Fimbriation of Pseudomonas cepacia - Infection and Immunity

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Dec 5, 1991 - Frost, L. S., and W. Paranchych. 1977. Composition ... Paranchych, W., and L. S. Frost. 1988. ... Smith, A. L., P. D. Jenny, L. Langley, et al. 1989.
INFECrION AND IMMUNITY, May 1992, p. 2002-2007

Vol. 60, No. 5

0019-9567/92/052002-06$02.00/0 Copyright © 1992, American Society for Microbiology

Fimbriation of Pseudomonas cepacia MARGARETHE KUEHN,"12 KAREN LENT,1'2 JOEL HAAS,2,3 JIM HAGENZIEKER,2'3 MARGUERITE CERVIN,"12 AND ARNOLD L. SMITH1,2* Division of Infectious Disease' and Department of Pathology,3 Children's Hospital and Medical Center, and Department of Pediatrics, School of Medicine, University of Washington,2 Seattle, Washington 98105 Received 5 December 1991/Accepted 3 March 1992

Fimbriae (pili) on the surface of bacteria have been suggested to facilitate adherence to mucosal epithelial surfaces. Three Pseudomonas cepacia cystic fibrosis isolates were screened for their ability to agglutinate erythrocytes (HA), a characteristic of some fimbrial types. One strain, designated PC103, was HA', while another, PC109, was HA-. A fimbriated (f+) HA' derivative of PC109 (PCE213) was selected by repeated erythrocyte adsorption. The two HA+ strains were shown by transmission electron microscopy to possess fimbriae which averaged 4.8 1.36 nm in width and 200 to >2,100 nm in length (PCE213) and 3.4 to 11.4 nm in diameter and 280 to 720 nm in length (PC103). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins prepared from PC103, PC109, and PCE213 indicated that the putative fimbrial subunit had a mass of 16 kDa. Western blot (immunoblot) analysis of sheared cell supernatants indicated that the 16-kDa subunit from PC103 and PCE213 reacted with antibody to the P. aeruginosa PAK pilin subunit. Southern blot analysis of a Sall digest of PC103 DNA showed DNA fragments which hybridized to P. aeruginosa PAK probes containing either the pilin structural gene (piL4) or the pilin accessory genes (pilB, -C, and -D) but not the conserved N-terminal region of piL4. A 15-kb band was common to both hybridizations, indicating that this fragment contains the PC103 fimbrial gene cluster. These results indicated the presence of homology between P. aeruginosa PAK and PC103 fimbriae but also suggested that the P. cepacia fimbriae are not type IV-like. The importance of fimbriae in adherence to A549 cells (type II pneumocytes) was assessed with PC109 (f) and PCE213 (f). PCE213 had an approximately 20-fold-higher level of adherence to A549 cells than PC109. This suggested that fimbriation of P. cepacia is associated with increased adherence in vitro.

Pseudomonas cepacia is becoming a major problem in cystic fibrosis. Reports from the Centers for Disease Control showed an increase in the percentage of cystic fibrosis care centers reporting the isolation of P. cepacia from sputum (7). Reports have also shown an increase in the number of patients with cystic fibrosis dying because of P. cepaciainduced disease (7, 27). The virulence factors of P. cepacia have not been well studied (4). Some extracellular factors have been identified, including a protease (14), phospholipase C, and a hemolysin (16, 29). However, the mechanism by which P. cepacia colonizes the lower respiratory tract has not been well studied. Since adherence is the first step in colonization and bacterial fimbriae have been shown to be involved in adhesion, the present study was undertaken to characterize fimbriae from clinical cystic fibrosis isolates of P. cepacia. The role of fimbriae in adhesion was also examined by an in vitro adherence assay.

