1. Introduction
Enterohemorrhagic
Escherichia coli (EHEC) strains are food-borne pathogens that can cause different clinical conditions, such as self-limited diarrhea, hemorrhagic colitis, and systemic complications, such as hemolytic-uremic syndrome (HUS) [
1,
2,
3,
4]. One of the EHEC strain most frequently associated with severe human disease is
E. coli O157:H7 [
5].
EHEC enters the gastrointestinal tract, survives the acidic condition of the stomach, and reaches intestine, where adhesion to epithelial cells is the first step in the pathogenic cascade. It has been revealed the preferential binding to the follicle associated epithelium (FAE) of Peyer’s patches in the initial events of EHEC colonization, which could lead to the rapid contact of
E. coli O157:H7 with underlying human macrophages [
6]. However, scarce information is available about the interactions between EHEC and these host cells. EHEC O157 from clade 8 carries several virulence factors including Shiga toxin 2a and/or 2c (Stx2), cytolethal distending toxin V (CdtV), EHEC hemolysin (EHEC-Hly), and flagellin [
7,
8]. The Stx2 is encoded in a lambdoid bacteriophage [
9,
10], which is an efficient vector for the transfer of
stx and plays an important role in the evolution of new pathogens [
11,
12,
13]. As a result of prophage induction, host bacteria lyse release Stx2 and free phage particles that can infect other bacteria [
14,
15,
16,
17]. However, low levels of spontaneous phage induction can also occur. Transcription of
stx2 is highly dependent on induction of the phage lytic cycle, as it is mainly governed by the late phage promoter pR’ [
11]. In addition, it has been recently demonstrated that Stx2a and/or Stx2c from periplasmic space could be delivered by outer membrane vesicles (OMVs) [
7,
18]. A comprehensive understanding of early events during EHEC colonization that lead to HUS could aid in the development of new strategies to prevent and treat the disease.
One way to understand the pathogenesis of HUS is to reproduce host-pathogen interactions on an in vitro model. We have previously reported the ability of eukaryotic cells to recognize putative promoter-like sequences on
stx2 driving Stx2 expression by cell lines [
19]. Moreover, mouse in vivo transfection with
stx2 cloned into a prokaryotic plasmid (pStx2) showed
stx2 mRNA in the liver and Stx2 biological toxicity [
20]. Therefore, in this work we analyzed the hypothesis that human cell lines participate in Stx2 production after infection with EHEC strains. We first demonstrated that the 293T cell line transfected with pStx2 and transcribed mRNA corresponding to Stx2 A and B subunits, which results in Stx2 biologic activity in the supernatant. Then, we analyzed whether this process could take place in human macrophagic and intestinal epithelial (HCT-8) cell lines during EHEC infection, as an in vitro model closer to the in vivo physiopathologic condition. With this aim, both cellular lines were infected with EHEC O157:H7 isolated from a pediatric HUS patient, and a time course analysis of cellular as well as bacterial survival, Stx2 production,
stx2 transcription, and cytokine secretion was done. We found that both cell lines differ markedly in the cellular response to bacterial infection. In fact, we demonstrated that macrophages are able to internalize and kill EHEC. However, HCT-8 cells are not able to eliminate bacteria nor EHEC are able to kill epithelial cells. We analyzed the triggering of inflammatory response and searched eukaryotic
stx2 mRNA in both cell types after infection. The interaction between EHEC and human cells could control infection, but also contribute to host damage.
3. Discussion
While regulation of Stx expression has been extensively investigated in bacterial broth cultures, it remains poorly understood how toxin production is regulated in the complex environment of the human gut. We have previously demonstrated that biologically active Stx2 is produced in vivo after pStx2 transfection, driven by its own
stx2 promoter [
20]. This evidence raised the question about which is the involvement of host eukaryotic cells in Stx2 production and tissue injury during EHEC infections. Because the first cells to get into contact with EHEC bacteria in the human gut include epithelial cells, macrophages, and dendritic cells, our in vitro approach to simulate the cellular interactions during EHEC infection was the co-culture of a virulent EHEC strain with i) THP-1 derived macrophages or ii) HCT-8 epithelial cells.
