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Anaerobiosis-induced virulence of Salmonella enterica subsp. enterica serovar Typhimurium: role of phospholipase C signalling cascade

Madhu Khullar¹, Raman Deep Singh¹, Manu Smriti² and Nirmal Kumar Ganguly¹

Departments of Experimental Medicine and Biotechnology¹ and Medical Microbiology², Postgraduate Institute of Medical Education and Research, Chandigarh 160012, India

Correspondence Madhu Khullar madhu_khullar{at}hotmail.com

Received January 22, 2003
Accepted May 23, 2003

Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) can initiate entry into non-phagocytic epithelial cells by triggering certain signal transduction pathways, thereby allowing the pathogen to invade and establish a niche within host cells. Anaerobiosis has been shown to be an important inducer of the invasion process of S. Typhimurium. However, the effect of anaerobiosis on modulation of cell signalling cascades by S. Typhimurium is not known. In the present study, the phospholipase C signalling cascade was investigated in mice enterocytes, following interaction with S. Typhimurium grown under aerobic and anaerobic growth conditions. Significant increases in enterocyte intracellular calcium and inositol 1,4,5-triphosphate levels were observed on interaction with S. Typhimurium grown anaerobically compared with S. Typhimurium grown aerobically. An increased membrane/cytosolic ratio of protein kinase C was also seen with anaerobic S. Typhimurium in enterocytes compared with aerobic S. Typhimurium. These data suggest that anaerobically grown organisms are more efficient in initiating cell-signalling events than are aerobically grown bacteria. These enhanced cell signals may contribute to the increased virulence of S. Typhimurium grown anaerobically.

Present address: Director General, ICMR, New Delhi, India.

Abbreviations: IP₃, inositol 1,4,5-triphosphate; PKC, protein kinase C; PLC, phospholipase C.

	INTRODUCTION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) is a broad-host-range serotype that causes disease in humans, livestock, domestic fowl, rodents and birds (Rabsch et al., 2002). The initial site of infection by S. Typhimurium is the distal ileum, where these organisms are exposed to a relatively anaerobic environment (pO₂ of 5–40 mmHg). S. Typhimurium grown under anaerobic growth conditions have been found to be more virulent and invasive (Finlay et al., 1989; Lee & Falkow, 1990). Anaerobically grown S. Typhimurium have been shown to cause effective cytoskeletal rearrangements and morphological changes in infected HEp-2 cells (Galan, 1996). A number of Salmonella-secreted proteins, termed Sips (Salmonella invasive proteins) and Sops (Salmonella outer proteins), have been characterized and have been shown to participate in the invasion of epithelial cells (Hermant et al., 1995; Wood et al., 1996). A ‘hyperinvasive’ (hil) locus has also been identified in the genome of S. Typhimurium, which is involved in the anaerobic induction of invasion (Lee et al., 1994).

Bacterial pathogens are known to exploit host-cell machinery to their advantage by activating signal transduction events within host cells. It has been shown that these signalling events play an important role in mediating invasion of non-phagocytic cells by bacteria (Falkow et al., 1992). Bliska et al. (1993) reported that S. Typhimurium could initiate uptake into non-phagocytic cells by activating cell signalling events in these cells. A specific type-III secretion system consisting of Inv–Spa complex located on a pathogenicity island had been proposed to be involved in signalling, uptake and invasion of S. Typhimurium (Kaniga et al., 1995; Galan, 1999). Membrane ruffling, cytoskeletal rearrangements and intracellular calcium fluxes have been observed in mammalian cells following activation of epidermal growth factor receptor (Pace et al., 1993), which also serves as a receptor for Salmonella typhi. Interaction of S. Typhimurium with HEp-2 cells was also observed to be accompanied by a marked increase in [Ca²⁺]_i (Ginnochio et al., 1992) and was found to be necessary for internalization of bacteria (Brumell et al., 1999), suggesting that these cell signalling events are important determinants in bacterial pathogenicity. Exposure of S. Typhimurium to anaerobic conditions has been observed to enhance its virulence (Singh et al., 2000) and invasiveness (Lee & Falkow, 1990), indicating anaerobiosis to be an important environmental cue for adhesion, invasion and intracellular survival of the bacterium. S. Typhimurium can also initiate uptake into non-phagocytic cells by initiating host-cell signalling cascades, thereby allowing the organism to establish a protected niche and to pass through the intestinal epithelial cell membrane barrier (Galan & Curtiss, 1989; Bliska et al., 1993). Since anaerobic growth conditions are known to increase virulence of S. Typhimurium, in the present study, we have examined the effect of anaerobiosis on host-cell signalling events mediated by the phospholipase C (PLC-) signalling cascade when these bacteria are grown under anaerobic conditions.

	METHODS

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Bacterial strain and growth conditions.

