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Keywords:

  • Salmonella typhi;
  • Vi-antigen;
  • Invasion;
  • Type 1 fimbria;
  • Outer membrane protein

Abstract

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

We generated nonfimbriated mutants from both Vi-positive and -negative Salmonella typhi to analyze the role of type 1 fimbriae and Vi-antigen in bacterial invasion. A Vi-defective mutant of S. typhi GIFU 10007-3 was more invasive than the wild-type strain GIFU 10007. The wild-type strain expressing Vi-antigen did not agglutinate both Saccharomyces cerevisiae and human erythrocytes but Vi-defective mutants were able to agglutinate S. cerevisiae and human erythrocytes. Nonfimbriated mutants from Vi-negative GIFU 10007-3 lost the ability to adhere to S. cerevisiae but still could agglutinate human erythrocytes. The Vi-negative mutant increased secreted proteins and became 5-fold more invasive than the wild-type strain. Nonfimbriated Vi mutants became 50–120-fold more invasive than the wild-type GIFU 10007. To determine why nonfimbriated Vi mutants still agglutinate human red blood cells, we searched bacterial proteins that could bind human blood-type antigens. We finally identified a candidate 37 kDa outer membrane protein that recognized fucosyl-galactose, a structure common to blood type A, B and H antigens.


1Introduction

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

The invasive mechanism of Salmonella has been studied extensively through the analysis of S. typhimurium[1, 2]. It is now understood that Sip proteins, which are secreted through the type III secretion apparatus, play a key role in the invasion of S. typhimurium and a similar mechanism is expected for S. typhi and S. dublin[3]. However, specific bacterial and epithelial receptors that trigger the type III secretion system have not yet been found [4]. The epidermal growth factor receptor was once considered a candidate for Salmonella entry [5], but activation of the receptor was not a specific event [6].

Several reports suggested that type 1 fimbriae (98 min of S. typhimurium chromosome) contributed to adhesion of S. typhimurium and invasion because a nonfimbriated mutant was less adhesive and invasive [6, 7]. A conflicting report [8] suggested that the type 1 fimbriae of S. typhimurium did not contribute to colonization and virulence [9]. The incongruity between the two results might be explained partially by early chemical mutagenesis methods [9]. However, seven different adhesins and at least five fimbriae other than type 1 have been reported in S. typhimurium[9, 10]. Moreover, a second type 1-like fimbrial lpf operon was found on the chromosome (78 min) of S. typhimurium and reported to contribute to the adhesion on mouse Peyer's patches. However, the invasiveness was not compared directly to the wild-type strain although the lpf mutant had about 5-fold increased 50% lethality when administered orally to mice. Despite extensive study, it is not clear which fimbrial operon contributes to the invasiveness of S. typhimurium.

In S. typhi, however, only the type 1 fimbrial operon was found and the newly identified lpf operon was not found on the S. typhi chromosome [9]. Thus the contribution of type 1 fimbriae in S. typhi invasiveness could be analyzed easily. Although it has been reported that the type 1 fimbriae of S. typhi contributed to virulence [10], we noticed in our preliminary work that the fimA-positive and Vi-positive S. typhi strain GIFU 10007 did not adhere to Saccharomyces cerevisiae. We expect that the fimA gene was not expressed in our S. typhi strain or was not functional. However, the Vi-deleted mutant GIFU 10007-3 agglutinated with S. cerevisiae and was hyperinvasive. This agglutination was suppressed by mannose. Therefore, we decided to analyze the roles of Vi-antigen and type 1 fimbriae in invasion. We constructed nonfimbriated mutants from both Vi-positive and -negative strains and studied their invasiveness.

2Materials and methods

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

2.1Strains

The bacterial strains used in this study are listed in Table 1.

