INFEcrION AND IMMUNITY, Sept. 1992, p. 3652-36570019-9567/92/093652-06$02.00/0Copyright © 1992, American Society for Microbiology

Identification of a P, Integrin on Mycobacteriumavium-Mycobacterium intracellulare

SAVITA P. RAO,' KURT R. GEHLSEN,2 AND ANTONINO CATANZARO1*Department ofMedicine, Pulmonary and Critical Care Division, University of California,

San Diego, San Diego, California 92103-8374,1 and California Institute ofBiological Research, La Jolla, California 920372

Received 30 January 1992/Accepted 15 June 1992

Mycobacterium avium-Mycobacterium intracellulare (MAI) is an opportunistic intracellular pathogen respon-sible for the highest incidence of disseminated bacterial infection in patients with AIDS. Treatment of theinfection is extremely difficult and has shown limited efficacy. A critical event in the initiation of a variety ofbacterial infections involves the adherence of bacteria to host cell surfaces. In the present study, we have shownthat MAI organisms bind avidly to extracellular matrix proteins such as laminin, collagen I, and fibronectin inan in vitro attachment assay. Immunoblot analysis of a sonicate of MAI with polyclonal antibodies againstdifferent integrin receptors indicated that the sonicate cross-reacts with polyclonal antibodies against a humanlaminin-binding integrin, c3,31, and a human fibronectin-binding integrin, a0l although it is reactive withonly the j13 subunit in the case of both antisera. Antibodies against the a3I1 and a5I3 integrins specificallyinhibited the binding of MAI to laminin, collagen I, and fibronectin by 70 to 97%, depending on the ligand,suggesting that the attachment ofMAI to these extracellular matrix proteins may be mediated by a 13, integrin.Furthermore, the attachment ofMAI to laminin, collagen I, and fibronectin was found to be cation dependent.MAI may use this and other 131-containing integrins to adhere and penetrate through basement membranestructures that underlie host cell linings. An understanding of the mechanism of attachment and a definition ofthe adhesive molecules on the surface of MAI may open up new approaches to the prevention of seriousinfection caused by this organism.

Mycobacterium avium-Mycobacterium intracellulare(MAI) is of renewed interest today because of the promi-nence of disseminated infection caused by this organism inpatients with AIDS (22, 39). In normal or nonimmunocom-promised hosts, disease due to this organism is rare. How-ever, when it does occur, the clinical manifestations areprimarily pulmonary (23). Treatment of MAI infection isdifficult in patients with normal immune responses and evenmore difficult in patients with immunosuppression inducedby human immunodeficiency virus (2). However, recentclinical trials with amikacin-containing regimens (10) andregimens containing macrolides (12) offer new hope. MAI iscommonly found in fresh water, soil, and air (3). The actualsource of an infection caused by MAI is usually not known,although it is thought to be acquired by ingestion or inhala-tion because of its ubiquitous distribution in the environment(11). Relatively little is known regarding the mode of attach-ment of MAI to cell surfaces, which is apparently the mostcritical event in the initiation of a bacterial infection, onceMAI has gained entry into the host.

Bacterial adherence has grown into one of the most active,if not the most exciting, areas of study in the field ofinfectious diseases. A variety of adhesive structures,broadly referred to as adhesins, have been described on thesurfaces of microorganisms, which in turn are thought tobind the microorganism to complementary adhesive struc-tures on the surfaces of host cells (4, 5). Some pathogenicorganisms have also developed proteinaceous surface struc-tures, such as the fimbriae (pili) of Escherichia coli (6). Suchstructures play a role in the interaction of the organism witheucaryotic host cells. Recent studies have shown that sev-

* Corresponding author.

eral pathogenic bacteria, including Staphylococcus aureus,Streptococcus pyogenes, and E. coli, bind to components ofthe basement membrane such as laminin (Lm) and collagen(Col) (21, 29, 35, 36, 38). In addition, S. aureus and E. colibind to many of the adhesive glycoproteins present in bloodand in the extracellular matrix such as fibronectin (Fn) (15,26, 27), vitronectin (Vn) (9), and fibrinogen (27). Treponemadenticola, a spirochete responsible for periodontal diseasesin humans, has been shown to bind to Fn and Lm. Thisbinding in turn has been implicated in the pathogenicity ofthe organism (13). From these studies, it appears thatadhesion of pathogens to host cell surfaces or basementmembrane structures may serve as an essential first step inthe pathogenesis of the diseases caused by these organisms.Consequently, the presence of different kinds of adhesivemolecules on microorganisms for complementary structuresin the host is widely encountered.The present investigation was undertaken to study the

