Microbial Pathogenesis 1991 ; 11 : 39-46
Cleavage of chimpanzee secretory immunoglobulin A by Haemophilus influenzae IgAl protease Michael F . Cole and Cynthia A . Hale Department of Microbiology, Georgetown University, School of Medicine, Washington, D .C . 20007, U .S .A .
(Received March 21, 1991 ; accepted March 24, 1991)
Cole, M . F. (Dept of Microbiology, Georgetown University, School of Medicine, Washington, D .C . 20007, U .S .A .) and C . A . Hale . Cleavage of chimpanzee secretory immunoglobulin A by Haemophilus influenzae IgAl protease . Microbial Pathogenesis 1991 ; 11 : 39-46 . Immunoglobulin (Ig)A proteases synthesized by human mucosal pathogens have a unique specificity for human IgA and will not cleave IgA from other species . In contrast, animal pathogens have not reliably been shown to cleave IgA of the animals they infect . This lack of an animal model has prevented an understanding of the importance of IgA1 proteases as virulence factors . One strategy to develop an animal model would be to identify a species capablt- of infection by a human IgA-producing pathogen whose IgA was susceptible to cleavage by IgAl protease of that bacterium . The chimpanzee can be infected with Haemophilus influenzae and is closely related immunologically to man . For these reasons it was sought to determine whether chimpanzee secretory IgA (SIgA) is susceptible to cleavage by IgA1 protease of H . influenzae. This report shows that chimpanzee SIgA can indeed be cleaved at the hinge region by H. influenzae IgA1 protease into Fab, and (Fc,) 2 • SC fragments . The susceptibility of chimpanzee SIgA to IgAl protease of a human pathogen could serve as the basis of an animal model to determine the importance of IgA1 protease in pathogenesis . Key words : SIgA ; IgAl protease; Haemophilus influenzae ; chimpanzee .
Introduction The immunoglobulin (1g)A1 proteases are extracellular constitutive endopeptidases produced by a number of human mucosal pathogens and commensal oral bacteria that cleave the human IgAl subclass at the hinge region into Fab,, and Fc « fragments . IgAl proteases synthesized by human pathogens have extraordinary specificity for human IgA . Although Kornfeld and Plaut' mentioned that IgA from the chimpanzee and gorilla are substrates for IgAl proteases, until recently 2 no experimental data had been provided to support their contention . Interestingly, as yet, no animal pathogens have reliably been shown to produce analogous proteases that cleave IgA of the animals they infect . Therefore, no animal model has so far been described in which the role of IgAl protease in pathogenesis can be studied . A central unresolved question regarding the importance of IgAl protease as a virulence factor of mucosal pathogens concerns the effect of cleavage of serum IgA, and more importantly secretory IgA (SIgA), on antibody activity. Attempts to compare antibody activity before and after cleavage with IgAl protease have been difficult to conduct for two main reasons . 0882-4010/91/070039+08 $03 .00/0
© 1991 Academic Press Limited
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First, it is difficult to obtain human secretory or serum IgA with high naturally-occurring antibody activity of known specificity . Second, no animal model exists in which high titer specific IgA antibody can be induced by active immunization that is susceptible to cleavage by IgA1 protease . In order to study the effect of cleavage of secretory and/or serum IgA on antibody activity, advantage has been taken of pathological monoclonal IgAl paraproteins with known specificity or low titer naturally-occurring SIgA antibodies reactive with oral bacteria .' -6 However, the results of these studies are equivocal . In order to understand the role of IgA1 protease in pathogenesis and its effect on antibody function an animal model is essential . To this end, advantage was taken of close immunological relatedness of man and the great apes . This report describes the cleavage of chimpanzee SIgA by the IgA1 protease of Haemophilus influenzae .
Results Cleavage of the chimpanzee SIgA was detected using methods, conditions and criteria previously established to demonstrate cleavage of human IgA by various IgAl proteaseproducing human pathogenic bacteria . The chimpanzee SIgAl was cleaved into Fab and Fc fragments by the human pathogen H. influenzae ATCC 9006 . Analysis of the digest by immunoelectrophoresis (IEP) using antibodies directed against a heavy chains and K and ) light chains revealed the generation of arcs of non-identity corresponding to Fab, and Fca . An arc with mobility identical to that of the SIgA zero time control remained, representing intact SIgA2 that was resistant to cleavage (Fig . 1) . Aliquots of the reaction mixture removed after increasing incubation times and analyzed by I EP revealed the progressive cleavage of the SIgA1 . Evidence of hydrolysis
Fig . 1 . Immunoelectrophoresis of chimpanzee SIgA after incubation with H . influenzae washed cells for 0 or 72 h . The troughs contained antisera specific for human heavy (a) and light (K and A) chains in combination or alone . Well 1 : intact SIgA (zero incubation time) . Wells 2 and 3 : SlgA after 72 h incubation .
