The Neisseria type 2 IgA1 protease cleaves LAMP1 and promotes survival of bacteria within epithelial...

The Neisseria type 2 IgA1 protease cleaves LAMP1 andpromotes survival of bacteria within epithelial cells

Lan Lin, 1 Patricia Ayala, 1 Jason Larson, 1 MarthaMulks, 5 Minoru Fukuda, 3 Sven R. Carlsson, 4 CarolineEnns 2 and Magdalene So 1*1Department of Molecular Microbiology and Immunology,L220 and 2Department of Cell and DevelopmentalBiology, Oregon Health Sciences University, 3181 SWSam Jackson Park Road, Portland, Oregon 97201-3098,USA.3Glycobiology/Carbohydrate Chemistry Program, TheBurnham Institute, La Jolla Cancer Research Center,10901 North Torrey Pines Road, La Jolla, California92037, USA.4Department of Medical Biochemistry and Biophysics,University of Umea, S-901 87 Umea, Sweden.5Department of Microbiology, Michigan State University,East Lansing, Michigan 48824, USA.

Summary

Infection of human epithelial cells by Neisseria men-ingitidis (MC) and Neisseria gonorrhoeae (GC) incre-ases the rate of degradation of LAMP1, a major integralmembrane glycoprotein of late endosomes and lyso-somes. Several lines of evidence indicate that theneisserial IgA1 protease is directly responsible forthis LAMP1 degradation. LAMP1 contains an IgA1-like hinge region with potential cleavage sites forthe neisserial type 1 and type 2 IgA1 proteases. Neis-serial type 2 IgA1 protease cleaves purified LAMP1 invitro . Unlike its wild-type isogenic parent, an iga ¹

mutant of N. gonorrhoeae cannot affect LAMP1 turn-over and its growth in epithelial cells is dramaticallyreduced. Thus, IgA1 protease cleavage of LAMP1 pro-motes intracellular survival of pathogenic Neisseriaspp.

Introduction

Neisseria meningitidis (MC) and Neisseria gonorrhoeae(GC) are the two pathogenic members of the Neisseriafamily of bacteria. They are closely related, sharing > 80%homology at the nucleic acid level. MC is a major cause ofbacterial meningitis in young children and adults; GC isthe agent of gonorrhoea. MC and GC colonize the mucosal

epithelium, while MC is thought to also interact with brainendothelium as it can cross the blood–brain barrier.

Studies of early events in Neisseria/host interactionsusing organ cultures (McGee et al., 1981; Stephens, 1989;Stephens et al., 1983) have identified key early events ina neisserial infection. MC and GC adhere to non-ciliatedcells of the epithelium and induce elongation of the micro-villi at the site of contact. They form a close associationwith the host surface and the region of contact enlargesuntil the bacteria are surrounded by host membrane. Thebacteria enter the apical side of the cells, travel throughthe cell and exit into the subepithelial stroma. Shortlyafter colonization, the cilia of neighbouring uncolonizedciliated cells stop beating and the cells slough from the tis-sue. This cell-type-specific toxicity is thought to be mediatedby TNFa produced by secretory cells in response to shedbacterial components such as lipooligosaccharide andpeptidoglycan (Gregg et al., 1981; Melly et al., 1981; 1984).

Several bacterial components are important in Neis-seria/host-cell interactions. Piliation, per se, and PilC, ahigh-molecular-weight pilus-associated protein, are impor-tant for adhesion of GC and MC to host cells (Rudel et al.,1992; 1995; Nassif et al., 1993; 1994). Certain Class I pilinvariants (Lambden et al., 1980; Rudel et al., 1992; Virji etal., 1992a; Nassif et al., 1993) and Opa/Class 5 variants(Virji et al., 1993; Kupsch et al., 1993; Waldbeser et al.,1994) promote adhesion and/or invasion, while Opc, anOpa-like protein present in some MC strains, enhancesadhesion and contributes to tissue tropism (Virji et al.,1992b; 1993).

All pathogenic neisseriae constitutively express andsecrete one of two closely related types of IgA1 proteaseswith different specificities for the hinge region of the humanIgA1 subclass of immunoglobulins (Plaut et al., 1975;Pohlner et al., 1987; Mulks and Shoberg, 1994). Type 1protease cleaves at a specific proline/serine (P/S) bond,while type 2 protease cleaves at a proline/threonine (P/T)bond in the IgA1 hinge. A given strain produces only onetype of IgA1 protease. The specificity of this enzyme formucosal immunoglobulins and their production only bythe pathogenic neisseriae argue for their importance inbacterial virulence. Infection studies using human fallo-pian-tube tissue segments with a GC iga¹ mutant indi-cated that the IgA1 protease does not influence the earlyphases of adherence or invasion in the absence of sec-retory IgA1 (Cooper et al., 1984). This mutant was notassessed for any role it might play at later stages of infection.

Molecular Microbiology (1997) 24(5), 1083–1094

Q 1997 Blackwell Science Ltd

Received 6 February, 1997; revised 15 April, 1997; accepted 21April, 1997. *For correspondence. E-mail [email protected]; Tel.(503) 494 6840; Fax (503) 494 6862.

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Neisseriae appear to enter cells by a phagocytic mech-anism, although the precise nature of the invasion processis unknown. Most phagosomes are targeted to fuse withlysosomes, which are acidic, terminal degradative com-partments in the endocytic route. This compartmentcontains hydrolytic enzymes and other microbicidal com-pounds and plays an important role in defending thebody against invading microbes. Many intracellular bac-teria have developed various means to modify their phago-somal environments in order to avoid lysosome killing. Forexample, Chlamydia psittaci (Zeichner et al., 1983) andLegionella pneumophila (Horwitz, 1993) block phago-some–lysosome fusion, while L. pneumophila (Horwitzand Maxfield, 1984), Toxoplasma gondii (Sibley et al.,1985), and Mycobacterium avium (Sturgill-Koszycki et al.,1994) attenuate the pH of their phagosomal environment.To date, the molecular mechanisms used by microbes tomodify their phagosomes are unknown.