early events that take place during the establishment ofinfection due to MAI. Several strains of Mycobacteriumbovis BCG, three strains of Mycobactenum tuberculosis,and four other mycobacterial species, including M. avium,have been shown to bind Fn through a trypsin-sensitive cellsurface component(s) (32). However, the binding mole-cule(s) involved was not identified. We have shown thatMAI binds to extracellular matrix proteins, such as Lm,collagen I (Col.T), and Fn. We investigated the role ofintegrins in the attachment of MAI to extracellular matrixproteins, since this family of cell-surface proteins is knownto mediate cell-substratum and cell-cell adhesion by bindingto components of the extracellular matrix in several eucary-otic cell types (1, 33). In addition, integrins have been shownto be present in invertebrates and fungi (30). In the presentstudy, we have been able to demonstrate that the binding of

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MAI to extracellular matrix proteins such as Lm, Col.I, andFn is mediated by a 1 integrin.

MATERIALS AND METHODS

Strain of MAI and culture conditions. MAI obtained fromthe American Type Culture Collection (ATCC 25291; Rock-ville, Md.) was cultured in Middlebrook 7H11 broth (DifcoLaboratories, Detroit, Mich.) at 37°C in 5% CO2 for 7 to 14days. Cultures were vigorously agitated once a day. Thebacteria were harvested by centrifugation for 15 min at 1,000x g and washed two times in phosphate-buffered salinecontaining 1 mM each CaCl2 and MgCl2 (Dulbecco's PBS[DPBS]). The pellet was resuspended in the same buffer atan optical density at 600 nm (OD6.) of 0.2 and then used inthe attachment assay. Pseudomonas aeruginosa and Strep-tococcus viridans were obtained from the microbiologylaboratory at the Medical Center, University of California,San Diego, San Diego.Attachment assay. Lm, Col.1, Fn, and Vn at 50, 100, 200,

and 300 ,ug/ml were coated (10 ,ul per well) on Terasaki plates(Miles Laboratories, Inc., Naperville, Ill.) for 1 h at 37°Cafter which they were incubated overnight at 4°C. Humanserum albumin (HSA) at the same concentrations and 0.5%ovalbumin (Sigma Chemical Co., St. Louis, Mo.) were usedas controls. To some wells, DPBS was added instead of thesample. After incubation, the plates were washed two timeswith DPBS and blocked with 0.5% ovalbumin (10 ,ul perwell) for 1 to 2 h at 37°C to decrease nonspecific binding.After blocking, the plates were once again washed twotimes, and 10 p,l of MAI at an OD6w of 0.2 was added to eachwell. The plates were centrifuged at 500 x g for 5 min in aBeckman (model TJ-6) table-top centrifuge and incubated at37°C for 30 min. The unbound MAI organisms were removedby washing three times with DPBS, and the bound bacteriawere fixed with 2% glutaraldehyde for 5 min. The number ofMAI bound in a given field in each well was counted by usingan Olympus IMT-2 inverted microscope (magnification,x400). Human Lm was isolated by monoclonal antibodyaffinity chromatography of pepsin-digested human placentaaccording to previously published protocols (14). HumanFn, Col.I, and Vn were obtained from Telios PharmaceuticalInc., San Diego, Calif. All samples were done in triplicate,and each experiment was run at least three times. Thenumber of MAI bound to DPBS wells was consideredbackground binding and subtracted from the values obtainedfor controls as well as samples. Data shown are the means oftriplicate values, and the results obtained are representativeof three experiments.

Effect of time on the attachment of MAI to Lm, Col.1, andFn. To determine the effect of time on the binding of MAI toLm, Col.1, and Fn, bacteria at an OD6. of 0.2 were added toplates coated with each of these extracellular matrix proteinsat 100 p,g/ml and incubated for 10, 30, and 60 min in theattachment assay described above. At the end of each timepoint, the plates were washed to remove the unbound MAIand then fixed. The number of bacteria bound in a given fieldin each well was determined as described above.