In the present work we demonstrated that eukaryotic 293T cells transfected with a pStx2 are able to generate mRNA corresponding to A and B subunits of Stx2, and most importantly, this mRNA has the capacity to translate a biologically active Stx2 protein in the eukaryotic context. Then, we tested the hypothesis that eukaryotic cells participate in stx2 transcription upon interaction with the 125/99 strain. When mRNA was purified from macrophages at 24 h post-infection, we detected stx2-A transcripts only using cDNA synthesized with random primers as a template by RT-qPCR. These results suggest that Stx2 is produced by internalized bacteria. Since polyadenylation is a characteristic of eukaryotic mRNA, the fact that no stx2-A transcripts were amplified with cDNA synthesized with oligo (dT) rules out the stx2 transcription by macrophages. When mRNA was purified from HCT-8 cells we could not detect any stx2 signal even when transcripts from the housekeeping gene were identified. In conclusion, after EHEC infection of macrophages or HCT-8 cells we only detected mRNA of bacterial origin within macrophages.
When analyzed biological interaction between macrophages and bacteria, we observed a significant death of macrophages after infection with the 125/99 strain. This effect was time- and Stx2-dependent, but independent of which bacteria produced the Stx2. That is, pathogenic 125/99 or non-pathogenic C600 carrying Stx-phage (C600Φ933W) induced a similar viability loss of macrophages.
Simultaneously, we found that macrophages were able to phagocyte all bacterial strains and kill them. However, we observed a higher number of non-pathogenic C600 compared to 125/99 bacteria within macrophages at 2 h post-infection, independently of Stx2-production, in spite of Poirier et al. [
21] showing that both Stx types inhibit EHEC uptake by macrophages. The lower intake of the EHEC strain compared to non-pathogenic bacteria by macrophages suggests that some of the multiple pathogenic factors expressed by EHEC strains, apart from Stx2, modulate phagocytosis by macrophages. This is in agreement with previous reports that have demonstrated that pathogenic bacteria (i.e., EPEC and EHEC) inhibit phagocytosis through several mechanisms, i.e., via inhibition of PI3K activity by proteins codified by the type III secretion system [
25,
26]. In the same line of evidence, it has been reported that EspF [
27], EspB [
28], EspJ [
29], and EspH [
30] inhibit EHEC uptake by macrophages. In spite of the lower intake of pathogenic bacteria, all strains were able to survive within macrophages after 24 h, showing similar numbers of living pathogenic and non-pathogenic bacteria (CFU/mL) in cell lysates at that time. This is in agreement with other authors who reported that macrophages die not only by EHEC infection [
31], but also that EHEC can survive and multiply within human macrophages up to 24 h [
21,
31]. Besides, Stx1/Stx2 is released during this time point [
21,
32]. In line with these results, the time course analysis of Stx2 in the culture supernatant from infected macrophages showed a significant Stx2 production up to 48 h.
Previous studies revealed that macrophages derived from THP-1 express Gb3 on their membrane [
33,
34]. As consequence, these cells are able to respond to Stx1/Stx2 [
35,
36], inducing cytokine/chemokine production, ribotoxic stress, and death of macrophages [
22]. Thus, to further elucidate the Stx2-dependent mechanism responsible for macrophage death, we treated cultures with an anti-Stx2 neutralizing antibody since the beginning of 125/99 infection [
37]. We observed that Stx2-killing effect on macrophages was not blocked by neutralizing antibodies, although Stx2 activity in these supernatants was specifically and effectively blocked by this antibody, as was demonstrated by Vero assay. In addition, antibody treatment was able to neutralize a CD100% (33 pg/mL) of rStx2 added in the culture medium. These results suggest that other mechanisms than those mediated by free Stx2 prevailed in determining macrophage death upon challenge with 125/99. While several killing mechanisms have been described for cells sensitive to extracellular Stx2, the results described herein led us to hypothesize that killing of macrophages at early times was mediated, at least in part, by intracellular Stx2 produced by bacteria that still remain viable. In this regard, exogenous Stx induces protein synthesis inhibition, but also apoptosis via intrinsic and extrinsic pathways in many cell types [
34,
38]. However, intracellular Stx2 produced by phagocytized bacteria might be another mechanism for killing macrophages and evading host immune response.
