherent Inc., Santa Clara, CA). The R-phycoerythrin (PE) fluorochrome was excited using the 491 nm laser and fluorescence quantified through a 585/42 nm bandpass filter. The APC fluorochrome was excited using the 640 nm laser and fluorescence quantified through a 676/29 nm bandpass filter (Semrock, Rochester, NY).

Statistical analysis

Statistical analysis was performed using SigmaStat 3.5 for Windows (Systat Software, San Jose, CA). One-way analysis of variance (anova) was used to compare CD154 gene transcription from control and treated cell with 200 ng mL−1 of SEA for 8 h. We also preformed the same analyses after incubation of splenocytes with increasing concentrations of SEA or SEB. The experiments were repeated at least three times and results with P < 0.05 were considered statistically significant.

Results

In our previous work (Rasooly & Do, 2009), we demonstrated that we can quantify active SEA in food after 48 h using splenocyte proliferation response. In this study, we recognized that there are very early molecular responses to SEA leading to CD4+ T-cell proliferation. To develop biomarkers for early detection of active SEA, we attempted to identify which differential expression genes are altered after short-term SEA exposure. Naïve CD4+ T cells were exposed to 200 ng mL−1 of SEA for 8 h, and changes in gene expression induced by the toxin by cDNA were measured by microarray gene expression. We validated the array results by quantitative real-time RT-PCR. Our microarray results showed that the mRNA transcript levels of the cell surface receptor CD154 were statistically significant; 3.2-fold higher in CD4+ T cell treated with SEA than in the control (data not shown). RT-PCR results reported as relative to the commonly used housekeeping gene HPRT were consistent with the findings from the microarray studies, although with smaller fold changes but statistically significant. The results show that SEA enhanced CD154 gene transcription by 1.6-fold above the level of control.

CD154 protein expression after short-term stimulation of CD4+ T cells with SEA

The molecular data suggested that CD154 expression was upregulated at the transcriptional level in CD4+ T cells treated with SEA. To demonstrate that this upregulation enhanced the kinetics of CD154 protein expression after short-term stimulation with SEA, we incubated CD4+ T cells with SEA for 2, 4, and 6 h, stained with anti CD4 and anti CD154 antibodies, and analyzed by flow cytometry. Control untreated noninduced cell were used to determine where the markers would be placed (Fig. 1a). The upper-right quadrant beyond the marker contains events that are double positive for both CD4+ and CD154. Our results demonstrated that short-term ex vivo stimulation of primary naïve CD4+ T cells with SEA induced the expression of the T-cell surface receptor CD154 and that the expression of CD154 increased over the 6-h SEA stimulation.

Figure 1

Kinetics of CD154 protein expression in CD4+ T cells after stimulation with SEA. CD4+ T cells were spiked with 5 µg mL−1 of SEA for 2, 4, and 6 h followed by flow cytometry measurement of CD154 expression.

CD154 differential gene expression for quantitative determination of biologically active SEA

Our data suggested that CD154 can be used as an early biomarker for SEA exposure. To evaluate the sensitivity of the assay and to determine whether increasing concentrations of SEA induced CD154 differential protein expression, we incubated naïve CD4+ T cells with increasing concentrations of SEA (1 ng mL−1, 200 ng mL−1, and 5 µg mL−1). After incubation for 6 h, the cell surface expression levels of CD154 were analyzed by flow cytometry. Our results (Fig. 2) showed for the first time that short-term ex vivo stimulation of primary naïve CD4+ T cells with SEA induced the expression of the T-cell surface receptor CD154 in dose-dependent response. The data suggested that there is a correlation between time of exposure, SEA concentration, and CD154 protein expression, and that SEA can be detected at 1 ng mL−1.

Figure 2

Quantitative determination of biologically active SEA. Increasing concentrations of SEA were incubated with CD4+ T cells. After 6 h, expression of CD154 was measured by flow cytometry.

CD154 is strongly induced by SEA and to a lesser extent by SEB

Our data suggested that low concentration of SEA induced elevated expression of CD154 and that this biomarker can be used for detection of one class of SEs. To assess whether CD154 could also be used to quantify the presence of biologically active SEB, which is genetically very diverse from SEA and shares a low degree (31%) of amino acid sequence homology with SEA (Balaban & Rasooly, 2000), we incubated naïve CD4+ T cells with two concentrations of SEB (200 ng mL−1 and 5 µg mL−1). SEA (200 ng mL−1) was used as a positive control. CD154 protein expression was analyzed after incubation for 6 h. As shown in Fig. 3a, negative control culture without the toxin showed the expression of 0.1% of the cell surface receptor CD154. The sensitivity of SEB detection was lower than SEA: At 200 ng mL−1, SEB induced CD154 protein expression on only 0.3% of CD4+ T cells. On the other hand, at the same concentration, SEA induced CD154 expression on 5% of the CD4+ T-cells. At 5 µg mL−1, SEB induces CD154 expression of only 1% of CD4+ T cells.