Effect of antibodies against different integrin receptors onthe adherence ofMAI to Lm-, Col.l-, and Fn-coated surfaces.To test the ability of anti-integrin antibodies to block attach-ment of MAI to extracellular matrix protein-coated surfaces,the following was done. MAI organisms were washed twotimes with DPBS and resuspended in the same buffer at anOD6w of 0.2. The bacterial suspension was divided intoseveral aliquots, and each aliquot was incubated with the

appropriate antibody or normal rabbit serum (NRS) at a 1:50dilution or with an equal volume of DPBS (as a control) for1 h at room temperature on an end-over rotator. MATpreincubated with these reagents was then added to platescoated with Lm, Col.1, and Fn (each at 100 ,ug/ml), and thenumber of bacteria bound in each case was determined.Rabbit polyclonal antibodies against the human Fn receptor,a5pl, and the human Vn receptor, a0I33, were obtained fromTelios Pharmaceutical Inc. Antibodies against the humanLm receptor, a3p1, were generated by Kurt R. Gehlsen andhave been previously characterized (17). Rabbit antibodiesagainst an extract of human monocytes (anti-crude mono-cyte extract [CME]) raised in our laboratory, as well asNRS, were used as control antisera.Immunoblot analysis of MAI sonicates with different inte-

grin receptor antisera. A sonicate of MAI was prepared bythe protocol of Lamb and coworkers (28). Briefly, MAIorganisms were washed two times with DPBS, resuspendedin the same buffer, and sonicated for 15 min at 100 W with1-min rests between 1-min bursts. The sonic extract wascentrifuged at 10,000 x g for 30 min at 4°C, and the cell-freesupernatant was collected. A sonicate of E. coli prepared bythe same protocol was used as a control.The sonicates were subjected to sodium dodecyl sulfate-

10% polyacrylamide gel electrophoresis (SDS-PAGE) undernonreduced conditions and electrophoretically transferred tonitrocellulose filters by using the Tris-glycine buffer system(pH 8.3). The filters were blocked in a 10% solution of milkproteins in lx Tris-buffered saline (pH 8.0) and then incu-bated with the appropriate antibody solution (1:500). Thebound antibodies were detected with goat antibodies torabbit immunoglobulin G that were conjugated to alkalinephosphatase (1:1,000) and developed by using the BCIP/NBT phosphatase substrate system (Kirkegaard and PerryLaboratories, Inc., Gaithersburg, Md.).

Effect of EDTA on the adherence of MAI to Lm-, Col.I-,and Fn-coated surfaces. MAI organisms were washed andresuspended at an OD6. of 0.2 as described earlier. Thebacterial suspension was preincubated with EDTA at a finalconcentration of 20 mM for 1 h at room temperature on anend-over rotator. As a control, an equal volume of DPBSwas added to another aliquot of the bacterial suspension, andthis mixture was incubated under identical conditions. MAIorganisms were then added to plates coated with 100 jig eachof Lm, Col.I, Fn, and HSA per ml as well as 0.5% ovalbu-min. The number ofMAI bound in each case was determinedas described earlier.

RESULTS

MAI organisms adhere to surfaces coated with Lm, Col.1,and Fn. Adhesion of pathogens to proteins and glycoconju-gates on host cell plasma membranes or to components ofthe extracellular matrix is thought to be a critical early stepin the initiation of infection. We studied the attachment ofMAI to surfaces coated with Lm, Col.I, Fn, and Vn (Fig. 1).The bacteria were found to bind avidly to Lm, Col.I, and Fn,while binding to Vn was only 50 to 60% of the bindingobserved to these other proteins. Binding to HSA andovalbumin, which were used as controls, was minimal.MAI organisms were found to bind to Lm and Fn in a

dose-dependent manner up to 300 pg/ml. On the other hand,attachment to Col.I was dose dependent only up to 100p.g/ml. At concentrations of 200 and 300 ,ug/ml, the numberof MAI bound to Col.I was less than that observed at 100,ug/ml but still significantly higher than that bound to the

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100120

100

c

.0

.0Rciz

80 F80

60

40

c

0

.o60

° 40

z

1 234 1 234 1 234 1 234 1 234 0.5%

HSA Lm Col. I Fn Vn Ova

FIG. 1. Attachment of MAI to surfaces coated with Lm, Col.I,Fn, and Vn. MAI at an OD600 of 0.2 was added to wells coated with50 (bars 1), 100 (bars 2), 200 (bars 3), and 300 (bars 4) ±g of HSA,Lm, Col.I, Fn, and Vn per ml. MAI at the same concentration wasalso added to wells coated with 0.5% ovalbumin (Ova). The plateswere incubated at 37°C for 30 min, washed to remove the unboundbacteria, and fixed with glutaraldehyde. The number of bacteriabound to the different proteins was determined as described inMaterials and Methods.