In parallel, supernatants from THP-1 derived macrophages incubated with pathogenic or non-pathogenic strains were tested for inflammatory response by measuring cytokine secretion. Macrophages showed the highest amounts of IL-1β after infection with any bacteria at 24 h. Interestingly, the production of IL-1β was similarly triggered by 125/99, the non-pathogenic C600 bacteria and the 125/99ΔStx2, which expresses all the same pathogenic factors than 125/99 except Stx2. In this sense, it has been previously reported that other pathogenic factors of EHEC strains, such as enterohemolysin encoded in the pO157 virulence plasmid, were involved in IL-1 β secretion [
39]. Although IL-1β release is generally associated with death of macrophages, it has been recently demonstrated that bacterial lipopolysaccharides (LPS) induce inflammasome-mediated release of IL-1β from living human cells [
40]. In contrast, only the 125/99 strain induced LDH release. LDH and IL-1β levels in supernatants indicated that while infection of macrophages with all strains (125/99, 125/99ΔStx2, or C600) induced IL-1β release, the only one that was associated with a lytic mechanism was 125/99. These results prevent us from ruling out that other lytic mechanisms such as pyroptosis has been involved during 125/99 infection in a Stx2-dependent or independent pathway. Altogether, the simultaneous release of IL-1β and Stx2-dependent cell death triggered by EHEC strain is a very inflammatory condition that could influence the outcome of intestinal infections.
While several previous in vitro studies demonstrated that human macrophages secrete IL-1β following Stx treatment in an NLRP3 inflammasome-dependent manner [
35,
41,
42], scarce reports have analyzed IL-1β secretion following in vitro EHEC infection. In the context of an EHEC infection, Stx2 produced within macrophages could be adding an additional lethal stimulus.
When we similarly analyzed the biological consequences of interaction between HCT-8 cells and bacteria, we found that HCT-8 cells did not diminish viability even after 72 h of infection with 125/99 bacteria. Although this result is not entirely surprising because HCT-8 cells express low levels of Gb3, several authors have reported that they are sensitive to Stx1/Stx2, in a lesser degree compared to Vero cells or human macrophages [
43,
44]. In parallel, a significant drop in the number of living bacteria since 24 h was observed, in agreement with the fact that they are non-invasive but extracellular living and media-contained gentamicin (20 µg/mL).
Surprisingly, regardless of the different interaction between bacteria and macrophages or HCT-8, we found similar amounts of Stx2 in supernatants from both cell lines, approximately 200–500 pg/mL at 24 h of incubation. These results probably indicate that EHEC-HCT-8 cells interaction leads phage to enter the lytic cycle and release Stx2 to the supernatant. Although we did not rule out that gentamicin triggers Stx2-phage induction, previous reports have also shown that the interaction between the EHEC-Caco-2 cell line induces Stx2 production [
32].
On the other hand, we showed that in vitro infection of HCT-8 cells with 125/99 strain triggers IL-8 secretion in line with previous reports [
45,
46]. Because IL-8 induces recruitment, activation, and migration of neutrophils to the intestine, it could contribute to disruption of epithelial integrity, increasing Stx absorption and thereby the pathogenicity of EHEC [
47].
In conclusion, we failed to demonstrate that 125/99-infected human cell lines transcribe the
stx2 sequence. However, we could not throw away the hypothesis that the
stx2 sequence that may reach host cells by direct bacterial-mammalian interactions as well as by other pathogenic delivery pathways could be processed by eukaryotic translation machine in vivo. In this regard, several recent reports have shown that OMV naturally formed during bacterial infection and released from EHEC can be internalized by eukaryotic cells [
48], acting as delivery vehicles for bacterial virulence factors [
49,
50]. Particularly, it has been demonstrated that OMV from EHEC broth cultures contain Stx2-DNA [
7,
8,
51].
