Antibodies by Haemophilus influenzae - Infection and Immunity

INFEcrION AND IMMUNITY, Aug. 1993, p. 3375-3381

Vol. 61, No. 8

0019-9567/93/083375-07$02.00/0 Copyright ©) 1993, American Society for Microbiology

Paradoxical Effect of Pilus Expression on Binding of Antibodies by Haemophilus influenzae JANET R. GILSDORF,1* MICHELLE TUCCI,1 LARRY J. FORNEY,2 WENDY WATSON,' CARL F. MARRS,3 AND ERIC J. HANSEN4

Pediatrics and Communicable Diseases, University ofMichigan, Ann Arbor, Michigan 48109Microbial Ecology, Michigan State University, East Lansing, Michigan 488242; Department of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, Michigan 48109-20293; and Department of Microbiology, University of Texas Southwestern Medical Center at Dallas, Dallas, Te-xas 752354

Department of

0244'; Center for

Received 6 November 1992/Accepted 17 May 1993

Haemophilus influenzae type b (Hib) pili are surface proteins that are associated with the ability of Hib to attach to human epithelial cells. Like pilus expression of other bacteria, expression of Hib pili undergoes phase variation. We observed that Hib in the piliated phase (Hib p+) bound monoclonal antibodies directed against six conserved, surface-exposed, nonpilus Hib outer membrane epitopes to a greater extent than Hib in the nonpiliated phase (Hib p-). However, after extended incubation, p+ and p- cells bound these antibodies in a similar fashion. The differential in nonpilus antibody binding to p+ and p- Hib was not related to the presence of the type b capsule. In addition, Hib p+ organisms whose pilin gene was insertionally inactivated and did not produce pili and Hib in the nonpiliated phase bound the nonpilus Hib antibodies similarly. Hib p+ and porganisms did not differ in their binding of anti-type b capsule antibody, and the binding was specific for the epitopes recognized by the antibodies. In complement-dependent bactericidal assays, the nonpilus antibodies killed Hib p+ more effectively than Hib p-. The increased binding to, and killing of, Hib p+ by a variety of nonpilus antibodies may be important for host defense against invasive Hib. The initial step in the pathogenesis of most bacterial respiratory infections is the adherence of the pathogen to respiratory epithelial cells, which is mediated in part by bacterial appendages called pili or fimbriae. Following adherence to the respiratory epithelial cells and subsequent colonization of the respiratory mucosa, potential pathogens may invade the mucosal barrier, with resultant bacteremia. In normal hosts, most of the circulating bacteria are rapidly cleared. Previous studies (13, 20) have shown that piliated organisms are cleared more readily from the circulation than nonpiliated organisms, which has been attributed to increased susceptibility of piliated bacteria to antibody-independent phagocytosis (2, 3, 24, 26). The interactions of the respiratory pathogen Haemophilus influenzae and mammalian hosts have been extensively studied and serve as an excellent model of the pathogenesis of infections caused by respiratory flora. The pili of H. influenzae type b (Hib), which mediate adherence to respiratory epithelial cells, have been well described (5, 8, 22, 27) and are multimeric proteins composed of subunit polypeptides, pilins, of about 200 amino acids (9). Like expression of adherence pili of other bacteria, the expression of pili by Hib undergoes phase variation (5); however, the environmental signals and molecular mechanisms resulting in variable pili expression are unknown. Previous studies have shown that piliated Hib (Hib p+) binds and is killed by antibodies directed against pili, whereas nonpiliated Hib (Hib p-) is not (21). In this report, we detail our observations that piliated Hib also binds and is killed by monoclonal antibodies directed against nonpilus Hib outer membrane components to a greater extent than

*

nonpiliated Hib. This nonpilus antibody bactericidal activity may be important in host defense against invasive Hib. MATERIALS AND METHODS

