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0022-1767/90/1446-2253$02.00/0 Vol. 144.2253-2257.No. 6 . March 15. 1990 Printed in U.S.A.
T H E JOURNAL OF h " N 0 L O C Y
Copyright 0 1990 by The American Assoclatlon of lmmunoioglsts
DEGRADATION OF IgA PROTEINS BY Pseudomonas aeruginosa ELASTASE' LOUIS W. HECK,'+ PATRICIA G. ALARCON,*ROSEM. KULHAVY,+KAZUYUKIMORIHARA,* MICHAEL W. RUSSELL,' AND JIRI F. MESTECKY"' F r o m t h e D e p a r t m e n t sof *Medicine and 'Microbiology. The University of A l a b a m a a t B i r m i n g h a m S c h ooofl Medicine, B i r m i n g h a m , A L35294; a n d t h e ' T o h o P h a r m a c e u t i c a l C o m p a n y , L t d . , K y o t o , J a p a n
Human colostral IgA andmyelomaproteins of especially of S-IgA a t mucosal surfaces, results in the could still both IgAl and IgA2 subclasses were susceptible to generation of free Faba fragments, then these cleavage by Pseudomonas aeruginosaelastase. De- bind to bacterial surface Ag. In the case of certain IgAl tailed analysis of the cleavage products of I g A mye- protease-producing bacteria, it has been postulated that loma proteins revealed complete degradation of Fab such surface-bound Faba fragments could block access with no evidence of intact Fab fragments as inter- of other specific immune factors and lead to invasive mediate cleavage products. In contrast, both IgAl infection ( 1 2, 13).At the same time, lack of the Fcaregion andIgA2proteinswere resistant to cleavage by would lessen the effectivenessof the IgA in inhibition of alkaline protease from P . aeruginosa. The suscep- adherence (14). Thus, the proteolytic cleavage of Ig of tibility of human IgA proteins to elastase suggests these isotypes could interfere with theirprotective funca mechanism by which P . aeruginosa might evade tion and may allowcolonization followed by bacterial the potentially protective function of IgA by produc- invasion. Our study was to investigate the degradation of ing this enzyme. different purified IgA proteins by Pseudomonas elastase and alkalineprotease.
Pseudomonas aeruginosa is a major pathogen among patients with cystic fibrosis, burns, trauma, and granulocytopenia and is a common etiologic agent in nosoco( 1 ) . The mial,gram-negative,necrotizingpneumonias host resistance against P. aeruginosa seems to depend mainly on the production of specific opsonic antibodies as well as on increased phagocytic activity (2).Paradoxically, however, certain patients with cystic fibrosis are not protected from progressive lung injury despite the presence of large amounts of serum antibodies to this pathogen (3-6). Mucosal surfaces of the body represent a major contact area between environmental microbial agents and the IgA is the major Ig isotype of many host's immune system. human body fluids such as tears, saliva. colostrum, and respiratory anddigestive tract secretions whereit constitutes a front line of defense against microorganisms(7). In addition, IgA constitutes about 15%of the Ig fraction of human serum(7).In several microbial systems, S-IgA3 prevents the attachment and colonization of organisms on epithelial surfaces (8, 9). The mechanisms by which P. aeruginosa circumvents normal mucosal immunity are currently not well understood. Most strains of P. aeruginosa produce extracellular proteases that cleave both human IgG (10, 1 1 ) and IgA (10).If this cleavage, Received for publication August 1 1, 1989. Accepted for publication December 1 1. 1989. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked aduertlsement in accordance with 1 8 U.S.C. Section 1734 solely to indicate thls fact. This work was supported by Cystic Fibrosis Research Development Grant from the Cystic Fibrosis Foundation and Grants AI-10854 and AI18745. 'Address correspondence to Louis Heck, M.D.. University of Alabama at Birmingham, LHR 442. University Station. Birmingham. AL 35294. Abbreviations used in this paper: S-IgA. secretory IgA; PE, Pseudomonas elastase: PAP, Pseudomonas alkaline protease; SAT. staphylococcal a-toxin.
