Amino-acid sequences encoded by the autB genes were predicted after alteration of the number of AAGC repeat units to create the correct reading frame and, in some cases, by removal of frameshift mutations and premature stop codons. The cleavage site of the N-terminal signal sequence was predicted using publicly available web tools (http://www.cbs.dtu.dk/services/SignalP/). The mature protein was then used for phylogenetic analysis using the neighbor-joining with bootstrap analysis (100 replications) considering distances based on p-distance with the available MEGA software version 6.0 (http://www.megasoftware.net/) (Tamura et al., 2013). For secondary and tertiary structure predictions, the public web-based programs PsiPred (Buchan et al., 2013) and PHYRE2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index), respectively, were used. Alignment of protein sequences was performed in MAFFT version 7 (http://mafft.cbrc.jp/alignment/server/). AutB variants were defined based on phylogeny of the N-terminal part of the passenger. Each variant was considered when the bootstrap value of the precedent node was 100, the distance within the group was lower than the overall distance for AutB, and the distance from other groups higher than the overall distance for AutB.

Meningococcal strains used in this study include reference strains MC58 (Tettelin et al., 2000), FAM18 (Bentley et al., 2007), α14 (Schoen et al., 2008), and α153 (Schoen et al., 2008). HB-1 (Bos and Tommassen, 2005) and BB-1 (Arenas et al., 2013a) are unencapsulated derivatives of H44/76 and B16B6, respectively. A panel of 102 meningococcal strains isolated from patients suffering from meningococcal disease in the Netherlands was already described (Arenas et al., 2015). All meningococcal strains were grown at 37°C on GC medium base (Difco) supplemented with IsovitaleX (Becton Dickinson) at 37°C in a candle jar overnight. To grow the bacteria in liquid cultures, bacteria were collected from GC plates and diluted in tryptic soy broth (TSB) (Beckton Dickinson) to an OD₅₅₀ of 0.1 and incubated in 25-cm² polystyrene cell culture flasks or 125-ml square media bottles with constant shaking at 110 rpm. Escherichia coli strains used in this study were DH5α and BL21(DE3) (Invitrogen), which were grown in Lysogeny broth (LB) or LB agar at 37°C.

For all strains, media were supplemented with the antibiotics kanamycin (100 μg ml⁻¹), chloramphenicol (25 μg ml⁻¹) or ampicillin (100 μg ml⁻¹) when required and with 0.1 mM of isopropyl-β-D-1-thiogalactopyranoside (IPTG) to induce gene expression.

Segments of autB were amplified by PCR from bacteria grown on plates with primers listed in Table S1 using High Fidelity Polymerase (Roche Diagnostics GmbH, Germany) or DreamTaq-DNA Polymerase (Fermentas, UK). PCR products were visualized in agarose gels stained with ethidium bromide. When required, they were purified with the PCR Clean-Up System (Promega Corporation) and sequenced at the Macrogen sequencing service (Amsterdam). Large sequences were assembled using the SeqMan II software (DNAstart) using at least two independent PCR reactions.

For cloning, PCR fragments were obtained from DNA of strain HB-1 using primers described in Table S1. PCR fragments were purified and digested with restriction enzymes (Fermentas, UK) for which sites were included in the primers, to be subsequently cloned in appropriate vectors. To generate an autB knockout construct, PCR fragments from immediately upstream and downstream of the autB gene were cloned into pKOnhbA-kan (Arenas et al., 2013a). In the resulting plasmid, called pKOautB-kan, these segments flank a kanamycin-resistance cassette.

To generate a plasmid for the production of a part of the passenger domain of AutB of strain HB-1 in E. coli, the corresponding PCR product was cloned into pET16b (Invitrogen) resulting in plasmid pET16b-AutBp. To generate plasmids for expression of AutB variants in N. meningitidis, the entire autB genes from isolates 2081107 and α153 were amplified and cloned into plasmid pFPIORF₁ (Arenas et al., 2013b), resulting in pENAutB₁ and pENAutB₂, respectively. N. meningitidis and E. coli were transformed with intact or linearized plasmids following standard protocols (Arenas et al., 2015) and transformants were selected on solid medium supplemented with appropriate antibiotics. The proper generation of knockout mutants or the presence of the plasmid was verified by PCR. In addition, protein synthesis was confirmed by Western blotting or gene expression was confirmed by RT-PCR as described below.