Bacteria. Hib strain M42 and its piliated phase variant,

M43p+, and Ela (Eagan) and its piliated phase variant, Elap+, have been described previously (21, 27). Hib strain DL42 has been described previously (4). Its piliated phase variant, DL42p+, was obtained by hemagglutination selection (21). Preliminary experiments showed no differences in the growth characteristics of piliated strains M43 and Ela and their nonpiliated variants under the conditions used in these studies. Mutated Hib strains Elap/mut and M43p/mut contain a kanamycin cassette inserted into the pilin genes and do not express pili. To construct these mutants, the recombinant cosmid pcosXV12, which carries a 16-kb fragment of Hib strain Eagan DNA containing the pilin gene at its approximate midpoint (7) was digested with BamHI to separate the 16-kb insert from the cosmid vector. The 16-kb fragment was ligated into the BamHI site in the polylinker of pGEM7, and the resulting subclone was named pBB16A. pBB16A was then digested with BglII, which makes a single cut in the plasmid within the pilin gene. A kanamycin resistance gene cassette with BamHI ends was isolated from the plasmid pWW84 (23) and ligated into the BglII site to create an insertion mutation within the Hib pilin gene. The resulting plasmid, pBB16/K, was amplified in Escherichia coli HB101. Cells of piliated Hib strains Eagan p+ and M43p+ were made competent by the method of Herriott et al. (16), and 1 ,ug of pBB16/K plasmid DNA was used to transform 1 ml of competent cells. The transformants were selected on brain heart infusion agar with 4 ,ug of NAD, 10 ,ug of hemin, and 25 ,ug of kanamycin per ml. To verify allelic exchange, genomic

Corresponding author. 3375

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TABLE 1. Antibodies used in binding studies Antibody

Antigena

Surface exposed

Antibody type or source

Reference

6G12 7C8 3E12 2F4 3E9 9F5 6C3 4C4 Anticapsule

98K P1

Yes Yes Yes Yes Yes No Yes Yes Yes

Purified monoclonal Purified monoclonal Monoclonal supernatant

18 11 Unpublished data 4 14 14 Unpublished data 12

P1 P2 P2 P2 PAL LOS PRP

a PAL, peptidoglycan-associated lipoprotein; LOS, lipo-oligosaccharide; PRP, b Hyland Laboratories.

DNA isolated from the transformants was digested with both BglII, which cuts outside the pilin gene and once within the pilin gene (the site of the Kanr cassette insertion), and PmlI, which cuts once outside the pilin gene. Southern blot analysis of the digested DNA probed with an -3.5-kb fragment containing the pilin gene and -2.5 kb of upstream sequence shows that the BglII site in the pilin gene has been obliterated and the other BglII and PmlI fragments increased in size by about 1.2 kb, the size of the kanamycin cassette, thus confirming allelic exchange. Kanamycin-resistant Hib colonies were tested by hemagglutination (21) and reactivity with antipilus antibody (8) and were found to have lost the piliated phenotype. Nonencapsulated variants of piliated (c-p+) and nonpiliated (c-p-) Ela were identified by colonial morphology on Levinthal agar and confirmed by India ink staining. Monoclonal antibodies. The monoclonal antibodies used in the assays are described in Table 1. Antibody binding assays with immobilized antigen. The binding assays were performed by enzyme-linked immunosorbent assay (ELISA) as previously described (8). Samples of the bacterial suspension (50 pl each of Elap+ [3.7 x 104 CFU], Elap- [4.4 x 104 CFU], M43p+ [4.4 x 10" CFU], and M42p- [4.4 x 104 CFU] per well) or purified pili (1.25 mg per well) (1) were incubated overnight in coating buffer (pH 9.6) in microtiter plate wells, and the wells were blocked for 1 h with 200 ,u of phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA). After a washing, 50 RI of the antibodies was incubated in the prepared wells for 1 h, purified monoclonal antibodies were diluted to 5.2 ngl,u in blocking buffer, and monoclonal antibodies in culture supernatant were tested undiluted. After another washing, 50 ,u of rabbit anti-mouse immunoglobulin G antibodies conjugated to horseradish peroxidase (Sigma Chemical Co., St. Louis, Mo.) and diluted 1:2,000 in PBS was added to each well. The reactions were developed as previously described (8). To measure binding of the anticapsular antibodies, rabbit polyclonal anticapsular antiserum (Hyland Laboratories, Costa Mesa, Calif.) was diluted 1:500 in blocking buffer and goat anti-rabbit immunoglobulin G (Cappel Laboratories, Malvern, Pa.) diluted 1:2,500 was used as the second antibody. In preliminary experiments, we monitored the relative binding of piliated and nonpiliated Hib to the microtiter plates by comparing the total protein content of the bacterium-coated wells. Bacteria were applied to microtiter wells as described above, but the wells were washed with PBS, and the blocking step with protein-containing buffer was omitted. The total protein content of each well was quantitated by the colorimetric bicinchoninic acid protein microassay (Pierce,

Purified monoclonal Purified monoclonal Monoclonal supernatant Monoclonal supernatant Purified monoclonal

Polyclonaib polyribose-ribitol phosphate.