Table 1.  Bacterial strains and plasmids used in this study
Strain or plasmidDescriptionSource or reference
S. typhi
GIFU 10007Wild-type, Vi-positive strain, human isolate[11]
GIFU 10007-3ViaB-deleted mutant from GIFU 10007, Vi−[12]
GIFU GGS447vipR::cat; Vi mutant from GIFU 10007, Vi−[13]
GIFU 3P336Vi−, ΔfimA (Δ404 bp) from GIFU 10007-3This study
GIFU 3P340Vi−, ΔfimAI (Δ747 bp) from GIFU 10007-3This study
GIFU 3P341Vi−, ΔfimAI (Δ534 bp) from GIFU 10007-3This study
GIFU 3P335Vi+, ΔfimA (Δ404 bp) from GIFU 10007This study
GIFU 3P339Vi+, ΔfimAI (Δ747 bp) from GIFU 10007This study
GIFU 3P342Vi+, ΔfimAI (Δ534 bp) from GIFU 10007This study
S. typhimurium
GIFU 12142wild-type, from human blood[15]
E. coli
XL1-BluerecA1 lac endA1 gyrA96 thi hsdR17 supE44 relAI (F+proAB laqIqlacZΔ M15 Tn10 (Tetr)Promega
SY327Δ(lac-pro) arg E(Am) nalA recA56 λpir; Rifr[18]
Plasmids
pGEM-T Easycloning vector for PCR productsPromega
pKNG101suicide vector; oriR6K, strAB, sacRB; ampicillin- and streptomycin-resistant, sucrose-sensitive[14]
pGBM 1522-kb PCR product of fimAI was cloned to pGEM-T EasyThis study
pGBM 153404 bp of fimA sequence was removed from pGBM 152This study
pGBM 154747 bp of fimAI sequence was removed from pGBM 152This study
pGBM 155534 bp of fimAI sequence was removed from pGBM 152This study

2.2Construction of S. typhi fimAI mutants

The PCR primers N335: 5′-CATGGATCCATCAGAGAGAAGATTCCATCTG-3′ and N345: 5′-CATGGATTCAGCGTTATTTTCGATCCATGA-3′, were designed to amplify the fimAI gene of S. typhi (PCR condition: denaturing at 95°C, annealing at 50°C, and extension at 74°C, for 30 cycles). A BamHI site was introduced into the 5′-end of each primer. These primers were used to amplify a 2.0-kb DNA fragment from S. typhi GIFU 10007 that was then cloned into pGEM-T Easy vector (Promega), designated pGBM152. The plasmid (pGBM152) was digested with NspV and then by exonuclease III and mung bean nuclease. Both ends were blunted using Klenow fragment, and the fragment was circularized (Deletion Kit, Takara Corp.). Plasmids pGBM153 (about 400 bp deleted) and pGBM154 (about 750 bp deleted) were made using this method. A third plasmid, pGBM155, was constructed by digesting the fimAI PCR product with XcmI and AgeI to remove 534 bp that included portions of the 3′-terminus of fimA and 5′-terminus of fimI (Fig. 1). Primers N320 and N321 were used to confirm the fimA deletion. Primers N320 (5′-TAATA-ATTCAAACGGAGCCG-3′) and N321 (5′-CATCAAAACAAGCCCCACTA-3′) were also used to detect the fimAI deletion. Plasmids pGBM153, 154, and 155 were digested with BamHI cloned in the suicide vector pKNG101 [14] and electroporated into Vi-positive S. typhi GIFU 10007 and Vi-negative S. typhi GIFU 10007-3. After electroporation, ampicillin- and streptomycin-resistant colonies were selected, and the colonies were plated on sucrose plates to select fimA or fimAI deletion mutants that resulted from a double crossover event. These mutants are listed in Table 1. Deletion of the fimAI gene in these mutants was confirmed by direct sequencing of the PCR products.

image

Figure 1. Construction of nonfimbriated mutants. Two kilobases of the fimAI gene were amplified from S. typhi GIFU 10007 and subcloned into pGEM-T Easy. After deletion of internal sequences, three plasmids were constructed. A: 404-bp fimA deletion plasmid pGBM153. B: 747-bp deletion plasmid pGBM154, and 534-bp deletion plasmid pGBM155. The plasmids were subcloned into suicide vector pKNG101 and electroporated into Vi-positive S. typhi GIFU 10007 and Vi-deficient S. typhi GIFU 10007-3 to generate fimAI mutants.

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2.3Cell culture and invasion assay

The human epithelial cell line Intestine 407 was used in this study. The invasion assay was described previously [15]. Briefly, bacteria were grown overnight at 37°C in LB broth in 5% CO2 without shaking. Stationary-phase bacteria were subcultured by diluting 1:100 into fresh LB broth for another 4.5 h. Bacterial concentration was adjusted to 0.3 at OD600 with Hanks' balanced salt solution. The cell monolayer was prepared by loading 1×105 cells into a plastic 96-well plate in Eagle's minimal essential medium and cultured overnight. 40 μl of the bacterial suspension was added to each well. The mixture was incubated for 30 min at 37°C in 5% CO2. The method for counting intracellular bacteria has been described previously [10]. Briefly, extracellular bacteria were killed with kanamycin and intracellular bacteria were plated on LB agar after lysing host cells.

