Surfactant Protein a Resists the Bactericidal Effects of Pulmonary

Bordetella pertussis Lipopolysaccharide Resists the Bactericidal Effects of Pulmonary Surfactant Protein A

This information is current as Lyndsay M. Schaeffer, Francis X. McCormack, Huixing Wu of September 29, 2021. and Alison A. Weiss J Immunol 2004; 173:1959-1965; ; doi: 10.4049/jimmunol.173.3.1959 http://www.jimmunol.org/content/173/3/1959 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2004 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Bordetella pertussis Lipopolysaccharide Resists the Bactericidal Effects of Pulmonary Surfactant Protein A1

Lyndsay M. Schaeffer,* Francis X. McCormack,† Huixing Wu,† and Alison A. Weiss2*

Surfactant protein A (SP-A) plays an important role in the innate immune defense of the respiratory tract. SP-A binds to lipid A of bacterial LPS, induces aggregation, destabilizes bacterial membranes, and promotes phagocytosis by neutrophils and macrophages. In this study, SP-A interaction with wild-type and mutant LPS of Bordetella pertussis, the causative agent of whooping cough, was examined. B. pertussis LPS has a branched core structure with a nonrepeating trisaccharide, rather than a long-chain repeating O-Ag. SP-A did not bind, aggregate, nor permeabilize wild-type B. pertussis. LPS mutants lacking even one of the sugars in the terminal trisaccharide were bound and aggregated by SP-A. SP-A enhanced phagocytosis by human monocytes of LPS mutants that were able to bind SP-A, but not wild-type bacteria. SP-A enhanced phagocytosis by human neutrophils of LPS-

mutant strains, but only in the absence of functional adenylate cyclase toxin, a B. pertussis toxin that has been shown to depress Downloaded from neutrophil activity. We conclude that the LPS of wild-type B. pertussis shields the bacteria from SP-A-mediated clearance, possibly by sterically limiting access to the lipid A region. The Journal of Immunology, 2004, 173: 1959–1965.

urfactant protein A (SP-A)3 is one of four known protein ponent of LPS suggests a mechanism for interaction with bacterial components of the pulmonary surfactant. Previous studies membranes (10). have demonstrated that SP-AϪ/Ϫ (knockout) mice have The LPS of organisms such as E. coli consists of a lipid A S http://www.jimmunol.org/ normal baseline pulmonary function (1, 2); however, when chal- domain, a relatively conserved core oligosaccharide, and an O-side lenged with a variety of viruses, bacteria, and fungi, these animals chain of repeating sugars. Bordetella pertussis, the causative agent proved to be more susceptible to respiratory infections (3, 4). SP-A of human whooping cough, and several other Gram-negative re- is a member of the collectin (collagen-like lectin) family of pro- spiratory pathogens, such as Moraxella catarrhalis, Neisseria teins. Like other collectins, including mannose-binding protein and meningitides, and Haemophilus influenzae, have an abbreviated surfactant protein D, SP-A participates in recognition and clear- LPS structure (14). B. pertussis lacks O-side chain (Fig. 1), and ance of microorganisms (for reviews, see Refs. 5 and 6). The in its place has a nonrepeating trisaccharide consisting of ␣-N- shared structural features of collectins include a globular carbohy- acetylglucosamine, ␤-2-acetamido-3-acetamido-2,3-dideoxy-man- drate recognition domain (CRD) at the C terminus, an amphipathic nuronic acid, and ␤-l-2-acetamido-4-methylamino-fucose (15, 16). by guest on September 29, 2021 helical neck region, an extended collagen-like domain, and a disulfide Electrophoresis of B. pertussis LPS reveals two distinct bands, bonded N-terminal region. SP-A monomers fold as a triple helix referred to as bands A and B (16). The slower migrating band A is through their collagen-like domains to form trimers, and six trimers composed of full-length LPS containing lipid A, core oligosaccha- assemble into a bouquet of tulips structure, which resembles C1q, the ride, and the terminal trisaccharide. Band B migrates faster and is first component of complement (7). The crystal structure of the composed of lipid A and core oligosaccharide, but lacks the tri- C-terminal domains of SP-A has been reported recently (8). saccharide (16). The simple structure of the B. pertussis LPS and SP-A can bind to a variety of microorganisms, including Esch- the availability of LPS-mutant forms of B. pertussis provide a erichia coli and Pseudomonas aeruginosa (9Ð11), induce aggre- unique opportunity to study the molecular basis of the interaction gation (9, 10, 12), and promote phagocytosis (9, 11, 13). The struc- between LPS and SP-A. tural basis of these functions is not completely understood, but The sugars of the terminal trisaccharide of B. pertussis are syn- evidence that SP-A binds via its CRD region to the lipid A com- thesized and attached to the core by the enzymes encoded in the 12-gene wlb operon (17, 18). A number of mutations have been introduced into this operon, leading to the generation of mutant strains lacking one, two, or all three of the terminal sugars. WlbH Departments of *Molecular Genetics, Biochemistry, and Microbiology, and †Internal Medicine, Division of Pulmonary and Critical Care Medicine, University of Cincin- lacks the terminal sugar (17, 18), MLT7 appears to lack the last nati, Cincinnati, OH 45267 two sugars (19), and WlbG and WlbL lack all three sugars (17, 18). Received for publication July 22, 2003. Accepted for publication May 24, 2004. Homology studies have suggested that WlbL fails to produce the The costs of publication of this article were defrayed in part by the payment of page first sugar in the trisaccharide, while WlbG retains the ability to charges. This article must therefore be hereby marked advertisement in accordance produce this sugar, but fails to transfer it to the surface of the with 18 U.S.C. Section 1734 solely to indicate this fact. bacterial cell (18). 1 This work was supported by National Institutes of Health Grants RO1 AI45715 (to Allen et al. (20) have identified and mutated the waaF gene of A.A.W.) and HL61612 (to F.X.M.). B. pertussis. This gene is responsible for the addition of a heptose 2 Address correspondence and reprint requests to Dr. Alison A. Weiss, Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati, 231 molecule at an early step in the synthesis of the core oligosaccha- Albert Sabin Way, ML 0524, Cincinnati, OH 45267-0524. E-mail address: ride (20). Mutation of this gene results in a truncated LPS molecule [email protected] with only lipid A, a single ketodeoxyoctulosonic acid (Kdo) res- 3 Abbreviations used in this paper: SP-A, surfactant protein A; BG, Bordet-Gengou; idue, and the first heptose residue of the core oligosaccharide (20). CRD, carbohydrate recognition domain; FHA, filamentous hemagglutinin; H-H, HBSS buffered with 10 mM HEPES; Kdo, ketodeoxyoctulosonic; MOI, multiplicity The LPS from WaaF mutants is smaller than band B LPS and fails of infection; SS, Stainer-Scholte; SS-BG, SS broth overlaid on BG agar. to react with mAbs against band B.

