Variable processing of the IgA protease autotransporter at the cell surface of Neisseria...

transcript

Variable processing of the IgA proteaseautotransporter at the cell surface of Neisseriameningitidis

Virginie Roussel-Jazede,13 Jesus Arenas,13 Jeroen D. Langereis,14Jan Tommassen1 and Peter van Ulsen1,2

Correspondence

Jan Tommassen

J.P.M.Tommassen@uu.nl

Peter van Ulsen

J.P.van.Ulsen@vu.nl

Received 15 July 2014

Accepted 20 August 2014

1Department of Molecular Microbiology, Institute of Biomembranes, Utrecht University,3584 CH Utrecht, The Netherlands

2Department of Molecular Microbiology, Institute of Molecular Cell Biology, VU University,1081 HV Amsterdam, The Netherlands

As with all classical monomeric autotransporters, IgA protease of Neisseria meningitidis is a

modular protein consisting of an N-terminal signal sequence, a passenger domain and a C-

terminal translocator domain (TD) that assists in the secretion of the passenger domain across the

outer membrane. The passenger of IgA protease consists of three separate domains: the protease

domain, the c-peptide and the a-peptide that contains nuclear localization signals (NLSs). The

protease domain is released into the extracellular milieu either via autocatalytic processing or via

cleavage by another autotransporter, NalP, expression of which is phase-variable. NalP-mediated

cleavage results in the release of a passenger that includes the a- and c-peptides. Here, we

studied the fate of the a-peptide when NalP was not expressed and observed strain-dependent

differences. In meningococcal strains where the a-peptide contained a single NLS, the a-peptide

remained covalently attached to the TD and was detected at the cell surface. In other strains, the

a-peptide contained four NLSs and was separated from the TD by an IgA protease autoproteolytic

cleavage site. In many of those cases, the a-peptide was found non-covalently associated with the

cells as a separate polypeptide. The cell surface association of the a-peptides may be relevant

physiologically. We report a novel function for the a-peptide, i.e. the binding of heparin – an

immune-modulatory molecule that in the host is found in the extracellular matrix and connected to

cell surfaces.

INTRODUCTION

Proteins that are secreted by Gram-negative bacteria haveto cross the bacterial cell envelope, which consists of theinner membrane, the periplasm containing the peptidogly-can layer and the outer membrane. The family of classicalmonomeric autotransporters constitutes a widespreadsecretion system among the Gram-negatives (Celik et al.,2012). These autotransporters are modular proteins thatconsist of an N-terminal signal sequence for transportacross the inner membrane via the Sec system, a C-terminal

translocator domain (TD) and, in between, the secretedpassenger domain (Grijpstra et al., 2013; van Ulsen et al.,2014). The TD inserts into the outer membrane, where itforms a 12-stranded b-barrel and facilitates the transport ofthe passenger across that membrane (Oomen et al., 2004;Roussel-Jazede et al., 2011; Saurı et al., 2011). Secretionfurther requires the Bam complex, which constitutes themachinery that inserts outer membrane proteins into theouter membrane (Jain & Goldberg, 2007; Voulhoux et al.,2003).

The overall three-domain organization is a general featureof autotransporters, but these domains can be furtherorganized into separate subdomains (Fig. 1a). For example,in the canonical autotransporter IgA protease of Neisseriagonorrhoeae, encoded by the iga gene, the secreted pas-senger domain is further processed into a protease domain(~ 106 kDa), a c-peptide (~3.1 kDa) and an a-peptide (~45 kDa) (Pohlner et al., 1987). Similarly, our laboratoryhas shown that the passenger domains of the autotran-sporters App and AusI of Neisseria meningitidis consist of

3These authors contributed equally to this work.

4Present address: Laboratory of Pediatric Infectious Diseases, Depart-ment of Pediatrics, Radboud UMC, 6500 HB Nijmegen, The Netherlands.

Abbreviations: NLS, nuclear localization signal; TD, translocator domain.

The GenBank/EMBL/DDBJ accession numbers for the new sequencesare GU190730-GU19070733 and KJ653448–KJ653452.

Seven supplementary figures and two supplementary tables areavailable with the online Supplementary Material.

Microbiology (2014), 160, 2421–2431 DOI 10.1099/mic.0.082511-0

082511 G 2014 The Authors Printed in Great Britain 2421

at least two subdomains (van Ulsen et al., 2003, 2006). TheTDs may also consist of several subdomains, e.g. the TDs ofIgA protease of N. gonorrhoeae and AIDA-I of Escherichiacoli consist of a surface-exposed linker peptide, which issensitive to externally added proteases, and a membrane-embedded b-core (Klauser et al., 1993; Konieczny et al.,2001). This b-core is protected against proteases andcompares to the crystallized TDs of other autotransporters(Roussel-Jazede et al., 2011).

In N. meningitidis, two different and mutually competingproteolytic events were found to be involved in the releaseof the passenger of IgA protease, resulting in the release oftwo different variants of the protein (van Ulsen et al.,

2003). Autoproteolytic cleavage results in the release ofthe protease domain as a separate polypeptide. Alterna-tively, proteolytic processing can be mediated by anotherautotransporter, NalP, whose expression is prone to phasevariation (Roussel-Jazede et al., 2013; van Ulsen et al.,2003). NalP-released IgA protease passengers include theprotease domain and the a-peptide (Fig. 1a).