controls. Binding of MAT to Vn-coated wells was not dosedependent. The number of bacteria bound was found todecrease with increasing concentrations of Vn. Since thebinding of MAI to Vn was not significant compared with thatobserved with Lm, Col.I, and Fn, Vn was not included inother experiments. In addition, Lm, Col.l, and Fn were usedat concentrations of 100 p,g/ml in all further experiments.The binding of two other bacterial strains, P. aermginosa

and S. viridans, to Lm, Col.I, Fn, and Vn was examined inthe attachment assay. In the case of both organisms, bindingto all of these proteins was the same as that observed in theDPBS wells and that in the control (HSA and 0.5% ovalbu-min) wells (data not included) and therefore appears to benonspecific.Attachment of MAI to Lm, Col.I, and Fn increases with

increasing time. MAI was incubated with Lm, Col.I, and Fnfor different periods of time (10, 30, and 60 min). Attachmentto Lm was found to increase linearly up to 60 min (Fig. 2).With Col.I, maximum binding was achieved at 30 min, afterwhich no further increase was observed. While substantialbinding was observed at 10 min in the Lm- and Col.I-coatedwells, the number of MAT bound to Fn-coated wells at thistime point was only slightly more than that observed in thecontrol wells. However, at 30 min, the number of MAIbound to Fn was found to be in the range observed for Lmand Col.I at the same time point. At 60 min, there was nofurther increase in the number of MAI bound. Binding toHSA and ovalbumin showed no significant changes withtime. On the basis of these results, an incubation time of 30min was used for all further experiments.

Antibodies against human Lm receptor (%P,) and humanFn receptor (cx31) inhibit attachment of MAI to Lm, Col.I,and Fn. Since MAI organisms were found to adhere to Lm,Col.I, and Fn, attempts were made to inhibit this attachmentwith antisera against different integrin receptors which areknown to bind to extracellular matrix proteins (1, 33). MAIorganisms preincubated with different antisera were used inthe attachment assay. Preincubation of MAI with anti-a301

20 _

0

0 10 20 30 40 50 60 70

Time (min)FIG. 2. Time dependence of binding of MAI to Lm, Col.I, and

Fn. MAI was added to wells coated with 100 1Lg of Lm (V), Col.I(O), Fn (U), and HSA (V) per ml and to a well coated with 0.5%ovalbumin (0), and the wells were incubated for 10, 30, and 60 min.At the end of each time point, the plates were washed to remove theunbound bacteria and fixed with glutaraldehyde. The number ofbacteria bound to the different extracellular matrix proteins at eachof the time points was determined.

strongly inhibited attachment of the bacteria to Lm (72%),Col.I (88%), and Fn (77%) (Fig. 3). Antibodies against ao513were also found to inhibit attachment of MAI to Lm (97%),Col.I (97%), and Fn (92%). On the other hand, anti-a,i3 didnot appear to strongly inhibit MAI attachment to theseproteins, showing only 37% inhibition to Lm, 12% inhibitionto Col.I, and 34% inhibition to Fn. Inhibition of attachment

100

75

c

0

0

Ci

50

25

0

Lm Col.l Fn

FIG. 3. Inhibition of attachment of MAI to Lm-, Col.I-, andFn-coated surfaces with anti-a3p3 and anti-ao,5l. MM was preincu-bated with DPBS (bars 1), NRS (bars 2), anti-CME (bars 3),anti-a301 (bars 4), anti-aL501 (bars 5), and anti-a.433 (bars 6) and thenused in the attachment assay. All antisera and NRS were used atdilutions of 1:50. The number of MAI bound to Lm, Col.I, and Fn ineach case was determined.

_

I/1I T

I = II

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120

116 Kd -

84 Kd -

n 100

0m 80

2 600

6i 40z

58 Kd -

48 Kd -

20

036 Kd -

26 Kd -

Antia3f1

Anti°a5p1

Anti°(VP3

AntiCME

NRS

FIG. 4. Immunoblot analysis of MAI sonicates with antiseraagainst different integrin receptors. A sonic extract of MAI wassubjected to SDS-10% PAGE under nonreduced conditions, trans-ferred to nitrocellulose, and incubated with different anti-integrinreceptor antisera. All antisera and NRS were used at dilutions of1:500. The bound antibodies were detected with goat antibodiesagainst rabbit immunoglobulin coupled to alkaline phosphatase(1:1,000).

of MAI to the above proteins by anti-CME was <35%, andthat by NRS was <12%.