agar and suspended to a density of 106 cells per ml, and 1 ml of 3% rabbit erythrocytes was added to 1 ml of bacterial culture. The erythrocytes were allowed to settle and immediately subcultured on M9 agar. This selection was repeated 13 times, and a resultant HA' organism (titer of 1:8) was selected and designated PCE213. P. aernginosa PAK (fimbriated) has been previously described (26). P. aeruginosa NP is P. aeruginosa PAK with a tetracycline resistance cassette inserted into the structural gene for the fimbrial subunit (21). P. aeruginosa Ni is P. aeruginosa PAK with a similarly inactivated gene for RpoN (6). P. cepacia strains were grown on M9 minimal medium (1) supplemented with 0.2% Casamino Acids (M9CA) (BBL Microbiology Systems, Cockeysville, Md.) and 2 jig of glucose per ml; subcultures were performed on the same medium solidified with 2.5% agar (BBL). P. aeruginosa PAK was grown on MacConkey's agar plates, and P. aeruginosa NP and Ni were grown on MacConkey's agar plus 50 ,ug of tetracycline per ml. Haemophilus influenzae R906 has been previously described (2). HA assay. A bacterial inoculum of 109 CFU was suspended in 30 RI of phosphate-buffered saline (PBS) in 96-well microtiter plates (Coming Glass Works, Coming, N.Y.). An equal volume of a 3% solution of rabbit erythrocytes in PBS was added, and the mixture was incubated at room temperature for approximately 20 min. Erythrocyte agglutination was determined by inspection of the suspension with a hand lens. The HA titers of PC103, PC109, and PCE213 were determined by performing serial twofold dilutions of the initial inoculum. Outer membrane protein (OMP) preparations. Cultures of PC103, PC109, and PCE213 were grown overnight at 37'C with shaking in 500 ml of M9CA. The cells were pelleted at

MATERIALS AND METHODS

Chemicals. All chemicals were obtained from Sigma (St. Louis, Mo.) unless otherwise indicated. Bacterial strains and media. P. cepacia PC103 and PC109 were selected from among 48 clinical cystic fibrosis isolates sent to our laboratory from cystic fibrosis care centers in the United States. PC103 was chosen because it had hemagglutination (HA) activity while PC109 did not. PCE213 was derived from PC109 as previously described (24). Briefly, PC109 was harvested from M9 medium solidified with 2% *

Corresponding author. 2002

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8,000 x g for 10 min, washed in 25 ml of cold 30 mM Tris-HCl (pH 8.0), repelleted, and resuspended in 20% sucrose-30 mM Tris-HCl (pH 8.0). RNase and DNase were added (0.25 mg of each). The cells were then passed through a French press twice at 20,000 lb/in2. Lysozyme was added (2 mg), and the cells were incubated on ice for 30 min. The cells were pelleted at 20,000 x g for 10 min, and the supernatant was diluted to 10 ml with 30 mM Tris-HCl (pH 8.0). The supernatant was centrifuged at 48,000 x g for 60 min at 4°C to pellet the proteins. The pellet was resuspended in 4 ml of 2% Triton X-100 in 30 mM Tris-HCI (pH 8.0) and incubated for 30 min at room temperature. The solution was diluted to 10 ml with 30 mM Tris-HCl (pH 8.0), and the proteins were harvested by centrifugation at 48,000 x 'g for 90 min at 4°C. The pellet was resuspended in deionized H20 and analyzed on a 10% polyacrylamide gel. Ammonium sulfate precipitation. PCE213 was grown overnight in M9CA, and the cells were pelleted at 8,000 x g for 10 min. The pellet was resuspended in 40 ml of 0.1 M Tris-HCl (pH 8.0) and sheared in a Sorvall Omni-Mixer at a setting of 2.5 for 15 min. The solution was then cooled to 4°C for 30 min and centrifuged at 25,000 x g for 60 min. Ammonium sulfate was added to the supernatant to a concentration of 20% and then cooled to 4°C for 30 min. The proteins were pelleted by centrifugation at 15,000 x g for 20 minutes at 4°C. Ammonium sulfate was added to the supernatant to a concentration of 40% and incubated at 4°C for 30 min, and the proteins were pelleted at 15,000 x g for 20 min. The 20 to 40% ammonium sulfate fraction was analyzed on a 10% polyacrylamide gel. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described by Laemmli (12). Gels were run on an SDSPAGE apparatus (Hoefer Scientific Instruments, San Francisco, Calif.) at 150 V for 4 to 6 h. The gels were then either stained with Coomassie brilliant blue R-250 (Bio-Rad Laboratories, Richmond, Calif.) and destained with 10% methanol-10% acetic acid or transferred to an Immobilon membrane (0.45-,um pore size; Millipore, Bedford, Mass.) for Western blot (immunoblot) analysis. Western blot analysis. Sheared supernatants were prepared for Western blot analysis by passing an overnight broth culture (M9CA) several times through a 27-gauge needle and centrifuging the culture at 12,000 x g for 10 min. The supernatant was designated the sheared supernatant, and the pellet was designated the sheared pellet. Sheared supernatants and sheared pellets of P. cepacia PC103, PC109, and PCE213 and P. aeruginosa PAK, NP, and Ni were separated on an SDS-12% polyacrylamide gel and transferred to an Immobilon membrane (0.45-,um pore size) by the method of Towbin et al. (28) with a Hoefer electrotransfer apparatus overnight at 2 mA. The membrane was incubated in blocking buffer (3% bovine serum albumin in PBS) at room temperature for 1 h with gentle shaking. The blocking buffer was removed, and the blot was incubated with P. aeruginosa PAK antipilin antibody (26) diluted 1:1,000 in blocking buffer for 4 hours at room temperature. This reagent contains antibody against flagellin as well as pilin. The blot was then washed three times with blocking buffer to remove unbound antibody and incubated with goat anti-mouse IgG-horseradish peroxidase conjugate (BioRad), diluted 1:1,000 in blocking buffer, at room temperature for 1 h with gentle shaking. The blot was then washed three times with blocking buffer and three times with PBS to remove unbound conjugate. Bands were visualized with horseradish peroxidase color reagent (Bio-Rad) (6 mg dissolved in a mixture of 2 ml of methanol, 10 ml of 20 mM