M A T E R I A L SA N DM E T H O D S
Preparation o f d ~ e r e n IgA t proteins. Three of the IgA proteins used in these studies were purified from the sera of patients with IgA myeloma using sequential ammonium sulfate precipitation, ionexchange chromatography, and gel filtrationchromatography as describedindetail (15). Colostral S-IgA wasisolatedfromfresh human colostrum and early milk (up to 4 days postpartum] and IgA myelomaproteinsincluded: purified as described(15).The 1gAl-K (Pet.,predominantlymonomeric], lgA1-K (Ham.,predominantlypolymeric),and IgA2-Xof A2m(2)allotype (Fel.. predominantly polymeric). The purity of each 1gA protein was assessed using immunoelectrophoresis and double immunodiffusion as described (15). The proteins were stored frozen at -2OOC in PBS containing 0.01% sodium azide. Before the degradation studies. they were dialyzed against 0.05 M Tris hydrochloride (pH 7.6) containing 0.15 M NaCl and 0.001 M CaCl, and adjusted to a final concentration of 1 mg/ml. Preparation of IgA proteins with SAT-binding activity.Two 1gA myeloma proteins with SAT-binding activity (16). one (Tin) being predominantlypolymeric lgA1-X and theother(Fug)monomeric l g A l - ~ were , purified by chromatography on DEAE-Sepharose and on jacalin-agarose. as described in detail (17). Purity assessed by ELlSA was >99.9%. and the preparations revealed only bands corH and L chains on SDS-PAGE after respondingtotheexpected reduction (17). Preparation of P E and PAP. PE and PAP were isolated from the culture media of P . aeruginosa strains IF0 3455 and IF0 3080, respectively, and characterized as previously reported (18. 19).Both of these enzymes were standardized for protein content (20). assayed for elastinolytic and caseinolytic activity (21).and stored frozen at -70°C at a final concentration of 500 @/ml in small aliquots. PE cleaved a standard insoluble elastin substrate suspended in agarose (1 fig of PE resulted in a ring diameter of 9.5 mm after 24 h a t 37°C) whereas PAP did not cleave this substrate. In the azocasein assay, PE gave a greater absorbance at 336 nm (1pg of PE resulted in A = 1.4 after 30 min a t 37°C) than PAP (1 Ng of PAP resulted in A = 0.54, after 30 min at37°C). Degradation of dgferent IgA t y p e s b yPE and PAP. The different IgA proteins were incubated with or without PE or PAP a t different enzyme:substrate ratios (1:5-1:lOO for PE and 1:5 for PAP] for 4 h a t 37OC. Limit cleavage at the greatest enzyme/substrate ratios was seen at 4 h and this incubation time period was used. The PE and PAP samples incubated alone were subjected to identical digestion conditions used for the IgA samples. The reactions were stoppedby adding an equal volume(50to 7 0 pl) of twice concentrated denaturing buffer (0.125 M Tris hydrochloride. pH 6.8 with 4%SDS. and 20%
Pseudomonas ELASTASE DEGRADATION O F IgA
glycerol) followed by boiling the samples for 2 min. For reducing spective enzyme. conditions. 