We are interested in understanding how the pathogenicNeisseria spp. escape lysosome killing in human epithelialcells. To this end we have examined the interactions ofintracellular Neisseria spp. with LAMP1, a major integralmembrane glycoprotein of late endosomes and lyso-somes that is thought to play a role in maintaining thestability of these compartments. Here we report thatinfection of human epithelial cells by the pathogenicNeisseria spp. increases the rate of LAMP1 degradation,that this process is due to cleavage of LAMP1 by theNeisseria type 2 IgA1 protease and that this hydrolysisultimately promotes bacterial survival within humanepithelial cells.

Results

LAMP1 levels are reduced in Neisseria-infected cells

Immunofluorescence microscopy studies were initiated toexamine interactions between the pathogenic neisseriaeand LAMP1. A431 cells (a human epithelial cell line derivedfrom columnar epithelium of the endocervix) were infectedfor 4, 8 and 22 h with MC strain 8013.6 at an MOI (multipli-city of infection; ratio of bacteria to epithelial cell) of 1, andexamined by microscopy. Results indicate that the amountof LAMP1 decreased in infected cells compared to theuninfected control (compare Fig. 1, A and G). This reduc-tion in the LAMP1 signal is due to a general decrease inthe amount of LAMP1 protein, not to any alteration of theMAb-reactive epitope (see below). Infected cells werehealthy, as judged by several parameters (see below).Similar results were also observed in cells infected withthree other MC strains and three GC strains (Table 1). Inaddition, Neisseria infection resulted in reduced LAMPlevels in two other human epithelial cell lines, HEC-1-Band T84 (data not shown). These data strongly suggest

that reduction of LAMP1 levels is an important biologicalactivity of the neisseriae.

LAMP1 levels in Neisseria-infected cells were alsoassessed by Western blotting. A431 cells were infectedfor various lengths of time with MC 8013.6 at an MOI of1 and total cell proteins were blotted using the LAMP1MAb and, as an internal control, with an anti-b-tubulinMAb. Results from this experiment indicate that a reduction

Q 1997 Blackwell Science Ltd, Molecular Microbiology, 24, 1083–1094

Fig. 1. Double immunofluorescence microscopy of MC-infectedA431 cells. A431 cells were infected with MC strain 8013.6 for 4 h(C and D), 8 h (E and F) or 22 h (G and H), and stained withprimary rabbit anti-MC Mab and anti-LAMP1 Mab, and secondaryTRITC-conjugated goat anti-rabbit and BODIPY-conjugated goatanti-mouse. A, C, E and G, LAMP1 staining; B, D, F and H, MCstaining. A and B, uninfected cells.

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in LAMP1 levels is first visible between 12 and 18 h post-infection (Fig. 2a). In a separate experiment, LAMP1 levelswere quantified in A431 cells infected with MC 8013.6 for22 h. Signals from the Western blot were scanned andquantified using the NIH Image System program. A com-parison of LAMP1 values, after normalization to theirrespective b-tubulin values, shows that in this experimentLAMP1 was reduced <40%. Data from several experi-ments indicate that LAMP1 levels can be reduced 20–50%, depending on the MOI. Similar results were alsoobtained from A431 cells infected with GC strain MS11A(data not shown).

MC and GC do not infect all cells in a culture. Dependingon the MOI, age of the inoculum and other undefined con-ditions, the extent of infection in any given experiment canvary between 40 and 90%. This variation in infectivityexplains the variation in LAMP1 values observed. In theexperiment shown in Fig. 2B LAMP1 was reduced<40% compared to the uninfected control and the levelof infection was <60%. Therefore, in a given infectedcell LAMP1 would be reduced by more than 40%. Thisdecrease can sometimes be dramatic (see Fig. 1G). Inall experiments, the amount of b-tubulin was nearly iden-tical in infected and uninfected cultures, strongly suggest-ing that MC infection does not affect the microtubulenetwork (also see below).

Neisseria infection does not affect the generalmorphology of, or cause extensive damage to, infectedcells

All strains used for these studies, with the exception of15063G, require 6–8 h for internalization (data not shown).Furthermore, a low MOI was used for these studies (from1 to 10) to avoid cytotoxicity upon prolonged infection.The decrease in LAMP1 at later stages of infection istherefore unlikely to have resulted from an indirect toxiceffect brought on by bacterial growth. However, infectedcells were examined for damage to their plasma mem-branes. A431 cells were infected with MC 8013.6 for22 h, then trypsinized and stained with trypan blue andthe number of stained and unstained cells was quantified.The percentage of cells excluding trypan blue was nearlyidentical in infected and uninfected cells (> 85%), indicat-ing that the plasma membranes of 22 h infected cells areintact (data not shown).

The effect of MC on the morphology of actin and micro-tubule networks was also investigated. The actin- andmicrotubule-based cytoskeletal systems are importantfor the structural integrity of the cell and play importantroles in organelle position and movement (Langford, 1995;Lippincott-Schwartz et al., 1995). Actin is involved in adhe-sion of the cell to the substratum, maintenance of cellpolarity and cell mobility (Hitt and Luna, 1994; Mays etal., 1994). Microtubules and their motor proteins maintainthe spatial organization of various organelles and facilitatethe delivery of transport intermediates between theseorganelles (Terasaki et al., 1986; Cooper et al., 1988;


Table 1. Relevant characteristics of Neisseria strains used in thisstudy.

