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Keywords:

  • Salmonella typhi;
  • Vi antigen;
  • Macrophage;
  • CD14;
  • Intracellular growth

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

We examined the intracellular survival of Vi-capsulated (lipopolysaccharide; (LPS)-masked) and Vi-deleted (LPS-exposed) Salmonella typhi strains inside macrophage cell lines. Growth of LPS-exposed S. typhi was inhibited in both mouse and human macrophage cell lines. However, the LPS-exposed strain survived in a CD14-deficient mouse macrophage cell lines. Wild-type S. typhi strain, which expressed the Vi antigen and masked LPS, survived in the resting human macrophage cell line. When the Vi-capsulated S. typhi entered the cells, the production of tumor necrosis factor-α (TNF-α) was suppressed. In contrast, S. typhimurium and LPS-exposed S. typhi stimulated the macrophages to produce a high level of TNF-α.


1Introduction

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

Studies on the invasion mechanisms and pathogenesis of Salmonella typhi have accumulated during the past 10 years [1–7]. The results of these studies suggest that the invasion mechanisms of S. typhimurium and S. typhi are similar [6]. However, the data accumulated thus far have failed to explain why the two taxonomically similar serovars cause different infections in humans. This is mainly because of poor recognition of the role of Vi antigen. S. typhi and S. typhimurium taxonomically belong to a single species; the two are differentiated by surface antigens, such as the Vi, lipopolysaccharide (LPS), and flagellar antigens. Vi antigen of S. typhi was poorly expressed when cultured in ordinary laboratory media.

During the last several years, our laboratory has focused attention on the study of the Vi gene structure and its regulation [8, 9], because the Vi capsule is only expressed on the surface of virulent S. typhi. As a result of these studies, we are able to control the expression of the Vi antigen to produce the maximum antigen level. Vi-negative S. typhi has been shown to be extremely serum sensitive; more than 99% of the cells were lysed within 40 min after exposure to human serum [10]. In our preliminary experiments, we found that both capsulated and uncapsulated S. typhi strains internalized into phagocytes at almost equal levels but their final fates differed. To study this difference between capsulated and uncapsulated S. typhi, we focused attention on the CD14 antigen expressed on the macrophage, that recognizes the LPS of gram-negative bacteria. Using both CD14-positive and -negative cultured macrophage cell lines, we analyzed invasiveness, intracellular replication, and tumor necrosis factor-α (TNF-α) production by the macrophages.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

2.1Bacterial strains and culture

The Vi-positive strain used was wild Salmonella typhi GIFU 10007 [11], and the negative strain was the Vi-deletion mutant GIFU 10007-3 [12], which is derived from GIFU 10007. S. typhimurium GIFU 12142 was isolated from the blood of a septic patient. All bacteria were cultured in a 5% carbon dioxide atmosphere at 37°C overnight in a culture medium (SOB agar): 20 g tryptone, 5 g yeast extract, 10 mM NaCl, 2.5 mM KCl, 1.5% agar, 10 mM MgCl2, and 10 mM MgSO4 in 1 liter of deionized water. This plate was found to be suitable for expression of Vi antigen at its maximum level.

2.2Phagocytes and cell lines

THP-1 cells derived from human monocytic leukemia were purchased from Dainippon Seiyaku (Osaka, Japan). Murine macrophage cell lines, J774.1 and J7.DEF3 [13] derived from J774.1 were kindly provided by Dr. Kirikae (Department of Microbiology, Jichi Medical School, Tochigi, Japan). The J7.DEF3 cell line lost the LPS binding capacity and was defective in expression of CD14 antigen. This property was confirmed using anti-mouse CD14 monoclonal antibodies after Western blot analysis of the membrane component (Dr. T. Kirikae, personal communication). All cell lines were maintained in RPMI1640 containing 10% fetal bovine serum (FBS) in 5% CO2. Activated THP-1 cells were cultured with either recombinant gamma interferon (rIFN-γ, 100 U/ml) or S. typhi LPS (200 ng/ml) for 48 h. rIFN-γ was purchased from Wako Pure Chemical Industries (Osaka, Japan). S. typhi LPS was extracted from GIFU 10007 using the phenol extraction method [14].