2-ME was added to a final concentration of 2% before Degradation oflgA proteins by PE. The different IgA boiling. SDS-PACE (5 to 20%) was performed by a modification of proteins displayed varying susceptibilitiesto degradation the method of Laemmll (22). and the protein visualized after Coomassie blue staining. The m.w. standards were purchased either inbyPE (Fig. 1). In general. the myeloma proteins were dividually from Sigma Chemfcal Company (St.Louis. MO) and Boehmore susceptible to proteolytic cleavage than S-IgA under IN) or as a mixture ringer Mannheim Biochemicals (Indianapolis. the incubation conditions used in this study. TheIgA1-K from Bio-Rad Laboratories(Richmond, CA) andconsisted of the proteins, Pet and Ham (Fig. 1 A ) and IgA2-X, A2m(2) profollowing proteins: rabbit muscle myosin (200.000 Da) and phosphoDa). tein, Fel (Fig. 1B. lanes 10 to 1 4 ) were more extensively rylase b (94.000Da). Escherichia colf8-galactosidase (1 17.000 BSA (67.000 Da). hen egg OVA (43.000 Da) and lysozyme (14.000 cleaved inadose-dependent manner than S-lgA. Low Da). bovine E carbonic anhydrase(30.000Da). and soybean trypsin m.w. cleavage fragments of these three proteins are seen inhibitor (21.OOO Da). Degradation of IgA proteins with SAT-binding activity. Antion Coomassie blue staining of the unreduced SDS-PAGE. SAT IgA antibodies at 100 rg/ml were mixed with a n equal volume In contrast, S-IgA was susceptibleto cleavage by PE only of PE a t 1 to 500 pg/ml and incubated for 4 to 20 h at 37°C. The at thehighest enzyme/substrate ratio withthe formation digestion was stopped by the addition of EDTA to IO-' M. Control of cleavage fragments less thanM, 21,000 (Fig. 1B. f a n e incubations were performed with the omission of PE. and with PE in the presence of 10 mM EDTA. to show that EDTA satisfactorily 4). inactivated the PE and prevented the enzyme from destroying the The PE digests were also separated using SDS-PAGE of the Ag in the subsequentELISA. For comparison, similar aliquots run under reducing conditions to better delineate cleavIgA antibodieswereincubatedwithlgAlproteasederivedfrom Haernophilus influenzae serotype b (donated by M. Kilian a n d J. age of the polypeptide chains. PE cleaved the H chains of Reinholdt. Royal Dental College. Aarhus.Denmark).Thedigests all the IgA proteins (Fig. 2, l a n e s 4 , 7,10. and 13) as in PBS containing 10 mM EDTA and were then diluted 100-fold noted by a decrease in the staining intensity of the H 0.15%Tween 20. and triplicate100-p1 aliquots were tested byELISA chainband. In addition, the secretorycomponent of in microtlter plates coated with avidin and biotinylated SAT (17). Intact bound IgA antibodies were detected using mouse monoclonal S-IgA was also cleaved by this enzyme (Fig. 2, lane 10). antibody specific for a determinant within the Fc (Cn3 domain) of However. the L chains of the two IgA-K proteins demonhuman IgA (clone 6E2C1: kindly donated by Dakopatts. Glostrup. strated variable susceptibility to proteolysis by PE. For Denmark). followed by peroxidase-conjugated rabbit anti-mouseIgC example, IgA1-KHam protein but not Pet protein (Fig. 2, (Dakopatts) which was absorbed against human lg. diluted 1/1000. as well and applied for 4 h each. To detect bound Fab-like fragments f a n e s 4 and 7. respectively) was extensively degraded as intact IgA. peroxidase-conjugated anti-human K or X L chain. as suggesting that proteolytic susceptibility to PE may reside appropriate. (Dakopatts) diluted 1/1000. was applied for 4 h. Color in the primary structure within the variable region of the was developed with 0.5 mg/ml o-phenylenediamine. 0.1 mM H,O, in 0.1 M citrate-phosphate pH 5. the reaction stopped by the addition L chains. Degradation of the L chain of both S-IgA and nm in a Titertek IgA2 Fel was alsonoted under our incubation conditions of 1 M H,SO,. and absorbance was measured at 492 Multiskan (Flow Laboratories. Inc.. McLean. VA) photometer.