Antisera against a301, a041 avP3, NRS, and CME weretested for binding to Lm, Col.I, and Fn by an enzyme-linkedimmunosorbent assay to rule out the possibility of cross-reactivity of these antisera with the extracellular matrixproteins used in our experiments. No binding was observed(data not included), suggesting that anti-a3131 and anti-ao3linhibit attachment of MAI to extracellular matrix proteins bybinding to the bacteria and not to the substrate. On the basisof these observations, attachment of MAI to Lm, Col.1, andFn appears to be specifically inhibited by antisera againstot313 and a5pl. These results suggest that attachment of MATto these extracellular matrix proteins may be mediated by an

integrin receptor on the bacteria.Antibodies against a3p4 and o1h1 cross-react with MAI

sonicates. Sonicates of MAI were analyzed for the presenceof integrin receptors by immunoblotting experiments usingantisera against different integrin receptors (Fig. 4). Antibod-ies against a3,1 were found to cross-react with the sonicateshowing a major band at 120 kDa, which is identical to themolecular size of the P, subunit of the human Lm receptor(17). Two minor bands with molecular sizes of 40 and 60 kDawere also observed. However, no bands were observed inthe molecular size range of the known a3 chain. Removal ofnonspecific antibodies by incubation of anti-a3P1 with the 40-and 60-kDa bands of MAI sonicates immobilized on nitro-cellulose strips or by incubation with denatured E. coliproteins did not remove antibodies against the lower-molec-ular-weight proteins. These proteins may be degradationproducts of 1 or MAI variants of this subunit which havesome sequences in common with the known human a3413. Aband of 120 kDa was also observed with antibodies against

Ova HSA Lm Col. 1 Fn(0.5%)

FIG. 5. Inhibition of attachment of MAI to Lm, Col.I, and Fn byEDTA. MAI organisms were preincubated with 20 mM EDTA andthen added to plates coated with 100 Fg each of HSA, Lm, Col.I,and Fn per ml. Cells were also added to wells coated with 0.5%ovalbumin (Ova). Closed bars, number of bacteria bound in theabsence of EDTA; open bars, number of bacteria bound in thepresence of EDTA.

a%P1. In addition, two more bands with molecular sizes of105 and 58 kDa were observed. The 105-kDa band may be anisoform or a degradation product of the 1 band. The 58-kDaband appears to be nonspecifically recognized since it wasalso observed in blots probed with NRS, which does notinhibit attachment of MAT to Lm, Col.I, or Fn (Fig. 3). Thislower-molecular-weight band, therefore, does not appear toplay a role in the attachment of MAT to these extracellularproteins. No band with a molecular weight corresponding tothat of the a5 chain of the known Fn receptor was observed.Antisera against a,43 and against human monocyte extract,which was used as a control, showed no cross-reactivity. Inaddition, to rule out the possibility of nonspecific cross-reactivity, a sonicate of E. coli was probed with anti-a31,3and was not found to cross-react (data not shown). Theseresults suggest the presence of a ,13-like integrin on MAI,which may mediate its attachment to the extracellular matrixproteins described earlier. In addition, these data also sug-gest the possibility of an evolutionary conservation of atleast some sequences in both procaryotic and eucaryotic1-like Lm-binding integrins.Inhibition of attachment of MAI to Lm, Col.I, and Fn by

EDTA. Integrins are known to require cations for binding totheir ligands (20). The requirement for cations such as Ca2"or Mg2+ in the attachment ofMAI to the extracellular matrixproteins was studied (Fig. 5). MAT organisms were preincu-bated with 20mM EDTA and then incubated with Lm, Col.I,and Fn in the attachment assay. Attachment to Lm, Col.I,and Fn was inhibited by 55, 63, and 47%, respectively.Binding to HSA and 0.5% ovalbumin was not inhibited byEDTA. These data suggest that attachment of MAI to Lm,Col.L, and Fn is cation dependent and again imply thepossible role of integrins in the interaction of MAI with theextracellular matrix.

DISCUSSION

With the drastic increase in the number of individualssuffering from AIDS, the treatment of systemic bacterialinfection due to MAI in these patients has become a serious

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medical concern. Information regarding the critical earlyevents in the establishment of infection such as entry into thesusceptible host and attachment to host cell surfaces or

extracellular substrates is limited. In the present study, we

have shown that MAI binds to extracellular matrix proteinssuch as Lm, Col.I, and Fn and that integrin receptors on thebacterial surface may be involved in the attachment.