FIGURE 1. Schematic of the LPS of wild-type B. pertussis and LPS mutants. B. pertussis LPS is comprised of lipid A and core oligosaccharide. However, instead of a long, repeating O-side chain, B. pertussis LPS terminates in a simple trisaccharide composed of ␣-N-acetylglucosamine, ␤-2- acetamido-3-acetamido-2,3-dideoxy-mannuronic acid, and ␤-l-2-acetamido-4-methylamino-fucose. The structure of full-length band A LPS (A) and the truncated band B LPS (lacking the terminal trisaccharide) (B) are indicated on the right. The WaaF mutant has an abbreviated core structure consisting of Kdo and a single heptose residue. A Bvg mutant, BP347, fails to efﬁciently attach the trisaccharide and has been reported to express mixed (band A and B) LPS (19).

Using wild-type, Wlb, and WaaF mutant strains of B. pertussis, gen, Carlsbad, CA). This plasmid was digested with HindIII and a 3-kb we have examined the LPS structural elements required for SP-A HindIII fragment from pUW2138, encoding gentamicin resistance, and the to bind, aggregate, opsonize, and kill B. pertussis. OriT region (to allow for mobilization from E. coli into B. pertussis), or gent/ OriT, was inserted to generate plasmid pLMS023 (Fig. 2). LMS023 was mobilized into B. pertussis by triparental mating, as previously described (37). A

Materials and Methods kanamycin- and gentamicin-resistant colony was selected and screened for loss Downloaded from Bacterial strains and growth conditions of band A LPS by Western blot using a mAb to band A LPS, G10F8C3 (33). Plasmids and bacterial strains used in this study are described in Table I. Mutation of the adenylate cyclase toxin gene B. pertussis strains containing a plasmid encoding GFP introduced by elec- troporation (22) were used, except where indicated. B. pertussis strains Adenylate cyclase toxin mutants were generated, as previously described, were grown on Bordet Gengou (BG) agar (BD Biosciences, Sparks, MD) using suicide plasmid pKC109 (26). Loss of adenylate cyclase toxin ex- or in Stainer-Scholte (SS) broth overlaid on BG agar (SS-BG), as described pression was confirmed by Western blotting using mAb 9D14 (36). previously (30). All other strains were grown on Luria-Bertani agar plates. http://www.jimmunol.org/ Antibiotics were used at the following concentrations: gentamicin sulfate, Isolation and characterization of LPS 30 ␮g/ml; nalidixic acid, 30 ␮g/ml; kanamycin, 50 ␮g/ml; chloramphen- ␮ ␮ Mutant and wild-type LPS was isolated by incubating boiled bacteria in icol, 15 g/ml; and ampicillin, 100 g/ml. PBS with 0.1 mg/ml proteinase K (Sigma-Aldrich, St. Louis, MO) for 1.5 h SDS-PAGE and immunoblotting at 56¡C, followed by acetone precipitation. The LPS was separated on 16% SDS-PAGE, followed by silver staining using the Bio-Rad silver stain kit SDS-PAGE was performed by the method of Laemmli (31) with the mod- (Bio-Rad, Hercules, CA), according to the manufacturer’s instructions. Pu- ifications of Peppler (32). Proteins were transferred to Immobilon-P poly- rified LPS for ELISA was prepared using a modification of the Tri-Reagent vinylidene difluoride membranes (Millipore, Bedford, MA) and probed (Molecular Research Center, Cincinnati, OH) method previously reported with the primary Ab at 1/1000. mAbs were: band A LPS, G10F8C3 (33), by Yi and Hackett (38). Following Tri-Reagent extraction, samples were or BL-2 (34); band B LPS, BL-8 (35); adenylate cyclase toxin, 9D14 (36). treated with proteinase K, as above, and then re-extracted with Tri-Reagent by guest on September 29, 2021 to ensure removal of all bacterial protein. Generation of a waaF mutant strain Human SP-A The WaaF strain was generated in a BP338 background using a similar strat- egy to that used previously (20). Primers 5Ј-CACCGACACCGGCAGCAT Human SP-A was purified from lung washings of patients with the lung GACGAGGATG-3Ј and 5Ј-GGTCGCGCAGCACCTTGCCGGTCATGGG-3Ј disease alveolar proteinosis, as previously described (29). SP-A prepara- were used to generate an 859-bp internal fragment of the waaF gene by PCR. tions contained 140Ð190 pg of LPS/␮g of SP-A, as determined by the The PCR product was cloned into the TOPO 2.1 TA cloning vector (Invitro- Limulus Amebocyte Lysate (BioWhittaker, Rockland, MD).