2.4Salmonella binding to human red blood cells

The binding of Vi-positive and -negative S. typhi to human red blood cells and S. cerevisiae was studied. A 1% human red blood cell suspension was prepared in phosphate buffered saline (PBS) and mixed with an equal volume of bacterial suspension (McFarland No. 2). After 2 h shaking at room temperature, agglutination was observed.

2.5Outer membrane and secreted protein analysis

S. typhi strains were grown in LB broth for 18 h at 37°C and washed with PBS. Cells were sonicated and centrifuged at 10 000×g for 15 min to remove intact cells. The supernatant was centrifuged at 100 000×g for 1 h to precipitate the cell envelopes. Finally, inner membranes were solubilized with 1% sodium lauroyl sarcosinate. The suspension was centrifuged at 100 000×g for 2 h to collect outer membrane proteins (OMP). The pellet was suspended in PBS and analyzed by SDS-PAGE.

Secreted protein analysis followed the method described by Wood et al. [3]. Briefly, S. typhi strains were grown in LB broth to an optical density of 0.5 at 600 nm. The culture was centrifuged, and the supernatant was filtered through a 0.45 μm pore filter. To precipitate proteins, trichloroacetic acid was added to a final concentration of 10%.

2.6Carbohydrate binding to Salmonella proteins

After performing SDS-PAGE, proteins were transferred to PVDF membrane (Bio-Rad). Filters were blocked for 30 min in PBS containing 5% skim milk. Filters were incubated with carbohydrate biotinylated polymeric (BP) probes (Seikagaku Corp., Osaka) overnight at room temperature. After rinsing twice in washing buffer (8.4 mM Na2HPO4·12H2O, 5.1 mM KH2PO4, 116.4 mM NaCl, 0.05% Tween-20, filters were incubated with streptavidin at a final concentration of 10 mg ml−1 for 1.5 h and subsequently washed twice in washing buffer. Filters were incubated with biotinylated alkaline phosphatase for 1.5 h. Blots were rinsed with washing buffer, and bound carbohydrates were detected using the AP conjugates substrate kit (Bio-Rad). The human blood type A-trisaccharide BP probe, type B-trisaccharide BP probe and Led-trisaccharide (H type 1) BP probe were used.

2.7Absorption of OMPs with blood type antigens

100 μl of each trisaccharide BP solution (10 μg ml−1 in PBS) was transferred to a streptavidin-coated microplate (Boehringer Mannheim) and incubated for 2 h at room temperature. The plate was washed with PBS three times. OMPs was added to wells of the microplate and incubated overnight at room temperature and the supernatant was analyzed by SDS-PAGE.

2.8Analysis of N-terminal amino acid of OMPs

The amino acid sequence from the N-terminus of an OMP was determined by automatic peptide sequencer (ABI 491, Procise, Applied Biosystems). After SDS-PAGE of OMPs, proteins were blotted onto a PVDF membrane. A protein of interest was excised from the membrane and applied to the automatic protein sequencer.

3Results

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

3.1Confirmation of nonfimbriated mutants

PCR products of the fimAI gene from the wild-type S. typhi and mutant are shown in Fig. 2.

image

Figure 2. PCR amplification of deleted fimAI gene. fimA1 deletion of Vi-positive S. typhi GIFU 10007 and Vi-deficient S. typhi GIFU 10007-3 were confirmed by PCR. A: Lane 1, GIFU 10007-3; lane 2, a ampR, strR colony (first recombinant); lane 3, GIFU 3P336 (second recombinant). B: Lane 1, GIFU 10007-3; lane 2, ampR, strR colony; lane 3, GIFU 3P340. C: Lane 1, GIFU 10007-3; lane 2, ampR, strR colony selection; lane 3, GIFU 3P341. M1 and M2, markers.

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After electroporation of three fimAI mutated plasmids into wild-type S. typhi, single recombinant mutants were selected on ampicillin and streptomycin plates. Most of the colonies contained both the native and mutated fimAI. They were then seeded on sucrose plates to select double recombinant strains. Ampicillin- and streptomycin-sensitive colonies that grew on sucrose agar were analyzed for fimAI gene mutation. Most of the ampicillin-sensitive colonies were double recombinants.