S. Typhimurium (virulent strain 1402/84) (Singh et al., 2000) used in the present study was obtained from the National Escherichia coli and Salmonella Center, Central Research Institute, Kasauli, India. The bacterial culture was checked for purity by biochemical methods, serotyping and by inoculation on xylose lysine deoxycholate and MacConkey agar plates. Bacterial strains were stored as lyophilized ampoules and fresh cultures were started from the stock every month. S. Typhimurium was grown under aerobic and anaerobic conditions in brain heart infusion (BHI) broth and BHI agar. Anaerobic conditions were maintained in a Macintosh anaerobic chamber by artificially filling the chamber with a mixture of 85 % nitrogen, 10 % hydrogen and 5 % carbon dioxide. The chamber was evacuated and flushed three times with the gas mixture as described above and the culture was incubated under static conditions at 37 °C. Bacteria were harvested in stationary phase (>18 h and < 24 h). Resazurin indicator remained colourless under these conditions. Aerobic conditions were obtained by static incubation of bacteria at 37 °C for 18 h.

Signal transduction assays.

In vitro signal transduction assays were carried out in enterocytes following incubation with S. Typhimurium grown under anaerobic (An. S. Typhimurium) and aerobic (Ae. S. Typhimurium) conditions.

Isolation of enterocytes.

Enterocytes were isolated from mice by the method of Laux et al. (1986). Briefly, the intestines were flushed with cold PBS, pH 7.4, and incubated with PBS containing DTT (pH 7.4) at 37 °C for 15 min, after tying the loose ends of the intestine. This was followed by incubation of intestines with PBS containing 1.5 mM EDTA, 0.5 mM DTT (pH 7.4). The solution inside the intestines was collected and centrifuged at 3000 r.p.m. for 10 min and the pellet was collected and suspended in Tris/mannitol buffer (2 mM Tris/HCl, 50 mM mannitol, pH 7.2). The viability of the cells was checked by staining with Trypan blue (0.2 %) and the purity of cells was determined under phase-contrast microscope. Enterocytes having more than 90 % viability were chosen for experimental purposes and were divided into the following groups. Group I was a control group, and signal transduction assays were assayed in enterocytes without interaction with S. Typhimurium. Group II included enterocytes incubated with Ae. S. Typhimurium, and group III included enterocytes incubated with An. S. Typhimurium.

Measurement of intracellular calcium ([Ca²⁺]_i) levels.

[Ca²⁺]_i in enterocytes was determined using Fura-2 AM (2 µmol per 10⁶ cells) (Sigma) (Chang et al., 1986). Briefly, isolated enterocytes in HEPES buffer (pH 7.2) were loaded with Fura-2 AM (dissolved in DMSO) and incubated with Ae. S. Typhimurium or An. S. Typhimurium for different time intervals (0, 5, 10, 20 and 30 min). Enterocytes were centrifuged and resuspended in HEPES buffer and placed on ice. Fluorescence measurements (excitation wavelength, 340 nm; emission wavelength, 510 nm) were made in a Kontron spectrofluorimeter (model SFM25). The basal fluorescence measurement of test cuvette was designated as F.

The intracellular free calcium was calculated from the formula:

[Ca²⁺]_i = 224 x (F - F_min/F_max - F),

where 224 is the K_d of Fura-2 AM. F_min and F_max were measured by permeabilizing the cells with 10 µl 10 mM digitonin and measuring fluorescence at zero Ca²⁺ and at saturating Ca²⁺ concentrations, respectively.

Measurement of inositol 1,4,5-triphosphate (IP₃) levels.

IP₃ turnover in enterocytes was measured using ³H-labelled myo-inositol (Sugimoto et al., 1984). Enterocytes (1 x 10⁶ cells ml^-1 in 20 mM Tris/HCl, pH 7.4) were incubated for different time periods (0, 1, 5, 10, 20, 30 and 45 min) with Ae. S. Typhimurium or An. S. Typhimurium at 37 °C (Paerregaard et al., 1991). Enterocytes without stimulation served as a control. Subsequently, enterocytes were treated with 1 ml LiCl (10 mM) for 30 min and incubated with 0.5 µCi ³H-myo-inositol for 30 min at 37 °C.

The enterocyte suspension was centrifuged at 2000 g for 10 min to wash off excess labelled inositol and enterocytes were resuspended in 1 ml Tris/HCl (20 mM, pH 7.4). The suspension was treated immediately with 2 vols ice-cold 20 % perchloric acid and kept on ice for 20 min. Proteins were removed by centrifugation at 2000 g for 20 min at 4 °C. Siliconized glassware was used in further steps to minimize losses of inositol phosphates. The pH of the supernatant was brought to 7.5 with ice-cold 5 M KOH on ice and the supernatant was centrifuged at 2000 g for 20 min at 4 °C to remove KClO₄ precipitate.