Table I. Bacterial plasmids and strains used in this study

Strain Plasmid Phenotypea Reference

pUW2138 Gentr, OriT in pBluescript SKϩ 21 pGB5P1 Kanr, GFP in pBBRMCS-3 22 pCW505 Kanr, Gentr, GFP in pBBRMCS-2 23 pCW382-6 Kanr,Cmr, GFP in pBBRMCS-1 24 pRK2013 ColE1 replicon, P-plasmid mobilization, Kanr 25 pKC109 Gentr, OriT, 2.1 kb of adenylate cyclase toxin in Bluescript KSϩ 26 pLMS023 859 bp of waaF, Gentr, OriT in pBluescript KSϩ, ampr, Kanr This study BP338 pGB5P1 Tohama I, Wild-type LPS, Nalr, Kanr, GFP 27 BP536 pCW505 BP338, Wild type LPS, Nalr, Kanr, Strepr, GFP 17 BP347 pCW505 BP338 with Tn5 insertion in bvg, avirulent, Nalr, Kanr, Gentr, GFP 27 BPM3183 pCW505 BP338 with Tn5 lac insertion in adenylate cyclase toxin, Nalr, Kanr,Cmr, GFP 28 MLT7 pCW505 BP338 with mini Tn5 insertion in LPS operon, Nalr, Kanr, Gentr, GFP 19 WlbG pCW382-6 BP536, ⌬wlbG::Kan; Nalr, Kanr, Strepr,Cmr, GFP 17, 18 WlbH pCW382-6 BP536, ⌬wlbH::Kan; Nalr, Kanr, Strepr,Cmr, GFP 17, 18 WlbL pCW382-6 BP536, ⌬wlbL::Kan; Nalr, Kanr, Strepr,Cmr, GFP 17, 18 WaaF pCW382-6 BP338, waaF::pLMS023, Nalr, Kanr, Gentr,Cmr, GFP This study MLT7 cyc- pCW382-6 MLT7, cyc::pKC109, Nalr, Kanr, Gentr,Cmr, GFP This study WlbG cyc- pCW382-6 WlbG, cyc::pKC109, Nalr, Kanr, Gentr, Strepr,Cmr, GFP This study WlbL cyc- pCW382-6 WlbL, cyc::KC109, Nalr, Kanr, Gentr, Strepr,Cmr, GFP This study Kpn Klebsiella pneumoniae, human fecal isolate, susceptible to SP-A-mediated killing 29

a Gentr, gentamicin resistant; OriT, origin of transfer P conjugation; Cmr, chloramphenicol resistant; Kanr, kanamycin resistant; Nalr, nalidixic acid resistant; Strepr, streptomycin resistant; ampr, ampicillin resistant; cyc, adenylate cyclase toxin operon. The Journal of Immunology 1961

Aggregation by SP-A Staining and preparation of slides

Bacteria were grown overnight in SS-BG and suspended to an OD600 of 0.6 Following phagocytosis, samples were incubated with ethidium bromide, in SS salts. SS contains sufﬁcient calcium (0.135 mM) to promote calcium- as previously described (22). Slides were observed by ﬂuorescence mi-

dependent SP-A activity. SS salts are SS broth lacking L-cystine, FeSO4, croscopy under a GFP filter. The numbers of adherent and internalized ascorbic acid, nicotinic acid, and glutathione; this medium will not support bacteria were counted for 100 phagocytes per coverslip, performed in trip- replication of the bacteria. Purified human SP-A was then added to a final licate, with at least three independent repetitions. concentration of 5 ␮g/ml. Samples were incubated at 37¡C, shaking at 150