A fragment of the AutB passenger domain of strain HB-1 was purified as described (Arenas et al., 2015). Briefly, the recombinant polypeptide, corresponding to amino-acid residues 151–278 of the mature AutB protein, with an N-terminal His-tag was produced in E. coli BL21(DE3) carrying pET16b-AutBp after induction with IPTG and purified as inclusion bodies. The protein band corresponding to the recombinant protein was excised from SDS-polyacrylamide gel electrophoresis (SDS-PAGE) gels and used for the production of rabbit antiserum at Eurogentec (Liège, Belgium). The concentration of the purified protein was determined with the BCA assay kit (Thermo Fisher Scientific, Rockford, IL, USA) and its purity was estimated in SDS-PAGE gels.

To obtain RNA, cells from exponentially growing cultures were collected by centrifugation for 10 min at 5000 rpm in an Eppendorf Centrifuge 5424, adjusted to an OD₅₅₀ of 4, and resuspended in trizol (Invitrogen, U.K.). Then, 200 μl of chloroform were added per ml of trizol, followed by centrifugation at 5000 rpm for 30 min. The resulting upper layer was mixed with an equal amount of ice-cold 75% ethanol. Next, RNA was isolated using the Nucleospin RNA II kit (Macherey-Nagel, U.S.A.) according to the manufacturer's instructions. The resulting solution was treated with Turbo DNA free (Ambion, Germany) for 1 h at 37°C to remove genomic DNA followed by inactivation of the DNase according to the recommendations of the manufacturer. The resulting pure RNA was used immediately to generate cDNA using the Transcriptor High Fidelity cDNA Synthesis Kit (Roche, The Netherlands). RNA, cDNA, and chromosomal DNA were used as templates in PCRs to determine the generation of specific transcripts with primers listed in Table S1. PCRs started with an incubation for 5 min at 95°C, followed by 30 cycles of 30 s at 95°C, 30 s at 65°C and 10 s at 72°C. Reactions ended with 10 min of incubation at 72°C.

Whole cell lysates, supernatants and cell envelopes were prepared and adjusted as previously described (Arenas et al., 2015). Protein concentrations were determined with the BCA assay kit. Before SDS-PAGE, samples were diluted 1:1 in double-strength sample buffer and heated for 10 min at 100°C. Proteins separated on gels were stained with Coomassie brilliant blue G250 or transferred to nitrocellulose membranes. These membranes were next blocked with phosphate-buffered saline (PBS) containing 0.1% (v/v) Tween 20 and 0.5% (w/v) non-fat dried milk (PBS-T-M), and then incubated with primary antibodies and subsequently with horseradish peroxidase-conjugated goat anti-rabbit IgG or anti-mouse IgG antibodies (Biosource International), as previously described (Arenas et al., 2006). All incubations were performed for 1 h and followed by three washes for 15 min with PBS-T-M. Blots were developed with the Pierce ECL Western Blotting Substrate. The monoclonal antibody MN2D6D directed against RmpM and the antiserum directed against fHbp were generously provided by the Netherlands Vaccine Institute (Bilthoven, The Netherlands) and by GlaxoSmithKline (Rixensart, Belgium), respectively. The antisera directed against the α-peptide and the translocator domain of IgA protease were from our laboratory collection (Roussel-Jazédé et al., 2014).

Proteinase K accessibility assays were performed as described (Arenas et al., 2015) with few modifications. Briefly, bacteria recovered from a culture grown for 4 h in TSB were adjusted to an optical density at 550 nm (OD₅₅₀) of 1 and incubated with 2 μg ml⁻¹ of proteinase K (Fermentas) for 1 h at 37°C, after which the protease was inactivated with 2 mM phenylmethylsulfonyl fluoride Sigma-Aldrich). Cells were harvested by centrifugation and protein degradation was examined by SDS-PAGE and Western blotting.

For biofilm formation and settling experiments, bacteria were initially grown in TSB with or without IPTG and adjusted to a similar OD₅₅₀. Settling experiments were performed as described (Arenas et al., 2015). Biofilm formation was analyzed under static conditions in polystyrene plates and in a flow-cell model as described before (Arenas et al., 2013a, 2015), with some modifications. Where indicated, 100 μg ml⁻¹ of DNase I was added to the cultures during biofilm formation. For the flow-cell model, pictures were taken at different time intervals during biofilm formation. After 16 h, biofilms were stained with LIVE/DEAD stain (Life Technologies Europe BV, the Netherlands) dissolved in Dulbecco's PBS (DPBS) (Lonza, USA) as suggested by the manufacturer. Structural parameters of the biofilms were analyzed using COMSTAT (Heydorn et al., 2000)/MATLAB R2010b software (The MathWorks) program, using eight image stacks randomly generated of each sample. For statistical analysis, data from at least three independent experiments performed in duplicate were used. Statistical comparisons of all biofilm parameters were calculated using an unpaired t-test with GRAPH PAD v 6.0 (Graph Pad Software, Inc).