H. inf/uenzae IgAl protease cleaves chimpanzee SIgA
41
Fig . 2 . Immunoelectrophoresis of chimpanzee SIgA after incubation with H. inf/uenzae washed cells for 0, 1 .5, 3, 22 and 72 h . BC (buffer control) indicates H. inf/uenzae incubated with PBS . The top well contained normal human serum (NHS) . Each trough contained antisera specific for human heavy (a) and light (K and A) chains .
of the SIgA1 was detected after as little as 1 .5 h of incubation (Fig . 2) . Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SIDS-PAGE) conducted under reducing conditions resolved the chimpanzee SIgA zero time control into secretory component (SC), a doublet corresponding to the a1 and a2 heavy chains and a single band representing the K and ,. light chains (Fig . 3, lane B) . Analysis of the SIgA by SDS-PAGE, after 72 h of incubation, demonstrated cleavage of the a1 heavy chain and the appearance of Fd, and Fc . heavy chain fragments (Fig . 3, lane C) . The apparent molecular masses of the component chains and fragments of the chimpanzee SIgA, based on six replicate gels, were SC, 88800 Da ; a1 and a2 chains, 60400 Da ; Fc x , 31 400 Da and light chains, 29300 Da . The identity of the component chains and fragments was confirmed by Western blotting using antisera to human SC, a heavy chains and K and A light chains (Fig . 4) . Densitometric analysis of the blot confirmed the presence of the heavy chain doublet and the reduction of only the upper band (a1) following incubation with H. influenzae . That the heavy chain doublet represented al and a2 subclasses in chimpanzee SIgA was confirmed by affinity chromatography of the SIgA using the lectin Jacalin . Jacalin has been employed to separate human IgA1 and IgA2 subclasses based on its ability to bind galactose terminal oligosaccharides on IgA1 that are absent on IgA2 .' Chimpanzee SIgA bound by Jacalin and eluted with galactose yielded heavy chains with a mobility identical to that of the upper band of the SIgA heavy chain doublet . This SIgA fraction was susceptible to cleavage by H. influenzae Rd - IgA1 protease (Fig . 5, lane 3) and was considered to represent SIgA1 . In contrast SIgA unbound by Jacalin was resistant to IgA1 protease from H . inf/uenzae Rd - (Fig . 5, lane 4) and
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215--
M,
14 .4--
Fig . 3 . SDS-PAGE of reduced chimpanzee SIgA incubated for 0 and 72 h with H. influenzae washed cells . Lane A : standards (kDa) . Lane B : intact SIgA (zero incubation time) . Lane C : cleaved SIgA (72 h incubation time) . Lane D : H. influenzae incubated with PBS (buffer control) .
Fig . 4 . Western blot of chimpanzee SIgA before and after cleavage by H. influenzae IgAl protease . The identity of the fragments was confirmed by probing with antibodies specific for SC, heavy (a) and light (K and A) chains in combination (A) ; SC alone (B) ; heavy (a) chains (C) and light (K and A) chains (D) . In each panel lane 1 represents intact SIgA (incubated in PBS) and lane 2 SIgA cleaved at the hinge region (incubated with H. influenzae) . In (A) lane 3 represents the supernatant obtained from centrifuging H. influenzae that had been incubated in PBS (buffer control) .
H. inf/uenzae IgAl protease cleaves chimpanzee SIgA
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21.5o14AO'
Fig . 5 Western blot of chimpanzee SIgA1 and SIgA2 after overnight incubation with H. influenzae Rd (iga') and its IgAl protease deficient isogenic mutant (iga - ) . Blot probed with antibodies specific for a chains . Lane 1 : SlgA1 untreated . Lane 2 : SIgA2 untreated . Lane 3 : SIgA1 incubated with iga' . Lane 4 : SlgA2 incubated with iga' . Lane 5 : SIgA1 incubated with iga - . Lane 6 : SIgA2 incubated with iga - . Lane 7 : iga' incubated with PBS . Note difference in mobility of a1 and a2 heavy chains and cleavage of al heavy chains in lane 3 .
had heavy chains with the same mobility as the lower band of the SIgA doublet and was therefore considered to represent SIgA2 . The failure of the IgAl protease deficient isogenic mutant of H. inf/uenzae Rd - to cleave SIgA1 (Fig . 5, lane 5) provided further evidence that the cleavage of the chimpanzee SIgA was the result of IgAl protease and not other bacterial proteases .
Discussion There are two central questions concerning the IgAl proteases . First, are the IgAl proteases important virulence factors and second, if so, what is (are) the mechanism(s) by which they contribute to pathogenesis? To date the lack of an animal model has made these questions difficult, if not impossible, to answer . The ability of H. influenzae to infect the chimpanzee and the susceptibility of chimpanzee SIgA to cleavage by the IgAl protease of this bacterium should now permit questions concerning the role of IgAl protease as a virulence factor and in pathogenesis to be addressed . The ability of H. inf/uenzae to colonize the upper respiratory tract and cause disease in the chimpanzee was first described in the early 1930s . 8 Since then a wide variety of sub-human primates have been employed as models of H. influenzae infection .' Recently, isogenic mutants of H. influenzae have been constructed that lack IgAl protease . 10 The utilization of such isogenic mutants of non-encapsulated H. influenzae should make it possible to determine whether IgAl protease is a virulence factor without the risk of serious invasive disease in the chimpanzee . Alternatively, the