In N. gonorrhoeae, which does not encode a functionalNalP (van Ulsen et al., 2001; van Ulsen & Tommassen,2006), the a-peptide of IgA protease was detected as aseparate polypeptide in the extracellular medium (Pohlneret al., 1987). In N. meningitidis, the involvement ofNalP may indicate different processing and the fate of

(b) NLS 4

MS11 (12+13 kDa)

NLS 1/2

FA1090 (23+9 kDa)

PP SP PP SP

NLS 3/4

NLS 1/2 NLS 3/4

NalPPP SP

PP SP PP SP NalP

H44/76 (30 kDa)

MC58 (44+14 kDa)

SP PP AP

Approximate NalP

cleavage site

α-peptide

(12–44 kDa)

γ -peptide

(3 kDa)

linker

(14 kDa)

TD: linker+β -core

(45 kDa)

α-peptide+linker+β -core

(62 kDa)

protease domain

(106 kDa)

signal peptide

(3 kDa)

β -core

(31 kDa)

Fig. 1. Schematic overview of the variousdomains and subdomains of neisserial IgAproteases (not drawn to scale). (a) Full-lengthIgA protease with the possible positionsof autocatalytic processing sites and theirsequences and the approximate position ofthe NalP cleavage site indicated. Arrowheadsindicate the potential positions of NLSs,whereby the open arrowhead shows theposition of the NLS in a-peptides with only asingle NLS present. (b) Schematic representa-tion of the differences between a-peptides andlinker regions of different N. meningitidis andN. gonorrhoeae strains. The arrowheads andthe colours of the a-peptide and linker regionmatch those in (a). The lighter boxes indicatethe positions of repeats sequences: a nona-peptide repeat ([RKAAELLAK] and variantsthereof; Jose et al., 2000) in the a-peptide,one or two copies of the peptide[VSESVDTSDKQPQDNTELHEKYEN] in thelinker and one to four copies of a tetra-peptiderepeat ([QAAA] or [QAVA]) at the C terminus.

V. Roussel-Jazede and others

2422 Microbiology 160

the a-peptide, when not released by NalP, is unknown.Also, the organization and appearance of the TD after therelease of the passenger variants is unknown. However, theTD does show pore activity in vitro (Roussel-Jazede et al.,2011) and its accumulation in the outer membrane,therefore, might affect the membrane’s integrity. Weinvestigated the fate of the a-peptide and the TD ofmeningococcal IgA proteases in the presence and absenceof NalP. The studies revealed novel cell-associatedfragments of IgA protease, but also showed strain-dependent differences. This differential processing mayhave functional implications, as the a-peptide has beenimplicated in biofilm formation (Arenas et al., 2013),contains nuclear localization signals (NLSs) that functionin vitro (Pohlner et al., 1995) and binds heparin, as shownin the present study.

METHODS

Bacterial strains and growth conditions. N. meningitidis strains

H44/76, its unencapsulated derivative HB-1, 2996 and B16B6, and the

iga : : kan and nalP : : kan mutant derivatives thereof, have been

described previously (van Ulsen et al., 2003; Bos & Tommassen,

2005; Arenas et al., 2013). Other strains of N. meningitidis and

Neisseria gonorrhoeae used are listed in Table S1 (available in the

online Supplementary Material) and are from our laboratory

collection (van Ulsen et al., 2006). To create a nalP : : cam iga : : kan

double mutant of strain B16B6, nalP was first disrupted by allelic

exchange through transformation with plasmid pKOnalP-cat (Arenas

et al., 2013), which carried a nalP allele in which an internal 2112

bp fragment was replaced by a chloramphenicol-resistance gene.

Subsequently, the iga gene was knocked out by allelic exchange using

the iga : : kan plasmid described previously (Vidarsson et al., 2005).

The strains were grown on GC agar (Oxoid) supplemented with Vitox

(Oxoid) at 37 uC in candle jars and liquid cultures were grown in

tryptic soy broth (TSB; Gibco-BRL) at 37 uC with mild shaking.

Where indicated, N. meningitidis strains H44/76 and B16B6 were

grown in RPMI 1860 medium (Gibco-BRL) supplemented or not

with 5 % FCS (PAA Laboratories). To obtain heat-inactivated FCS,

samples were incubated for 30 min at 56 uC. E. coli strains

BL21(DE3) (Invitrogen) and DH5a were grown in lysogeny broth.

Antibiotic concentrations added for plasmid maintenance were

100 mg ampicillin ml21 and 25 mg chloramphenicol ml21 for E. coli,

and 10 mg chloramphenicol ml21 for N. meningitidis. When

appropriate, IPTG was added at a final concentration of 1 mM to

induce gene expression.

Sequencing and sequence comparison. DNA fragments encoding

the a-peptide and the linker peptide of the IgA proteases of various

N. meningitidis strains were amplified by PCR from genomic DNA

using primers IgA1 (59-CCCGATTGTACAATCCTTATGCCGA-39)

and IgA4 (59-GCCCAAAGTAAGGCCGGTTTGGA-39) based upon

iga of N. meningitidis strain MC58. Genomic DNA was prepared

from bacteria that were resuspended in water to OD550~2.0. The

suspension was boiled for 5 min and then centrifuged at full speed in

a microfuge. The supernatant was used as template DNA for the PCR,

which was performed as described (van Ulsen et al., 2001). Resulting

PCR fragments were cloned into pCRII-TOPO (Invitrogen) and

sequenced (GenBank accession numbers GU190730–GU190733 and

KJ653448–KJ653452). Sequence alignments were performed with the

deduced amino acid sequences and using the CLUSTAL Omega

program at https://www.ebi.ac.uk/Tools/msa/clustalo/ (Sievers et al.,

2011).