Figure 3

(a) SEA induces higher CD154 protein expression at lower concentration than SEB. The effect of increasing concentrations of SEB (200 ng mL−1 and 5 µg mL−1) or SEA (200 ng mL−1) on the percentage of CD4+ T cells expressing CD154. Flow cytometry shows that after 6 h of incubation, the sensitivity of SEB detection was lower than SEA. Without the toxins, only 0.1% of the T cells are induced to express CD154. At 200 ng mL−1, SEB induced CD154 protein expression is 0.3% of CD4+ T-cells. At 200 ng mL−1, SEA induced CD154 expression is 5%. (b) Splenocytes cell strongly proliferate with exposure to SEA and to a lesser extent with SEB. Splenocytes cells were spiked with increasing concentrations of SEA or SEB. After incubation for 48 h, newly synthesized DNA was measured by BrdU incorporation. Error bars represent standard errors, and an asterisk indicates significant differences (P < 0.05) between SEA or SEB treatment and control.

Figure 4

Detection of SEA in milk. Fifteen microlitres of milk spiked with increasing concentrations of SEA were added to 185 µL of 1 × 106 cells mL−1. After incubation for 6 h, expression of CD154 was measured by flow cytometry.

We also performed the same analyses using BrdU splenocyte proliferation assay. The findings were consistent with the flow cytometry results, that is, SEA was able to stimulate splenocyte cells at a much lower concentration than SEB. Consequently, the level of SEA detection was at least 10 000 times lower than SEB (Fig. 3b).

Expression of CD154 can be used for rapid detection of active SEA in food

We spiked milk with increasing concentrations of SEA. Our results showed that 6 h post-spiking, the CD154 protein on the CD4+ T-cell surface was upregulated proportionate in a dose-dependent manner. This result demonstrated that we can detect active SEA in milk after a short exposure to the toxin and suggests application in other food matrices.

Discussion

The current test to detect active SEs is an in vivo monkey or kitten bioassay (Bergdoll, 1988; Bennett, 2005). Those procedures have low sensitivity and poor reproducibility. Enzyme-linked immunosorbent assays (ELISA) and MS have been developed for several SEs (Bennett, 2005; Dupuis et al., 2008). However, those immunological methods cannot distinguish between active and inactive toxin, and antibodies can further react with components in a food sample to give false-positive results (Park et al., 1992). Previously, we developed a sensitive cell proliferation–based activity assay that can detect SEA in 48 h (Rasooly & Do, 2009). While this cell proliferation–based activity assays are very sensitive with good reproducibility, this assays are slow, requiring 48 h of incubation. To develop faster detection methods for active toxin, we study the early molecular responses to the toxins. To demonstrate this approach, we used SEA, which is associated with 75% of food-borne staphylococcal outbreaks (Vernozy-Rozand et al., 2004); we identified CD154 as a molecular marker that is differentially expressed soon after exposure to active SEA. And we are able to demonstrate that CD154 activity can be detected in < 6 h. To prevent down regulation of CD154 expression, anti CD40 blocking antibody was added during the stimulation to inhibit the interaction of CD154 with its receptor CD40. To detect CD154 protein expression on CD4+ T cells, PE-conjugated anti-CD154 antibody was added and the cell surface expression levels of CD154 were analyzed by flow cytometry. The results show that we can use the CD154 protein expression for early detection of SEA in milk and potentially other food matrices.

Interestingly SEB, which is genetically very diverse from SEA, was shown here not to induce high levels of CD154. Higher SEB concentrations are needed to induce CD154 protein expression. This might be due to the fact that SEA and SEB induced different TCR V-beta; SEB induces V-beta 12, 13.2, 14, 17, 20 and SEA induces V-beta 5.2, 5.3, 7.2, 9, 16, 18, 22 (Thomas et al., 2009). It was shown that staphylococcal toxins are divided in two groups based on amino acid homology, one group consisting of the staphylococcal toxins SEA, SEE, and SED. The other group consists of SEB and SEC. In general, the amino acid homology between members of the two groups is 22–33% (Balaban & Rasooly, 2000). Because SEA is able to induce high level of CD154 protein expression, we predict that other members of this group will induce CD154 protein expression as well. The data presented here suggest that CD154 is an early molecular marker of SEA in ex vivo exposure.

Acknowledgements

We thank Daphne Tamar and Sharon Abigail for helpful discussions.

Footnotes

  • Editor: Jacques Schrenzel

References

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