Nonfimbriated mutants of Vi-negative GIFU 10007-3 were confirmed by adherence to S. cerevisiae. All three mutants lost adherence capacity. They were also less adherent to the three human red blood cell ABH types than the parent. PCR products from the mutants reflected the expected deletion in the fimAI gene as shown in Fig. 2. Deletion of the fimAI gene in Vi-negative mutants GIFU 3P336 and GIFU 3P340 was confirmed by sequencing. Four hundred and four base pairs of the fimA open reading frame (ORF) were deleted (positions 85–488). The DNA between position 83 of the fimA gene and position 200 of the fimI gene was removed from GIFU 3P340.

3.2Invasiveness of Vi-positive and -negative fim mutants

The Intestine 407 epithelial cell line was used for the invasion assay (Fig. 3). Vi mutant GIFU 10007-3 was shown to be 5-fold more invasive than the wild-type strain. The fimbriae-deleted mutants constructed from Vi-positive GIFU 10007 were 2–5-fold more invasive than the wild-type strains. The three fimbriae-deleted double mutant strains from Vi-negative mutant GIFU 10007-3 were hyperinvasive. Strains GIFU 3P336, GIFU 3P340 and GIFU 3P341 were 60–120-fold more invasive than the Vi-positive wild-type strain GIFU 10007.

image

Figure 3. Invasiveness of nonfimbriated mutants into Intestine 407 cells. Lane 1, GIFU 10007; lane 2, GIFU 3P335; lane 3, GIFU 3P339; lane 4, GIFU 3P342; lane 5, GIFU 10007-3; lane 6, GIFU GGS447; lane 7, GIFU 3P336; lane 8, GIFU 3P340; lane 9, GIFU 3P341; lane 10, S. typhimurium GIFU 12142. Results of yeast agglutination and human red blood cell agglutination are indicated in the upper area of the figure. Error bar, 1 S.D.

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3.3Binding to yeast and red blood cells

The Vi-negative S. typhi strain agglutinated with red blood cells prepared from all human blood ABO types (A, B, AB and O type), whereas the Vi-positive wild-type strain GIFU 10007 did not agglutinate with red blood cells as shown in Fig. 3. All three fimbriae mutants lost the ability to agglutinate with S. cerevisiae but were still able to agglutinate human red cells.

3.4Carbohydrate binding to a Salmonella OMP protein

To identify factors that bind to human red blood cells, we studied many carbohydrate binding proteins that are present on the surface of red blood cells and the M cells of Peyer's patches.

Eight oligosaccharides were examined (data not shown), and three human blood type antigen determinants bound strongly to Vi-deficient S. typhi GIFU 10007-3. Whole cell proteins, outer membrane proteins and secreted proteins were prepared to analyze their binding to human blood type-antigen determinants as shown in Fig. 4. A 37-kDa protein band bound specifically to blood type A determinant carbohydrate (Fig. 4). Outer membrane proteins were prepared to identify the location of the 37-kDa protein (Fig. 5). The 37 kDa was a major portion of the OMP from S. typhi and S. typhimurium and was found to bind human blood type antigen determinants A, B and H. Nine amino acids determined from the N-terminal sequence of the 37-kDa protein were AEIYNKDGN. This was a commonly found sequence in OmpF and OmpC of S. typhi and S. typhimurium. The 37-kDa protein was absorbed by blood type antigens as shown in Fig. 5. Secreted proteins did not bind any of the three human blood type trisaccharide antigens (data not shown).

image

Figure 4. Binding of human blood type antigen determinants to Salmonella proteins. A: SDS-PAGE pattern of whole cell protein. B: Binding of biotinylated human blood type A trisaccharide probe to a 37-kDa protein. Lane 1, GIFU 10007 (Vi-positive); lane 2, GIFU 3P336 (Vi-negative, fimA); lane 3, GIFU 10007-3 (Vi-negative); lane 4, S. typhimurium GIFU 12142.