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importance of IgAl protease in virulence could be determined by actively immunizing chimpanzees with purified IgAl protease prior to challenge with H. influenzae . Studies on the mechanisms by which IgAl proteases may contribute to pathogenesis have thus far been limited to indirect in vitro experiments on the effect of IgAl protease on IgA antibody activity . These studies have employed monoclonal human serum IgAl paraproteins with either rheumatoid activity' or specificities for the bacterial antigens streptolysin 0, x staphylolysin and streptococcal hyaluronidase . 4 However, the microorganisms that elaborate IgAl proteases are mucosal pathogens that encounter secretory IgA which is known to be more resistant to proteolysis than serum IgA as a result of the covalent binding of secretory component ." Unfortunately, naturallyoccurring SIgA antibodies in human external secretions are present at low titers as a result of the large volumes of secretions . Furthermore, exocrine IgA is composed of a relatively greater proportion of the IgA2 subclass than serum IgA . Depending on the external secretion the ratio of SIgA1 to SIgA2 varies from 3 :1 to 1 :1 . 12 For these reasons it has proved difficult to employ naturally-occurring SIgA antibodies to determine how cleavage by IgAl protease affects antibody activity .' The results of such studies concerning the antigen-binding competence of Fab, fragments have been equivocal . Plaut and co-workers' reported the loss of rheumatoid factor and cold agglutinin activities of five human IgA paraproteins after incubation with IgAl protease from Neisseria gonorrhoeae and Streptococcus sanguis. In contrast, Mallet et al.' using naturally-occurring SIgA antibodies, and Mansa and Kilian, 4 using IgAl myeloma proteins, found that Fab, fragments retained their antigen binding capacity . It is clear that high titer SIgA antibodies with known antibody specificity that are susceptible to cleavage by IgA protease from human mucosal pathogens would be a far more suitable substrate for studying the effect of protease on IgA antibody activity . Such SIgA antibodies have been induced in this laboratory by peroral immunization'of pregnant chimpanzees and partitioned into Al and A2 subclasses by Jacalin affinity chromatography . The susceptibility of chimpanzee SIgA to cleavage by the IgAl protease of the human pathogen H. influenzae clearly reflects close homology between the aminoacid sequence and tertiary structure of the hinge region of the human and chimpanzee IgAl heavy chains . The primary structure of chimpanzee al heavy chains has yet to be reported, but it should now be possible to obtain a consensus sequence of the hinge region of the cleaved al chains . In summary, the finding that chimpanzee SIgAl is susceptible to cleavage by the IgA1 protease of a human mucosal pathogenic bacterium opens up the possibility of directly studying the role of IgAl proteases as virulence factors and in the pathogenesis of disease . Experiments directed to these ends are currently in progress .
Materials and methods
Animals. Prepartum mammary secretions were obtained by manual expression from a multiparous chimpanzee (Ch .334, Lulu), maintained at the New York University Medical Center's Laboratory for Experimental Medicine and Surgery in Primates (LEMSIP) . Husbandry and breeding procedures in this colony have been previously described . 13
Purification of S/gA from chimpanzee breast mi/k . Chimpanzee breast milk was diluted with an equal volume of saline and centrifuged for 2 h at 16300xg at 4°C . The liquid above the sedimented milk solids and below the fat was removed and adjusted to pH 4 .2 by the dropwise addition of 2% acetic acid with stirring, to precipitate casein . The casein was packed by centrifugation for 1 h 16300xg at 4°C . The supernatant whey was removed and neutralized with 1 N NaOH .
H . influenzae IgAl protease cleaves chimpanzee SIgA
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SIgA was purified from the whey by high-performance liquid chromatography (HPLC) using a Waters system (Millipore, Milford, Massachusetts) equipped with a preparative Bio-Sil TSK 250 600x21 .5 mm gel filtration column coupled with a Bio-Sil TSK 75x21 .5 mm guard column (Bio-Rad, Richmond, California) . The mobile phase was 200 men sodium phosphate buffer, pH 6 .8 and the flow rate 2 .25 ml/min . Eluted proteins were detected by absorbance at 280 nm . The purity of the SIgA preparation was determined by SDS-PAGE as described below . The concentration of the SIgA preparation was determined by absorbance at 280 nm using an extinction coefficient E11%"' of 13 .8 . Separation of SlgA1 and SlgA2 subclasses . The purified SIgA was separated into the SIgA1 and SIgA2 subclasses by affinity chromatography using the lectin Jacalin . s Bacteria . H. influenzae ATCC 9006, serotype a, and a serotype d strain, Rd - and its IgAl protease deficient (iga - ) isogenic mutant were used in this study . the iga' parent and the isogenic iga - mutant were provided by Dr Andrew Wright, Tufts University, Boston, Massachusetts . The microorganisms were cultivated on chocolate agar plates for 24 h at 37 ° C in a candle jar . Cleavage of chimpanzee S/gA . Cleavage of chimpanzee SIgA was accomplished using a method similar to that described by Frandsen et al." Confluent growth of H. influenzae ATCC 9006 was removed from the surface of chocolate agar plates using a sterile cotton swab moistened with sterile 0 .067 M phosphate buffered saline (PBS), pH 7 .4 . The bacteria were suspended in 1 .5 ml of PBS at an optical density of 1 .0 at 660 nm and sedimented by centrifugation for 3 min at 7300xg . The packed bacteria were resuspended in 3 .0 ml of SIgA at a concentration of 1 .8 mg/ml . Immediately, 250 yl of the SIgA-bacteria suspension were removed, centrifuged for 5 min