rpm, and the OD600 was determined at the indicated times. Statistical analyses Binding of SP-A to LPS All experiments were performed at least in triplicate. Data were analyzed using Student’s t test. The ability of SP-A to bind directly to LPS was examined by ELISA. Brieﬂy, Pro-bind 96-well plates (BD Biosciences) were coated with 1 ␮g Results of puriﬁed LPS in coating buffer (1.59 g of Na CO , 2.93 NaHCO /L in 2 3 3 Generation of a waaF mutation in B. pertussis H2O, pH 9.6) for2hat37¡C. Between all steps, wells were washed with PBS with 0.5% Tween. Wells were blocked with PBS with 1% BSA for 30 Disruption of the B. pertussis waaF gene, responsible for addition min at 37¡C, incubated with 50 ␮g/ml SP-A in wash buffer for1hat37¡C, of the second heptose in LPS biosynthesis, results in a deep-rough and probed with a 1/1000 dilution of a polyclonal rabbit anti-human SP-A Ab (39) for1hatroom temperature, followed by alkaline phosphatase- LPS, containing the Kdo residue and a single heptose residue of conjugated goat anti-rabbit Ab (Valeant Pharmaceuticals, Costa Mesa, the core oligosaccharide (20). To generate a waaF mutant in the CA). The reaction was developed using the Sigma Fast p-nitrophenyl phos- BP338 background, suicide plasmid pLMS023 containing an phate substrate (Sigma-Aldrich). Absorbance was read at 405 nm. Wells

859-bp internal fragment of the waaF gene (Fig. 2A) was intro- Downloaded from were repeated in triplicate, and three independent experiments were duced into BP338. Colonies were screened for loss of band A LPS performed. expression by Western blot, and LPS isolated from a band A neg- Microscopic examination of SP-A killing ative strain was analyzed by SDS-PAGE. The mutant expressed a single band of LPS that migrated faster than both band A and B Bacterial membrane integrity was assessed by staining with the membrane- impermeant fluorescent probe, propidium iodide (Molecular Probes, Eu- LPS (Fig. 2B), consistent with the truncated LPS of a waaF gene, OR). Bacteria (not containing plasmid-encoded GFP) were harvested mutant (20). http://www.jimmunol.org/ from 16-h BG or Luria-Bertani agar plates and suspended in SS salts at an ␮ OD600 of 0.07. Samples were incubated with unlabeled SP-A at 100 g/ml for1hat37¡C with shaking in a 5-ml polypropylene tube. An equivalent volume of propidium iodide diluted in SS salts was added to a final concentration of 30 ␮M, and samples were stained for 15 min in the dark. Bacteria were pelleted, suspended in SS salts to one-tenth the original volume, and 7 ␮l was mounted on a glass slide. Samples were observed by phase-contrast microscopy to identify bacterial cells and under fluorescent microscopy using the Texas Red filter (excitation 560 nm, emission 630 nm) to assess viability. by guest on September 29, 2021 Bacterial cultures for phagocytosis studies Growth conditions can influence virulence factor expression. To address this issue, phagocytosis studies were performed using bacteria harvested from both broth and agar cultures. For liquid cultures, bacteria expressing GFP were grown overnight in SS-BG, pelleted, and suspended in fresh SS

broth, and the OD600 was determined. Bacteria were washed three times in HBSS (BioWhittaker) buffered with 10 mM HEPES (Sigma-Aldrich)

(H-H) and suspended to an OD600 of 1.0 in H-H. For agar-grown cultures, bacteria were grown for 24 h on BG agar plates and harvested in SS broth

using a Dacron ﬁber-tipped swab. The OD600 was determined, and the bacteria were pelleted and suspended to an OD600 of 1.0 in H-H. For SP-A opsonization, SP-A was added to bacteria at 30 ␮g/ml and incubated for 15 min at 37¡C, while control bacteria were incubated without SP-A. The samples were diluted to 400 ␮l and added to wells containing adherent phagocytes.

Phagocytosis by monocytes Monocytes were isolated from the blood of healthy adult volunteers, as previously described (24), and suspended to 6 ϫ 106/ml in H-H with au- tologous serum (0.1%). One-milliliter samples were added to sterile 24- well tissue culture dishes containing glass coverslips and incubated for 1 h

at 37¡C, 5% CO2, to allow for adherence. Bacteria were added at a multiplicity of infection (MOI) of ϳ5; the plates were centrifuged for 5 min at FIGURE 2. Characterization of LPS expression. A, Suicide plasmid 640 ϫ g to facilitate contact between bacteria and adherent monocytes and pLMS023 containing nt 73Ð932 of waaF was mobilized into wild-type B. incubated for1hat37¡C with 5% CO to allow phagocytosis to occur. 2 pertussis, and colonies were selected for kanamycin and gentamicin resis- Phagocytosis by neutrophils tance, indicative of integration into the B. pertussis chromosome. B, Silver- stained SDS-PAGE of B. pertussis LPS, in which the wild-type strain, Neutrophils were isolated from the blood of healthy adult volunteers, as BP338, expresses predominantly band A LPS, and BP347, the Bvg mutant, ϫ 5 - previously described (22), and suspended to 2 10 /ml in HBSS (Bio expresses predominantly band B LPS. LPS from the WaaF mutant fails to Whittaker) buffered with 10 mM HEPES (Sigma-Aldrich) and BSA (0.25%). One-milliliter samples were plated and incubated to allow for comigrate with either band A or B. C, Band A and B expression. LPS from adherence, as described above. Bacteria were added to achieve an MOI of whole bacteria was separated on 16% SDS-PAGE gels and transferred to ϳ5, centrifuged for 5 min at 640 ϫ g to facilitate contact between bacteria polyvinylidene diﬂuoride membranes. A and B, Immunoblot probed with

and adherent neutrophils, and incubated for1hat37¡C with 5% CO2 to mAb to band A; band B, immunoblot probed with mAb to band B. WT, allow phagocytosis to occur. wild-type strain BP338; BvgϪ, strain BP347. 1962 B. pertussis LPS AND SP-A