Supernatant was applied on Amprep TM mini columns (SAX 100 mg; Amersham), pre-conditioned with 2 ml 1.0 M KHCO₃ and 10 ml double-distilled water. Inositol phosphates were then eluted by stepwise addition of (i) 0.05 M KHCO₃, (ii) 0.10 M KHCO₃ and (iii) 0.17 M KHCO₃ according to the manufacturer's instructions. Elution with 5 ml 0.17 M KHCO₃ corresponds to IP₃. An aliquot (1 ml) of the 0.17 M KHCO₃ eluate was suspended in 7 ml Bray's scintillation fluid. Incorporated radioactivity was determined by using a liquid scintillation counter (Rackbeta 1214).

Measurement of protein kinase C (PKC) levels.

PKC translocation and its activity in cytosolic and membrane fractions of enterocytes were determined as described by Howcroft et al. (1988). Briefly, enterocytes (1 x 10⁶ cells ml^-1) in serum-free RPMI-1640 (pH 7.2) were incubated with Ae. S. Typhimurium or An. S. Typhimurium for 30 min at 37 °C. Stimulation was stopped by adding 5 ml ice-cold Hanks’ balanced salt solution (HBSS) followed by centrifugation at 2000 g for 10 min at 4 °C. The pellet was resuspended in 1 ml HBSS and lysed in 6–8 vols Tris/EGTA buffer (1 mM) by sonication at 1–16 mA (three bursts of 5 s each). The homogenate was centrifuged at 100 000 g for 1 h at 4 °C. The supernatant provided the cytosolic fraction for PKC assay. The pellet was then suspended in 1 ml Tris/EGTA containing 0.2 % Triton X-100 (v/v) to solubilize the membrane fraction. The suspension was centrifuged at 100 000 g for 1 h at 4 °C and the supernatant was used for estimation of PKC activity in the membrane fraction. Aliquots (100 µl) were drawn from the cytosolic and membrane fractions for PKC assay. PKC activity was assayed in a reaction mixture containing 20 mM Tris/HCl, 5.6 mM DTT, 10 mM MgCl₂, 4.5 mM CaCl₂, 3.0 mM EGTA, 0.6 mM EDTA (pH 7.5), 60 µg phosphatidyl serine ml^-1, 6 µg diolein ml^-1, 1 mg histone III-S ml^-1 and 50 µmol [-³²P]ATP (0.5 µCi, specific activity 3000 Ci mmol^-1). The reaction was stopped by adding 0.8 ml ice-cold 10 % TCA (w/v). The reaction mixture was filtered through Whatman filter paper (0.45 µm). Filters were washed three times with 5 ml 5 % TCA (w/v) and suspended in 7 ml Bray's scintillation fluid. Radioactivity incorporated was determined as outlined above. PKC activity was calculated by subtracting the amount of ³²P incorporation into histone in the presence of EGTA and the absence of added phosphatidyl serine, diolein and Ca²⁺ from the amount of ³²P incorporated in the presence of phosphatidyl serine, diolein and Ca²⁺.

Statistical analysis.

Results were expressed as means±SD and were compared by one-way analysis of variance (ANOVA) in multiple groups and by Student's unpaired t-test between two groups. P < 0.05 was considered statistically significant.

	RESULTS AND DISCUSSION

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Enterocyte [Ca²⁺]_i

Changes in [Ca²⁺]_i in enterocytes upon interaction with An. S. Typhimurium and Ae. S. Typhimurium are shown in Fig. 1. A significant increase (P < 0.01) in [Ca²⁺]_i was observed in enterocytes stimulated with An. S. Typhimurium compared with Ae. S. Typhimurium. Moreover, the maximal increase in [Ca²⁺]_i was seen at 10 min with An. S. Typhimurium compared with 20 min with Ae. S. Typhimurium.

View larger version (33K): [in this window] [in a new window]   Fig. 1. [Ca2+]i within enterocytes (control, open bars) and upon interaction with aerobic (hatched bars) and anaerobic (filled bars) S. Typhimurium. Values are means ± SD of three experiments conducted in triplicate. **P < 0.01 (anaerobic vs aerobic); *P < 0.05 (anaerobic/aerobic vs control) (one-way ANOVA).

IP₃ levels

A significant and time-dependent increase in IP₃ was observed in enterocytes treated with either An. S. Typhimurium or Ae. S. Typhimurium compared with untreated enterocytes (P < 0.001). However, the increase in IP₃ was significantly greater with An. S. Typhimurium compared with Ae. S. Typhimurium (P < 0.01) (Fig. 2).