2003

TABLE 1. HA titers of P. cepacia strains HA at indicated titer' Strain

PC103 PC109

PCE213

1:1

1:2

1:4

1:8

1:16

+

+

+

-

+

+

+

-

-

-

+

+

+

1:32 -

-

a + indicates hemagglutination; + indicates weak hemagglutination.

Tris-HCI plus 500 mM NaCl [pH 7.5], and 60 ,u1 of 30% H202). Development was stopped by washing the blot with deionized H20. Isolation of chromosomal DNA. Chromosomal DNA was isolated from P. aeruginosa PAK as previously described (26). Chromosomal DNA was isolated from P. cepacia strains by the method of Scordilis et al. (23). Chromosomal DNA was isolated from H. influenzae R906 by the method of Hull et al. (5). Southern blot analysis. Chromosomal DNA (2 ,ug) from P. aenuginosa PAK, H. influenzae R906, and P. cepacia PC103 was digested with SalI as per the manufacturer's directions (GIBCO BRL, Gaithersburg, Md.). Bands were separated on a 1% agarose gel and transferred to a nylon membrane (Magna NT; Micron Separations, Inc.) by the method of Southern (25). Hybridization was carried out at 42°C in 50% formamide (GIBCO BRL) with using either the HindIII fragment from P. aeruginosa PAK (26), which contains the pilin structural gene (pilA); the 4-kb XbaI fragment from P. aeruginosa PAK, which contains the pilin accessory genes (piB, -C, and -D) (17); or the 126-bp PstI fragment from the N terminus of pilA (8). All probes were obtained from S. Lory, Department of Microbiology, University of Washington. Hybridizable bands were detected as per the manufacturer's directions with a Genius Non-Radioactive DNA Labeling and Detection Kit (Boehringer Mannheim, Indianapolis, Ind.). The blots were washed twice for 5 min each time in 0.2x SSC-0.1% SDS and twice for 15 min each time at 68°C in 0.1x SSC-0.1% SDS (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate). Electron microscopy. Carbon-coated Formvar grids were floated on PBS and touched to the surface of colonies grown on solid media. Negative staining was performed as described by McGee et al. (13), and the grids were examined and photographed with a Zeiss EM9 electron microscope. The photograph images were analyzed with the Sigma Scan program (Jandel Scientific) and compared with images of reference particles. RESULTS