Characterization of LPS expression from wild-type and LPS mutants Previous studies have shown that although the LPS of wild-type B. pertussis, BP338, consists of almost entirely band A LPS, some strains fail to complete their LPS molecules and express a mixture of band A and B LPS (19). Band A expression was examined by Western blot (Fig. 2C). Only the wild-type strain, BP338, reacted with Ab BL-2, a mAb specific to band A (34). No band A expression was observed for BP347, the Bvg mutant (Fig. 2C, BvgϪ). Bvg mutants lack a transcription factor that regulates virulence factor expression (27). BP347 was previously reported to express a mixture of band A and B LPS (19), and the lack of band A expression led us to characterize the LPS expressed by other mutants used in this study. As expected, none of the LPS mutants produced full-length band A LPS (Fig. 2C). Band B expression was characterized using mAb BL-8 (35) specific for band B LPS. The wild-type strain reacted only weakly with the band B mAb (Fig. 2C, band B), consistent with Fig. 2B, in which this strain produced predominantly full- Downloaded from length LPS. In contrast, the Bvg mutant, which failed to produce band A LPS, showed strong reactivity with the band B mAb. Mu- tants WlbG and WlbL, which are only capable of producing band B LPS, also displayed strong reactivity with mAb BL-8. Band B was produced by mutants WlbH and MLT7, but to a lesser extent than the WlbG and WlbL mutants, consistent with previous studies http://www.jimmunol.org/ suggesting that these mutants produce band B LPS, as well as truncated band A LPS (17Ð19). The WaaF mutant showed no reactivity with the band B monoclonal, confirming this mutant is unable to produce full-length core oligosaccharide. FIGURE 3. Aggregation of B. pertussis in the presence of SP-A. Bac-

teria were suspended to an OD600 of 0.6 in SS salts. Puriﬁed human SP-A Binding and aggregation of B. pertussis in suspension by SP-A was added to a ﬁnal concentration of 5 ␮g/ml. Samples were incubated at To examine the ability of SP-A to promote aggregation, bacterial 37¡C with shaking. OD600 was read at sequential time points and reported

as a percentage of the starting OD . Mutant strains compared with the by guest on September 29, 2021 suspensions were incubated with or without SP-A in SS salts, and 600 appropriate parental wild-type (WT) controls, either BP338 (A) or BP536 the OD was monitored. In the absence of SP-A, none of the strains (B), are shown. Data represent mean ϩ SEM. All mutants had a statistically examined demonstrated a significant drop in OD600 (data not Ͻ significant (p 0.05) difference in OD600 from wild type by 1 h. C, Bind- shown), indicating that the bacteria remained in suspension ing of SP-A to purified LPS from wild type (BP338) and the WlbG mutant throughout the course of the experiment. This same trend was ob- strain of B. pertussis. Wells were coated with purified LPS and incubated served for two strains expressing wild-type LPS, BP338 (Fig. 3A) with SP-A at 50 ␮g/ml or buffer alone (control) and probed, as described. Indicates a statistically significant ,ء .and BP536 (Fig. 3B), in the presence of SP-A. However, a drop in Absorbance at 405 nm is reported (p Ͻ 0.05) difference from the LPS control. OD600 was seen after the addition of SP-A for all of the LPS mutants, indicating that SP-A induced the formation of aggregates of bacteria large enough to settle out of suspension. These results indicate that SP-A is unable to aggregate wild-type B. pertussis; Effects of SP-A on attachment and phagocytosis of B. pertussis however, strains lacking one or more of the three terminal sugars by human monocytes of the LPS molecule are rapidly aggregated in the presence Previous studies have demonstrated a role for SP-A in enhancing of SP-A. the uptake of a variety of opsonized particles by human monocytes To determine whether aggregation was due to a direct interac- and macrophages (13, 40, 41). We examined the effects of SP-A tion between SP-A and the LPS of B. pertussis, we performed an opsonization on the adherence and phagocytosis of wild-type and ELISA to measure the amount of SP-A bound by purified LPS. LPS-mutant strains of B. pertussis by human monocytes (Fig. 4). The LPS of wild-type and the WlbG mutant strains of B. pertussis As seen in previous studies (24), phagocytosis of unopsonized was purified and used to coat wells of a Pro-bind assay plate. The B. pertussis by human monocytes is relatively inefficient, with less hydrophobic coating ensures that the purified LPS bound to the than one internalized bacterium per monocyte when experiments plates in the correct orientation. Wells were incubated with either are conducted at an MOI of 5 (Fig. 4). In the absence of SP-A, SP-A (50 ␮g/ml) or wash buffer and probed with Ab to SP-A. For internalization of wild-type and mutant bacteria was similar. Ad- LPS from the wild-type strain, the absorbance when SP-A was dition of SP-A did not affect the number of wild-type bacteria present was not different from wells incubated with buffer alone internalized by human monocytes. In contrast, a 2-fold increase in (Fig. 3C). In contrast, a significant increase ( p Ͻ 0.05) in absor- phagocytosis was observed for the LPS mutants opsonized with bance was observed with LPS from the WlbG strain in the pres- SP-A ( p Ͻ 0.05). No differences were observed in the number of ence of SP-A. These results indicate that SP-A bound to the mutant extracellular adherent bacteria (data not shown). LPS, but not to the wild-type LPS. SP-A has been reported to promote phagocytosis by opsoniza- Taken together, these data indicate that the terminal trisaccha- tion and by directly stimulating phagocytic cells (42). The finding ride of the B. pertussis LPS molecule blocks the binding of SP-A that the presence or absence of SP-A had no effect on phagocytosis to wild-type B. pertussis. of the wild-type strain indicates that direct stimulation of host cells The Journal of Immunology 1963