Relevant Characteristics

Strain pili Opa Opc capsule

IgA1proteasetype

LAMP1decreasea

MC 8013.6 þb ND ¹c Gp C type 2 þ

MC 7972 þd ND ND Gp C type 1 þ

MC 8064 þd ND ND Gp C ND þ

MC 130.77 ND ND ND Gp A ND þ

GC MS11A þb ¹ ¹ ¹ type 2 þ

GC MS11C3 þb ¹ ¹ ¹ type 2 þ

GC MS11A-300e ¹ ¹ ¹ ¹ type 2 þ

GC MS11A-14.1f þ ¹ ¹ ¹ type 2 þ

GC 15063G þd þg ¹ ¹ ND þ

GCM740 þd ¹ ¹ ¹ type 2 þ

GCM740D4 þd ¹ ¹ ¹ ¹ ¹

N. perflava ¹ ND ND ¹ ¹ ¹

N. polysaccharea ¹ ND ND ¹ ¹ ¹

a. Determined by fluorescence microscopy; at 18–22h post-infection.b. pilE sequences differ (Hagblom et al., 1985; Nassif et al., 1993).c. opc gene is absent from this strain (X. Nassif, personalcommunication).d. pilE sequence not determined.e. Non-piliated derivative of MS11A; pilE1::Erm; DpilE2 (Merz et al.,1996).f. opaH ¹ ; invasion frequency decreased approx. sixfold (Waldbeseret al., 1994).g. Opa profile not characterized.ND, not determined.

Fig. 2. LAMP1 levels in A431 cells infected with MC strain 8013.6.A. LAMP1 levels in A431 cells infected for various lengths of time.B. LAMP1 levels in uninfected (U) A431 cells and cells infectedwith MC strain 8013.6 (I). Equal amounts of total cell proteins wereseparated by SDS–PAGE and probed with anti-b-tubulin oranti-LAMP1 Mabs.

IgA1 protease-mediated cleavage of LAMP1 promotes intracellular survival of Neisseria 1085

Lippincott-Schwartz et al., 1991). A431 cells were infectedwith MC 8013.6 for 22 h, stained for actin using phalloidin(Fig. 3, A and B) or for microtubules using a MAb to b-tubu-lin (Fig. 3, C and D). Examination of stained cells revealedthat the gross arrangement of both cytoskeletal actin andmicrotubules was identical in infected and uninfected cells.Taken together, our results indicate that the reduction inLAMP1 is not due to a decrease in the integrity of theplasma membrane or cytoskeletal network.

Neisseria infection affects the half-life of LAMP1

The decrease in LAMP1 levels in infected cells may becaused by perturbations in the biosynthetic or turnoverrate of this protein. The synthesis of LAMP1 was firstexamined. A431 cells were infected with MC 8013.6 for22 h and labelled with [35S]-methionine. At various timesthe cells were lysed and the labelled LAMP1 was immuno-precipitated and counted. The results (Fig. 4A) show thatthe biosynthetic rate of LAMP1 is identical in infected anduninfected cells over a 90 min period.

The turnover rate of LAMP1 was then examined. A431

cells were infected with MC 8013.6 at an MOI of 5. At 22 hpost-infection, cells were labelled with [35S]-methioninefor 90 min, washed free of label and incubated with label-free medium. The amount of LAMP1 was determined forthe next 80 min. LAMP1 was immunoprecipitated from thelysates, separated by SDS–PAGE and autoradiographed.Signals from the gel were quantified and the LAMP1values in infected and uninfected cells were expressedrelative to the 0 h value (Fig. 4B). Results indicate thatthe rate of LAMP1 turnover in infected cultures wasincreased significantly compared to uninfected controlsand that it followed second-order kinetics. In the uninfectedcontrol, the turnover rate was constant over the entirechase period. In infected cells, this rate was nearly twiceas rapid in the first 30 min of the chase period comparedto the control. Thereafter it was similar to that for unin-fected cells. In an infected culture, the rate of LAMP1 turn-over is a reflection of the turnover in infected and uninfectedcells. The increased rate at the beginning of the chaseperiod mostly reflects the rate of turnover in the largepopulation (90%) of infected cells. The rate observed atlater time points mostly reflects the rate of turnover in


Fig. 3. Fluorescence microscopy of cytoskeletal networks in uninfected (B and D) or 22 h MC infected (A and C) A431 cells. Cells werestained for actin with phalloidin (A and B) or for tubulin with primary anti-b-tubulin Mab and secondary BODIPY-conjugated goat anti-mouse(C and D).

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the uninfected population of cells. The high infectivity inthese experiments would explain the almost 1-log differ-ence in LAMP1 levels between infected and uninfectedcells at the later time points. Increased LAMP1 turnoverwas also observed in experiments using lower MOIs. Inthese experiments, the rate of turnover was less dramatic,as would be expected in a less infected culture (data notshown).

The increased rate of degradation of LAMP1 (Fig. 4B)did not affect the biosynthetic rate (Fig. 4A) for the follow-ing reason. LAMP1 biosynthesis and turnover are likely tooccur in different compartments. The half-time of deliveryof newly synthesized LAMP1 to lysosomes is approx.60 min (Carlsson and Fukuda, 1992). In the biosynthesisexperiment, the cells were assayed for only 90 min. There-fore, increased LAMP1 turnover would have a visible effecton the biosynthesis measurements only at later time points.

A small decrease in the early time points would occurwithin the error bars. Taken together, our results stronglysuggest that the decrease in LAMP1 levels in MC-infectedcells is due to accelerated degradation of this glycoprotein.