Bacterial surface receptors involved in binding to extra-cellular matrix proteins have been identified for severalpathogens (16, 29, 34-36). In the case of bacteria such as thestaphylococci and streptococci, binding to Fn in woundedtissues with the help of these receptors may represent a

mechanism of adherence, enabling tissue colonization anddevelopment of an infection. Fn receptors of parasitic pro-tozoa such as Entamoeba histolytica and Trypanosoma cruzihave also been identified and are thought to play a role inhost tissue invasion (31, 37). Recent studies with otherpathogenic protozoa such as Tnichomonas vaginalis andTrichomonas foetus have revealed the presence of Fn- as

well as Lm-binding sites on their surfaces (7), which are

involved in the pathogenesis of the microorganism by medi-ating attachment to host cells (8). These studies clearlysuggest the role played by microbial receptors for hostextracellular matrix proteins in the establishment of patho-genesis.Our experiments suggest that a P, integrin on the surface

of MAI mediates attachment of the organism to proteins ofthe extracellular matrix. Immunoblot analysis of MAI soni-cates with polyclonal antibodies against a human Lm recep-

tor containing the Oa3 and subunits and a human Fnreceptor containing the a5 and 1 subunits showed that bothantisera recognize the P, subunit. Although no band corre-

sponding to the a3 or a5 subunit was observed, it is possiblethat the bacteria may contain a different a subunit. Further-more, it is well known that a subunits, in general, are lessimmunogenic and do not generate antibodies comparable tothose of the 1 subunit. Recognition of the bacterialsubunit by antiserum raised against a human 31-containingintegrin suggests that at least some regions of this subunitwere strongly conserved during evolution. Recognition of a

mammalian cell Lm receptor by antibodies to bacterialLm-binding proteins has also been reported in the case of S.aureus (18). Binding of MAI to Lm, Col.I, and Fn could bespecifically inhibited with anti-a3p1 as well as with antibod-ies to ax5p1. This further substantiates the observation that a

13l-like integrin on MAI may be responsible for attachment tothese components of the extracellular matrix. The a subunitof integrins is known to have cation-binding sites which are

required for activity (20). Although an a subunit could not beidentified in sonicates of MAI with the antisera used in thepresent study, attachment of the bacteria to these proteinswas found to be cation dependent. Immunoblot analysis ofMAI sonicates with polyclonal antisera against the cytoplas-mic domains of a2 and O3 subunits also showed no cross-

reactivity (data not included). Attempts are being made toidentify and characterize the a subunit on MAI.As reviewed earlier, several bacterial receptors for host

extracellular matrix proteins have been identified and char-acterized. However, none of these receptors have beenshown to be integrins. Although proteins that cross-reactimmunologically with antisera against a synthetic peptidecorresponding to the cytoplasmic domain of the chickenintegrin subunit have been detected in many vertebrates,invertebrates, and fungi (30), our studies suggest, for the firsttime, the presence of an integrinlike molecule on a bacterialsurface. It is quite possible that this versatile group of

adhesion molecules may play a role in the attachment ofMAI to host tissues. The observations made in the presentstudy may be significant when the clinical manifestations ofMAI infection in AIDS patients are considered. There iswidespread dissemination, including infestation of the or-gans of the reticuloendothelial system (25) and those of thegastrointestinal system (11, 40). In addition, MAI organismshave been shown to be present in body fluids, respiratorysecretions, urine, and stool (19, 24). On the basis of thesefindings, it would be reasonable to speculate that MAI mayinitially attach to basement membrane structures such asLm, Col, and Fn with the help of the ,1 integrin, penetrateand multiply in the underlying tissues, gain access to thelymphatic system or bloodstream of the host, and spreadthroughout the body, resulting in systemic infection. On theother hand, since MAI is known to be an intracellularpathogen (41), it may also be phagocytosed by macrophagesonce it has attached, resulting in intracellular infection.

ACKNOWLEDGMENT

This work was supported by the University of California Univer-sitywide AIDS Research Program.

REFERENCES

1. Albelda, S. M., and C. A. Buck. 1990. Integrins and other celladhesion molecules. FASEB J. 4:2868-2880.

2. Armstrong, D., J. W. M. Gold, J. Dryjanski, E. Whimbey, B.Poisky, C. Hawkins, A. E. Brown, E. Bernard, and T. E. Kiehn.1985. Treatment of infections in patients with the acquiredimmunodeficiency syndrome. Ann. Intern. Med. 103:738-743.