to remove the bacteria and the supernatant frozen at -20°C . This sample served as the zero time control . The remaining suspension was incubated at 37°C . Additional 250 µl aliquots of bacterial suspension were removed after 5, 10, 15, 30 and 45 min, and 1, 1 .5, 2, 3, 22 and 72 h . As before, each aliquot was centrifuged to remove the bacteria and the supernatant frozen at -20°C . In order to control for the presence of bacterial components released into the supernatants during incubation, H. inf/uenzae ATCC 9006 was incubated for 72 h with PBS alone (buffer control) . Cleavage of chimpanzee S/gA1 and S/gA2 . Cleavage of the SIgA subclasses was performed essentially as described above except that each subclass was incubated overnight with H. influenzae Rd - and its IgAl deficient isogenic mutant . Detection of cleavage products of chimpanzee SIgA . The chimpanzee SIgA digested by H. influenzae ATCC 9006 was examined by IEP, SDS-PAGE and Western blotting . The SIgA subclasses incubated with H . influenzae Rd - and its IgAl protease deficient isogenic mutant were examined by Western blotting . Immunoelectrophoresis of the digest was performed in 2% agarose in 0 .04 M barbital buffer, pH 8 .2 . The IEP patterns were developed using either a mixture of the IgG fraction of rabbit anti-human a heavy chains and x and A light chains (Dako, Santa Barbara, California) in the trough or anti-x and anti-light chain antibodies placed in separate troughs to permit positive identification of Fab, and Fc, fragments . Washed and dried IEP plates were stained with Coomassie Brilliant Blue R-250 (Bio-Rad) . SDS-PAGE was carried out using a Mini Protean II system (Bio-Rad) with a 10% separation gel and a 5% stacking gel . The digests were reduced by boiling for 10 min in 2% SDS, 20% sucrose, 2 mm ethylenediaminetetraacetic acid (EDTA), 80 mm dithiothreitol (DTT), 20 mm Tris, pH 8 .9 . The gels were stained with Coomassie Brilliant Blue R-250 . The apparent molecular weights of the SIgA fragments were determined by reference to molecular weight standards (Bio-Rad), using the method of Weber and Osborn . 15 In order to confirm the identity of the bands generated after reduction of the cleaved chimpanzee SIgA, Western blotting with specific antisera to component human SIgA chains (SC, 2, x and ).) was performed with a duplicate gel . Western blotting was performed using a Mini trans-Blot electrophoretic transfer cell (Bio-Rad) according to the manufacturer's instructions .
The authors thank Dr C . James Mahoney, Laboratory for Experimental Medicine and Surgery in Primates, New York University Medical Center, for providing chimpanzee breast milk necessary
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for this study and for helpful discussions and Dr Andrew Wright, Department of Molecular Biology and Microbiology, Tufts University, School of Medicine, for providing the parent and IgAl deficient isogenic strain of H. influenzae.
References 1 . Kornfeld SJ, Plaut AG . Secretory immunity and the bacterial IgA proteases . Rev Infect Dis 1981 ; 3 : 521-34 . 2 . Proctor M, Manning PJ . Production of immunoglobulin A protease by Streptococcus pneumoniae from animals . Infect Immun 1990 ; 58: 2733-7 . 3 . Mallet CP, Boylan RJ, Everhart DL . Competent antigen-binding fragments (Fab) from secretory immunoglobulin A using Streptococcus sanguis immunoglobulin A protease . Caries Res 1984 ; 18 : 201-8 . 4 . Mansa B, Kilian M . Retained antigen-binding activity of Fab fragments of human monoclonal immunoglobulin Al (IgAl) cleaved by IgAl protease . Infect Immun 1986 ; 52 :171-4 . 5 . Plaut AG, Gilbert JV, Wistar R, Jr. Loss of antibody activity in human immunoglobulin A exposed to extracellular immunoglobulin A proteases of Neisseria gonorrhoeae and Streptococcus sanguis . Infect Immun 1977 ; 17 :130-5. 6 . Reinholdt J, Kilian M . Interference of IgA protease with the effect of secretory IgA on adherence of oral streptococci to saliva-coated hydroxyapatite . J Dent Res 1987 ; 66: 492-7 . 7 . Kondoh H, Kobayashi K, Hagiwara K . A simple procedure for the isolation of human secretory IgA of IgAl and IgA2 subclass by a jack fruit lectin, jacalin, affinity chromatography . Mol Immunol 1987 ; 24 : 1219-22 . 8 . Doches AR, Mills KC, Knerland Y . Variation of H. influenzae during acute respiratory infection in the chimpanzee . Proc Soc Exp Biol NY 1932 ; 30 : 314-16 . 9 . McClure HM . Bacterial diseases of nonhuman primates . In : Montali RJ, Migaki G, eds . The comparative pathology of zoo animals . Washington : Smithsonian Press 1980; 197-218 . 10 . Bricker J, Mulks M, Moxon ER, Plaut AG, Wright A . Physical and genetic analysis of DNA regions encoding the immunoglobulin A proteases of different specificities produced by Haemophilus influenzae . Infect Immun 1985 ; 47 : 370-4 . 11 . Lindh E . Increased resistance of immunoglobulin A dimers to proteolytic degradation after binding of secretory component . J Immunol 1975; 114 : 284-6 . 12 . Delacroix DL, Dive C, Rambaud JC, Vaerman JP . IgA subclasses in various secretions and in serum . Immunology 1982; 47 : 383-5 . 13 . Mahoney CJ . A monogamous system of breeding chimpanzees in a controlled environment . 16th TABS Congress : The standardization of animals to improve biomedical research, production and control, San Antonio 1979 . Develop Biol Standard 1980; 45 : 73-82 . 14 . Frandsen EVG, Reinholdt J, Kilian M . Enzymatic and antigenic characterization of immunoglobulin Al proteases of Bacteroides and Capnocytophagia spp . Infect Immun 1987 ; 55 : 631-8 . 15 . Weber K, Osborn M . The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 1969 ; 244 : 4406-12 .