FIGURE 4. Effects of SP-A on internalization of B. pertussis by human monocytes. Bacteria were grown on BG agar. Monocytes isolated from healthy adult volunteers were allowed to adhere to glass coverslips for 1 h. FIGURE 6. Effects of SP-A on internalization of adenylate cyclase Bacteria alone or incubated with SP-A were added at an MOI of 5, and toxin mutants of B. pertussis by human neutrophils. Bacteria were grown phagocytosis was allowed to occur for 1 h. Samples were prepared in on BG agar, and phagocytosis assays were performed as described in Fig. Indicates a statistically signiﬁcant (p Յ 0.05) difference from bacteria ,ء .triplicate for each experiment, and at least three independent experiments 4 .Indicates a statistically signiﬁcant (p Ͻ 0.05) difference alone. Data represent mean Ϯ SEM ,ء .were performed from bacteria alone. Data represent mean Ϯ SEM.

enylate cyclase toxin double mutants to examine the effects of Downloaded from by SP-A is not responsible for the increase in phagocytosis that SP-A on phagocytosis in the absence of this toxin. The adenylate was observed. cyclase toxin gene was disrupted by insertion of the suicide plas- The bacterial adhesin filamentous hemagglutinin (FHA) has mid pKC109. Phagocytosis of the LPS/adenylate cyclase toxin been shown to mediate bacterial attachment to human cells (23, double mutants was compared with both the wild-type strain and 43). FHA exists as a membrane-bound protein, and in addition, strain BPM3183, which has a Tn5-lac insertion in the adenylate soluble FHA is released into the culture supernatant by a proteo- cyclase toxin locus (28) (Fig. 6). SP-A did not promote internal- http://www.jimmunol.org/ lytic event mediated by the B. pertussis protease, SphB1 (44). Pre- ization of either wild-type BP338 or the adenylate cyclase toxin vious studies (24) examined phagocytosis of broth-grown bacteria, mutant, BPM3183, both of which express wild-type LPS. How- which were centrifuged and washed several times. Washing could ever, addition of SP-A caused a statistically significant increase in influence the ratio of surface-associated FHA to soluble FHA. We internalization of the LPS/adenylate cyclase toxin double mutants found that bacteria harvested from agar displayed increased ad- compared with internalization in the absence of SP-A. These herence to monocytes compared with the broth-grown cells (data phagocytosis studies were repeated using human monocytes. not shown). However, phagocytosis of broth-grown cells was sim- Phagocytosis of LPS/adenylate cyclase toxin double mutants in the ilar to that seen in Fig. 4 for agar-grown cells, and while addition presence or absence of SP-A was similar to that seen for the LPS of SP-A promoted internalization of LPS mutants, it did not alter single mutants, consistent with previous studies (24) demonstrat- by guest on September 29, 2021 internalization of the wild-type strain. ing that adenylate cyclase toxin inhibits phagocytosis by neutro- Adenylate cyclase toxin antagonizes SP-A-mediated phagocytosis phils, but not by monocytes. of B. pertussis by human neutrophils Opsonization with SP-A did not increase phagocytosis of wild- type B. pertussis or the LPS mutants by neutrophils (Fig. 5). How- ever, the adenylate cyclase toxin of B. pertussis elevates cAMP levels in eukaryotic cells and has been shown to cripple the phagocytic abilities of human neutrophils (23, 45). Phagocytosis is increased several-fold for strains that fail to express the toxin or in the presence of neutralizing Abs to adenylate cyclase toxin (23, 45). Because the presence of functional adenylate cyclase toxin could impair SP-A-mediated opsonization, we generated LPS/ad-

FIGURE 7. Membrane permeability of SP-A-treated bacteria. Bacteria were incubated alone (A) or with SP-A (B), and assessed for membrane permeability using propidium iodide, a membrane-impermeable dye, at ϫ630 magnification using a Texas Red filter. Samples were photographed with phase-contrast microscopy (Phase) to show the location of all bacteria FIGURE 5. Effects of SP-A on internalization of B. pertussis by human present in the field, and under fluorescence microscopy to show the loca- neutrophils. Bacteria were grown on BG agar. Human neutrophils were tion of organisms permeabilized by propidium iodide (PI). Filled arrows isolated from healthy adult volunteers and allowed to adhere to glass cov- indicate aggregates; open arrows indicate the location of the same aggre- erslips for 1 h. Phagocytosis assays were performed as described in Fig. 4. gates under fluorescence microscopy. K. pn, K. pneumoniae; WT, B. per- Data represent mean Ϯ SEM. tussis BP338. 1964 B. pertussis LPS AND SP-A