3. Barksdale, L., and K.-S. Kim. 1977. Mycobacterium. Bacteriol.Rev. 41:217-372.

4. Beachey, E. H. 1981. Bacterial adherence: adhesin-receptorinteractions mediating the attachment of bacteria to mucosalsurfaces. J. Infect. Dis. 143:325-345.

5. Beachey, E. H., and H. S. Courtney. 1989. Bacterial adherenceof group A streptococci to mucosal surfaces. Respiration55(Suppl. 1):33-40.

6. Beachey, E. H., C. S. Giampapa, and S. N. Abraham. 1988.Adhesin receptor-mediated attachment of pathogenic bacteriato mucosal surfaces. Am. Rev. Respir. Dis. 138:S45-S48.

7. Benchimol, M., C. Batista, and W. DeSouza. 1990. Fibronectin-and laminin-mediated endocytic activity in the parasitic proto-zoa Tnchomonas vaginalis and Trichomonas foetus. J. Submi-crosc. Cytol. Pathol. 22:39-45.

8. Casta e Silva Filho, F., W. DeSouza, and J. D. Lopes. 1988.Presence of laminin-binding proteins in trichomonads and theirrole in adhesion. Proc. Natl. Acad. Sci. USA 58:8042-8046.

9. Chhatwal, G. S., K. T. Preissner, G. Mulhler-Berghaus, and H.Blobel. 1987. Specific binding of the human S protein (vitronec-tin) to streptococci, Staphylococcus aureus, and Escherichiacoli. Infect. Immun. 55:1878-1883.

10. Chiu, J., J. Nussbaum, S. Bozzette, J. G. Tilles, L. S. Young, J.Leedom, P. N. R. Heseltine, A. McCutchan, and The CaliforniaCollaborative Treatment Group. 1990. Treatment of dissemi-nated Mycobactenium avium complex infection in AIDS withamikacin, ethambutol, rifampin and ciprofloxacin. Ann. Intern.Med. 113:358-361.

11. Damsker, B., and E. J. Bottone. 1985. Concise communications.Mycobacterium avium-Mycobactenum intracellulare from theintestinal tracts of patients with acquired immunodeficiencysyndrome: concepts regarding acquisition and pathogenesis. J.Infect. Dis. 151:179-181.

12. Dautzenberg, B., C. Truffot, S. Legris, M.-C. Meyohas, H. C.Berlie, A. Mercat, S. Chevret, and J. Grosset. 1991. Activity ofclarithromycin against Mycobacterium avium infection in pa-tients with the acquired immune deficiency syndrome. Am.Rev. Respir. Dis. 144:564-569.

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V,1INTEGRIN OF M. AVIUM-M. INTRACELLULARE 3657

13. Dawson, J. R., and RI P. Ellen. 1990. Tip-oriented adherence ofTreponema denticola to fibronectin. Infect. Immun. 58:3924-3928.

14. Engvall, E., G. E. Davis, K. Dickerson, E. Rouslabti, and S.Varon. 1986. Mapping of domains of human laminin usingmonoclonal antibodies: localization of the neurite-promotingsite. J. Cell Biol. 103:2457-2565.

15. Froman, G., L. M. Switalski, A. Faris, T. Wadstrom, and M.Hook. 1984. Binding of Escherichia coli to fibronectin. J. Biol.Chem. 259:14899-14905.

16. Froman, G., L. M. Switalski, P. Speziale, and M. Hook 1987.Isolation and characterization of a fibronectin receptor fromStaphylococcus aureus. J. Biol. Chem. 262:6564-6571.

17. Gehisen, K. R., K. Dickerson, W. S. Argraves, E. Engvall, andE. Rouslahti. 1989. Subunit structure of a laminin-binding inte-grin and localization of its binding site on laminin. J. Biol.Chem. 264:19034-19038.

18. Gloria, F. A. M., C. R. W. Carneiro, L. Gomes, and J. D. Lopes.1988. Monoclonal antibodies to Staphylococcus aureus laminin-binding proteins cross-react with mammalian cells. Infect. Im-mun. 56:1580-1584.

19. Greene, J. B., G. S. Sidhu, S. Lewin, J. F. Levine, H. Masur,M. S. Simberkoff, P. Nicholas, R. C. Good, S. B. Zolla-Pazner,A. A. Pollock, M. L. Tapper, and R. S. Holzman. 1982. Myco-bacterinum avium-intracellulare: a cause of disseminated lifethreatening infection in homosexuals and drug abusers. Ann.Intern. Med. 97:539-546.

20. Hemler, M. E. 1990. VLA proteins in the integrin family:structure, function, and their role in leukocytes. Annu. Rev.Immunol. 8:365-400.