2.3Detection of bacterial surface antigen

S. typhi GIFU 10007 and GIFU 10007-3, cultured on SOB agar plates, and S. typhi GIFU 10007, cultured on heart infusion agar plates, were stained for the detection of Vi antigen and O9 antigen. The Vi antigen was detected using rabbit anti-Vi polyclonal antibody and cy-5 labeled goat anti-rabbit IgG as a second antibody. The O9 antigen was detected using rabbit anti-O9 polyclonal antibody and fluorescein isothiocyanate (FITC)-labeled goat anti-rabbit IgG as a second antibody. Bacterial DNA was stained with propidium iodide (PI). Bacteria were stained with three fluorescent dyes: cy-5, FITC, and PI. The bacteria were then observed by confocal laser microscopy (MRC-1024, BioRad, Tokyo, Japan).

2.4Monoclonal antibodies and immunocytochemical staining of THP-1 cells

Mouse monoclonal antibodies (mAb), 2E1(anti-human CD32) were purchased from Cosmo Bio Industry (Tokyo, Japan). 2LPM19c (anti-human CD11b), 002 (anti-human CD64), and TÜK4 (anti-human CD14) were purchased from dako (DAKO Japan Co., Ltd., Kyoto, Japan). TC-8B1 (anti-human HLA-DR) [15] and TB1-2B3 (anti-human CD35) were prepared by one of the authors (T. Takami). Cell surface antigens were detected by indirect immunocytochemical staining using Vectastain ABC reagent (Vector Lab., Burlingame, CA, USA).

2.5Phagocytosis and replication assay

Cultured cells were washed three times with an excess amount of phosphate-buffered saline (PBS) to wash out any serum and then suspended in Hanks' solution at a concentration of 1×106 cells/ml. The bacteria and phagocytes were mixed at a ratio of 50 bacteria to 1 phagocyte. After incubation at 37°C for 1 h, the mixture was centrifuged and washed three times with PBS. To determine the initial number of phagocytosed bacteria, the medium was then changed to RPMI 1640 containing 10% FBS and gentamicin (50 μg/ml), so that the extracellular bacteria were killed during additional 30 min incubation. To release intracellular bacteria, the cells were then lysed with 1 ml of 1% Triton X-100, and 100 μl of lysate was diluted and spread on heart infusion agar plates. The number of colonies were counted after incubation for 24 h at 37°C. To determine the intracellular replicated bacteria, the cells were incubated in RPMI 1640 containing 10% FBS and gentamicin (50 μg/ml) for additional overnight incubation at 37°C. The cells were then lysed and intracellular bacteria were measured by the same procedure.

2.6Measurement of TNF-α

TNF-α in the culture supernatant was assayed by commercial enzyme-immune assay (Predict TNF-α, Genzyme, Cambridge, MA, USA). Briefly, 2×106 of THP-1 cells activated by rIFN-γ were infected with the bacterial strains and were incubated in 200 μl of RPMI1640 containing 10% FBS. After 6, 24, and 48 h of incubation at 37°C, 100 μl of each culture supernatant was assayed.

3Results

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

3.1Detection of bacterial surface antigen cultured on SOB agar

S. typhi GIFU 10007 cultured on an SOB agar plate reacted only with anti-Vi antibody and not with anti-O9 antibody. In the case of S. typhi GIFU 10007 cultured on an heart infusion agar plate, some cells reacted with either anti-Vi antibody or anti-O9 antibody, and some cells reacted with both (Fig. 1, lane C).

image

Figure 1. Staining images of Vi and O9 antigens of S. typhi cultured in different media. Lane A: Vi-positive S. typhi GIFU 10007 cultured on SOB agar plate under 5% CO2. Lane B: Vi-negative S. typhi GIFU 10007-3 cultured on SOB agar plate under 5% CO2. Lane C: Vi-positive S. typhi GIFU 10007 cultured on heart infusion agar plate under aerobic condition. A.1, B.1, and C.1 are images of cells that were stained with PI, a DNA stain. All cells were stained. A.2, B.2 and C.2 are images of cells in the same field of vision as A.1, B.1, and C.1 respectively, but show only those with the Vi antigen, which were stained with cy-5. A.3, B.3, and C.3 are also images of cells in the same field of vision as A.1, B.1, and C.1 respectively, but show only those with the O9 antigen, which were stained with FITC. Magnification was ×1500 each image.

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3.2Surface markers of THP-1 cells

The surface markers on resting and activated THP-1 cells were compared (Table 1). After staining with mAbs, the number of cells which expressed these markers were counted under a light microscope. The percentages of positive cells are shown in Table 1. More than 80% of the THP-1 cells in the resting stage expressed CD11b, CD32, CD35, and HLA-DR, whereas only 25 and 30% of the THP-1 cells in the resting stage expressed CD14 and CD64, respectively. Most THP-1 cells in the resting stage did not express CD14. However, after exposure to either LPS or rIFN-γ for 48 h, 90% of the cells expressed CD14. We also observed the increase in CD64 (FcγR I) expression on THP-1 cells after stimulation with either LPS or rIFN-γ. The invasion assay was carried out under serum-free conditions to avoid the influences of natural antibodies and CD64.