IgA1 production correlates with the ability to reduceLAMP1 levels

Many neisserial virulence factors could potentially reduceLAMP1 levels. Thirteen Neisseria strains differing in theproduction of a series of virulence factors were examinedfor their ability to reduce LAMP1 levels in A431 cells(Table 1). These strains differed in pilin type and in pilus,Opa, Opc and capsule expression. Two strains (Neisseriaperflava and Neisseria polysaccharea) were non-patho-genic. Of the virulence factors examined, only the produc-tion of IgA1 protease correlates with the ability of theorganisms to reduce LAMP1 levels.

The Neisseria IgA1 protease cleaves LAMP1 in vitro

LAMP1 is an approx. 120 kDa protein present in the mem-brane of late endosomes and lysosomes (reviewed byFukuda, 1991). It is heavily glycosylated and the carbo-hydrate groups are thought to play an important role in pro-tecting the protein and ultimately the compartments fromdegradation by lysosomal acid hydrolases (Barriocanalet al., 1986; Fukuda, 1991). The protein consists of twoglycosylated segments of similar size separated by a pro-line–serine-rich region with striking homology to the hingeregion of the human IgA1 subclass of immunoglobulins(Viitala et al., 1988). All pathogenic Neisseria spp. secreteone of two types of IgA1 protease. Present in the humanLAMP1 hinge region are potential cleavage sites for thetype 1 and type 2 neisserial IgA1 proteases (reviewedby Mulks and Shoberg, 1994). In late endosomes and lyso-somes, the LAMP1 hinge is situated in the lumenal face ofthe organelle and thus is a potential target for these pro-teases. The human IgA1 hinge region is: CPVPSTPPT-PSPSTPP2TPSP1SCC; the hLAMP1 hinge region is:P1SP2TTAPPAPP1SP1SP1SPVPKSP1S, where 1 indicatesa potential type 1 IgA1 protease cleavage site and 2 indi-cates a potential type 2 IgA1 protease cleavage site.

The presence of potential IgA1 protease cleavage sitesin LAMP1 and the location of these sites in the inner (lume-nal) face of late endosomes and lysosomes strongly sug-gest that the neisserial IgA1 protease may play a role inthe rapid turnover of this protein.

The ability of the neisserial IgA1 protease to cleaveLAMP1 was investigated. Purified human LAMP1 wasincubated with purified Neisseria type 2 IgA1 protease,at an enzyme-to-substrate ratio of <66 to 1, in pH 7.5,6.5 or 5.0 buffer, or with buffer alone, at 378C for 4 h. The


Fig. 4. LAMP1 synthesis (A) and turnover (B) in uninfected (opencircles) and MC-infected (closed circles) A431 cells. To determinethe effect of infection on LAMP1 biosynthesis, approximately equalnumbers of uninfected and 22 h infected cells were labelled with[35S]-methionine for various lengths of time, lysed, and the lysateswere immunoprecipitated using the anti-LAMP1 Mab and counted.To determine LAMP1 turnover, approximately equal numbers ofuninfected (open squares) and 22 h infected (closed squares) cellswere labelled with [35S]-methionine for 90 min, chased for the indi-cated lengths of time, lysed and the lysates immunoprecipitatedusing the anti-LAMP1 Mab. Equal amounts of each precipitate wereseparated by SDS–PAGE and autoradiographed. Signals from thegel were quantified in the PhosphorImager PSI-Mac and the NIHimage system program version 1.57.


samples were separated by SDS–PAGE and LAMP1 wasdetected by Western blotting using a rabbit polyclonal anti-body to LAMP1. In all samples containing IgA1 protease,the amount of full-length LAMP1 decreased and an addi-tional broad band migrating at <60 kDa appeared(Fig. 5). The hinge region in LAMP1 is situated near thecentre of the molecule (Mattei et al., 1990) and IgA1 pro-tease cleavage at this site would generate two fragmentsof similar molecular weight. Therefore, the <60 kDa bandprobably contains both IgA1 protease cleavage products.IgA1 protease was able to partially hydrolyse LAMP1 inall three buffer conditions, although its activity was mostefficient in pH 7.5 buffer. In contrast, it did not cleavebovine serum albumin after overnight incubation (datanot shown). These results indicate that LAMP1 cleavagewas IgA1-protease specific. Taken together, our resultsdemonstrate that IgA1 protease is able to cleave LAMP1in vitro although these particular reactions were relativelyinefficient. They also demonstrate that hydrolysis probablyoccurs at the LAMP1 hinge, and that the enzyme is activeat the pH found on the cell surface (pH 7.5) and in lateendosomes (pH 6.5) and lysosomes (pH 5.0).

The two high-molecular-weight proteins near the top ofthe gel (Fig. 5) are probably LAMP1 dimers. They reactedwith the anti-LAMP1 antibody and their signals decreasedin the presence of IgA1 protease. Moreover, purifiedLAMP1, in a detergent-free solution, has been observedto form dimers in vitro (Carlsson and Fukuda, 1989).

The specific activity of IgA1 protease in pH 7.5 buffer onthe IgA1 and LAMP1 substrates was determined. For thispreparation of IgA1 protease, the specific activity of theenzyme for IgA1 was 10.4 6 2.2 mg substrate cleavedmin¹1 mg¹1 enzyme. For LAMP1 it was 0.038 6 0.007 mgsubstrate cleaved min¹1 mg¹1 enzyme. Under these condi-tions, the enzyme hydrolysed human IgA1 more efficiently(<273-fold) than LAMP1. There are several possibleexplanations for this result. IgA1 and LAMP1 are very

different glycoproteins, residing in different microenviron-ments. The conditions for this assay, optimized for cleav-age of IgA1, may not be optimal for LAMP1. Also, unlikethe immunoglobulin molecule which has a rigid structure(Underdown and Schiff, 1986), the two domains flankingthe LAMP1 hinge can move freely relative to each other(Carlsson and Fukuda, 1989) and in certain configurationsmay mask the protease cleavage site. Finally, LAMP1 inits native state is anchored to the late endosomal and lyso-somal membranes and purified LAMP1 may not have thesame conformation as membrane-bound LAMP1. Thus,enzyme access to the hinge of purified LAMP1 may beimpeded. Indeed, IgA1 protease is much more active onLAMP1 in cell lysates than on purified LAMP1 (data notshown).