Plasmid construction. The iga gene of N. meningitidis strain H44/76

was amplified by PCR from genomic DNA using primers based upon

iga of N. meningitidis strain MC58 and cloned in pCRII-TOPO,

yielding pCRT_iga_H44/76. DNA fragments encoding the a-peptide

(aa 1005–1182) and the TD (b-core plus linker peptide; aa 1183–

1561) were obtained by PCR using the primer couples IgaAstart

(59-CCATATGAGCCCGCAGGCAAATCAAGCCGA-39)/IgaAend (59-

GAAGATCTGGGCGGTGCCGGCAGAGTAGAT-39) and IgATD (59-

GCATATGCCGCAAGCCGATGCGTCAG-39)/IgAFend, respectively,

and pCRT_iga_H44/76 as template DNA. PCR fragments were cloned

into pCRII-TOPO. The fragments encoding the a-peptide and the TD

were excised from the pCRII-TOPO constructs with NdeI and BglII,

and ligated into NdeI/BamHI-digested pET11a, yielding pET_IgA-

APH44 and pET_IgA-TDH44, respectively.

Collection of cells and supernatants. Cells were harvested by

centrifugation (4500 g, 5 min) and resuspended in PBS (pH 7.6) to

OD550 10. The culture supernatants were centrifuged again (16 000 g,

5 min) to remove residual cells. Then, the protein content was

precipitated by adding ice-cold TCA to a final concentration of 5 %

and incubation for at least 30 min at 4 uC. Samples were centrifuged

(16 000 g, 20 min), and the pellets were washed with 90 % acetone

and dissolved in PBS. Relative to the OD550 of the original cultures,

the precipitated proteins from the culture supernatant fractions

were 10-fold more concentrated than the cell lysates. The protein

preparations were mixed with an equal volume of twofold con-

centrated sample buffer and boiled for 10 min.

Electrophoresis and immunoblotting. Protein samples were run on

10 % (w/v) SDS-PAGE gels and blotted onto a 0.45 mm Protran

membrane (Schleicher & Schuell). Unspecific binding of antibodies to

the filters was prevented by overnight incubation in blocking buffer[PBS containing 0.5 % non-fat dried milk (Nutricia) and 0.1 % Tween

20 (Merck)]. The sera were diluted 1 : 5000 or 1 : 20 000 in the same

buffer and applied for 1 h to the blots. After washing the blots four times

for 5 min with blocking buffer, the blots were incubated with goat anti-

rabbit IgG conjugated to horseradish peroxidase (Biosource) diluted

1 : 10 000 in blocking buffer for 1 h. Binding of antibodies was

visualized by chemiluminescence using an ECL kit (Amersham).

Antisera. The antisera against the TD of NalP (Oomen et al., 2004) and

against IgA protease of strain HF13 (Vidarsson et al., 2005) have been

described previously. To raise antisera against the a-peptide and TD of

IgA protease of strain H44/74, recombinant proteins were purified from

IPTG-induced E. coli BL21(DE3) carrying plasmids pET_IgA-APH44

and pET_IgA-TDH44 as described previously (van Ulsen et al., 2003),

and sent to Eurogentec to raise the polyclonal rabbit antisera.

Antibodies specifically recognizing the a-peptide of IgA protease were

further purified from the anti-IgA protease a-peptide antiserum by

affinity purification. Recombinant a-peptide was run on a 10 % SDS-

PAGE gel and electroblotted. The a-peptide band was localized on the

blot by staining in 0.1 % Ponceau S/5 % acetic acid and cut out. The

blot slice was pre-incubated in blocking buffer and then incubated for

1 h with antiserum, diluted 1 : 100 in blocking buffer. After extensive

washing, the specifically bound antibodies were eluted by incubating

the slice with 0.2 M glycine (pH 3.0) for 5 min. The elution buffer

was neutralized by adding 50 ml 1 M Tris/HCl (pH 8.0) and then

diluted 10-fold in blocking buffer before use.

Immunofluorescence microscopy. Exponential-phase cultures

were centrifuged at 4500 g for 5 min and the cells were prepared

for immunofluorescence microscopy as described previously (van

Ulsen et al., 2006).

Protease accessibility assays. Bacterial cells were harvested by

centrifugation (7000 g, 10 min). Pellets were washed with PBS

Variable processing of neisserial IgA protease

http://mic.sgmjournals.org 2423

(pH 7.6) and resuspended in 10 mM Tris/HCl (pH 7.6) and 10 mMMgCl2, to OD600 1.0, and incubated on ice for 10 min. Aliquots of500 ml of the cell suspensions were incubated with 20 mg trypsin ml21

or 20 mg proteinase K ml21 (Roche) for 30 min on ice. Proteaseactivity was stopped by adding 5 mg PMSF ml21 (Sigma) and furtherincubation for 30 min on ice. Cells were then harvested bycentrifugation and analysed by immunoblotting.

Cell envelope preparation. Cells were harvested by centrifugation(7000 g, 10 min) and the pellet was frozen at 220 uC for at leastovernight. After thawing, the pellet was resuspended in 50 mM Tris/HCl (pH 8.0) and 2 mM EDTA, and then disrupted by sonication for265 min in a Branson sonifier at full power whilst on ice with 5 minbetween the steps. Lysates were cleared from unbroken cells bycentrifugation (1400 g, 10 min). The membrane fraction (mainlyouter membranes) was pelleted by centrifugation (8 min, 100 000 g)and resuspended in 5 mM Tris/HCl (pH 7.6).

Heparin affinity chromatography. The binding of the a-peptide ofIgA protease to heparin was analysed using a 1 ml HiTrap heparinHP column (GE Healthcare). Aliquots of 50 ml culture super-natants of HB-1 and HB-1iga : : kan were dialysed twice against 1 l10 mM sodium phosphate (pH 7.0) at 4 uC for 2 days. Then,100 mg DNase I (Sigma) ml21 was added, and the sample wasincubated for 3 h at 37 uC and then centrifuged at 13 000 g for10 min at 4 uC. The resulting supernatant was loaded onto a pre-washed heparin column and the columns were washed with 40 volsphosphate buffer (pH 7.2). Finally, the heparin-bound protein waseluted with phosphate buffer containing 1.0 M NaCl. The proteincontent of the culture supernatant and of fractions from the heparincolumn were concentrated by TCA precipitation and analysed byimmunoblotting.