Cleavage of chimpanzee secretory immunoglobulin A by Haemophilus influenzae IgAl protease Michael F . Cole and Cynthia A . Hale Department of Microbiology, Georgetown University, School of Medicine, Washington, D .C . 20007, U .S .A .
(Received March 21, 1991 ; accepted March 24, 1991)
Cole, M . F. (Dept of Microbiology, Georgetown University, School of Medicine, Washington, D .C . 20007, U .S .A .) and C . A . Hale . Cleavage of chimpanzee secretory immunoglobulin A by Haemophilus influenzae IgAl protease . Microbial Pathogenesis 1991 ; 11 : 39-46 . Immunoglobulin (Ig)A proteases synthesized by human mucosal pathogens have a unique specificity for human IgA and will not cleave IgA from other species . In contrast, animal pathogens have not reliably been shown to cleave IgA of the animals they infect . This lack of an animal model has prevented an understanding of the importance of IgA1 proteases as virulence factors . One strategy to develop an animal model would be to identify a species capablt- of infection by a human IgA-producing pathogen whose IgA was susceptible to cleavage by IgAl protease of that bacterium . The chimpanzee can be infected with Haemophilus influenzae and is closely related immunologically to man . For these reasons it was sought to determine whether chimpanzee secretory IgA (SIgA) is susceptible to cleavage by IgA1 protease of H . influenzae. This report shows that chimpanzee SIgA can indeed be cleaved at the hinge region by H. influenzae IgA1 protease into Fab, and (Fc,) 2 • SC fragments . The susceptibility of chimpanzee SIgA to IgAl protease of a human pathogen could serve as the basis of an animal model to determine the importance of IgA1 protease in pathogenesis . Key words : SIgA ; IgAl protease; Haemophilus influenzae ; chimpanzee .
Introduction The immunoglobulin (1g)A1 proteases are extracellular constitutive endopeptidases produced by a number of human mucosal pathogens and commensal oral bacteria that cleave the human IgAl subclass at the hinge region into Fab,, and Fc « fragments . IgAl proteases synthesized by human pathogens have extraordinary specificity for human IgA . Although Kornfeld and Plaut' mentioned that IgA from the chimpanzee and gorilla are substrates for IgAl proteases, until recently 2 no experimental data had been provided to support their contention . Interestingly, as yet, no animal pathogens have reliably been shown to produce analogous proteases that cleave IgA of the animals they infect . Therefore, no animal model has so far been described in which the role of IgAl protease in pathogenesis can be studied . A central unresolved question regarding the importance of IgAl protease as a virulence factor of mucosal pathogens concerns the effect of cleavage of serum IgA, and more importantly secretory IgA (SIgA), on antibody activity. Attempts to compare antibody activity before and after cleavage with IgAl protease have been difficult to conduct for two main reasons . 0882-4010/91/070039+08 $03 .00/0
© 1991 Academic Press Limited
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M . F . Cole and C . A . Hale
First, it is difficult to obtain human secretory or serum IgA with high naturally-occurring antibody activity of known specificity . Second, no animal model exists in which high titer specific IgA antibody can be induced by active immunization that is susceptible to cleavage by IgA1 protease . In order to study the effect of cleavage of secretory and/or serum IgA on antibody activity, advantage has been taken of pathological monoclonal IgAl paraproteins with known specificity or low titer naturally-occurring SIgA antibodies reactive with oral bacteria .' -6 However, the results of these studies are equivocal . In order to understand the role of IgA1 protease in pathogenesis and its effect on antibody function an animal model is essential . To this end, advantage was taken of close immunological relatedness of man and the great apes . This report describes the cleavage of chimpanzee SIgA by the IgA1 protease of Haemophilus influenzae .
Results Cleavage of the chimpanzee SIgA was detected using methods, conditions and criteria previously established to demonstrate cleavage of human IgA by various IgAl proteaseproducing human pathogenic bacteria . The chimpanzee SIgAl was cleaved into Fab and Fc fragments by the human pathogen H. influenzae ATCC 9006 . Analysis of the digest by immunoelectrophoresis (IEP) using antibodies directed against a heavy chains and K and ) light chains revealed the generation of arcs of non-identity corresponding to Fab, and Fca . An arc with mobility identical to that of the SIgA zero time control remained, representing intact SIgA2 that was resistant to cleavage (Fig . 1) . Aliquots of the reaction mixture removed after increasing incubation times and analyzed by I EP revealed the progressive cleavage of the SIgA1 . Evidence of hydrolysis
Fig . 1 . Immunoelectrophoresis of chimpanzee SIgA after incubation with H . influenzae washed cells for 0 or 72 h . The troughs contained antisera specific for human heavy (a) and light (K and A) chains in combination or alone . Well 1 : intact SIgA (zero incubation time) . Wells 2 and 3 : SlgA after 72 h incubation .