The epithelial cell line NCI-H292 (ATCC CRL 1848) was cultured in RPMI 1640 medium supplemented with 5% fetal calf serum, which was heat inactivated before use (1 h at 56°C). All cell-culture medium components were purchased from Laboratories PAA. The cells were cultured at 37°C in a humidified atmosphere containing 5% CO₂ and maintained in 25-cm² tissue-culture flasks (Nunc) until ~80% confluence was reached.

Bacterial adherence was determined as previously described (van Putten and Paul, 1995). In short, 2 days before the assays, NCI-H292 cells from 4 to 25 passages were seeded in 24-well plates. Bacteria grown overnight on plate were suspended in HEPES buffer (10 mM HEPES, 145 mM NaCl, 5 mM KCl, 5 mM glucose, 1 mM CaCl₂ and 1 mM MgCl₂, pH 7.2), washed by centrifugation (1500 g, 10 min) and incubated with cultured cells at a multiplicity of infection of 100 for 3 h. Non-adherent bacteria were removed by sequential washings with DPBS, and the viable adhering bacteria were counted by determining the colony-forming units (CFU) after disruption of the cell layer with 1% saponin diluted in DPBS for 15 min and subsequent mechanic homogenization.

Passage of cell layers was assayed as described previously (van Schilfgaarde et al., 1995) with modifications. In short, cells were grown on transparent tissue culture inserts with 1-μm pores (model no. 353104; Falcon) placed in 24-well plates (Falcon) containing 0.5 ml of medium. The culture medium was refreshed every 2 days. After 6 days, the transepithelial resistance of the cellular layers was measured with the Millicell-ERS Resistance system (Millipore, Bedford, Mass.), and the insert was moved to a new well containing 300 μl of RPMI in the basal compartment with 100 μg/ml of DNase I for avoid biofilm formation. Before starting the infection, the culture medium above the insert was replaced, and the cellular layers were then infected with ~10⁷ CFU for in total 12 h. After 3 h, the medium in the upper compartment was replaced to avoid medium acidification, and the number of CFU in the basal compartment was determined at regular time intervals.

The nucleotide sequences of the autB genes of disease isolates 2081107, 2061551, and 2070077 were deposited in GenBank ID (KT367782, KT367783, and KT367784, respectively).

BLASTn searches identified the autB gene in available genome sequences of the pathogenic Neisseria spp. N. meningitidis and N. gonorrhoeae but not in those of non-pathogenic species, such as N. lactamica (Table 1 and Table S2). The gene was also identified in some strains of Haemophilus spp. (Table 1 and Table S2). Figure 1 illustrates the position of the autB gene in representative genomes. The autB gene is ubiquitous in both pathogenic Neisseria spp. with the exception of one meningococcal strain, i.e., strain 053442. In this strain, as well as in N. lactamica, the flanking genes are preserved, but the autB gene is substituted by a 1415-bp sequence. Interestingly, the sequences flanking the autB gene in H. influenzae strains, including the 5′ end of the holB gene and the 3′ end of the tmk gene are also present as intergenic regions flanking autB in N. meningitidis and N. gonorrhoeae (Figure 1). This indicates that autB and the flanking regions may have been transferred en bloc from Haemophilus to Neisseria as suggested previously (Davis et al., 2001). However, many Haemophilus strains do neither contain the autB gene (Table 1), nor the alternative region found in N. lactamica (Figure 1 and data not shown). Interestingly, a sequence containing the 3′ ends of the autB and tmk genes is tandem repeated in H. haemolyticus (Figure 1), indicating that this region is prone to genetic recombination.