(Fig. 2, l a n e s 10 and 13). Degradation of IgA proteins by PAP. The formation of a few cleavage fragments of the three myeloma proPurified IgA proteins displayed a migration pattern on teins were found only at the highest enzyme/substrate SDS-PAGE run under nonreducing conditions (Fig. 1 , A ratio (Fig. 3. f a n e s 4. 6,and 10)after incubating the IgA and B. f a n e s 3 and 9)which reflected their polymeric proteins withPAP. No further cleavage of the IgA proteins and monomeric forms. Both enzymes, PE and PAP, mi- was observed after optimizing PAP activity (28) by ingrated a s single bands in this electrophoresis systemwith creasing the pHof the reaction mixture to 8.0, adding respective M,of 33.000 (Figs. 1and 2,l a n e 2) and 50,000 CoCl, to a final concentration of 0.01 and 0.001 M, or (Fig. 3. f a n e 2) confirming the homogeneity of each re- prolonging the incubation time to 8 h. No significant RESULTS
9 10 11 1213
1 0 11 12 1 3 1 4
Figure I . SDS-PACE run under nonreducing conditions of 50 & o f IgAl-Pet ( A . lanes 3 to 8).IgAl -Ham ( A . lanes 9 to 1 4 ) .S-IgA (E.lanes 3 to 8). or IgAZ-Fel (B. lanes 9 to 1 4 ) after incubating with varying concentrations of PE at 37°C for 4 h. L a n e 1 . protein M, standards. M, X 1000 shown on the left: lane 2. 10 p g of PE alone: lanes 3 and 9.undigested proteins: lanes 4 to 8 and 10 to 14. proteins digested with 10. 5. 188.8.131.52. and 0.5 p g of PE. IgAl myeloma proteins ((Pet and Ham) in A ) and colostral S-lgA and IgA2 myeloma protein ((Fei)in respectively). In lanes 4 through 8 and 10 through 14 are 50 pg of Pet igAl and 50 pg of Ham IgA 1 . respectively. incubated with 10. 5. 2.5. 1 .O. and 0.5 p g of PE ( A ) .In lanes 4 through 8 and 10 through 14 are 50 @of S-igA and 50 p g of Fel IgA2. respectively. incubated with 10. 5. 2.5.1 .O. and 0.5 p g of PE ( R ) .
200 F i g u r e 2 . SDS-PAGE rununderreduc-
ing conditions of IgA proteins (50 p g ) after incubation with PE (10 or 1.0 pg) at 37°C for 4h. Lane I . proteinm.w.standards: fane 2.10 p g of PE alone. Lanes 3 . 4 and 5. IgAl-Pet digested with 0. 10. 1 p g of PE: fanes 6.7.and 8. IgAl-Ham digested with 0. 10. 1 pg of PE: fanes 9. 10 and 1 1 , S-lgA digested with 0. 10. 1 pgof PE: fanes 12. 13 and 14. IgA2-Fel digested with 0. 10. 1 pg of PE. In fanes 3. 6.9.and 12 are 50p g of IgAlPet and Ham. S-lgA. and lgA2Fel alone. In lanes 4. 7. 10. and 13 are 50pg of Pet. Ham. S-lgA. and Fel with 10 p g of PE. In fanes 5. 8. 1 1 . and 1 4 are 50 p g of Pet. Ham S-IgA. and Fel with 1 p g of PE. SC. secretory component.
2 3 4
F i g u r e 3 . SDS-PAGE run under nonreducing ( A )and reducing ( B ) conditions after incubating different IgA proteins with PAP 200( 10 and pg) 1 at 37°C for 4 h.Lane 1 . protein m.w. standards: fane 2. 10 pg of PAP. A . 94L a n e s 3 a n d 4 .IgAl-Pet digested with 0 and 67- W 10 pg of PAP: fanes 5 and 6. IgAl-Ham digested with 0 and 10 pg of PAP: fanes 7 and 8.S-IgA digested with 0 and 10 pg of PAP: fanes 9 and 10. IgA2-Fel digested with 0 and 10 pg of PAP. B. Lanes 3. 4 . and 5. IgAl-Pet digested with 0. 10. 1 p g ofPAP: fanes 6.7.and 8. IgAl-Ham digested with " . I 210, 10. 1 pg of PAP; lanes 9. 10. and 1 1 . S 14IgA digested with O. 10. 1 pg of PAP: fanes 12. 13. and 1 4 . IgA2-Fel digested with 0. 1 2 3 4 5 6 7 8 9 1 0 10. 1 p g of PAP.