HA is associated with the presence of fimbriae on P. cepacia. HA of erythrocytes is a characteristic associated with certain types of fimbriae (10). Therefore, 48 clinical cystic fibrosis isolates ofP. cepacia were screened for HA activity. One isolate, PC103, was found to hemagglutinate rabbit erythrocytes with a titer of 1:32 (Table 1). Another isolate, PC109, which was HA-, was also selected. Since it was possible that PC109 expressed fimbriae at a low frequency in culture, an HA' derivative was selected by repeated (13 times) adsorption to rabbit erythrocytes. This derivative was designated PCE213 and had an HA titer of 1:8. These three strains were then examined for the presence of fimbriae by transmission electron microscopy. Fimbriae were not detectable on the HA- strain, PC109 (Fig. 1A).

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KUEHN ET AL.

A

INFECT. IMMUN.

_C_

_

_4.

~

~

~

-5"

.X9.1

o.le IJ M~ FIG. 1. Transmission electron micrographs of PC109 (A), PCE213 (B), and PC103 (C). Note peritrichous fimbriae on PCE213 and polar fimbriae on PC103 (arrows). The bacteria were grown overnight on M9CA plates and negatively stained. Magnification, x99,000.

However, the HA' derivative, PCE213, possessed approximately 50 peritrichous fimbriae per cell and was also flagellated (Fig. 1B). These fimbriae measured 4.8 ± 1.36 nm in diameter and 200 to >2,100 nm in length. This differed from the naturally HA' strain PC103, which had from one to three polar fimbriae (Fig. 1C), measuring 3.4 to 11.4 nm in diameter and from 280 to 720 nm in length, per cell. The fimbriae from both strains appeared to have hollow cores and were similar to fimbriae seen in other gram-negative bacilli. These data suggested that the presence of fimbriae was associated with HA activity. Identification of the fimbrial subunit. The fimbriated derivative PCE213 was developed from the nonfimbriated strain PC109 in order to aid in the identification of the fimbrial subunit. The OMP profile of PC109 was compared to those of PCE213 and PC103 (Fig. 2A). There was a marked increase in the amount a 40-kDa protein and a moderate increase in the amounts of a 20- and a 16-kDa protein (arrows) in strains PC103 and PCE213 compared with PC109. Apart from the increased amounts of the 40-kDa protein, the OMP profile of PCE213 was very similar to that of PC103. Since PCE213 was also flagellated, the fimbrial subunit had to be differentiated from the flagellin subunit. PCE213 was grown to mid-log phase, harvested, and sheared in a Sorvall Omni-Mixer for 10 min at 4°C. After centrifugation at 15,000 x g for 10 min, a 20 to 40% ammonium sulfate fraction was examined by SDS-PAGE (Fig. 2B). The analysis showed that this fraction was enriched in a protein with the relative mobility of 16 kDa. This protein was chosen as the putative fimbrial subunit. It was unlikely that this was the flagellin subunit because there were 50 or more fimbriae per cell but only one flagellum per cell, as shown by electron microscopy. Thus, the relatively larger amount of the 16-kDa protein made it the more likely candidate for being the fimbrial subunit. The molecular mass (16 kDa) was in the range of molecular masses of reported fimbrial subunits seen in other gram-negative bacilli, including P. aeruginosa (22). In addition, 12 P. cepacia flagellar antigens were examined by SDS-PAGE. The flagellins were divisible into two groups:

a group of four with molecular masses of 31 kDa and a group of eight with molecular masses of 45 kDa (15). These mobilities differed from that of the putative fimbrial subunit (16 kDa) but are within the range of the 40-kDa protein seen in the PCE213 OMP profile. Homology between the P. cepacia 16-kDa protein and P. aeruginosa PAK pilin subunit. The presence of homology between the fimbrial subunits of P. cepacia and P. aeruginosa was examined by Western blotting with polyclonal P. aeruginosa PAK antifimbrial antibody and by Southern blot

A

1 kDa 200 97 68-

43-

2 3

B 4 -- 40

26 -

26-

1814-

kDa 200 976843-

4- 20

18-

*- 1 6

14-

4-16

FIG. 2. SDS-PAGE analysis of P. cepacia proteins. (A) OMP preparations from PC109 (lane 1), PCE213 (lane 2), and PC103 (lane 3) were separated on a 10% polyacrylamide gel. Arrows indicate the 16-, 20-, and 40-kDa proteins in the fimbriated strains PC103 and PCE213. (B) Ammonium sulfate (20 to 40%) cut of PCE213 showing the enrichment of the 16-kDa protein (arrow). Molecular mass standards are indicated on the left in each panel.