Influence of SP-A on bacterial viability gens, including M. catarrhalis, N. meningitides, and H. influenzae Previous work has shown that both SP-A and surfactant protein D (14), have short or absent O-chains and may, therefore, have sus- are able to directly inhibit the growth of some Gram-negative bac- ceptibilities to SP-A that differ from those observed for E. coli and teria via a mechanism that is distinct from their macrophage-de- P. aeruginosa. Studies of SP-A-resistant and susceptible bacteria pendent antimicrobial functions (29). Growth inhibition appears to may yield insights into the molecular basis of SP-A/LPS be due to permeabilization of the bacterial membrane by a CRD- interactions. mediated mechanism. To determine whether SP-A could kill B. References pertussis, bacterial viability was assessed using the membrane- 1. Ikegami, M., T. R. Korfhagen, J. A. Whitsett, M. D. Bruno, S. E. Wert, K. Wada, impermeant DNA-specific dye, propidium iodide. B. pertussis ap- and A. H. Jobe.1998. Characteristics of surfactant from SP-A-deficient mice. peared as small coccobacilli, smaller than Klebsiella pneumoniae Am. J. Physiol. 275:L247. 2. Korfhagen, T. R., M. D. Bruno, G. F. Ross, K. M. Huelsman, M. Ikegami, when viewed by phase microscopy, and most of the bacteria were A. H. Jobe, S. E. Wert, B. R. Stripp, R. E. Morris, S. W. Glasser, et al. 1996. viable following growth under control conditions (Fig. 7, Bacteria Altered surfactant function and structure in SP-A gene targeted mice. Proc. Natl. alone). Following incubation with SP-A, the SP-A-susceptible K. Acad. Sci. USA 93:9594. 3. LeVine, A. M., K. Hartshorn, J. Elliott, J. Whitsett, and T. Korfhagen. 2002. pneumoniae strain and the WlbG mutant formed aggregates, and Absence of SP-A modulates innate and adaptive defense responses to pulmonary many of the bacteria stained with propidium iodide, consistent influenza infection. Am. J. Physiol. Lung Cell. Mol. Physiol. 282:L563. with cell death (Fig. 7, Bacteria ϩ SP-A). In contrast, the wild- 4. Harrod, K. S., B. C. Trapnell, K. Otake, T. R. Korfhagen, and J. A. Whitsett. 1999. SP-A enhances viral clearance and inhibits inflammation after pulmonary type B. pertussis did not aggregate in the presence of SP-A and did adenoviral infection. Am. J. Physiol. 277:L580. not stain extensively with propidium iodide. These results suggest 5. McCormack, F. X., and J. A. Whitsett. 2002. The pulmonary collectins, SP-A and SP-D, orchestrate innate immunity in the lung. J. Clin. Invest. 109:707. Downloaded from that B. pertussis mutants that bind SP-A are susceptible to SP-A- 6. Holmskov, U., S. Thiel, and J. C. Jensenius. 2003. Collectins and ficolins: hu- mediated killing. moral lectins of the innate immune defense. Annu. Rev. Immunol. 21:547. 7. Holmskov, U. L. 2000. Collectins and collectin receptors in innate immunity. APMIS Suppl. 100:1. 8. Head, J. F., T. R. Mealy, F. X. McCormack, and B. A. Seaton. 2003. Crystal Discussion structure of trimeric carbohydrate recognition and neck domains of surfactant In this study, the ability of SP-A to mediate the clearance of B. protein A. J. Biol. Chem. 278:43254. 9. Hartshorn, K. L., E. Crouch, M. R. White, M. L. Colamussi, A. Kakkanatt, pertussis was examined. SP-A was unable to bind to or aggregate http://www.jimmunol.org/ B. Tauber, V. Shepherd, and K. N. Sastry. 1998. Pulmonary surfactant proteins B. pertussis strains expressing wild-type LPS. However, binding A and D enhance neutrophil uptake of bacteria. Am. J. Physiol. 274:L958. and SP-A-mediated aggregation were observed for five indepen- 10. Van Iwaarden, J. F., J. C. Pikaar, J. Storm, E. Brouwer, J. Verhoef, R. S. Oosting, dent mutants in the same biochemical pathway (LPS synthesis) L. M. van Golde, and J. A. van Strijp. 1994. Binding of surfactant protein A to the lipid A moiety of bacterial lipopolysaccharides. Biochem. J. 303:407. from two independent parental lines. Furthermore, ELISA studies 11. Manz-Keinke, H., H. Plattner, and J. Schlepper-Schafer. 1992. Lung surfactant with purified LPS suggest that SP-A binds directly to mutant, but protein A (SP-A) enhances serum-independent phagocytosis of bacteria by alveolar macrophages. Eur. J. Cell Biol. 57:95. not wild-type LPS. SP-A has been reported to bind to the lipid A 12. McNeely, T. B., and J. D. Coonrod. 1994. Aggregation and opsonization of type portion of LPS (10), and it is possible that one or more of the three A but not type B Hemophilus influenzae by surfactant protein A. Am. J. Respir. terminal sugars may sterically hinder access of SP-A to lipid A; Cell Mol. Biol. 11:114.