H. inf/uenzae IgAl protease cleaves chimpanzee SIgA
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Fig . 2 . Immunoelectrophoresis of chimpanzee SIgA after incubation with H. inf/uenzae washed cells for 0, 1 .5, 3, 22 and 72 h . BC (buffer control) indicates H. inf/uenzae incubated with PBS . The top well contained normal human serum (NHS) . Each trough contained antisera specific for human heavy (a) and light (K and A) chains .
of the SIgA1 was detected after as little as 1 .5 h of incubation (Fig . 2) . Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SIDS-PAGE) conducted under reducing conditions resolved the chimpanzee SIgA zero time control into secretory component (SC), a doublet corresponding to the a1 and a2 heavy chains and a single band representing the K and ,. light chains (Fig . 3, lane B) . Analysis of the SIgA by SDS-PAGE, after 72 h of incubation, demonstrated cleavage of the a1 heavy chain and the appearance of Fd, and Fc . heavy chain fragments (Fig . 3, lane C) . The apparent molecular masses of the component chains and fragments of the chimpanzee SIgA, based on six replicate gels, were SC, 88800 Da ; a1 and a2 chains, 60400 Da ; Fc x , 31 400 Da and light chains, 29300 Da . The identity of the component chains and fragments was confirmed by Western blotting using antisera to human SC, a heavy chains and K and A light chains (Fig . 4) . Densitometric analysis of the blot confirmed the presence of the heavy chain doublet and the reduction of only the upper band (a1) following incubation with H. influenzae . That the heavy chain doublet represented al and a2 subclasses in chimpanzee SIgA was confirmed by affinity chromatography of the SIgA using the lectin Jacalin . Jacalin has been employed to separate human IgA1 and IgA2 subclasses based on its ability to bind galactose terminal oligosaccharides on IgA1 that are absent on IgA2 .' Chimpanzee SIgA bound by Jacalin and eluted with galactose yielded heavy chains with a mobility identical to that of the upper band of the SIgA heavy chain doublet . This SIgA fraction was susceptible to cleavage by H. influenzae Rd - IgA1 protease (Fig . 5, lane 3) and was considered to represent SIgA1 . In contrast SIgA unbound by Jacalin was resistant to IgA1 protease from H . inf/uenzae Rd - (Fig . 5, lane 4) and
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215--
M,
14 .4--
Fig . 3 . SDS-PAGE of reduced chimpanzee SIgA incubated for 0 and 72 h with H. influenzae washed cells . Lane A : standards (kDa) . Lane B : intact SIgA (zero incubation time) . Lane C : cleaved SIgA (72 h incubation time) . Lane D : H. influenzae incubated with PBS (buffer control) .
Fig . 4 . Western blot of chimpanzee SIgA before and after cleavage by H. influenzae IgAl protease . The identity of the fragments was confirmed by probing with antibodies specific for SC, heavy (a) and light (K and A) chains in combination (A) ; SC alone (B) ; heavy (a) chains (C) and light (K and A) chains (D) . In each panel lane 1 represents intact SIgA (incubated in PBS) and lane 2 SIgA cleaved at the hinge region (incubated with H. influenzae) . In (A) lane 3 represents the supernatant obtained from centrifuging H. influenzae that had been incubated in PBS (buffer control) .
H. inf/uenzae IgAl protease cleaves chimpanzee SIgA
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Fig . 5 Western blot of chimpanzee SIgA1 and SIgA2 after overnight incubation with H. influenzae Rd (iga') and its IgAl protease deficient isogenic mutant (iga - ) . Blot probed with antibodies specific for a chains . Lane 1 : SlgA1 untreated . Lane 2 : SIgA2 untreated . Lane 3 : SIgA1 incubated with iga' . Lane 4 : SlgA2 incubated with iga' . Lane 5 : SIgA1 incubated with iga - . Lane 6 : SIgA2 incubated with iga - . Lane 7 : iga' incubated with PBS . Note difference in mobility of a1 and a2 heavy chains and cleavage of al heavy chains in lane 3 .
had heavy chains with the same mobility as the lower band of the SIgA doublet and was therefore considered to represent SIgA2 . The failure of the IgAl protease deficient isogenic mutant of H. inf/uenzae Rd - to cleave SIgA1 (Fig . 5, lane 5) provided further evidence that the cleavage of the chimpanzee SIgA was the result of IgAl protease and not other bacterial proteases .
Discussion There are two central questions concerning the IgAl proteases . First, are the IgAl proteases important virulence factors and second, if so, what is (are) the mechanism(s) by which they contribute to pathogenesis? To date the lack of an animal model has made these questions difficult, if not impossible, to answer . The ability of H. influenzae to infect the chimpanzee and the susceptibility of chimpanzee SIgA to cleavage by the IgAl protease of this bacterium should now permit questions concerning the role of IgAl protease as a virulence factor and in pathogenesis to be addressed . The ability of H. inf/uenzae to colonize the upper respiratory tract and cause disease in the chimpanzee was first described in the early 1930s . 8 Since then a wide variety of sub-human primates have been employed as models of H. influenzae infection .' Recently, isogenic mutants of H. influenzae have been constructed that lack IgAl protease . 10 The utilization of such isogenic mutants of non-encapsulated H. influenzae should make it possible to determine whether IgAl protease is a virulence factor without the risk of serious invasive disease in the chimpanzee . Alternatively, the