In brief, our results indicate that Stx2 is not involved in the uptake of bacteria by macrophages. Nevertheless, Stx2 produced within macrophages is majorly responsible for IL-1β release associated with a lytic mechanism. During EHEC infection, cells present in the intestine may contribute to pro-inflammatory cytokines release and bacterial production of Stx2. These effects could facilitate EHEC infection and induce intestinal cell damage. Further research in this area may help to develop strategies to interfere with early events in the gut and prevent HUS pathogenesis.
4. Materials and Methods
4.1. Bacterial Strains
E. coli O157:H7 strain 125/99 was isolated from a child with HUS. This strain was from clade 8, harbor
stx2a, but not
stx1 [
52,
53,
54]. The
E. coli 125/99ΔStx2 strain is a 125/99 isogenic strain that was mutated on Stx2-production and generously provided by Dr. Angel Cataldi et. al. [
55].
E. coli C600 and
E. coli C600Φ933W (C600Φ933W) were provided by Dr. Leticia Bentancor et. al. [
56]. C600Φ933W is a lysogenized C600 strain carrying the 933W bacteriophage. All strains were cultured overnight (ON) in Tryptic soy broth (TSB) (Difco, Le Point de Claix, France) at 37 °C. ON cultures were diluted 1:100 in RPMI 1640 medium (RPMI) (Gibco, Invitrogen, San Diego, CA, USA) and grown at 37 °C for 2 h. Cultures were centrifuged and bacterial pellets were resuspended in fresh medium. Concentrations of bacteria were determined by measuring absorbance at an optical density of 600 nm.
4.2. Cell Lines and Cell Culture
The human monocyte cell line THP-1 (ATCC TIB202) was maintained in RPMI supplemented with 10% heat-inactivated fetal bovine serum (FBS) (Natocor, Córdoba, Argentina), antibiotics (100 U/mL penicillin/streptomycin) (EMEVE Microvet SRL Laboratories, Buenos Aires, Argentina), 0.05 mM β-mercaptoethanol (Sigma, St Louis, MO, USA), and 4 mM L-glutamine (EMEVE Microvet SRL Laboratories, Buenos Aires, Argentina). THP-1 cells were differentiated to macrophages by addition of 10 ng/mL phorbol 12-myristate 13-acetate (PMA) (Sigma, St Louis, MO, USA) for 48 h. To confirm macrophage-like differentiation, PMA-treated and not-treated THP-1 cells were labeled with PECy5-conjugated anti-human CD14 antibody (Clone RMO52) (Beckman Coulter, Brea, CA, USA) or the PECy5 Mouse IgG2a, κ isotype control (Biolegend, San Diego, CA, USA). The fluorescence was measured on 10,000 events by using the Cell Quest program on a FACSCalibur (Beckton Dickinson, San Jose, CA, USA).
Vero cells and the human ileocecal carcinoma cell line HCT-8 (ATCC CCL-244) were grown in RPMI supplemented with 10% heat-inactivated FBS and antibiotics. 293T cells (human embryonic kidney cells) were grown in Eagle’s Minimum Essential Medium (DMEN) (Gibco, Invitrogen, San Diego, CA, USA) supplemented with 10% heat-inactivated FBS and antibiotics. All the cell lines were grown at 37 °C under 5% CO2 in a humidified atmosphere.
4.3. Infection Assay
THP-1 derived macrophages were seeded at 5 × 105 cells per well in 24-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) for viability assays and functional studies or 2 × 106 cells per well in 6-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) for RNA isolation and then bacteria were added to the cell monolayer at a MOI of 10. The plate was centrifuged briefly to synchronize phagocytosis and incubated for 20 min (0 h). Afterward, infected macrophages were washed and fresh medium containing 100 µg/mL of gentamicin (Richet, Buenos Aires, Argentina) was added to kill extracellular bacteria. The supernatants were collected and fresh medium containing 20 µg/mL of gentamicin and 5% heat-inactivated FBS was added after 2 h incubation and every 24 h post-infection. To determine the number of surviving bacteria in the supernatants, pellets obtained by centrifugation were resuspended in phosphate-buffered saline (PBS) and plated onto LB agar. To determine the number of intracellular bacteria in the infected macrophages, a 5 min treatment with 200 µL 0.1% Triton X-100 (Sigma, St Louis, MO, USA) was used to lyse eukaryotic cells at 2, 24, and 48 h. Then, 10-fold dilutions of the lysates were plated onto LB agar and the number of bacteria was determined by CFU. In parallel, to evaluate the Stx2-cytotoxic activity inside the infected macrophages, they were harvested with 400 µL PBS and lysed by four freeze-thaw cycles.