13. Tino, M. J., and J. R. Wright. 1996. Surfactant protein A stimulates phagocytosis by guest on September 29, 2021 however, these sugars may block binding to other structures as of specific pulmonary pathogens by alveolar macrophages. Am. J. Physiol. well. Mutant WlbH, lacking only the terminal sugar, was aggre- 270:L677. gated by SP-A. However, whether the terminal sugar is sufficient 14. Preston, A., R. E. Mandrell, B. W. Gibson, and M. A. Apicella. 1996. The lipo- oligosaccharides of pathogenic Gram-negative bacteria. Crit. Rev. Microbiol. for preventing SP-A binding is unclear, because the WlbH mutant 22:139. is inefficient at adding the terminal sugars to the core and produces 15. Caroff, M., J. Brisson, A. Martin, and D. Karibian. 2000. Structure of the Bor- detella pertussis 1414 endotoxin. FEBS Lett. 477:8. a considerable amount of band B LPS (Fig. 2C). 16. Peppler, M. S. 1984. Two physically and serologically distinct lipopolysaccharide The failure of wild-type B. pertussis LPS to be recognized by profiles in strains of Bordetella pertussis and their phenotype variants. Infect. SP-A not only protects the organism from the agglutinating func- Immun. 43:224. 17. Allen, A., and D. Maskell. 1996. The identification, cloning and mutagenesis of tions of SP-A, but also serves to protect the bacteria from SP-A- a genetic locus required for lipopolysaccharide biosynthesis in Bordetella per- mediated phagocytosis. SP-A has been reported to have direct tussis. Mol. Microbiol. 19:37. stimulatory effects on phagocytic cells (42); however, SP-A only 18. Allen, A. G., R. M. Thomas, J. T. Cadisch, and D. J. Maskell. 1998. Molecular and functional analysis of the lipopolysaccharide biosynthesis locus wlb from promoted phagocytosis of mutants capable of binding SP-A. These Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica. data suggest that enhanced phagocytosis of the LPS mutants was Mol. Microbiol. 29:27. 19. Turcotte, M. L., D. Martin, B. R. Brodeur, and M. S. Peppler. 1997. Tn5-induced due to the opsonic effects of SP-A, and not a direct stimulatory lipopolysaccharide mutations in Bordetella pertussis that affect outer membrane effect of SP-A on the monocytes. Wild-type LPS also protected function. Microbiology 143:2381. bacteria from SP-A-mediated phagocytosis by neutrophils, but 20. Allen, A. G., T. Isobe, and D. J. Maskell. 1998. Identification and cloning of waaF (rfaF) from Bordetella pertussis and use to generate mutants of Bordetella only in the absence of the B. pertussis adenylate cyclase toxin. spp. with deep rough lipopolysaccharide. J. Bacteriol. 180:35. Adenylate cyclase toxin has been shown to inhibit the phagocytic 21. Fernandez, R. C., and A. A. Weiss. 1998. Serum resistance in bvg-regulated capability of neutrophils (23, 45), and it was necessary to inacti- mutants of Bordetella pertussis. FEMS Microbiol. Lett. 163:57. 22. Weingart, C. L., G. Broitman-Maduro, G. Dean, S. Newman, M. Peppler, and vate the adenylate cyclase toxin of LPS mutants before any effect A. A. Weiss. 1999. Fluorescent labels influence phagocytosis of Bordetella per- of SP-A could be observed. tussis by human neutrophils. Infect. Immun. 67:4264. 23. Weingart, C. L., and A. A. Weiss. 2000. Bordetella pertussis virulence factors SP-A has been shown to kill bacteria by increasing membrane affect phagocytosis by human neutrophils. Infect. Immun. 68:1735. permeability (29). A mutant B. pertussis strain that was aggregated 24. Schaeffer, L. M., and A. A. Weiss. 2001. Pertussis toxin and lipopolysaccharide in the presence of SP-A was also susceptible to SP-A-mediated influence phagocytosis of Bordetella pertussis by human monocytes. Infect. Im- mun. 69:7635. permeability, as demonstrated by propidium iodide staining; how- 25. Figurski, D. H., and D. R. Helinski. 1979. Replication of an origin-containing ever, the wild-type strain was not affected by SP-A. We conclude derivative of plasmid RK2 dependent on a plasmid function provided in trans. that the antimicrobial activity of SP-A is ineffective against B. Proc. Natl. Acad. Sci. USA 76:1648. 26. Craig-Mylius, K. A., and A. A. Weiss. 1999. Mutants in the ptlA-H genes of pertussis strains expressing wild-type LPS. We speculate that Bordetella pertussis are deficient for pertussis toxin secretion. FEMS Microbiol. SP-A resistance contributes to the ability of B. pertussis to evade Lett. 179:479. 27. Weiss, A. A., E. L. Hewlett, G. A. Myers, and S. Falkow. 1983. Tn5-induced the immune defenses of the respiratory mucosa. It is of interest that mutations affecting virulence factors of Bordetella pertussis. Infect. Immun. the LPS of a number of other Gram-negative respiratory patho- 42:33. The Journal of Immunology 1965

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