Table 1.  Surface markers of resting and activated THP-1 cells after stimulation with LPS and rIFN-γ
AntigenCD11bCD14CD32CD35CD64HLA-DR
  1. Values indicate the percentage of positive cells. ND, not determined.

Resting802510010030100
+LPSND90NDND100ND
+rIFN-γND90NDND100ND

3.3The replication of S. typhi inside resting and activated human macrophage cell line THP-1 cells

In resting THP-1 cells, Vi-positive S. typhi GIFU 10007 and Vi-negative S. typhi GIFU10388 were internalized into THP-1 cells at almost the same level. S. typhimurium internalized to a greater extent than the other two strains. However, the difference was not statistically significant. In activated THP-1 cells, higher numbers of bacteria were engulfed (Fig. 2A). Vi-positive S. typhi GIFU 10007 and S. typhimurium GIFU 12142 replicated inside resting THP-1 cells. However Vi-negative S. typhi GIFU 10007-3 did not multiply inside the cells, although Vi-positive and Vi-negative strains were ingested at almost the same level. 90% of the THP-1 cells activated with 100 U/ml of rIFN-γ for 48 h expressed the CD14 antigen. Nevertheless, the number of bacteria internalized into activated THP-1 cells was higher than that into resting THP-1 cells; neither Vi-positive nor Vi-negative S. typhi survived in the activated THP-1 cells. Only S. typhimurium survived inside the cells (Fig. 2B). However, its replication was extensively suppressed when compared to its multiplication in resting THP-1 cells.

image

Figure 2. Percentage of internalized bacteria (A) and the number of intracellular replicated bacteria in resting and activated THP-1 cells (B). The percentage of bacteria recovered after 1 h infection relative to the initial inoculum (A). Y-axis represents the number of intracellular bacteria (CFU) in 100 μl of cell lysate after 24 h incubation (B). Vi(+); S. typhi GIFU10007, Vi-positive strain, Vi(−); S. typhi GIFU 10007-3, Vi-negative strain, and STM; S. typhimurium GIFU 12142. Each experiment was performed at least 6 times. Error bar represents standard error.

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3.4Replication of S. typhi in mouse macrophage cell line J7.Def3

Resting J774.1 cells and J7.DEF3 cells engulfed similar amounts of both Vi-positive and Vi-negative bacteria (Fig. 3A). The Vi-positive strain multiplied inside resting J774.1 cells, but the Vi-negative S. typhi did not. However, this Vi-negative strain was able to multiply inside resting J7.DEF3 cells (Fig. 3B).

image

Figure 3. Percentage of internalized bacteria (A) and the number of intracellular replicated bacteria in resting J774.1 cells and in resting J7.DEF3 cells (B). The percentage of bacteria recovered after 1 h infection relative to the initial inoculum (A). Y-axis represents the number of intracellular bacteria (CFU) in 100 μl of cell lysate after 24 h incubation (B). Vi(+); S. typhi GIFU10007, Vi-positive strain, Vi(−); S. typhi GIFU 10007-3, Vi-negative strain; and STM; S. typhimurium GIFU 12142. Each experiment was performed at least 6 times. Error bar represents standard error.

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3.5Production of TNF-α of THP-1 cells after S. typhi invasion

We observed differences between the Vi-positive and negative strains in the induction of TNF-α production with activated THP-1 cells. In activated THP-1 cells, S. typhimurium and Vi-negative S. typhi induced high level of TNF-α production at 48 h. Vi-positive S. typhi induced less TNF-α production than the Vi-negative strain at 6, 24, and 48 h of incubation (Fig. 4).

image

Figure 4. TNF-α production of activated THP-1 cells after ingested bacteria. (◯) S. typhi Vi (+) GIFU 10007; (▵) S. typhi Vi(−) GIFU 10007-3; (□) S. typhimurium GIFU 12142. Each test was performed three times. Error bar represents standard error.