To neutralize the cytotoxic effect of extracellular Stx2 on the infected macrophages, medium supplemented with anti-Stx2 neutralizing antibody or non-neutralizing antibody to a final concentration of 10 nM was added [
37]. For RNA extraction macrophages from two different wells were combined and lysed in TRIzol (Invitrogen Life Technologies, San Diego, CA, USA) at 24 h post-infection. RNA from non-infected macrophages was isolated as a control.
4.4. Adhesion Assay
HCT-8 cells were cultivated at 2.5 × 105 cells/well in 24-well plates for viability assays and functional studies or 2 × 106 cells per well in 6-well plates for RNA isolation. Cells were washed with PBS, then bacteria were added at an MOI 10 and the culture plate was centrifuged briefly to synchronize adhesion. After incubation for 3 h at 37 °C, non-adherent bacteria were removed by five washes with PBS and fresh medium containing 20 µg/mL of gentamicin was added (0 h). Subsequently, the supernatants were collected and fresh medium containing 20 µg/mL of gentamicin was renewed every 24 h. For quantitative bacterial adherence assays, cell monolayer was gently scraped off with 200 µL 1X-Triton, and the number of adhered bacteria was determined by CFU as described before. In parallel, to evaluate the Stx2-cytotoxic activity inside infected HCT-8 cells, they were harvested with 400 µL PBS and lysed by four freeze-thaw cycles. For RNA extraction, HCT-8 cells from two different wells were combined and lysed in TRIzol at 24 h post-infection. RNA from non-infected HCT-8 cells was isolated as control.
4.5. Stx2- cytotoxic Activity on Vero Cells
The supernatants or lysates collected, at the mentioned time points, from the different cell lines (macrophages, HCT-8 and 293T cells) were tested for Stx2-cytotoxic activity on Vero cells as previously described [
57]. Briefly, Vero cells were grown in RPMI supplemented with 10% heat-inactivated FBS and antibiotics in 96-wellplates. One in two dilutions of lysates and supernatants were added to each well containing 2 × 10
4 Vero cells. Cells were incubated at 37 °C in 5% CO
2 for 48 h. Cells were washed, stained with crystal violet dye, and read on a Microwell System reader 230S (Organon, Teknika, OR, USA) with a 570 nm filter.
The specificity of cytotoxicity of supernatants derived from 293T cells transfected with pStx2 (SN-pStx2) was evaluated in parallel by pre-incubating these samples with an anti-Stx2 neutralizing antibody for 1 h at 37 °C and for 1 h at 4 °C [
37]. Known concentrations of rStx2 (Phoenix Lab, Tufts University, Boston, MA, USA) were used to estimate the Stx2 concentration.
4.6. Cell Viability Assay
Viability of macrophages and HCT-8 cells was examined using a 3-(4,5-DimethylthiaZA-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma, St Louis, MO, USA) assay as previously described [
58]. Briefly, RPMI with 1:10 volume of MTT solution (5 mg/mL) was added to cell monolayers and incubated at 37 °C for 4 h. The reaction was stopped with acid-isopropanol (100 µL of 0.04 N HCI in isopropanol) and mixed thoroughly to dissolve the formazan crystals. After a few minutes at room temperature to ensure that all crystals were dissolved, the solution was transferred to 96-well plates (Greiner Bio-One GmbH, Frickenhausen, Germany) and analyzed by measuring the absorbance on a Microwell System reader 230S (Organon, Teknika, OR, USA) at 540 and 720 nm. The 720 nm absorbance value (background) was subtracted from the 540 nm absorbance to get a more exact measurement.