9 10 11 1 2 1 3 1 4
cleavage of the IgA proteins H and L chains or of the SC of S-IgA were seen (Fig. 3 B ) . Deg r a d a tio n o f lgA ant i -SAT ant i bodiby e s PE. When IgA 1 Tin or IgA 1 Fug weredigested with excess PE for 20 h. no remnant of Ag-binding IgA could be detected with either the anti-Fc monoclonal. or the anti-L chain reagents (Table I). Digestion byPE was completely abrogated by these criteria in the presence of EDTA, which
1 0 1 111321 4
showed that anyresidual PE could not interfere with the assay for binding to SAT-coated plates. Treatment of IgA 1 Tin with smaller amountsof PE. or for shorter times, resulted in incomplete digestion, butthe loss of reactivity with both anti-Fc and anti-L chain reagents was similar (Table 11). Thus, although IgAl Tin and IgA1 Fug seemed more resistant to PE than other IgA proteins, intact Fablike fragments were not generated, even a s short-lived
TABLE I Digestion oJIgA anti-SAT by PE. compared with IgAl protease IgA Tln
IRA 1 protease
0.826 - b
lgA Fug Anll-X"
1.133 1.505 1.1 1 3 16 tO.0 k O . 0 12 +0.038 f0.006 1 . 11.183 91 1.586 f0.008+0.007 f0.010 f0.007 1.205 1.191 1.676 f O . 015 f O . 0 12 fO.001 0.694 1.580 0.186 f0.006 fO.001 f0.002 1.296 50.04 +O.O 14 f 0t.00.0070 5
0.730 fO.020 0.7 1 1 f0.027 1
Results are given a s mean absorbance t SE ( n = 3) obtained in ELISA with the stated reagents. Backgrounds (all c0.05) obtained in wells incubated without sampleswere subtracted. bunincubated controls. a
Pseudomonas ELASTASE DEGRADATION OF IgA
TABLE I1 Digestion of IgA I h Ttn by uarious concentrationsof PE for20 h
fragments resulting from cleavage of IgA by Pseudomonas proteases. Thus, this mechanismof evading the Mean OD-Bg 9% Loss host's immune system does not appear to operateP .with PE ( P E W AntiFca Anti-X Fca X aeruginosa. Nevertheless, the resultsof these studies are consistentwiththeconceptthat P . aeruginosa may 250 0 0 100 100 50 1.010 0.358 55 65 evade a major defense mechanism of mucosal surfaces 10 2.328 0.885 0 13 by producing an elastase capable of completely degrading 2.5 2.360 0.907 0 11 0 1.022 2.237 0 0 IgA. This could lead to the persistence of this pathogen on mucosal surfaces (colonization) and the potential for intermediate products.In contrast, treatmentof IgAl Tin tissueinvasion. Although numerousstudieshavereor IgA1 Fug with IgAl protease from H . influenzae re- ported high titers of antibodies toPseudomonas cellular sulted in substantial reduction of reactivity with the anti-and extracellular products in the sera of cystic fibrosis patients (3-6). the progressive pulmonary disease may FC monoclonal reagent, whereas reactivity with anti-L well be related to a localized host defense defect in the chainreagents was fullyretained,demonstratingthe microenvironment that allows persistentPseudomonas generation of Faba fragments (TableI). colonization facilitated by degradation of secretory antibodies that interfere with bacterial adherence. DISCUSSION
The data presented clearly demonstrate that PE posAcknowledgments. The authors thank M. Mays and S. sesses proteolytic activity against both IgA subclasses Reid for typing this manuscript. and S-IgA, supporting the findings of Doring et al. (10) REFERENCES who showed the degradationof one myeloma IgA protein and S-IgA. In addition, we have shown that PAP has little 1 . Bodey, G. P., R. Bolivar, V. Fainstein, and L. Jadeja. 1983. InfecIgA cleaving activity. This latter finding is a variance tions causedby Pseudomonas aeruginosa.Rev. Infect. Dis. 5:279. 