A GC iga¹ mutant cannot reduce LAMP1 levels ininfected cells

The involvement of the IgA1 protease in LAMP1 turnoverpredicts that an IgA1 protease-deficient strain would beunable to reduce LAMP1 levels in vivo. An iga¹ mutantwas therefore assessed for its ability to reduce LAMP1levels. GCM740 is a type 2 IgA1 protease-producing GCstrain and GCM740D4 is its isogenic protease-deficientmutant. Both are piliated and transparent (do not produceOpa). In GCM740D4, a 4.3 kbp deletion, beginning in theextreme 58 end of the open reading frame (ORF), hasremoved <93% of the iga coding sequence. GCM740D4has no IgA1 protease activity and the outer membrane


Fig. 5. Cleavage of purified human LAMP1 by Neisseria type 2IgA1 protease in vitro. A total of 3 ng of purified human LAMP1was incubated with 200 ng of purified Neisseria type 2 IgA1 pro-tease (þ) at 378C for 4 h in pH 5.0, 6.5 or 7.5 buffer, or with bufferalone (¹). LAMP1 was detected by Western blotting using theanti-LAMP1 polyclonal antibody. The arrow indicates the LAMP1cleavage products; the arrowhead indicates full-length hLAMP1.

Fig. 6. LAMP1 levels in uninfected (U) A431 cells and cellsinfected for 22 h with protease-producing GC strain GCM740 andGCM740D4, its genetically defined iga¹ mutant. Equal amounts oftotal cell proteins were separated by SDS–PAGE and probed withanti-b-tubulin or anti-LAMP1 Mabs. Signals from the blot werescanned and quantified using the NIH image system programversion 1.57. LAMP1 values were normalized to their internalb-tubulin signals and values for GCM740 and GCM740D4 wereexpressed relative to the value for uninfected cells. Resultsrepresent the average of five independent experiments. Error barsrepresent standard deviation.

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protein profiles of both parent and mutant are identical(Shoberg and Mulks, 1991). A431 cells were infectedwith GCM740 and GCM740D4 for 22 h and total cellularproteins from the cultures were detected on Westernblots using the anti-LAMP1 and anti-b-tubulin Mabs. Thesignals were quantified and the LAMP1 values fromeach strain were normalized to their respective internalb-tubulin values and compared. As expected, LAMP1levels in GCM740-infected cells were reduced (<20%;Fig. 6). In contrast, LAMP1 levels in GCM740D4 infectedcells were nearly identical to those in uninfected cells. Thesame results were obtained in HEC1-B cells infected withGCM740 and GCM740D4 (data not shown). These datasupport the in vitro cleavage results and demonstratethe importance of IgA1 protease in LAMP1 turnover invivo.

The GC iga¹ mutant fails to grow intracellularly

The intracellular growth rate of the GC iga¹ mutantGCM740D4 was assessed. A431 cells were infected for

12 h with GCM740 and GCM740D4 at an MOI of 5, atwhich time the cultures were treated with gentamicin(Gm) to kill extracellular bacteria. At 2-hr intervals afterremoval of the antibiotic, the cells were washed andlysed and the lysates were plated for intracellular colony-forming units (cfus). Results show that the intracellulargrowth rate of the iga¹ mutant, GCM740D4, is dramati-cally reduced compared to its wild-type GCM740 parent(Fig. 7). The number of intracellular GCM740D4 decreased<15-fold during the incubation time examined (10 h). In con-trast, the number of intracellular GCM740 cells increasedafter an initial decrease. Both strains have identical growthrates in vitro (Shoberg and Mulks, 1991) and adhere toand invade A431 cells at the same frequency (data notshown). The reduced cell counts of GCM740D4 shownhere therefore represent a decrease in its ability to repli-cate within cells. At the 0 h time point (termination of Gmtreatment), the actual intracellular cfus for GCM740 isapprox. sixfold greater than that for GCM740D4. This dif-ference is due to the fact that at this time, the cells havealready been infected for 12 h and therefore are expectedto yield fewer viable GCM740D4 cells. The increase inGCM740 cfus was not due to intracellular bacteria escap-ing into and subsequently growing in the supernatant, asplate counts of supernatants at each time point yieldedno detectable cfus. The initial decrease in intracellularbacteria has been observed in other experiments (ourunpublished data). Taken together, our results indicatethat IgA1 protease-mediated LAMP1 cleavage is impor-tant for the intracellular survival of the pathogenic Neis-seria spp.

Discussion

We have presented evidence that the IgA1 protease isimportant for the survival of pathogenic Neisseria spp.within epithelial cells. Infection of human epithelial cellsby MC and GC results in a decrease in cellular levels ofLAMP1, a major glycoprotein present in the membrane oflate endosomes and lysosomes. This decrease is observedin three human epithelial cell lines using four MC and threeGC strains, strongly suggesting that this activity is impor-tant to the biology of the neisseriae. The reduction ofLAMP1 is due to accelerated degradation of the glycopro-tein. Several lines of evidence indicate that the NeisseriaIgA1 protease is directly responsible for LAMP1 degrada-tion. LAMP1 contains an IgA1-like hinge region with poten-tial cleavage sites for the neisserial type 1 and type 2 IgA1proteases. Neisseria type 2 IgA1 protease cleaves puri-fied LAMP1 in vitro. A genetically-defined iga¹ mutantwhich does not produce IgA1 protease cannot affect theturnover rate of LAMP1 in infected epithelial cells. Further-more, its intracellular growth rate is dramatically reducedwhen compared to its protease-producing parent strain.