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importance of IgAl protease in virulence could be determined by actively immunizing chimpanzees with purified IgAl protease prior to challenge with H. influenzae . Studies on the mechanisms by which IgAl proteases may contribute to pathogenesis have thus far been limited to indirect in vitro experiments on the effect of IgAl protease on IgA antibody activity . These studies have employed monoclonal human serum IgAl paraproteins with either rheumatoid activity' or specificities for the bacterial antigens streptolysin 0, x staphylolysin and streptococcal hyaluronidase . 4 However, the microorganisms that elaborate IgAl proteases are mucosal pathogens that encounter secretory IgA which is known to be more resistant to proteolysis than serum IgA as a result of the covalent binding of secretory component ." Unfortunately, naturallyoccurring SIgA antibodies in human external secretions are present at low titers as a result of the large volumes of secretions . Furthermore, exocrine IgA is composed of a relatively greater proportion of the IgA2 subclass than serum IgA . Depending on the external secretion the ratio of SIgA1 to SIgA2 varies from 3 :1 to 1 :1 . 12 For these reasons it has proved difficult to employ naturally-occurring SIgA antibodies to determine how cleavage by IgAl protease affects antibody activity .' The results of such studies concerning the antigen-binding competence of Fab, fragments have been equivocal . Plaut and co-workers' reported the loss of rheumatoid factor and cold agglutinin activities of five human IgA paraproteins after incubation with IgAl protease from Neisseria gonorrhoeae and Streptococcus sanguis. In contrast, Mallet et al.' using naturally-occurring SIgA antibodies, and Mansa and Kilian, 4 using IgAl myeloma proteins, found that Fab, fragments retained their antigen binding capacity . It is clear that high titer SIgA antibodies with known antibody specificity that are susceptible to cleavage by IgA protease from human mucosal pathogens would be a far more suitable substrate for studying the effect of protease on IgA antibody activity . Such SIgA antibodies have been induced in this laboratory by peroral immunization'of pregnant chimpanzees and partitioned into Al and A2 subclasses by Jacalin affinity chromatography . The susceptibility of chimpanzee SIgA to cleavage by the IgAl protease of the human pathogen H. influenzae clearly reflects close homology between the aminoacid sequence and tertiary structure of the hinge region of the human and chimpanzee IgAl heavy chains . The primary structure of chimpanzee al heavy chains has yet to be reported, but it should now be possible to obtain a consensus sequence of the hinge region of the cleaved al chains . In summary, the finding that chimpanzee SIgAl is susceptible to cleavage by the IgA1 protease of a human mucosal pathogenic bacterium opens up the possibility of directly studying the role of IgAl proteases as virulence factors and in the pathogenesis of disease . Experiments directed to these ends are currently in progress .
Materials and methods
Animals. Prepartum mammary secretions were obtained by manual expression from a multiparous chimpanzee (Ch .334, Lulu), maintained at the New York University Medical Center's Laboratory for Experimental Medicine and Surgery in Primates (LEMSIP) . Husbandry and breeding procedures in this colony have been previously described . 13
Purification of S/gA from chimpanzee breast mi/k . Chimpanzee breast milk was diluted with an equal volume of saline and centrifuged for 2 h at 16300xg at 4°C . The liquid above the sedimented milk solids and below the fat was removed and adjusted to pH 4 .2 by the dropwise addition of 2% acetic acid with stirring, to precipitate casein . The casein was packed by centrifugation for 1 h 16300xg at 4°C . The supernatant whey was removed and neutralized with 1 N NaOH .
H . influenzae IgAl protease cleaves chimpanzee SIgA
45
SIgA was purified from the whey by high-performance liquid chromatography (HPLC) using a Waters system (Millipore, Milford, Massachusetts) equipped with a preparative Bio-Sil TSK 250 600x21 .5 mm gel filtration column coupled with a Bio-Sil TSK 75x21 .5 mm guard column (Bio-Rad, Richmond, California) . The mobile phase was 200 men sodium phosphate buffer, pH 6 .8 and the flow rate 2 .25 ml/min . Eluted proteins were detected by absorbance at 280 nm . The purity of the SIgA preparation was determined by SDS-PAGE as described below . The concentration of the SIgA preparation was determined by absorbance at 280 nm using an extinction coefficient E11%"' of 13 .8 . Separation of SlgA1 and SlgA2 subclasses . The purified SIgA was separated into the SIgA1 and SIgA2 subclasses by affinity chromatography using the lectin Jacalin . s Bacteria . H. influenzae ATCC 9006, serotype a, and a serotype d strain, Rd - and its IgAl protease deficient (iga - ) isogenic mutant were used in this study . the iga' parent and the isogenic iga - mutant were provided by Dr Andrew Wright, Tufts University, Boston, Massachusetts . The microorganisms were cultivated on chocolate agar plates for 24 h at 37 ° C in a candle jar . Cleavage of chimpanzee S/gA . Cleavage of chimpanzee SIgA was accomplished using a method similar to that described by Frandsen et al." Confluent growth of H. influenzae ATCC 9006 was removed from the surface of chocolate agar plates using a sterile cotton swab moistened with sterile 0 .067 M phosphate buffered saline (PBS), pH 7 .4 . The bacteria were suspended in 1 .5 ml of PBS at an optical density of 1 .0 at 660 nm and sedimented by centrifugation for 3 min at 7300xg . The packed bacteria were resuspended in 3 .0 ml of SIgA at a concentration of 1 .8 mg/ml . Immediately, 250 yl of the SIgA-bacteria suspension were removed, centrifuged for 5 min to remove the bacteria