2. Young. L. S. 1972. Human immunity to P. aeruginosa 11. Relationwith his previous report (10) thatPAP could extensively ship between heat-stable opsonins andtype-specific Iipopolysacchadegrade a myeloma IgA but not S-IgA. Also, in contrast rides. J . Infect. Dis. 126:277. 3. Doring. G., H. J. Obernesser, K. Botzenhart. B. Flehmig. N. Hoiby. to theirpreviousreport(10).wewereunabletofind and A. Hofmann. 1983. 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P. aeruginosa infections: persisting strated with other enzymesby Lindh (23). Alternately, it problems and current research to find new therapies.Ann. Intern. is possible that S-IgA may contain neutralizing antibodies Med. 82:819. that may inhibit PE, as shown earlier for IgAl proteases 7. Mestecky, J.. and J. McGhee. 1987. Immunoglobulin A (IgA): molecular and cellular interactions involved in IgA biosynthesis and imfrom other bacteria (24). Interestingly, pancreatic elasmune response.Adu. Immunol. 40: 153. tase has been demonstrated to cleave a n IgA myeloma 8. Svanborg Eden, C.. and A.". Svennerholm. 1978. Secretory Improtein but notS-IgA (25). munoglobulin A and C antibodies prevent adhesion of Escherichia coli to human urinary tract epithelialcells. Infect. Irnmun. 22790. Most strains of P . aeruginosa produce PE and PAP that 9. Williams, R. C., and R. J. Gibbons. 1972. Inhibition of bacterial may be important in tissue invasion and necrosis (26adherence by secretory immunoglobulinA: a mechanism of antigen disposal. Science 177597. 28). For example, PE proteolytically inactivates C com10. Doring. G., H. J. Obernesser. and K. Botzenhart. 1981. Extracelluponents and C-derived peptides (29). a-1-proteinase inlar toxins of P. aeruginosa. 11. Effect of two proteases on human hibitor (30).IgG (1 1). airwaylysozyme (31). a n d type 111 immunoglobulins IgC, IgA and secretory IgA. Zentralbl. Bakteriol. Parasitenk. fnfectionskt. Hyg. Abt. I Orig. Reihe A 249:89. and IV collagens (32).In addition, both PE and PAP may 11. Holder, I. A., and R. Wheeler. 1984. Experimental studies of the be involved in basement membrane injury and degradapathogenesis of infections dueto Pseudomonas aeruginosa elastase: a n IgC protease. Can.J. Microbial. 30: I 1 18. tion by degrading laminin (33). M.. and J. Reinholdt. 1986. A hypothetical model for the Proteolytic cleavageof S-IgA by PE a t mucosal surfaces 12. Kilian, development of invasive infection due to IgAl protease-producing would eliminate a major host defense mechanism. Howbacteria. Adu. Exp. Med. Biol. 21 68: 1261. 13. Kilian. M.. J. Mestecky, and M. W. Russell. 1988. Defense mechaever, complete cleavage is not necessary to achieve this nisms involving Fc-dependent functions of immunoglobulin A and effect, as the secondary effector functionsof antibodies their subversionby bacterial immunoglobulin A proteases. Microbiol. depend largely on structures within the Fc region. This Rev. 52:296. is true alsoof S-IgA, one of whose known functionsis to 14. Reinholdt, J.. and M. Kilian. 1987. Interference of IgA protease with the effectof secretory IgA on adherence of oral streptococcito salivainhibit adherence to mucosal surfaces, a property that coated hydroxyapatite. J. Dent. Res. 66:492. Mestecky, J., and M. Kilian. 1985. 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Ebata. 1965. gitis (12, 13).However, in contrast to a previous report Pseudomonas aeruginosa elastase: isolation, crystallization. and preliminary characterization.J . Biol. Chem. 240:3295. (lo),we were unable to find evidence of residual Faba
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