The genetic characteristics of the autB gene in a panel of Neisseria and Haemophilus strains are listed in Table S2. Table 1 summarizes the numbers of strains in which the gene can be expressed. The number of AAGC nucleotide repeats is considerably lower in Neisseria spp. (3–19) than in Haemophilus spp. (17–30 repeats). In the meningococcal autA gene, the same repeat is present, but the range of copies is considerably larger (6–40) (Arenas et al., 2015) than in meningococcal autB. In 12 of the 117 meningococcal strains examined (10.3%), the autB gene is in frame at these repeats, but in 10 of them the gene is disrupted further downstream of the repeats by frame-shift mutations, premature stop codons or insertion of a transposase element (Table S2). Thus, only two strains (1.7%), i.e., α153 and LNP27256, putatively express autB (Table 1). In stark contrast, in the 105 meningococcal strains, in which the autB gene is out of phase at the AAGC repeats, the gene is further intact with only three exceptions (Table S2). Hence, all these strains can potentially express the gene after phase variation occurs, but, apparently, this is prevented by a very strong selection pressure. Due to downstream disruptions, none of the gonococcal strains can express autB, independent of number or repeats. Such disruptions are not present in the autB genes of the Haemophilus strains examined, and the gene is in frame in 28% of them (Table S2), but all strains can potentially express autB after slipped-strand mispairing. To summarize, autB can be expressed, but expression seems to be prone to phase variation at the AAGC repeats and to different genetic disruptions. In addition, these data indicate a strong selection against autB expression in Neisseria, but not in Haemophilus spp.

The autB gene from N. meningitidis reference strain MC58 is not expressed because the number of AAGC repeats renders the gene out of frame (Table S2). Deletion of two repeats results in an N-terminally extended ORF coding for a hypothetical protein of 674 amino-acid residues (aa). The signal peptide cleavage site in this protein was predicted between aa residues A₃₀ and V₃₁ resulting in a mature protein of 644 aa, which is similar to that of AutA (660 aa), and with a calculated molecular mass of 73.2 kDa. The passenger of AutB is constituted of two parts, a C-terminal linker domain (aa 231–338), which shows homology to the autochaperone domain of other ATs and could accordingly be modeled as a right-handed β-helix (data not shown), and an N-terminal domain. Secondary structure predictions indicate that this N-terminal part of the passenger contains two conserved α-helices (with approximate positions between aa 1–20 and 90–110 of the mature aa sequences) separated by a largely unstructured region of ~80 aa and followed by a segment that is rich in β-sheet but occasionally also contains some α-helix prediction (Figure S1). Rather similar secondary structure was predicted for the corresponding fragment of AutA (Figure S1). The passengers of AutB proteins each contain one or two pairs of cysteines, which probably form a disulfide bond, but they are located at different positions in the sequence (Figure S2). The translocator domain of AutB (aa 360–644) is predicted to form a 12-stranded β-barrel that is connected via an α-helix (aa 337–354) to the passenger.

To determine sequence conservation, the predicted mature AutB proteins from several Neisseria and Haemophilus strains were aligned. The alignments (see Figure S2 for representatives) revealed that the sequence of the translocator domain was better conserved than that of the passenger. The translocator domain also showed considerable similarity to that of AutA of MC58 (not included in the alignments, but see below). However, the passenger domain showed high sequence diversity (see Figure S2 for examples). To investigate this variability in more detail, we performed independent phylogenetic analysis for the N-terminal part of the passenger, the linker and the translocator domain of representative AutB proteins and AutA of MC58 (Figure 2). Indeed, the translocator domain showed limited variability as compared with the other two domains (compare overall mean distances of the separate domains of the AutB proteins). Although similar to the translocator domain of the AutB proteins, the translocator domain of AutA clustered in a different branch, demonstrating that AutA is a different AT, which is consistent with its distinct genomic location. When the N-terminal domain of the passengers was analyzed, three major branches were identified, here designated AutB1, AutB2, and AutB3. The assignment of the AutB proteins in the different strains to these clusters is shown in Table S2. AutA again formed a separate branch. Interestingly, the phylogenic relationships of the AutB proteins do not parallel the boundaries of the species in which these proteins are found, suggesting that specific variants did not evolve in one specific organism, but that there is regular exchange of the genes between these species. AutB1 is the predominant variant in three species, i.e., N. meningitidis, N. gonorrhoeae, and H. influenzae. The AutB3 variant was found in three Haemophilus spp. and the AutB2 variant is present in all species except in N. gonorrhoeae and H. parainfluenzae. Variant AutB3 showed a larger diversity in the N-terminal domain of the passenger than the other variants and, interestingly, clustered with the passenger of AutA, suggesting the exchange of sequences between both AT, but limited to this domain. Cluster analysis of the linker domain also indicated the presence of the three main branches with AutA being a separate cluster. The groups were constituted of the same autB genes as for the N-terminal domain of the passenger; therefore, the same nomenclature is maintained. However, the relation between the groups varied notably. In this part of the protein, AutB1 was closely related to AutB3 and only distantly related to AutB2. This analysis confirmed that the passenger is rather variable as compared with the translocator domain, which is in agreement with its expected surface exposure and accessibility to the immune system. In addition, this analysis reflects shuffling of different domains within the passenger as suggested before (Davis et al., 2001).