RESULTS

Fate of the a-peptide of IgA protease in a nalPmutant of strain HB-1

Approximately equal amounts of two forms of IgA proteaseare released into the extracellular medium of strain HB-1 –an unencapsulated derivative of H44/76. The smaller formof ~110 kDa, representing the IgA protease passengerwithout the a-peptide attached, is generated by autopro-teolytic activity, whilst the larger variant with an apparentmolecular mass of ~160 kDa in SDS-PAGE gels wasgenerated by NalP activity and represents the proteasedomain with attached a-peptide (van Ulsen et al., 2003).The calculated molecular mass of the larger variant is only~136 kDa, but the aberrant electrophoretic mobility islikely caused by the unusually high number of chargedresidues in the a-peptide rendering it very basic (Pohlneret al., 1995). To determine the fate of the a-peptide afterautocatalytic processing, we analysed whole-cell lysates andculture supernatants of strain HB-1 and its nalP : : kan andiga : : kan mutant derivatives. Immunoblots incubated withan antiserum directed against the TD revealed a band of~34 kDa in the whole-cell lysate of HB-1 (Fig. 2a). Thispolypeptide was much smaller than the TD of IgA proteaseof the gonococcal strain MS11, which has a calculated massof 45 kDa (Pohlner et al., 1987), but corresponded in sizeto the b-core of that TD (Klauser et al., 1993). In contrast,in the lysate of the nalP mutant, a ~72 kDa band was

detected with the same antiserum as well as with anantiserum directed against the a-peptide (Fig. 2a). Thisband therefore most likely represented a polypeptide con-sisting of the a-peptide fused via the linker peptide to theb-core, which together has a calculated molecular mass of62 kDa. The faint ~170 kDa band detected with the a-peptide antiserum (Fig. 2a, right panel) likely was full-length unprocessed IgA protease present in the cell lysates.When we complemented the nalP mutant with plasmidpEN300, carrying nalP under control of a lac promoter(van Ulsen et al., 2003), the separate b-core fragment wasdetected in cell lysates after induction of NalP synthesiswith IPTG, whereas the non-induced cells showed the~72 kDa TD with the a-peptide attached (Fig. 2b).

Next, two other N. meningitidis strains, i.e. 2996 andB16B6, and their nalP : : kan mutant derivatives wereanalysed. As in strain HB-1, a band of ~33–34 kDa thatreacted only with the antiserum against the TD wasdetected in whole-cell lysates of the WT strains, whereas a~73–75 kDa band was detected in the cells of the nalP-mutant derivatives that reacted with both antisera directedagainst the TD and against the a-peptide (Fig. 2c). In theB16B6 nalP : : kan mutant, an additional ~37 kDa bandwas detected with the antiserum directed against the TD(Fig. 2c); this band might have resulted from processing byanother protease present at the cell surface. Unexpectedly, afaint band of ~75 kDa in the lysate of WT 2996 reactedwith the anti a-peptide serum, suggesting that NalP mightbe less active in this strain or switched off by phasevariation in a portion of the cells. The expression of nalPin strain 2996, indeed, appeared relatively low on immu-noblot (Fig. S1a), but analysis by immunofluorescencemicroscopy indicated that this was not due to phasevariation in a part of the population, as most cells appearedto express the protein (Fig. S1b). Taken together, theseresults indicated that the a-peptide of IgA proteaseremained fused to the TD when NalP was not expressed.

In addition to IgA protease, NalP also processes theautotransporters App and AusI (van Ulsen et al., 2003,2006). The passenger of App also includes an a-peptide. Toanalyse the fate of the a-peptide of App in the absence ofNalP, similar immunoblotting experiments were per-formed on whole-cell lysates of HB-1 and its nalP : : kanand app : : kan mutant derivatives as described above forIgA protease. The results showed the accumulation of a~60 kDa band that reacted with both antisera directedagainst the TD and against the a-peptide of App (Fig. S2),indicating that the a-peptide of App also remainedattached to the TD in absence of NalP.

Variations in autocatalytic processing of IgAprotease between meningococcal strains

The results for the N. meningitidis strains described abovedeviated from those described for the IgA protease of N.gonorrhoeae MS11, whose TD after passenger release wasreported to be ~ 45 kDa and to consist of two subdomains:

the linker peptide of ~14 kDa and the b-core of ~34 kDa(Klauser et al., 1993; Pohlner et al., 1987). To verify thoseresults, we analysed the membrane-associated IgA proteasedomains of two gonococcal strains, FA1090 and MS11.Blots containing cell envelopes of these strains wereincubated with antiserum directed against the TD andrevealed bands of ~45 and ~50 kDa, respectively (Fig. S3),in agreement with the calculated molecular masses of 40and 45 kDa, respectively. These bands did not react withthe anti-a-peptide antiserum (results not shown).

The differences in processing of IgA protease between N.meningitidis and N. gonorrhoeae can be explained only inpart by the absence of a functional NalP in N. gonorrhoeae(van Ulsen et al., 2001; van Ulsen & Tommassen, 2006).Whilst this absence may explain why the linker peptide isnot released from the b-core in the IgA protease of N.gonorrhoeae, it fails to explain the observation that the a-peptide remained fused to the TD in nalP mutants of N.meningitidis. IgA protease of N. gonorrhoeae was reportedto contain three autocatalytic processing sites, i.e. betweenthe protease domain and the c-peptide, between the c-peptide and the a-peptide, and between the a-peptide andthe linker peptide (Fig. 1). Inspection of the sequence of IgAprotease from strain H44/76 (Budroni et al., 2011; Piet et al.,2011) revealed that the autocatalytic cleavage site betweenthe a-peptide and the linker peptide was missing (Fig. S4).This explained why the a-peptide remains covalentlyassociated with the TD when nalP was not expressed.