21. Holderbaum, D., G. S. Hall, and L. A. Ehrhart. 1986. Collagenbinding to Staphylococcus aureus. Infect. Immun. 54:359-365.

22. Horsburgh, C. 1991. Mycobacterium avium complex infectionin the acquired immunodeficiency syndrome. N. Engl. J. Med.324:1332-1338.

23. Iseman, M. 1989. Mycobacterinum avium complex and the nor-mal host. N. Engl. J. Med. 321:896-898.

24. Kiehn, T. E., F. F. Edwards, and P. Brannon. 1985. Infectionscaused by Mycobacterium avium complex in immunocompro-mised patients: diagnosis by blood culture and fecal examina-tion, antimicrobial susceptibility tests, and morphological andseroagglutination characteristics. J. Clin. Microbiol. 21:168-173.

25. Klatt, E. C., D. F. Jensen, and P. R. Meyer. 1987. Pathology ofMycobacterium avium-mntracellulare in acquired immunodefi-ciency syndrome. Hum. Pathol. 18:709-714.

26. Kuusela, P. 1978. Fibronectin binds to Staphylococcus aureus.Nature (London) 276:718-720.

27. Kuusela, P., T. Vartio, M. Vuento, and E. B. Myhre. 1985.

Attachment of staphylococci and streptococci on fibronectin,fibronectin fragments, and fibrinogen bound to a solid phase.Infect. Immun. 50:77-81.

28. Lamb, F. I., N. B. Singh, and M. J. Colston. 1990. The specific18 kDa antigen of Mycobacterium leprae is present in Myco-bacterium habana and functions as a heat-shock protein. J.Immunol. 144:1922-1925.

29. Lopes, J. D., M. D. Reis, and R. R. Brentani. 1985. Presence oflaminin receptors in Staphylococcus aureus. Science 229:275-277.

30. Marcantonio, E. E., and R. 0. Hynes. 1988. Antibodies to theconserved cytoplasmic domain of the integrin I1 subunit reactwith proteins in vertebrates, invertebrates, and fungi. J. CellBiol. 106:1765-1772.

31. Onaissi, M. A., D. Afchain, A. Capron, and J. A. Grimaud. 1984.Fibronectin receptors on Trypanosoma cnrzi trypomastigotesand their biological function. Nature (London) 308:380-382.

32. Ratliff, T., J. A. McGarr, C. Abou-Zeid, G. A. W. Rook, J. L.Stanford, J. Aslanzadeh, and E. J. Brown. 1988. Attachment ofmycobacteria to fibronectin-coated surfaces. J. Gen. Microbiol.134:1307-1313.

33. Rouslahti, E. 1991. Integrins. J. Clin. Invest. 87:1-5.34. Speziale, P., M. Hook, L. M. Switalski, and T. Wadstrom. 1984.

Fibronectin binding to a Streptococcus pyogenes strain. J.Bacteriol. 157:420-427.

35. Speziale, P., G. Raucci, L. Visai, L. M. Switalski, R. Tlmpl, andM. Hook. 1986. Binding of collagen to Staphylococcus aureusCowan 1. J. Bacteriol. 167:77-81.

36. Switalski, L. M., P. Speziale, M. Hook, T. Wadstrom, and R.Timpl. 1984. Binding of Streptococcus pyogenes to laminin. J.Biol. Chem. 259:3734-3738.

37. Talamas-Rohana, P., and I. Meza. 1988. Interaction betweenpathogenic amebas and fibronectin: substrate degradation andchanges in cytoskeleton organization. J. Cell Biol. 106:1787-1794.

38. Visai, L., P. Speziale, and S. Bozzini. 1990. Binding of collagensto an enterotoxigenic strain of Escherichia coli. Infect. Immun.58:449-455.

39. Wallace, J. M., and J. B. Hannah. 1988. Mycobacteinum aviumcomplex infection in patients with acquired immunodeficiencysyndrome. Chest 93:926-932.

40. Wong, B., F. F. Edwards, T. E. Kiehn, E. Whimbey, H.Donnelly, E. M. Bernard, J. W. M. Gold, and D. Armstrong.1985. Continuous high grade Mycobactenum avium-intracellu-lare bacteremia in patients with acquired immunodeficiencysyndrome. Am. J. Med. 78:35-40.

41. Young, L. S. 1988. AIDS commentary: Mycobacterium aviumcomplex infection. J. Infect. Dis. 157:863-867.

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