To gain more insight in the evolution of autB, we calculated the GC content in the three regions in several autB genes. The GC content was similar in the regions encoding the N-terminal part of the passenger and the linker domain (35 ± 2%), but remarkably different for that encoding the translocator domain (44 ± 2%), which suggests that the passenger and the translocator domains have different evolutionary origins that are both different from the pathogenic Neisseria spp. with a GC content of 52% in their genome sequences. No differences were found in this respect between the three autB variants, suggesting that they evolved in the same species. In the autA gene, the GC content of the three regions was remarkably different, i.e., 40 ± 3, 52 ± 3, and 56 ± 1% for the N-terminal part of the passenger, the linker and the translocator domain, respectively. The two latter regions are closer to the average GC content in Neisseria genomes. Together, this analysis supports the hypothesis that both ATs have a different origin and suggests the exchange of genetic material between genes of different ATs in the region encoding the passenger.

To study phase variation of autB, we analyzed large sets of meningococcal disease isolates from the same genetic origin. If phase variation indeed occurs frequently, different isolates of the same cc should contain different numbers of repeats and, in the absence of selection pressure, ~33% should have the gene in the proper reading frame. We analyzed collections of cc213 and cc32 isolates, two hyper-invasive clonal complexes. The number of repeat units in the autA gene of these isolates was previously analyzed (Arenas et al., 2015) allowing us to compare phase variation in these two AT genes. In cc213 strain M01-240355, autB is in phase at the AAGC repeat but it is disrupted by a downstream frameshift mutation (Table S2). In the cc32 strains listed in Table S2 (n = 17), the autB gene is out of phase but it does not contain additional disruptions. All these genomes harbor an AutB1 variant (Table S2).

The repeat region of the autB gene was amplified by PCR and sequenced in a panel of 53 isolates of cc213 and 49 isolates of cc32 collected in The Netherlands between 2000 and 2010 from patients with meningococcal disease. The number of repeat units varied and ranged from 2 to 10 in autB and from 5 to 32 in autA (Table S3 and Figure 3). These results illustrate that slipped-strand mispairing at the AAGC repeats occurs in both genes and in both clonal complexes. However, in only 1 of the 49 cc32 isolates (2%), i.e., strain 2081107, and none of the 53 cc213 isolates the autB gene was in phase. This low frequency contrasts drastically with that observed for autA, where the gene was found to be in phase in 28% and 45% for cc213 and cc32 isolates, respectively (Figure 3), i.e., close to the 33% that would be expected in the absence of any selection pressure (Arenas et al., 2015). Thus, the results support the hypothesis that, although slipped-strand mispairing resulting in frameshifts does occur at the AAGC repeats in autB, there is a strong selection pressure against the occurrence of a number of repeats that would render the gene in frame in both clonal complexes. Further sequence examination revealed that cc213 isolates do not contain the same additional frameshift that disrupts the autB gene downstream of the AAGC repeats in strain M01-240355, suggesting that this feature is not conserved in cc213 strains. Therefore, the gene could potentially be expressed in this cc after slipped-strand mispairing. Interestingly, examination of the sequence after the repeats revealed that isolate 2041085 harbors an AutB2 variant, whilst all other cc213 isolates harbor an AutB1 variant, evidencing horizontal genetic transfer. The full-length autB gene was sequenced in three isolates of cc32, i.e., 2081107, which is in frame at the AAGC repeats, and 2061551 and 2070077, which are out of phase. All these isolates revealed only variation in the number of repeat units and no other genetic disruptions or amino-acid substitutions relative to that of strain MC58. Thus, the autB gene of isolate 2081107 is intact and, therefore, probably expressed.

Since the vast majority of isolates analyzed in Tables S2, S3 are from patients with invasive meningococcal disease, our selection of strains may not be representative for those residing in the nasopharynx. Thus, we analyzed the autB gene in the genome sequences of a collection of carriage isolates available in a public data base (http://pubmlst.org/software/database/bigsdb/). A total of 207 strains was analyzed. The number of repeat units in the autB gene ranged from 2 to 22. In total, 10.6% of these isolates had 3, 6, or 12 repeats that render autB in frame. However, in the majority of these strains, the gene is disrupted by downstream-located single-nucleotide insertions and only 2.4% of isolates (28871, 28881, 28942, 28955, 28959) had an undisrupted autB gene in frame. Hence, these data do not show obvious differences in possible autB expression between carrier and disease isolates.