Analysis of full-length IgA protease sequences in theGenBank database (n551) revealed a very high sequencediversity that concentrated in the a-peptide and linker

regions of the proteins (see Fig. S4 for examples and TableS2 for a list of sequences used). This variability includeddifferences in the numbers of NLSs, and repeats upstreamof and in between the NLSs, as well as in the linker. Basedon this variability, four different subtypes of a-peptidescould be distinguished (see Fig. 1b for a schematic repre-sentation), with limited variation within each subtype. Thevariability also included the presence or not of anautocatalytic cleavage site between the a-peptide and thelinker peptide. This cleavage site was present in the IgAproteases of all N. gonorrhoeae strains (n513), as well as ofmany N. meningitidis strains analysed (n525/38), includ-ing those from the sequenced genomes of MC58 and Z2491(Fig. S4). However, this site was not present in 13 of 38meningococcal isolates, including H44/76, B16B6 andFAM18, which were therefore all expected to have in theirmembranes a TD that included both the linker and a-peptide when nalP was in phase ‘‘off’’.

The sequence analysis suggested that many meningococcalstrains, similar to N. gonorrhoeae, should contain a TDconsisting of the b-core and linker, but lacking the a-peptide when NalP is not expressed. To confirm thispossibility, we analysed in total 20 N. meningitidis isolatesof which we had previously determined the nalP expressionstatus (phase ‘‘on’’ or ‘‘off’’) by sequencing of the poly-cytosine stretch where slipped-strand mispairing wasexpected to occur (van Ulsen et al., 2006; Table S1), andby immunoblotting and immunofluorescence microscopy(see Fig. S1 for examples). Immunoblots containing whole-cell lysates of these strains were then probed withantiserum against the TD of IgA protease. In 12 out of

(a) (b) (c)

HB-1 naIP::kan+pEN300

naIP– + – +– + – + – + – +

-1 iga:

150100

Anti-lgA TD anti-lgA α P Anti-lgA TD Anti-lgA α P Anti-lgA TD Anti-lgA α P

Fig. 2. The a-peptide of IgA protease remains fused to the TD in the absence of NalP. (a) Immunoblots of whole-cell lysates ofN. meningitidis HB-1 and its nalP : : kan and iga : : kan mutant derivatives incubated with antiserum directed against the TD(anti-IgA TD) or the a-peptide (anti-IgA aP) of IgA protease. (b) Similar analysis of lysates of the nalP : : kan mutant carryingpEN300. Expression of nalP from pEN300 was either induced with IPTG (+) or not (–) as indicated. (c) Immunoblots of whole-cell lysates of N. meningitidis strains B16B6 and 2996 and their nalP : : kan mutant derivatives. The positions of molecular massmarkers are indicated (in kDa).

the 13 NalP+ strains examined, a ~34 kDa band corres-ponding to the b-core was detected (see Fig. 3a forexamples), consistent with the expected release of thelinker and a-peptide. The exception was strain M992,which did express NalP (Fig. S1) but, nevertheless, showeda predominant ~75 kDa band corresponding to the TDassociated with the a-peptide, as well as bands of ~33–35 kDa that appeared to represent the expected b-core.Apparently, the strain encoded an IgA protease variant thatwas less-well recognized by NalP.

Three out of the seven meningococcal strains that did notexpress nalP revealed a major band of ~75 kDa reacting withthe anti-TD antiserum, as in the nalP mutant of strain HB-1(i.e. strains 35E, FAM18 and M990; see Fig. 3a). StrainFAM18 additionally showed a prominent 37 kDa band (Fig.3a), as the nalP mutant of strain B16B6 did (Fig. 2c); thesetwo strains are of the same clonal complex, i.e. cc11.Interestingly, four nalP phase ‘‘off’’ strains, i.e. S3446, 881710,13077 (Fig. 3a) and 881607 (result not shown), showed apredominant 45–50 kDa band, similar to N. gonorrhoeaestrains MS11 and FA1090, suggesting the presence of anadditional autocatalytic processing site between the a-peptideand linker of the IgA proteases of these strains. The sequenceof the corresponding DNA segments confirmed the presenceof this additional autocatalytic cleavage site downstream ofthe a-peptide (Fig. S5). We also analysed the correspondingsegment of strains 2996 and B16B6, which showed a ~75 kDaband similar to HB-1 (Fig. 2c), and of strain M992, whichshowed this band even when it expressed NalP (Fig. 3a). Asexpected, these sequences lacked the additional autocatalyticcleavage site (Fig. S5).