Rockford, Ill.), by methods recommended by the manufacturer. The color change in each well was quantitated with an automated ELISA reader spectrophotometer at 562 nm. Antibody binding assay with suspended antigen. To further circumvent the possibility of p+ and p- Hib adhering differently to the microtiter plate wells used in the immobilized antigen assays, we developed an antibody-binding assay using suspended antigen, Hib strain Ela. Monoclonal antibody 4C4 was labeled with biotin by the method of Heggeness and Ash (15). One hundred microliters of piliated or nonpiliated bacteria suspended in PBS containing 1% BSA to a transmission of 55% at 610 nm was mixed with 2.6 ,ug of biotin-labeled antibody in tubes blocked with 5% (wt/vol) dry skim milk in 0.1 PBS (pH 7.4) and incubated for 3 or 24 h. The bacteria were pelleted, washed in PBS four times, and resuspended in 100 ,u of PBS-1% BSA. Peroxidase-labeled avidin (Sigma Chemical Co.) (50 RI diluted 1:2,500 in PBS) was added and allowed to incubate for 1 h. The reactions were developed as previously described (8). Bactericidal assay. Bactericidal activity of nonpilus Hib outer membrane antibodies against piliated and nonpiliated Hib strains M43p+ and M42p- and Elap+ and p- was measured as previously described (21). In brief, 10 RI of log-phase Hib (1.5 x 105 CFU in Veronal-buffered saline) and 10 ,u of precolostral calf serum as a source of complement (21) were added to microtiter plate wells containing 60 ,u of monoclonal antibody (or fetal bovine serum in control wells). After 0, 45, and 180 min of incubation at 37°C, samples (5 pl for each time) of the mixture were plated on Levinthal agar. After overnight incubation, the CFU on each plate were counted and the bactericidal activity of the antibodies was calculated by the following formula: [preincubation CFU (0 min) - postincubation CFU (45 or 180 min)]/preincubation CFU (0 min) x 100. Controls included bacteria incubated with the monoclonal antibodies without complement and bacteria incubated with fetal bovine serum, with and without complement. To evaluate killing of Hib p+ and p- by nonpilus antibody in the same reaction mixture, we developed a mixed-organism bactericidal assay. These assays, using piliated Hib strains Elap+ and M43p+, nonpiliated strains Elap/mut and M43p/mut (which contain a kanamycin cassette interrupting the pilin gene), and monoclonal antibody 4C4, were identical to those described above, except that the pre- and postincubation samples were plated both on Levinthal agar and on Levinthal agar containing 25 ,g of kanamycin per ml to allow identification of the phenotypes of the survivors. Bacterial growth and degradation. To identify differences in the growth rates of piliated and nonpiliated strains, freshly grown piliated and nonpiliated Ela and M43 from Levinthal