Fig. 7. Intracellular growth curve of GCM740 (WT) andGCM740D4 (iga¹). The diagram at the top of the figure illustratesthe sequence of manipulations in this experiment and relates thetiming of the manipulations to the start of the growth curve. A431cells were infected for 12 h with GCM740 (open circles) andGCM740D4 (closed circles) at an MOI of 5 then treated with Gmfor 90 min to kill extracellular bacteria. Cells were then re-incubatedwith fresh DMEM without Gm. At various times cells were lysedand lysates were plated for intracellular cfus. Numbers of viableintracellular bacteria at each time point were normalized to the 0 hvalue. Note that at 0 h, the cultures have been infected for 12 h andtreated with Gm for 90 min. Each point is the average from fourindependent experiments. Time points for each experiment wereperformed in duplicate. Error bars represent standard deviation; forsome time points the standard deviation may be too small to bevisible.


All pathogenic Neisseria spp. produce one of two typesof IgA1 proteases with slightly different specificities for theIgA1 hinge region. LAMP1 contains five potential type 1IgA1 protease cleavage sites (P/S bond) and one type 2site (P/T bond). Our data strongly suggest that type 2 pro-tease cleavage of LAMP1 occurs at the single P/T bond inthe LAMP1 hinge. Further studies should establish whetherthe type 1 IgA1 protease recognizes any of the P/S bondsin this region.

In our experiments, the protease was more efficient incleaving IgA1 than purified LAMP1. It may not be relevant,however, to compare the activities of the enzyme on thesetwo substrates. As mentioned earlier, IgA1 and LAMP1are very different glycoproteins, residing in very differentenvironments in vivo. Therefore, the optimal cleavage con-ditions for these two substrates are likely to differ. Fromour data it can only be concluded that IgA1 protease doescleave LAMP1 in its hinge region and that, under the con-ditions of our assay, its activity on this substrate is lessefficient than on human IgA1.

LAMP1 is a major integral membrane protein of lyso-somes and late endosomes. Lysosomes are acidic com-partments (pH 5.0 or less). They contain acid hydrolasesthat function, at low pH, in the digestion of a wide varietyof macromolecules. Many lysosomal constituents are deliv-ered to this compartment via late endosomes, which aremildly acidic (pH 6.5) and also contain many acid hydro-lases. LAMP1 is thought to play an important role in main-taining the stability of these compartments, presumably byprotecting them from digestion by the resident hydrolyticenzymes (Barriocanal et al., 1986; Fukuda, 1991). Thus,even a partial reduction of LAMP1 levels in these compart-ments may modify them in such a manner as to promoteintracellular bacterial survival and growth. Our data indi-cate that LAMP1 levels are reduced 20–50% in infectedcultures. As mentioned earlier, neisseriae do not infectevery cell in a culture. Therefore, in a given infected cell,the amount of LAMP1 is probably much less than theexperimental value determined for the entire culture.

How IgA1 protease gains access to LAMP1 is unclear. Itmay be transported to LAMP1 compartments by the endo-cytic route. This enzyme is constitutively expressed andsecreted in large amounts by the pathogenic Neisseriaspp. (Pohlner et al., 1987; Mulks and Shoberg, 1994).IgA1 protease secreted by colonizing bacteria may beendocytosed and, like a variety of internalized com-pounds, delivered to late endosomes and lysosomes viavesicle fusion. IgA1 protease may also be delivered toLAMP1 compartments by bacteria entering the cell. Mostreports indicate that intracellular MC and GC are sur-rounded by a phagosomal membrane (Nassif and So,1995; Stephens et al., 1983; Weel et al., 1991) althoughone study found no such evidence (Shaw and Falkow,1988). If intracellular Neisseria spp. are indeed inside

phagosomes, and if these vesicles are capable of fusionwith late endosomes and/or lysosomes, then the IgA1protease secreted by intracellular bacteria would gainaccess to LAMP1 upon fusion of the phagosome withthese compartments.

Finally, LAMP1 may be cleaved by IgA1 protease at thecell surface. The majority of newly synthesized LAMP1 istransported from the Golgi directly to late endosomes andlysosomes (Carlsson and Fukuda, 1992). A small fraction,however, is first transported to the plasma membranebefore it is internalized and delivered to lysosomes (Carls-son and Fukuda, 1992). The hinge region of cell-surfaceLAMP1 is situated on the extracellular face of the plasmamembrane and therefore accessible to IgA1 proteasesecreted by colonizing bacteria. Future experiments shouldestablish which pathway(s) is important for transportingIgA1 protease to LAMP1. It is interesting to note that invitro IgA1 protease can cleave LAMP1 at the pH foundon the mucosal surface (pH 7.5), in late endosomes(pH 6.5) and lysosomes (pH 5.0).

We have shown that the IgA1 protease is important forthe ability of the pathogenic Neisseria spp. to survivewithin epithelial cells. It will be interesting to determinewhether these proteases also play a role in bacterial sur-vival within other cell types, including phagocytes. Severalother mucosal pathogens also produce IgA1 proteases:Haemophilus influenzae, Ureaplasma urealyticum, Clostri-dium ramosum, Bacteriodes melaninogenicus and severalstreptococcal species (reviewed by Mulks and Shoberg,1994). The exact function of these proteases is unknown.Recently, Hap, a H. influenzae protein with a region homo-logous to the catalytic domain of IgA1 protease, was shownto promote bacterial adherence and invasion in vitro (St.Geme et al., 1994). It will be interesting to determinewhether these bacterial IgA1 proteases play a similarrole in the intracellular survival of these pathogens.