Blots were also probed with the antiserum against the a-peptide (Fig. 3b). This antiserum recognized ~72–75 kDabands in the strains that were positive for a similarly sizedband using the antiserum directed against the TD, whichconfirmed that this band corresponded to the TD withassociated a-peptide. In addition, the antiserum recognizeda band of ~49 kDa in the lysates of nalP phase ‘‘off’’ strainsthat possessed an IgA protease with a confirmed additionalautoproteolytic cleavage site between the a-peptide and thelinker peptide (strains S3446, 881710 and 13077 in Fig. 3b;see also Fig. S5). This band, therefore, must represent thecleaved a-peptide that is of similar size in all these strains(calculated at 44 kDa) and contains four NLSs (Fig. S5).Apparently, these a-peptides were not released into themedium after they are autocatalytically cleaved from theTD, but remained non-covalently associated to the cellsurface. The cleaved a-peptide was also detected in the celllysate of the NalP-expressing strains MC58 and 2208, but,for unknown reasons, not in those of strains 892557 andM981 (Fig. 3b). In the IgA proteases that did not containan autoproteolytic cleavage site between the a-peptide andthe linker peptide, such as the IgA proteases of strains HB-1, B16B6 and 2996, a fragment consisting of the a-peptideand the linker was expected to be the result of a combinedautocleavage and NalP-mediated cleavage. Such a peptidewas not detected with the antiserum directed against the

a-peptide in the cell lysates of these strains (Fig. 2a, c).However, examination of the culture supernatant of strainHB-1 revealed the presence of this peptide, which migrated

+ + + + + +– – – – – – – NaIP

Anti-lgA TD

881710

892557

passenger+α+linker

passenger

Anti-lgA protease

Anti-lgA α P

α+linker

+ + + + +– – – – – – – NaIP

Anti-lgA α P

-1 naIP:

-1 iga:

881710

892557

Fig. 3. Different compositions of the membrane-associated IgAprotease fragments in N. meningitidis isolates. (a, b) Immunoblots ofisolated cell envelopes of a panel of 11 N. meningitidis strains thatexpressed nalP or not (as indicated above the blots). The blots wereprobed with antisera directed against the TD (anti-IgA TD; a) or thea-peptide (anti-IgA aP; b) of IgA protease. Whole-cell lysates of HB-1 and HB-1 nalP : : kan were added as controls. (c) Immunoblots ofconcentrated culture supernatants incubated with antisera directedagainst the passenger (top) or the a-peptide (bottom) of IgAprotease. Detected protein species are indicated. A fragment thatcorresponded to the a-peptide plus linker was detected in thesupernatant of HB-1 and not in that of HB-1 nalP : : kan.

at ~40–42 kDa (Fig. 3c), which was higher than the calcu-lated ~28 kDa, as observed for other a-peptide-containingbands. Interestingly, the a-peptides of the IgA proteases inthese strains contained only a single NLS (Fig. S5).

Taken together, our results indicate clearly that in N.meningitidis the membrane-associated IgA protease frag-ment that remains after the release of the passenger canexist in three different forms: (i) the ~34 kDa b-coreresulting from NalP-mediated processing, (ii) the 45–50 kDa TD consisting of the b-core with the linkerattached or (iii) the ~75 kDa form consisting of the TDwith linker, c- and a-peptides attached. The latter twoforms resulted from autoproteolytic cleavage. In addition,in several strains the autoproteolytically cleaved loose a-peptide remained non-covalently cell-associated.

Membrane-associated IgA protease fragmentsare stable and extend from the cell surface

The detection of the TD of IgA protease in its variousforms on immunoblots suggested that these fragmentswere not rapidly degraded and, therefore, could be func-tionally relevant. To verify their stability, exponentiallygrowing cultures of HB-1 and its nalP : : kan derivative wereincubated with chloramphenicol to block further proteinsynthesis, and the possible degradation of the TDs wasassessed over a time period of 4 h (Fig. 4a). The amountsof b-core and TD with a-peptide attached detected on blotsdid not decrease significantly within this period, confirm-ing their high stability in the outer membrane. In the nalPmutant, the a-peptide of IgA protease remained attached tothe TD during the incubation period, demonstrating that itwas not rapidly removed by other proteases. Similar experi-ments using antibodies against the TDs of App and NalPshowed that their TDs were also stable within the 4 h timeperiod, whilst the a-peptide of App also remained attachedto the TD in a nalP knockout background (Fig. S6).

When the a-peptide remained attached to the TD, it wasexpected to extend from the cell surface to interact withenvironmental factors. Cell-surface localization of the a-peptide of IgA protease on cells of HB-1 nalP : : kan wasconfirmed by immunofluorescence microscopy using theantiserum against the a-peptide (Fig. 4b). Furthermore,both the a-peptide attached to the TD as found in HB-1nalP : : kan in the absence of NalP and the cleaved and cell-associated a-peptide as found in MC58 were sensitiveto externally added proteinase K and trypsin (Fig. 4c andresults not shown), indicating their presence on the surfaceof the cells. Additionally, the externally added proteasesreduced the TDs to the size of the b-core.

a-Peptide of IgA protease binds heparin

The a-peptide of IgA protease was suggested recently to beinvolved in biofilm formation by binding extracellularDNA (Arenas et al., 2013). As DNA-binding proteins cangenerally be purified by affinity chromatography on

heparin columns, we considered the possibility that thea-peptide could also bind heparin. Concentrated culturesupernatants of strain HB-1 and its iga : : kan mutantderivative were applied to a heparin column, and thebound proteins were eluted and analysed by immunoblot-ting using the antiserum against the a-peptide (Fig. 5a).Indeed, the a-peptide was found to bind to the column andcould be eluted with high salt along with some smallerfragments that presumably represented degradation pro-ducts (Fig. 5a). Although the culture supernatant of strainHB-1 also contained a complete form of the IgA proteasepassenger that was released by NalP and consisted of theprotease domain, the a-peptide and the linker peptide (vanUlsen et al., 2003), this form was not detected in the eluateof the heparin column, most likely because the a-peptidewas released from the protease domain by autoproteolysisduring the time-consuming dialysis steps prior to applica-tion on the heparin column. Indeed, we observed that thea-peptide-containing version of the IgA protease passengerwas further processed into its separate subdomains duringprolonged incubations of supernatant fractions (Fig. S7).