EFFECT OF PILI ON ANTIBODY BINDING BY H. INFLUENZAE

VOL. 61, 1993

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UElapz

0.6 -M2p U W p-

~~~~~~~~~~~~~~~DL42

0

O0.4 0.0

4C4 anti-capsule 9E3 9F5 6C3 3E9 MONOCLONAL FIG. 1. Binding of antibodies directed against surface components of Hib to piliated and nonpiliated Hib. Piliated and nonpiliated Hib strains were bound to microtiter plate wells and reacted by ELISA with monoclonal antibodies specific for eight epitopes on five outer membrane structures of Hib and polyclonal antibodies against the type b capsule. The y axis shows OD at 490 nm. 6G12

7C8

3E12

2F4

agar were grown overnight (to stationary phase) in 50 ml of Levinthal broth at 37°C. One hundred microliters of the overnight broth culture was subcultured into 5 ml of fresh Levinthal broth and grown at 37°C in a shaking water bath. Samples of the broth cultures were taken hourly for 8 h and at 12 and 15 h, serially diluted in PBS, and immediately plated on Levinthal agar for colony counts.

RESULTS Using ELISA, we compared the ability of Hib in the piliated and nonpiliated phases to bind antibodies specific for several cell surface antigens, including outer membrane proteins (P1, P2, peptidoglycan-associated lipoprotein, and the 98-kDa protein), lipo-oligosaccharide, and the polyribose-ribitol phosphate capsule. The results (Fig. 1) show that monoclonal antibodies against highly conserved epitopes (6G12, 3E12, 2F4, 3E9, and 4C4) bound the three piliated strains to a greater extent than their nonpiliated phase variants. The epitope on P1 recognized by monoclonal antibody 7C8 is not well conserved and is found on approximately half of the Hib strains tested (11). Thus, the poor binding of antibody 7C8 to piliated Hib strain Elap+ compared with its binding to the other two piliated strains suggests that the epitope recognized by this monoclonal antibody is not present on strain Elap+. Furthermore, the poor binding of 7C8 also suggests that the enhanced binding of the other antibodies to Elap+ is specific rather than the result of nonspecific interactions, such as antibody binding by bacterial Fc receptors. The relatively poor binding of antibody 6C3 reflects its inability to bind to its surface-exposed epitope, peptidoglycan-associated lipoprotein. Antibody 9F5, specific for an epitope on P2 that is not surface exposed (14), bound to none of the strains and thus also serves as a control for nonspecific antibody binding. In contrast, the polyclonal antibodies directed against the polyribose-ribitol phosphate capsule found on all Hib strains bound similarly to both piliated and nonpiliated Hib. Although the monoclonal antibodies initially showed increased binding to the piliated strains compared with nonpiliated strains, after 6 h of incubation, these differences were minimal (Fig. 2). After 24 h of incubation, antibody binding did not differ between piliated and nonpiliated strains (data not shown). In addition, none of the monoclonal

antibodies bound to purified pili, even after extended incubation times (Fig. 2). To investigate the role of capsule in nonpilus antibody binding to their outer membrane targets, we tested binding of the nonpilus antibodies to type b organisms and nonencapsulated variants. The binding of the monoclonal antibodies (3E9 and 3E12) did not differ between encapsulated and nonencapsulated variants of piliated and nonpiliated Hib strain Ela (Fig. 3). To ensure that the increased reactivity of the monoclonal antibodies with the piliated organisms compared with their reactivity with nonpiliated organisms was not related to increased binding of piliated bacteria to the microtiter plates, the total protein content of the bacterium-coated wells was measured, and no differences were found between wells coated with piliated and those with nonpiliated organisms (nanograms of protein per well + standard deviation [n = 3]: Elap+, 85 + 13; Elap-, 83 ± 16; M43p+, 91 ± 11; and M42p-, 85 ± 16). In addition, the relative quantities of organisms adherent to the plates were determined by ELISA using a nonspecific rabbit antiserum (R10) that reacts with all Hib strains similarly (8). Mean optical density (OD) values after 2 h of incubation with R10 were 0.760 for Elap+, 0.766 for Elap-, 0.760 for M43p+, and 0.780 for M42p-. Furthermore, binding of antibody 4C4 to piliated and nonpiliated bacteria was tested in a suspension assay that did not depend on bacterial immobilization on microtiter wells. Results of these experiments showed increased binding of monoclonal antibody 4C4 to piliated Hib strain Eagan compared with binding to a nonpiliated variant after 3 h of incubation (OD = 0.606 + 0.070 versus 0.211 + 0.053; P < 0.0005). After 24 h of incubation, binding of piliated and nonpiliated strains was not significantly different (OD = 0.745 + 0.029 versus 0.683 + 0.032; p > 0.05). These three different experiments show no difference in the abilities of piliated and nonpiliated Hib to coat wells. Since different rates of bacterial growth and degradation could explain the differential binding of nonpilus antibodies by p+ and p- organisms, these parameters were assessed. Growth curves of piliated and nonpiliated Ela and M42 over 15 h of incubation in broth were identical. Alkaline phosphatase release after 24 h was 0.361 nmollpl for M43p+ compared with 0.350 nmol/,ul for M42p- (0.375 < P < 0.4) and 0.228 nmolV,l for Elap+ compared with 0.33 nmolV,l for Elap- (0.1 < P < 0.375), demonstrating no difference in the