Experimental procedures

Cell culture

A431 (obtained from S. Schmid) is a human endocervicalepithelial cell line. HEC-1-B (obtained from the AmericanType Culture Collection (ATCC)) is a human epithelial cellline derived from an adenocarcinoma of the endocervix.Cells were grown in Dulbecco’s Modified Eagle’s Medium(DMEM) supplemented with 5% heat-inactivated fetal bovineserum (FBS, Gibco). Cells were used for bacterial infectionexperiments between the 5th and 20th passages.

Bacterial strains

Information for all strains used in this study is shown inTable 1. All pathogenic strains used in this study, except forGC MS11A-300, are piliated (Pþ). N. meningitidis MC 130.77,serogroup A, was obtained from the ATCC. MC 7972, MC


1090 L. Lin et al.

8064 and MC 8013.6, belonging to serogroup C, were blood-stream isolates from patients from the Pasteur Hospital (pro-vided by X. Nassif ). MC 8013.6 is a capsulated, Pþ strainproducing high-adhesive pilin (Nassif et al., 1993); its Opaphenotype has not been determined. N. gonorrhoeae strainMS11A is Pþ and Opa¹ (Segal et al., 1986); 15063G is ahighly invasive Pþ, Opaþ clinical isolate (Waldbeser et al.,1994); its Opa proteins have not been characterized.GCM740 is Pþ, Opa¹. The construction and characterizationof GCM740D4, its iga¹, Pþ, Opa¹ derivative, has beendescribed in detail (Shoberg and Mulks, 1991). The iga muta-tion was generated by deleting 4.3 kb of the 58 end of the igagene, which removed <93% of the iga coding sequence.GCM740D4 has no protease activity and the outer membraneprotein profiles of GCM740 and GCM740D4 are identical. N.perflava and N. polysaccharea are commensal strains obtainedfrom the Center for Disease Control, Atlanta, Georgia, USA.

Infection and intracellular growth-rate experiments

Bacteria were passaged every 18–22 h on GCB agar plates(Difco) with supplements (Kellogg et al., 1968) as previouslydescribed (Segal et al., 1986). All strains used for infectionexperiments were passaged fewer than 10 times. A431cells were seeded on 18 mm square coverslips at a densityof 5–8 ×104 cells well¹1 24–36 h prior to infection. Cells werewashed with PBS, pH 7.4, incubated in DMEM/5% FBS andinfected with bacteria resuspended in the same medium.Bacteria were added at the stated multiplicity of infection(MOI). To determine intracellular growth rates of GCM740and GCM740D4, A431 cells were seeded in 24-well plates(Falcon), grown to <80% confluency, and infected with bac-teria for 12 h at an MOI of 5. Cells were washed four timeswith PBS, pH 7.4, and incubated with 0.5 ml fresh DMEM/5% FBS containing 20 mg ml¹1 Gm for 90 min at 378C in 5%CO2. Cultures were then washed four times with PBS andincubated with fresh DMEM without Gm. At various times,100 ml of the supernatants were plated on supplementedGCB agar for extracellular cfus. The cells were washed fourtimes with PBS, lysed with 0.5 ml of GCB containing 0.5%saponin (Aldrich) at room temperature for 15 min and thelysates were plated on supplemented GCB agar for intracel-lular cfus.

Antibodies

Polyclonal rabbit anti-MC antibodies were raised against wholeMC (L. Walbeser and A. Merz; this laboratory). The H4A3(anti-human LAMP1) and E7 (anti-b-tubulin) monoclonalantibodies were obtained from the Developmental StudiesHybridoma Bank; polyclonal antiserum to human LAMP1was generated as described previously (Carlsson et al.,1988); BODIPY 581/591-Phalloidin and BODIPY goat anti-mouse antibody were obtained from Molecular Probes; TRITCgoat anti-rabbit antibody was purchased from Pierce.

Immunofluorescence microscopy

Coverslips with uninfected or infected cells were fixed for25 min in Zamboni’s fixative (PAPF; Zamboni and Di Martino,

1967) at room temperature. After three washes with PBS,samples were permeabilized and blocked in immunofluores-cence blocking buffer (IFB; PBS with 3% goat serum, 0.01%azide, and 0.01% saponin). Primary antibodies were addedat the appropriate dilutions and incubated for 3 h at room tem-perature or overnight at 48C. Coverslips were then washedthree times with PBS, blocked with IFB for 20 min andincubated with secondary antibodies at a 1:200 dilution inthe dark for 30 min. Coverslips were further washed fivetimes with PBS and mounted in mounting buffer (5 mM Tris,pH 8.0, 20 mg ml¹1 n-propyl gallate, 90% glycerol). Stainedcells were examined using a Nikon Microphot FX at the statedmagnification.

Quantification of LAMP1 by Western blots

Cells were infected with bacteria as described above. Unin-fected and infected cells were washed three times with PBS,lysed with PBS/1% Triton-X100, and cell debris pelleted bycentrifugation at 27 200 × g for 15 min. The proteins in thesupernate were separated in an 8% SDS acrylamide geland transferred to nitrocellulose membrane (Ausubel et al.,1994). The membrane was incubated with the anti-LAMP1or anti-b-tubulin monoclonal or the anti-LAMP1 rabbit poly-clonal antiserum and developed using goat anti-mouse orgoat anti-rabbit alkaline phosphatase (Boehringer Mannheim)or sheep anti-mouse horseradish peroxidase (ECL Chemi-luminescence Kit, Amersham). LAMP1 and b-tubulin signalsfrom the blots were scanned and quantified using the NIHImage System V1.57 program. The LAMP1 value of eachsample was normalized to the internal b-tubulin value. Nor-malized LAMP1 values for infected samples were thenexpressed relative to the normalized LAMP1 value of theuninfected control.