TD-associated domains are subject to cleavage byserum components

In the course of experiments in which we investigated theinteraction of N. meningitidis with cultured eukaryotic celllines, we noticed that the culture medium could affect themembrane-associated domains of IgA protease. In particu-lar, we detected the presence of a ~43 kDa band in whole-cell lysates of B16B6 nalP : : kan and HB-1 nalP : : kan insteadof the expected band of ~75 kDa when these strains weregrown in RPMI medium supplemented with 5 % FCS, i.e.the medium used routinely in cell culture experiments (Fig.5b). This 43 kDa band was not a cross-reacting band derivedfrom the medium, as it was not detected in a B16B6nalP : : cam iga : : kan double mutant (Fig. 5b). The presenceof FCS in the culture medium appeared responsible for thegeneration of the 43 kDa band, as the expected 75 kDa bandrepresenting the a-peptide fused to the TD was observedafter growth in RPMI without FCS. Furthermore, samples ofcells grown in RPMI supplemented with FCS that was heat-inactivated for 30 min at 56 uC did show the 75 kDa bandalong with the 43 kDa band (Fig. 5b) and prolongedincubation of FCS at 56 uC to .1 h restored the 75 kDaband as the predominant band, whilst the 43 kDa bandwas barely detectable any more (result not shown). Similarresults were obtained when human serum instead of FCSwas used (results not shown). Thus, serum appeared toinclude a heat-labile proteolytic enzyme that cleaved the cell-associated TD plus a-peptide fragment of IgA proteasewithin the a-peptide and/or the linker peptide.

DISCUSSION

Expression in N. meningitidis strain H44/76 of the phase-variable autotransporter NalP results in the release of IgA

protease passengers extended with the a- and c-peptides(van Ulsen et al., 2003), and with the linker peptide (thisstudy). We also show here that a 34 kDa fragment remainsin the cell envelope. This fragment corresponds to the b-core (Klauser et al., 1993) consisting of a 12-stranded b-barrel and an a-helix that plugs its channel (Roussel-Jazedeet al., 2011). In the absence of NalP, a shorter variant ofthe IgA protease passenger is released via autocatalyticcleavage. Our results further show that the sizes and com-position of the secreted and membrane-bound domains ofIgA protease vary depending on the N. meningitidis strainanalysed. The secreted proteins may consist of the extendedpassenger, or the passenger, c-peptide and a-peptide, withor without linker peptide attached, as separate proteins.The membrane-bound protein may consist of either a TDas found in N. gonorrhoeae MS11, i.e. the b-core extendedwith the linker peptide, or as the separate b-core, or as theb-core extended with the linker and the a-peptide. Whichvariant exists is determined by the presence or absence ofautocatalytic processing sites, as well as the expressionstatus of the phase-variable nalP gene. The secreted andcell-associated domains of the autotransporter App showeda similar dependence on nalP expression (van Ulsen et al.,2003) (Fig. S2). Furthermore, the different autotransporterdomains that remain in the membrane appear very stable(Figs 4 and S6) and, therefore, mechanisms must exist toprevent the membrane from being filled up with theseprotein fragments. Possibly, N. meningitidis maintains a

fine balance between autotransporter production and thedilution of accumulated TDs by cell division. However, theorganism is also well known for its abundant release ofouter membrane vesicles. Thus, an alternative mechanismcould be that these also release the redundant outer-membrane-associated autotransporter fragments. Such amechanism is supported by a proteomics analysis thatindicated that autotransporter proteins were enriched inouter membrane vesicles over isolated membrane fractions(Lappann et al., 2013).

The NalP-mediated release of the a-peptide from the cellsurface could be a protective measure (Roussel-Jazedeet al., 2010). The a-peptide of IgA protease can elicitresponses of human T-cells and thus activate the immunesystem (Jose et al., 2000). Consistently, the a-peptide andlinker peptide are the most variable parts of IgA protease(Figs 1b, S4 and S5) – a feature often associated withimmunodominance. The NalP-mediated release of thesefragments from the cell surface could help meningococcito escape the immune response. In the absence of NalP,removal of the a-peptide occurs autocatalytically in manystrains of N. meningitidis and N. gonorrhoeae, due to thepresence of a processing site between the a-peptide andlinker. Arguing against an immunoprotective function ofthe release, either autocatalytically or via NalP, is perhapsour observation that the released a-peptide remained non-covalently associated with the cells in some meningococci,

(a) (b)

Time (h)

-1 naIP:

Anti-lgA TD

TD+linker+α

β -core

HB-1 naIP::kan

HB-1 iga::kan

100 50

– – + + Proteinase K

TD+linker+α

β -core

TD+linker

Anti-lgA TD

Anti-Fbp

Anti-lgA α P50

Fig. 4. Stability and surface exposure of membrane-associated autotransporter fragments. (a) Cells of N. meningitidis strainHB-1 and its nalP : : kan mutant derivative were incubated for the indicated time periods in the presence of 10 mgchloramphenicol ml”1. Cell envelopes were isolated and separated by SDS-PAGE and blotted. Blots were probed with anti-IgATD. (b) Immunofluorescence microscopy of cells of HB-1 and its nalP : : kan and iga : : kan derivatives using anti-IgA a-peptide(aP) antiserum and Alexa-labelled goat anti-rabbit IgG (green) as secondary antibody. Propidium iodine (red) was used as acounter-stain to detect the cells. (c) Cells of the nalP : : kan mutant derivative of HB-1 and of strain MC58 were treated withexternally added proteinase K. Immunoblots of whole-cell lysates were incubated with antisera directed against the TD and thea-peptide of IgA protease, and with an antiserum directed against the periplasmic protein FbpA to check the integrity of thecells. The protein species are indicated.

indicating that complete release from the cell surface maynot be that important.