3378

A

GILSDORF ET AL.

INFECT. IMMUN.

2F4

I~-

13z

1.0

w

a z

-J

4c 0

a

FIL 0

-j

I.

0

0.5

TIME (hr)

B

3E9

0.0

3E9

>.

MONOCLONAL ANTIBODIES

1.5-

FIG. 3. Binding of two monoclonal antibodies to encapsulated and nonencapsulated piliated and nonpiliated Hib strain Ela. Piliated and nonpiliated Hib phase variants possessing the type b capsule or nonencapsulated variants (c-) and p+ Hib strain Ela with its pilin gene insertionally inactivated (Elap/mut) were reacted with monoclonal antibodies 3E9 and 3E12. The y axis shows OD at 490 nm. Error bars show standard deviations (n = 3 to 5). P is >0.1 for Elap+ versus Elac-p+ and Elap- versus Elac-p- with both antibodies.

z LU -i

3E1 2

1.0

0

0.5-

TIME (hr) C

3E1

2

1.5 -

I.-

z

1.0-

a

4c F

0.5-

0

0

1

2

3

4

5

6

7

TIME (hr)

FIG. 2. Kinetics of binding of three monoclonal antibodies to piliated and nonpiliated variants of Hib and to purified pili. Piliated and nonpiliated Hib phase variants and purified pili were incubated for 0.5, 1, 2, 4, and 6 h with monoclonal antibodies 2F4 (A), 3E9 (B), and 3E12 (C). They axis shows OD at 490 nm (P < 0.05 for piliated versus nonpiliated Hib for each antibody, as measured by the Wilcoxon test). A, Elap'; A, Elap-; O, DL42p+; *, DL42p-; El, M43p+; *, M42p-; x, purified pili.

rates of bacterial degradation of piliated and nonpiliated isolates. The observation of increased nonpilus antibody binding to p organisms after prolonged incubation could be explained by a change in p- organisms to p+ after extended incubation. To demonstrate that the nonpiliated organisms remained nonpiliated, we tested organisms on the microtiter plates after 24 h of incubation with and without monoclonal antibody 3E12 for the presence of pili by reactivity with R19, the antipilus rabbit polyclonal antibody. Results showed that

after 24 h of incubation with 3E12, R19 bound well to p+ organisms (OD = 0.796) and did not bind to p- organisms (OD = 0.023). Similarly, after 24 h of incubation with 3E12, R19 also bound p+ organisms well (OD = 0.852) and did not bind to p- organisms (OD = 0.025). To define the role of the Hib pilin gene and its gene product in the enhanced binding of the nonpilus outer membrane antibodies to piliated strains, we tested the binding of monoclonal antibodies 3E9 and 3E12 (12) to a nonpiliated mutant of Hib strain Ela in which the pilin gene had been insertionally inactivated. After 1 h of incubation, the antibodies showed reduced binding to the mutant strain (Fig. 3) similar to their binding to the nonpiliated phase variants. After 24 h of incubation, no difference in the rates of antibody binding of piliated and nonpiliated organisms was seen (data not shown). The effect of the differential binding of the nonpilus outer membrane antibodies on their ability to kill piliated and nonpiliated Hib was tested in a complement-dependent bactericidal assay. After 45 min of incubation, the antibodies killed piliated M43p+ more readily than the nonpiliated phase variant, M42p- (Fig. 4). However, after 180 min of incubation, the killing rates for piliated and nonpiliated strains were similar (Fig. 4). Thus, initially, the outer nonpilus membrane antibodies both bound and killed piliated bacteria to a greater extent than nonpiliated bacteria, but after longer incubation, these differences were not significant. The piliated Hib strains Elap+ and M43p+ and their isogenic nonpiliated mutants containing insertionally inactivated pilin genes were also tested in the bactericidal assay. Monoclonal antibody 4C4 demonstrated enhanced killing of the piliated strain compared with the nonpiliated mutant after 45 min of incubation, but after 180 min of incubation,

EFFECT OF PILI ON ANTIBODY BINDING BY H. INFLUENZAE

VOL. 61, 1993

A 0 M43p+ U M42p-

100

80

-

-I *

40

20-

6G12

7C8

2F4 4C4 ANTIBODIES

FBS

6G12

7C8

2F4 4C4 ANTIBODIES

FBS

B 100

80 z

60

-i 2

P

40

20

FIG. 4. Bactericidal activity of nonpilus outer membrane antibodies against piliated and nonpiliated Hib. Piliated and nonpiliated Hib strains were incubated for 45 or 180 min with four monoclonal antibodies (6G12, 7C8, 2F4, and 4C4) directed against epitopes on four Hib outer membrane structures. (A) Percent killing of M43p+ versus M42p- at 45 min. P is