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4Discussion

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

In this study, we controlled Vi expression of S. typhi to achieve the maximum level by culturing the organisms on SOB agar under a 5% CO2 condition. The LPS of the S. typhi strain cultured under the above condition was well covered with Vi antigen (Fig. 1). This was substantiated by the fact that Vi-positive S. typhi could not be stained with O9 antibody. Using bacteria cultured under this condition, we carried out intracellular replication assays with Vi-expressed S. typhi GIFU 10007 and LPS-exposed S. typhi GIFU 10007-3. First, we used resting THP-1 cells in which only 25% of the cells expressed CD14 (Table 1). The LPS-masked strain, GIFU 10007, multiplied in the cells. However, the LPS-exposed strains, GIFU 10007-3, showed only a small amount of growth. (Fig. 2B). Next we used rIFN-γ-activated THP-1 cells. More than 90% of the activated THP-1 cells expressed CD14. In these cells, both the Vi-positive GIFU 10007 and the Vi-negative GIFU 10007-3 did not survive (Fig. 2B). However, LPS-exposed S. typhimurium GIFU 12142 did multiply, although its growth was suppressed in comparison to growth in resting THP-1 cells. From these data, we hypothesized that during entry, bacterial LPS stimulated the CD14 on the surface of THP-1 cells, which led to activation of THP-1 cells, and the bacteria were then killed. However Vi-positive S. typhi, whose LPS was masked, may not stimulate CD14 during its entry, so it could survive inside resting THP-1 cells.

In activated THP-1, both Vi-positive and Vi-negative S. typhi strains did not survive. In this case, we cannot explain this result with only our theory on the role of CD14. rIFN-γ activation might involve other mechanisms that lead to activation of other cytotoxic factors.

We performed intracellular replication assay to observe the influence of CD14 by using the CD14 defective cell line, J7.DEF3. In resting J774.1 cells, the growth of Vi-negative GIFU 10007-3 was suppressed. However, the strain multiplied inside resting J7.DEF3 cells (Fig. 3B). Furthermore, the Vi-negative GIFU 10007-3 also multiplied, even in rIFN-γ activated J7.DEF3 cells (data not shown).

As shown in Fig. 4, Vi-negative GIFU 10007-3 and S. typhimurium GIFU 12142 induced the production of more than 600 pg/ml of TNF-α within 48 h after infection, but Vi-positive GIFU 10007 induced less than 150 pg/ml of TNF-α.

From these data, we inferred that the bacteria covered with the Vi antigen did not stimulate phagocytes during their entry. We hypothesized that the factor that recognizes invading bacteria might be the LPS receptor, CD14, because, Vi antigen is a homopolymer of N-acetylgalactosamine-uronic acid, it can be found in any tissue of the human body. Purified Vi antigen did not induce oxygen radical production (data not shown). The bacteria covered with this antigen might not be recognized by human phagocytes. Another question arising from our results was why LPS-exposed S. typhimurium survived in activated macrophages in spite of poor pathogenesis in human adults. S. typhimurium was found to be serum resistant in our study (data not published). This observation has been reported by other researchers and the mechanism has been explained by its LPS structure [16, 17] and resistance to intracellular cytotoxic molecules, such as permeability increasing factor, hydrogen radicals, and defensin [18, 19]. Our results showed that proliferation of S. typhimurium in activated THP-1 cells was suppressed compared to resting THP-1 cells. The infected cells may have been stimulated through the CD14, producing many kinds of inflammatory signals, such as IL-1 [20], IL-6 [21], and TNF-α[22]. The activated macrophages may then have stimulated the T cells to produce IFN-γ. Through this IFN-γ, the resting macrophages might have shifted to the activated stage and suppressed S. typhimurium proliferation.

Vi-capsulated S. typhi stimulated the resting macrophages to produce only small amounts of TNF-α compared to uncapsulated S. typhi and S. typhimurium. Effector molecules were not efficiently produced from the Vi-capsulated S. typhi-infected macrophages. These macrophages might have remained in the resting stage, because even Vi-capsulated S. typhi could not survive in activated macrophages. Thus, S. typhi could have been internalized in the resting macrophages without stimulating CD14. This would have kept the host cell at a resting stage due to the expression of the maximum level of Vi antigen by the organisms inside the macrophages. We confirmed that the wild S. typhi GIFU 10007 strain expressed the maximum level of Vi antigen inside macrophages 24 h after infection (data not shown).

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References

This work was partially supported by a Grant in Aid for cooperation research (A) No. 07307004 given by the Ministry of Education of Science, Sports and Culture, Japan. We appreciated the critical reading of this manuscript by Dr. Esperanza Cabrera (De La Salle University, Manila, Philippines).

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  1. Top of page
  2. Abstract
  3. 1Introduction
  4. 2Materials and methods
  5. 3Results
  6. 4Discussion
  7. Acknowledgements
  8. References
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