Enhancement of T3SS activity by nitrate
In our previous study, maturation of the T3SS machinery under anaerobic conditions was achieved by the activation of specific anaerobic respiration using an electron acceptor as nitrate. To show the dependency of T3SS function and adherence capacity on nitrate, an EHEC wild-type strain was grown in LB medium containing various concentrations of nitrate under microanaerobic conditions at 37°C. As expected, the growth of EHEC was enhanced and reached a higher density in accord with the increase in the concentration of nitrate (Fig. 1A). To examine the maturation of T3SS, we compared the secretion of the effector protein EspB into the culture supernatant (Fig. 1B). Although EspB protein was produced in EHEC grown without nitrate (see Fig. 1B, 0 mM), EspB in culture supernatant was barely detected. The EspB protein level in the supernatant was increased in accordance with the increase in nitrate (Fig. 1B). Because EspB protein in whole cells was not increased by an increase in nitrate, this clearly indicates that T3SS function is activated by the presence of nitrate and that activity is dependent on the nitrate concentration. T3SS function is necessary for intimate attachment and hence microcolony formation on epithelial cells. The effect of various concentrations of nitrate on the efficiency of microcolony formation, which reflects the adherence capacity of cultured bacteria, was examined. After growth in LB medium containing various concentrations of nitrate, HeLa cells were infected for 90 min with the same number of bacteria (1x107 cfu) and further cultivated after washing out uninfected bacteria for 3.5 h. During this period, EHEC formed microcolonies originated from intimately attached single bacteria, whose adherence is dependent on T3SS function. Finally, the cells were washed and stained, and the number of microcolonies on HeLa cells was compared. The number of microcolonies per cell, which corresponds to number of intimately attached bacteria at the initial infection period, was increased in accordance with the increase in nitrate in preculture medium (Fig. 1C). Importantly, even at a low concentration of nitrate (100 mM), the effect was clearly shown. These results indicated that an increase in nitrate stimulates the activity of T3SS and the capacity for colony formation, as shown previously.
Nitric oxide increases the capacity of colony formation
Nitric oxide is a source of nitrate but is also harmful to bacteria. To examine the effect of nitric oxide on T3SS activity during growth under microaerobic conditions, the nitric oxide-producing agent NOR-4 was added to the medium for bacterial culture, and the bacteria were examined for colony-forming capacity. Then, 100 mM NOR-4 was added to the medium 18 h before inoculation, and EHEC was grown for 3 h under microaerobic conditions. The growth of EHEC in the presence of NOR-4 was not repressed but rather stimulated (Fig. 2A). The colony-forming frequency of EHEC was increased 3-fold compared to EHEC grown without NOR-4. Because maturation of the T3SS machinery by nitrate respiration is only dependent on nitrate reductase encoded by the narGHJI operon, to examine the necessity of nitrate respiration using NarGHJI nitrate reductase in enhanced colony forming capacity, the narGHJI mutant of EHEC was grown in medium containing NOR-4 and used for infection experiments as described previously. Although growth was stimulated by the presence of NOR-4, the colony-forming capacity of the mutant was not increased, and the capacity was comparable to the levels of wild-type and mutant EHEC grown without NOR-4 (Fig. 2B). In the presence of 100 mM NOR-4, nitrate was produced at approximately 100 mM at 18 h post addition. These results indicated that the presence of nitric oxide stimulates the EHEC colony-forming capacity through activation of T3SS machinery maturation.
The inflammatory response alters the environment to enhance the colony-formation capacity
The inflammatory response induces a variety of secretion factors, including nitric oxide. To explore the effect of the inflammatory response of host cells on the EHEC virulence capacity, the supernatant of cells stimulated to induce inflammation was inoculated with EHEC, and non-stimulated cells were infected. The Caco-2 cells were stimulated by a cytokine mixture (2000 U/ml IFN-g, 50 ng/ml IL-8b, 100 ng/ml IL-22) for 24 h, and culture supernatants were collected. EHECs grown under microaerobic conditions were incubated in the culture supernatant of Caco-2 cells for 1 h, and then, HeLa cells were infected. To allow the formation of microcolonies, after 90 min of initial infection, further growth was performed after washing out unattached bacteria. The colony-forming frequency was increased when EHEC was incubated in the supernatant of cells stimulated by cytokines compared to incubation in the supernatant of non-stimulated cells (Fig. 3A). The supernatant of cytokine-stimulated cells contains a variety of factors secreted by cells. To determine the contribution of nitric oxide to the enhancement of the colony-forming capacity of EHEC, an inhibitor of iNOS activity, aminoguanidine hydrochloride (AG), was added to the Caco-2 cells along with the cytokine mix. Although the colony-forming capacity of EHEC was increased by pre-growth in the cell culture supernatant of cytokine-stimulated cells, the addition of AG abolished the effect completely (Fig. 3B). To further examine the contribution of nitric oxide to the increase of EHEC colony-forming capacity, the same infection experiments were performed with the narGHJI mutant of EHEC. The frequency of colony formation by the mutant was not increased even after incubation in the supernatant of cells stimulated by cytokines (Fig. 3C). The nitrate concentration of the supernatant of cells stimulated by cytokines was approximately 50-100 mM (Fig. S1). These results indicated that cells involved in the inflammatory response secrete factor(s) enhancing the EHEC colony-forming capacity and that the factor was nitrate derived from nitric oxide.
The inflammatory response enhances the adherence capacity of EHEC
The inflammatory response in host cells is induced by bacterial components when bacteria closely contact cells. This finding suggested that the microenvironment surrounding the cells changes into a nitrate-rich one that enhances EHEC T3SS activity and accelerates microcolony-formation once host cells are stimulated by bacterial attachment. To explore this hypothesis, the effect of pre-exposure of epithelial cells with bacteria on secondary infection was examined. The HT-29 cells were exposed to heat-killed EHEC for 2 h, and then, the cells were infected with EHEC wild-type or narGHJI mutant for 3 h. The number of adherent wild-type bacteria was greater than that of the narGHJI mutant (Fig. 4). The reduction of adherent bacteria was not due to the difference of growth rate, because growth of EHEC in the supernatant of the stimulated cells was not affected by the narGHJI mutation. This could be explained by the availability of other nitrate reductase, such as NarZYWV. To confirm the involvement of nitric oxide production, the cells were incubated with aminoguanidine hydrochloride (AG), an inhibitor of iNOS activity, while exposed to heat-killed EHEC. The number of adherent wild-type bacteria was markedly decreased to a level comparable to the narGHJI mutant (Fig. 4). These results indicated that cells involved in inflammation provide a microenvironment enhancing EHEC adherence by increasing the nitrate concentration.