View larger version (32K): [in this window] [in a new window]   Fig. 2. IP3 concentration estimated by time-course incorporation of 3H-myo-inositol in enterocytes (control, open bars) and upon interaction with aerobic (hatched bars) and anaerobic (filled bars) S. Typhimurium. Values are means ± SD of two experiments in triplicate. **P < 0.01 (anaerobic vs aerobic); *P < 0.001 (anaerobic/aerobic vs control) (one-way ANOVA).

PKC activity

PKC activity was measured in cytosolic and membrane fractions of enterocytes following incubation with Ae. S. Typhimurium and An. S. Typhimurium and was expressed as a ratio of membrane/cytosolic activity (Fig. 3). A significant increase in the membrane/cytosolic ratio of PKC activity was observed in enterocytes treated with An. S. Typhimurium compared with those treated with Ae. S. Typhimurium (P < 0.05).

View larger version (24K): [in this window] [in a new window]   Fig. 3. PKC activity in enterocytes (control, open bars) and upon interaction with aerobic (hatched bars) and anaerobic (filled bars) S. Typhimurium. Values are means±SD of two experiments in triplicate. **P < 0.05 (anaerobic vs aerobic); *P < 0.01 (anaerobic/aerobic vs control) (one-way ANOVA).

Conclusions

In the present study, we investigated host-cell signalling events mediated by PLC- in enterocytes upon interaction with An. S. Typhimurium. We observed that An. S. Typhimurium caused a significant increase in [Ca²⁺]_i in enterocytes, compared with Ae. S. Typhimurium. This observed increase could result from either increased Ca²⁺ influx or increased mobilization of Ca²⁺ from intracellular stores. A significant increase in [Ca²⁺]_i caused by anaerobic bacteria was seen even in the absence of calcium from extracellular medium (M. Khullar, unpublished), indicating that An. S. Typhimurium triggered increased mobilization of [Ca²⁺]_i from intracellular stores. Moreover, S. Typhimurium has been shown to induce an inositol phosphate flux in infected epithelial cells (Galan & Curtiss, 1989). Thus, the increased mobilization of intracellular Ca²⁺ in our study might be due to the increased production of IP₃ by PLC-, which, in turn, could initiate the mobilization of Ca²⁺ from intracellular stores. Changes in [Ca²⁺]_i are an important cell signal leading to various cellular activities. Increases in [Ca²⁺]_i have been reported to play an important role in bacterial internalization by the formation of membrane ruffles so as to allow bacterial entry (Pace et al., 1993; Brumell et al., 1999). The induction of membrane ruffles is critical for entry of S. Typhimurium, since mutants unable to induce Ca²⁺ flux are severely impeded in their ability to enter cultured HEp-2 cells (Pace et al., 1993). We observed that enterocytes incubated with An. S. Typhimurium showed significantly higher IP₃ levels compared with those incubated with aerobic bacteria. This enhanced IP₃ response suggests specific and avid interaction between these bacteria and the enterocytes, which could lead to initiation of signal cascading via IP₃ and [Ca²⁺]_i. This interaction could be via specific ligand binding to a hitherto unknown receptor on enterocytes. Hence, the increased [Ca²⁺]_i in enterocytes observed in the present study may contribute to enhanced virulence and penetration of host cells by anaerobic bacteria.

Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP₂) by PLC- is accompanied by production of diacylglycerol (DAG) along with IP₃, and second messengers like DAG, Ca²⁺ and IP₃ are known to be potent activators of PKC (Galan, 1996). The activation of PKC results in the translocation of PKC activity from cytosol to membrane, thereby causing the phosphorylation of membrane-bound proteins (Gupta et al., 1999). We observed that interaction of An. S. Typhimurium with enterocytes resulted in increased translocation of PKC activity from cytosol to membrane compared with aerobic bacteria. PKC activation is also essential for diarrhoeagenic organisms, as it results in phosphorylation of specific membrane proteins responsible for the efflux of ions, electrolytes and water from intestinal epithelial cells, leading to the manifestation of diarrhoea (Ruschkowski et al., 1992; Ganguly & Kaur, 1996). Since S. Typhimurium does not cause diarrhoea in mice, its seems that activation of PKC in mice enterocytes may help in phosphorylation of membrane proteins involved in adhesion and entry of bacteria into these cells. The relative increase in PKC activation following interaction of anaerobic bacteria with enterocytes observed in our study suggests it to be an important cell signalling event aiding entry of the organism into the host cell by specific phosphorylation of specific membrane proteins.

In conclusion, we observed that anaerobic S. Typhimurium could initiate potent host-cell signal responses. These enhanced cell signalling responses may lead in turn to increased bacterial virulence and invasion of host cells.

	ACKNOWLEDGEMENTS

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This work was supported in part by an ICMR Senior Research Fellowship to R. D. S.

	REFERENCES

TOP INTRODUCTION METHODS RESULTS AND DISCUSSION ACKNOWLEDGEMENTS REFERENCES