and the supernatant frozen at -20°C . This sample served as the zero time control . The remaining suspension was incubated at 37°C . Additional 250 µl aliquots of bacterial suspension were removed after 5, 10, 15, 30 and 45 min, and 1, 1 .5, 2, 3, 22 and 72 h . As before, each aliquot was centrifuged to remove the bacteria and the supernatant frozen at -20°C . In order to control for the presence of bacterial components released into the supernatants during incubation, H. inf/uenzae ATCC 9006 was incubated for 72 h with PBS alone (buffer control) . Cleavage of chimpanzee S/gA1 and S/gA2 . Cleavage of the SIgA subclasses was performed essentially as described above except that each subclass was incubated overnight with H. influenzae Rd - and its IgAl deficient isogenic mutant . Detection of cleavage products of chimpanzee SIgA . The chimpanzee SIgA digested by H. influenzae ATCC 9006 was examined by IEP, SDS-PAGE and Western blotting . The SIgA subclasses incubated with H . influenzae Rd - and its IgAl protease deficient isogenic mutant were examined by Western blotting . Immunoelectrophoresis of the digest was performed in 2% agarose in 0 .04 M barbital buffer, pH 8 .2 . The IEP patterns were developed using either a mixture of the IgG fraction of rabbit anti-human a heavy chains and x and A light chains (Dako, Santa Barbara, California) in the trough or anti-x and anti-light chain antibodies placed in separate troughs to permit positive identification of Fab, and Fc, fragments . Washed and dried IEP plates were stained with Coomassie Brilliant Blue R-250 (Bio-Rad) . SDS-PAGE was carried out using a Mini Protean II system (Bio-Rad) with a 10% separation gel and a 5% stacking gel . The digests were reduced by boiling for 10 min in 2% SDS, 20% sucrose, 2 mm ethylenediaminetetraacetic acid (EDTA), 80 mm dithiothreitol (DTT), 20 mm Tris, pH 8 .9 . The gels were stained with Coomassie Brilliant Blue R-250 . The apparent molecular weights of the SIgA fragments were determined by reference to molecular weight standards (Bio-Rad), using the method of Weber and Osborn . 15 In order to confirm the identity of the bands generated after reduction of the cleaved chimpanzee SIgA, Western blotting with specific antisera to component human SIgA chains (SC, 2, x and ).) was performed with a duplicate gel . Western blotting was performed using a Mini trans-Blot electrophoretic transfer cell (Bio-Rad) according to the manufacturer's instructions .
The authors thank Dr C . James Mahoney, Laboratory for Experimental Medicine and Surgery in Primates, New York University Medical Center, for providing chimpanzee breast milk necessary
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M . F . Cole and C . A . Hale
for this study and for helpful discussions and Dr Andrew Wright, Department of Molecular Biology and Microbiology, Tufts University, School of Medicine, for providing the parent and IgAl deficient isogenic strain of H. influenzae.
References 1 . Kornfeld SJ, Plaut AG . Secretory immunity and the bacterial IgA proteases . Rev Infect Dis 1981 ; 3 : 521-34 . 2 . Proctor M, Manning PJ . Production of immunoglobulin A protease by Streptococcus pneumoniae from animals . Infect Immun 1990 ; 58: 2733-7 . 3 . Mallet CP, Boylan RJ, Everhart DL . Competent antigen-binding fragments (Fab) from secretory immunoglobulin A using Streptococcus sanguis immunoglobulin A protease . Caries Res 1984 ; 18 : 201-8 . 4 . Mansa B, Kilian M . Retained antigen-binding activity of Fab fragments of human monoclonal immunoglobulin Al (IgAl) cleaved by IgAl protease . Infect Immun 1986 ; 52 :171-4 . 5 . Plaut AG, Gilbert JV, Wistar R, Jr. Loss of antibody activity in human immunoglobulin A exposed to extracellular immunoglobulin A proteases of Neisseria gonorrhoeae and Streptococcus sanguis . Infect Immun 1977 ; 17 :130-5. 6 . Reinholdt J, Kilian M . Interference of IgA protease with the effect of secretory IgA on adherence of oral streptococci to saliva-coated hydroxyapatite . J Dent Res 1987 ; 66: 492-7 . 7 . Kondoh H, Kobayashi K, Hagiwara K . A simple procedure for the isolation of human secretory IgA of IgAl and IgA2 subclass by a jack fruit lectin, jacalin, affinity chromatography . Mol Immunol 1987 ; 24 : 1219-22 . 8 . Doches AR, Mills KC, Knerland Y . Variation of H. influenzae during acute respiratory infection in the chimpanzee . Proc Soc Exp Biol NY 1932 ; 30 : 314-16 . 9 . McClure HM . Bacterial diseases of nonhuman primates . In : Montali RJ, Migaki G, eds . The comparative pathology of zoo animals . Washington : Smithsonian Press 1980; 197-218 . 10 . Bricker J, Mulks M, Moxon ER, Plaut AG, Wright A . Physical and genetic analysis of DNA regions encoding the immunoglobulin A proteases of different specificities produced by Haemophilus influenzae . Infect Immun 1985 ; 47 : 370-4 . 11 . Lindh E . Increased resistance of immunoglobulin A dimers to proteolytic degradation after binding of secretory component . J Immunol 1975; 114 : 284-6 . 12 . Delacroix DL, Dive C, Rambaud JC, Vaerman JP . IgA subclasses in various secretions and in serum . Immunology 1982; 47 : 383-5 . 13 . Mahoney CJ . A monogamous system of breeding chimpanzees in a controlled environment . 16th TABS Congress : The standardization of animals to improve biomedical research, production and control, San Antonio 1979 . Develop Biol Standard 1980; 45 : 73-82 . 14 . Frandsen EVG, Reinholdt J, Kilian M . Enzymatic and antigenic characterization of immunoglobulin Al proteases of Bacteroides and Capnocytophagia spp . Infect Immun 1987 ; 55 : 631-8 . 15 . Weber K, Osborn M . The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 1969 ; 244 : 4406-12 .