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image

Figure 5. Binding of blood type A, B and H trisaccharide probes to 37-kDa OMP of S. typhi. Top: Lane 1, GIFU 10007 (Vi-positive); lane 2, GIFU 10007-3 (Vi-negative); lane 3, GIFU 3P336 (Vi-negative, fimA); lane 4, S. typhimurium GIFU 12142. Bottom: The OMP fractions of S. typhi were absorbed with human blood type A, B and H trisaccharide probes. Lane A, blood type A trisaccharide; lane B, blood type B trisaccharide; lanes C and D, blood type H trisaccharide; lane E, unabsorbed OMPs of GIFU 10007-3.

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3.5Secreted proteins of fimAI and viaB double mutants

To determine why the double mutants became hyperinvasive, we compared secreted proteins among wild-type strain and the mutants. Profiles of whole proteins (Fig. 4A) and OMPs (Fig. 5A) were not different from the wild-type strain GIFU 10007, however, secreted proteins were apparently increased in the double mutants as shown in Fig. 6.

image

Figure 6. Secreted proteins of nonfimbriated, Vi-negative mutants. Lane 1, wild-type GIFU 10007; lane 2, GIFU 3P335 (Vi-positive, fimA); lane 3, GIFU 3P339 (Vi-positive, fimAI); lane 4, GIFU 3P342 (Vi-positive, fimAI); lane 5, GIFU 10007-3 (Vi-negative, fimbriae-positive); lane 6, GIFU GGS447 (Vi-positive, fimAI); lane 7, GIFU 3P336 (Vi-negative, fimA), lane 8, GIFU 3P340 (Vi-negative, fimAI); lane 9, GIFU 3P341 (Vi-negative, fimAI); lane 10, S. typhimurium GIFU 12142. Secreted proteins were stained by silver stain.

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4Discussion

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

Vi expression is crucial for S. typhi to survive inside phagocytes, lipopolysaccharide-exposed S. typhi was recognized by infected phagocytes through CD14 and induced a strong immune response [15]. The Vi capsule is also an important factor in surviving complement attack [12]. However, its role in epithelial invasion was not clear. Through this study, we demonstrated that Vi-expressing S. typhi masked surface antigens, fimbriae and OMPs. We expected that the type 1 fimbriae of S. typhi would not function in S. typhi because wild-type S. typhi did not agglutinate yeast cells. However, the Vi-deleted mutant GIFU 10007-3 was able to agglutinate yeast, and this agglutination was inhibited by mannose (data not shown). These data prompted us to analyze nonfimbriated mutants. Nonfimbriated mutants lost the ability to agglutinate yeast cells but retained the ability to agglutinate human red blood cells. Surprisingly, these mutants were hyperinvasive as compared to the Vi-negative, fimbriae-positive GIFU 10007-3. Later, we found that Vi-negative single mutants and the nonfimbriated double mutants increased their secreted proteins (Fig. 6), while wild-type S. typhi GIFU 10007 and Vi-positive nonfimbriated mutants only secreted a 53-kDa protein (this was identified as flagellin, data not shown).

Because nonfimbriated double mutant still agglutinated human red blood cells, we searched for Salmonella proteins that functioned as an agglutination factor. After screening many carbohydrate molecules to agglutinate our double mutants of S. typhi, we concentrated on the human blood type antigens because the antigen determinants were reported to be expressed in both red blood cells and the M cells of Peyer's patches [16]. After analysis of whole cell proteins, we found a 37-kDa protein in the outer membrane fraction that specifically bound human blood type antigen (Fig. 5). This protein had an N-terminal sequence which was found in OmpF and OmpC of S. typhi. The protein bound blood type antigen determinants A, B and H. A common structure of the recognition molecules is fucosyl-galactose. This sugar antigen is found on the surface of M cells based on experiments with Urex europaeus-1 (UEA-1) [17]. We also confirmed that UEA-1 bound to mouse Peyer's patches (data not shown). This UEA-1 lectin is known to recognize fucosyl-galactose, which is a structure in the blood type H antigen determinant.

The 37-kDa protein was equally expressed in wild-type S. typhi, Vi-deficient strains and S. typhimurium. Therefore, we suggest that binding of bacteria to human blood type antigen determinant was masked by Vi-antigen and fimbriae in the wild-type strains.

In conclusion, Vi-antigen and type 1 fimbriae were not essential factors in the invasion of S. typhi, although the Vi-antigen was essential for survival in the blood stream and inside macrophages [12, 15]. S. typhi might alter the expression of different virulent factors in different infection sites.

Analysis of the function of this 37-kDa OMP in the invasion of S. typhi is under way.

References

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