4.7. Cytokine Assay
The supernatants of macrophages were collected at 2, 24, and 48 h post-infection and the concentration of human IL-1β was quantified by ELISA (BD Biosciences, Franklin Lakes, NJ, USA) following the manufacturer’s instructions and read on a Microwell System reader 230S (Organon, Teknika, OR, USA) with a 450 nm filter. Supernatants from PMA-differentiated THP-1 cells cultured in medium alone served as control for the spontaneous release of cytokine.
The supernatants of HCT-8 cells were collected at 24, 48, and 72 h post-infection and the levels of human IL-8 was quantified by ELISA (BioLegend, San Diego, CA, USA) following the manufacturer’s instructions and read on a Microwell System reader 230S (Organon, Teknika, OR, USA) with a 450 and 570 nm filter. The 570 nm absorbance value (background) was subtracted from the 450 nm absorbance. Supernatants from HCT-8 cells cultured in medium alone served as a control for the spontaneous release of cytokine.
4.8. Lactate Dehydrogenase (LDH) Assay
LDH released in the supernatants from macrophages was detected using a cytotoxicity detection kit (Pierce LDH Cytotoxicity Assay Kit), purchased from Thermo Fisher Scientific (Waltham, MA, USA). Data was expressed as Absorbance units (AU).
4.9. Plasmid Construction
The plasmid was constructed by standard cloning techniques, according to the NIH policy manual on Working Safely with Hazardous Biological Materials. The complete
stx2a sequence was amplified by PCR from total DNA from
E. coli C600Φ933W, using the primers
stx2Fw (5′-GAATTCATTATGCGTTGTTAG-3′) and
stx2R (5′-GAATTCTCAGTCATTATTAAACTG-3′), both containing an EcoRI restriction site [
18]. The resulting fragment (1413 bp) was cloned in a pGEM-T Easy vector (Promega Inc., Madison, WI, USA), generating the plasmid pStx2. This plasmid and pGEM-T religated vector were replicated in
E. coli DH5α competent cells in parallel and purified using the Wizard Plus Minipreps DNA purification system (Promega Inc., Madison, WI, USA) following standardized instructions.
4.10. Transfection Assay
293T cells were seeded at 6 × 105 cells/well in six-well culture plates. Cells were transfected with Polyfect reagent (QIAGEN Inc, Germantown, MD, USA). Briefly, 2 µg of plasmid pStx2 or pGEM-T religated vector were mixed with 15 µL of Polyfect reagent following the manufacturer’s instructions. After 10 min, the cells were incubated with the transfection mix (DNA-polyfect) using complete medium at 37 °C in 5% CO2. Supernatants were collected and cell monolayer were lysed in TRIzol for total RNA isolation 18 h later.
4.11. Quantitative RT-qPCR
For total RNA isolation, macrophages HCT-8 or 293T cells were lysed in TRIzol according to the manufacturer’s instructions. For all RNA samples, 1µg was treated with RQ1 RNase-free DNase (Promega Inc., Madison, WI, USA). Then cDNA was synthesized using Superscript III reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) and gene-specific primer (
stx2-A 5′-ACACAGGAGCAGTTTCAGACAG-3′ and
stx2-B 5′-GAATTCTCAGTCATTATTAAACTG-3′), random hexamers (Biodynamics SRL, Buenos Aires, Argentina) or oligo (dT) primers (Biodynamics SRL, Buenos Aires, Argentina) according to the manufacturer’s guidelines (Invitrogen Life Technologies, San Diego, CA, USA). cDNAs were amplified using Taq DNA polymerase (Invitrogen Life Technologies, San Diego, CA, USA) with SYBR green using an Eppendorf Mastercycler. Primers and PCR conditions for the
stx2-A subunit,
stx2-B subunit, and
SDHA are listed in
Table 1and
Table 2, respectively.
Data was analyzed with Realplex software using the relative standard curve method. Reactions were performed in duplicates or triplicates.
4.12. Statistical Analysis
Data are expressed as mean ± Standard Error of the Mean (SEM) and were analyzed by t-test, one-way ANOVA, or two-way ANOVA as indicated in legend, always using Tukey’s Multiple comparisons post-test. Data were analyzed using Graphpad Prism 8. p values less than 0.05 were considered statistically significant.