If the a-peptides are highly immunogenic, their ubiquitouspresence in the IgA proteases from N. meningitidis and N.gonorrhoeae implies an important role in bacterial physi-ology or virulence. The a-peptide contains NLSs and thereporter proteins fused to the a-peptide of IgA proteasefrom N. gonorrhoeae have been shown to target the nucleusof transfected cell lines (Pohlner et al., 1995), althoughfunctional implications of targeting IgA protease itself tothe nucleus remain to be demonstrated. Recently, weshowed that the a-peptide of meningococcal IgA proteaseis involved in biofilm formation, presumably by binding

extracellular DNA (Arenas et al., 2013). NLSs are char-acterized by their high number of positively chargedresidues that could explain their affinity for DNA or othernegatively charged surfaces. In this study, we showed thatthe a-peptide is able to bind heparin, which is negativelycharged as well. Heparin is a known ligand for bacterialadhesins (Duensing et al., 1999; Menozzi et al., 2002). Italso interacts with proteins that regulate the complementsystem, including factor H, C4b-binding protein and C1inhibitor (Yu et al., 2005), and attaching these factors viaheparin to the bacterial cell surface may enhance serumresistance (Duensing et al., 1999; Menozzi et al., 2002). N.meningitidis contains multiple heparin-binding proteins,including the Neisseria heparin-binding antigen NhbA,whose recruitment of heparin via an arginine-rich proteinsegment that resembles the NLSs in the a-peptides of IgAproteases has been shown to result in increased serumresistance (Serruto et al., 2010). Furthermore, heparinbound via the a-peptide to the bacterial surface might bindvitronectin, thus allowing for uptake of the bacteria intohost cells expressing vitronectin receptors (Duensing et al.,1999). In conclusion, the a-peptide of IgA protease appearsto have various functions dependent on whether it isreleased in association with the IgA protease into themilieu or whether it remains associated with the bacterialcell surface. Interestingly, several gonococcal strains, suchas strains MS11 and 1291, contain an extra NLS in thelinker region (Fig. 1b), which, in the absence of afunctional NalP, is never released into the medium of N.gonorrhoeae. Similarly, Haemophilus influenza IgA pro-teases (Poulsen et al., 1992) show a linker peptide sequencewith one or two potential NLSs that remains attached tothe b-domain. These observations underscore the potentialimportance of having these positively charged peptides onthe cell surface.

Sequence analysis of the a-peptides of 13 gonococcal and38 meningococcal strains revealed that they contain eitherone or four NLSs (Figs S4 and S5). In the meningococcalIgA proteases, all a-peptides that contain four NLSs arefollowed by an autoproteolytic cleavage site between the a-peptide and the linker domain. We observed that many ofthese a-peptides remained nevertheless associated asseparate polypeptides with the bacterial cell surface (Fig.3). We hypothesize that in these cases the a-peptides bindthe negatively charged lipo-oligosaccharides via their N-terminal NLS, whilst the C-terminal NLS remain availableto bind extracellular DNA or heparin. All meningococcalIgA proteases containing a single NLS are not followed byan autocatalytic processing site. Thus, these a-peptidesremain covalently associated to the cell surface via the TDunless NalP is expressed. The latter mechanism offersthe possibility to escape from the immune system whenantibodies against the a-peptide are elicited and/or toescape from biofilms in order to colonize new host tissues.Importantly, we observed that the TD-linked a-peptideswere removed from the cell surface by proteases present inserum (Fig. 5b). Therefore, this form of the protein

TD+linker+α

43-kDa

fragment

β -core

Anti-lgA TD

B16B6 HB-1

iganaIP+

+––

–––

–– +

150 lgA protease

Anti-lgA α P

α-peptide

lgA P + + + + + + ––

Fig. 5. Interaction of the IgA protease a-peptide with hostcomponents. (a) Concentrated culture supernatants of strainHB-1 and its iga : : kan mutant derivative were dialysed and loadedonto HiTrap heparin HP columns to detect binding. Variousfractions of the isolation procedure were analysed by immunoblot-ting with antiserum directed against the a-peptide (aP). First andsecond wash represent fractions obtained after washing thecolumn with 5 and 40 vols of buffer, respectively. The positions offull-length IgA protease and the a-peptide are indicated. (b) FCScomponents cleave the cell-associated a-peptide from the TD.Immunoblots of whole-cell lysates of N. meningitidis strainsB16B6 (left panel) and HB-1 (right panel) and their nalP : : kan

and nalP : : cam iga : : kan mutant derivatives were grown in RPMI,RPMI supplemented with 5 % FCS (+FCS) or RPMI supplemen-ted with FCS that had been heat-inactivated for 30 min at 56 6C(+HI FCS). Whole-cell lysates were analysed with antiserumdirected against the TD of IgA protease.

presumably has a role only during the colonization of thenasopharynx, and not after crossing the epithelial layer andreaching the bloodstream.

In conclusion, we demonstrate for the first time that the a-peptide of IgA protease of N. meningitidis can remainassociated with the bacterial cell surface either covalentlylinked via the TD or non-covalently associated as a separatepolypeptide. In this location, it may exert new functionssuch as stimulating biofilm formation by binding extra-cellular DNA (Arenas et al., 2013) or by binding heparin, asdemonstrated in this study.

ACKNOWLEDGEMENTS

We thank Willem ten Hove for plasmid construction. This work

was supported by the Research Council for Chemical Sciences (CW)

with financial aid from the Netherlands Organization for Scientific

Research (NWO) and by the Netherlands Organization for Health

Research and Development (ZonMw).

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Edited by: G. Thomas

Variable processing of the IgA protease autotransporter at the cell surface of Neisseria...

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