LAMP1 synthesis and turnover

To examine LAMP1 biosynthetic rate, uninfected and infectedcells were washed three times in PBS and incubated inDMEM/5% FBS containing 200 mCi ml¹1 [35S]-methionineand [35S]-cysteine (Dupont) at 378C in 5% CO2. At varioustimes, the labelled cells were washed three times with ice-cold PBS and lysed by incubating in ice-cold NET buffer(0.01 M Tris, pH 7.4, 0.15 M NaCl, 0.005 M EDTA) containing1% Triton X-100 and 1 mM PMSF for 5 min. Cell lysates werecentrifuged at 27 200 × g for 15 min and the supernatantswere collected for LAMP1 immunoprecipitation. To immuno-precipitate LAMP1, supernatants were incubated with mousemonoclonal anti-LAMP1 at 48C overnight on a shaker. Thenext day, samples were centrifuged at 27 200 × g at 48C, andthe supernatants incubated with protein A-conjugated Sepha-rose CL-4B (Sigma) for 3 h at 48C on a shaker. Samples werecentrifuged at 27 200 × g for 30 s and the pellets were washedwith washing buffer A (0.1 M Tris, pH 8.0, 0.1 M NaCl, 1.0 mMPMSF, 2 mM EDTA, pH 8.0, 1% Triton) three times and wash-ing buffer C (0.1 M Tris, pH 8.0, 0.1 M NaCl) twice. The pelletswere then resuspended in 30 ml of protein-sample buffer. Avolume of 5 ml of each sample was counted in a Beckman(LS 5000 TD) liquid scintillation counter.

To examine the LAMP1 turnover rate, cells were infected



for 22 h with bacteria, then washed with fresh medium. [35S]-methionine (200 mCi ml¹1) was added to the wells and cellsincubated for a further 90 min. Supernatants were removedand the cells were chased by incubation with label-freeDMEM/5% FBS for various lengths of time. (The 0 h timepoint in Fig. 4B represents the beginning of the chase period.)Cells were lysed, immunoprecipitated with the anti-LAMP1Mab as described above and the precipitates resuspendedin 30 ml of SDS-sample buffer. Equal volumes of each precipi-tate were separated by SDS–PAGE. The gels were pro-cessed for autoradiography and the signals on the gelswere quantified using a PhosphorImager PSI-Mac (MolecularDynamics) and analysed using the NIH Image V1.57 program.

Cleavage of human LAMP1 and human IgA1 by IgA1protease

Human LAMP1 was purified from chronic myelogenousleukemia cells (CML) as described previously (Carlsson andFukuda, 1989). The preparation is essentially homogeneous,as judged by silver staining of the preparation separated on apolyacrylamide gel. A total of 3 ng of LAMP1 was incubatedwith 200 ng of type 2 IgA1 protease (Boehringer Mannheim;purified from an Escherichia coli recombinant expressingthe GC type 2 iga gene) at 378C for 4 h. The pH 7.5 bufferwas essentially as recommended by the manufacturer: 10 mMTris, 0.1 mM EDTA, 0.002% BSA. The pH 6.5 buffer wasessentially as recommended: 20 mM Tris, 4 mM CaCl2, 4 mMMgCl2, 0.002% BSA (Mulks and Shoberg, 1994). The pH 5.0buffer comprised: 20mM Na-citrate, 0.002% BSA. The sampleswere boiled in the presence of 1 mM dithiothreitol (DTT),separated by 8% SDS–PAGE and analysed by Western blot-ting using an anti-LAMP1 polyclonal antibody and the West-ern blotting kit from Pierce. Specific activity of IgA1 proteasefor human IgA1 (purified as described by Mulks and Shoberg,1994) was determined using two different approaches. First,substrate was incubated with enzyme at a determined ratioin pH 7.5 buffer as described above, at 378C, for variouslengths of time. Second, substrate was incubated with varyingamounts of enzyme in pH 7.5 buffer, at 378C, for a fixed amountof time. Samples were separated by 10% SDS–PAGE andsilver stained. The gels were scanned and the amount offull-length substrate remaining in each sample was analysedusing the NIH Image V1.6 program. The specific activity ofthe enzyme for LAMP1 was similarly determined, with theexception that LAMP1 was visualized by Western blottingusing the polyclonal anti-LAMP1 antibody. Specific activitywas calculated based on the ability of the enzyme preparationto cleave 50% of the substate.

Note added in proof

Part of the results was presented at the Tenth InternationalPathogenic Neisseria Conference (Sept. 8–13, 1996, No.93, p. 293; edited by W. D. Zollinger, C. E. Frasch, and C. D.Deal).

Acknowledgements

We wish to thank Alex Merz for innumerable hours of thought-ful discussions and Michael Moody for his help in digital

imaging of confocal micrographs. The H4A3 (anti-LAMP1)and E7 (anti-b-tubulin) monoclonal antibodies were obtainedfrom the Developmental Studies Hybridoma Bank maintainedby the department of Pharmacology and Molecular Sciences,Johns Hopkins University School of Medicine, Baltimore,Maryland, USA, and the Department of Biological Sciences,University of Iowa, Iowa City, Iowa, USA, under contractN01-HD-6-2915 from the NICHD. This work was supportedby National Institutes of Health (NIH) grant AI32493 awardedto M.S. and NCI grant CA48737 to M.F.

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