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KDa

1

2

3

4

5 6

7

1

8 9

2

3

23.1 9.4-

ar

6.6

-

4.4

-

2.3 2.0

-

2005

4 2,100 nm in length, per cell. The fimbriae of both of these strains appeared to have hollow cores. These measurements were in the range of those found in other bacteria, including P. aeruginosa (3, 26). These observations suggested that the hemagglutinating activity is associated with the presence of fimbriae. However, a second attempt to derive a fimbriated derivative of PC109 yielded an organism that was HA- but exhibited autoagglutinating activity. These results suggested that P. cepacia can express to more than one type of fimbria. OMP preparations from PC103 and PCE213 were enriched in three proteins (16, 20, and 40 kDa) which were not present in the nonfimbriated strain PC109. Western blot analysis of sheared supernatants from the three P. cepacia strains showed that the 16-kDa protein reacted with P. aeruginosa PAK antipilin antibody, indicating that there is some antigenic homology between the two species of Pseudomonas. This differed from the results obtained by Saiman et al. (21), in which their two strains of P. cepacia appeared to bear no homology with P. aeruginosa PA01 fimbriae. However, P. aeruginosa PAK and PAO fimbrial subunits, although highly homologous between N-terminal residues 1 and 53, have little homology at the C terminus (30). It is possible that similar sequence variation exists among pilin genes of different P. cepacia strains, and any individual pilin probe from P. aeruginosa may not reveal a specific P. cepacia pilin gene. Homology with P. aeruginosa PAK at the genetic level was also found by Southern blot analysis. SalI digests of chromosomal DNA from PC103 contained hybridizable

bands when probed with either the 1.2-kb HindIII fragment containing the P. aeruginosa PAK fimbrial structural gene (26) or the 4.0 kb-XbaI fragment containing the pilin accessory genes (17). A 15-kb SalI fragment was found to hybridize with both probes. This result indicated that the PC103 fimbrial gene cluster is contained on a 15-kb Sall fragment. However, hybridizable bands were not found by using a probe from the conserved N-terminal region of piLA. Therefore, homology with P. aeruginosa PAK is probably in the C-terminal binding region as well as in the accessory genes. The role of fimbriae in P. cepacia adherence has not been well studied. Saiman et al. (20) have reported that P. cepacia adhered to bovine tracheal epithelial cells in a cell attachment assay. However, in this assay, the eukaryotic cells did not bind a protein of 16 kDa. We used an in vitro cell-binding assay that compared adherence of the nonfimbriated strain PC109 and its fimbriated derivative PCE213. PCE213 showed a 20- to 40-fold increase in adherence to A549 cells compared with PC109. This result agreed with previous results comparing mean residence times of PC109 and PCE213 (24). This suggested that in vitro, fimbriae enhanced adherence of P. cepacia to cells. It is also possible that other nonfimbrial adhesins are involved in adherence. The exact role of fimbriae in cell adherence is currently being examined. The data in this study have identified the fimbrial subunit of P. cepacia as a 16-kDa protein. P. cepacia fimbriae are similar to those seen in other bacteria and have homology to P. aeruginosa PAK fimbriae. Of biological importance is the finding that the presence of fimbriae increases the ability of P. cepacia to adhere to A549 cells in vitro. Future studies will determine the role that fimbriae play in in vivo adherence of P. cepacia. ACKNOWLEDGMENTS This work was supported by a grant from the Cystic Fibrosis Foundation. REFERENCES 1. Anderson, E. H. 1946. Growth requirement of virus-resistant mutants of Escherichia coli strain 'B'. Proc. Natl. Acad. Sci.

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