We next analyzed whether AutB is indeed synthesized in strains where the gene is in phase at the AAGC repeats and has no other genetic disruptions. Plasmids encoding the autB1 and autB2 genes from strains 2081107 and α153, respectively, were introduced in HB-1ΔautB. Although the autB gene is phase off in HB-1 (Table S2), we deleted the chromosomal copy of the gene to avoid genetic exchange with the autB gene on the plasmids. Western blot analysis showed a band with an apparent molecular weight of ~73 kDa in strain HB-1ΔautB carrying pENAutB₁ only when the strain was grown in the presence of IPTG (Figure 4A), thus identifying this band AutB₁. A band of similar size was detected in isolate 2081107, which contains an intact in-frame autB gene on the chromosome, but not in preparations of other strains where the autB1 gene is out of phase (Figure 4A). This result demonstrates that AutB is synthesized in isolate 2081107. Unfortunately, an autB mutant of this strain could not be created, because the strain was not transformable. No reaction was observed in strains HB-1ΔautB carrying pENAutB₂ (data not shown) or α153, which is expected to express AutB2 from the chromosome (Figure 4A), presumably due to lack of cross-reactivity of the antiserum, which was raised against a part of AutB1 that shows little sequence similarity to AutB2 (Figure S2). The antiserum did also not react with a preparation of strain α14 (Figure 4A), which expresses AutA, confirming the specificity of the antiserum.

To investigate if autB2 is expressed in α153, RT-PCR assays were done using primers targeting internal DNA fragments of autB1 and autB2. With chromosomal DNA from HB-1 and α153 as templates, the amplicons for autB1 and autB2 were according the expected size, i.e., 100 and 150 bp, respectively (Figure 4B). No amplicons were obtained from RNA preparations as templates (data not shown), but they were obtained from cDNA preparations of strains HB-1ΔautB containing pENAutB₁ and grown in presence of IPTG and α153, but not of strains HB-1 and the α153ΔautB mutant (Figure 4B). Primers for rmpM yielded an amplicon in all preparations, evidencing that all contained similar amounts of cDNA. The lack of detection of autB transcripts in strain HB-1 could be due to degradation of the mRNA by the presence of a premature stop codon resulting from the frameshift at the AAGC repeat region. Together, these data show that autB is expressed in at least some meningococcal strains and confirm that its expression is determined by phase variation at the AAGC repeat units.

Our Western blotting analysis detected the full-length AutB in cell envelope preparations (Figure 4A). To determine whether the passenger domain may be released from a proportion of the AutB molecules into the extracellular medium, whole cell lysates, cell envelopes and supernatants of strain HB-1ΔautB expressing AutB1 from pENAutB₁ were analyzed by SDS-PAGE and Western blotting. The expected 73-kDa band was detected in cell-envelope preparations but no band reacting with the antiserum was detected in supernatant preparations (Figure 5A). As a control, the secretion of IgA protease was also analyzed using specific antibodies against its translocator domain and the α-peptide, which is released into the milieu after cleavage by NalP (Roussel-Jazédé et al., 2014). As expected, the translocator domain and the α-peptide were detected in the cell envelope fraction and the culture supernatant, respectively (Figure 5A). Hence, the passenger of AutB is not released into the extracellular medium. To confirm its cell-surface exposition, we used proteinase K accessibility assays in intact bacteria (Figure 5B). The protease degraded AutB and fHbp, a surface-exposed lipoprotein that was analyzed as a positive control (Bos et al., 2014). In contrast, the periplasmically exposed outer membrane-associated protein RmpM remained unaffected, confirming the integrity of the outer membrane. Thus, the passenger of AutB is secreted but remains attached via the translocator domain to the cell surface.

Previously, we demonstrated that AutA has a role in autoaggregation and biofilm formation (Arenas et al., 2015). To investigate whether AutB also has a role in autoaggregation, settling assays were performed. These assays showed a clear effect of the of AutA production on bacterial autoaggregation (Figure S3A); however, such an effect was not observed when autB1 was expressed from plasmid in HB-1ΔautB, and also no difference between α153 and its autB mutant derivative was observed (Figure S3A). Thus, in contrast to AutA, synthesis of AutB does not induce strong autoaggregation.

A possible role of AutB in biofilm formation was first studied under static conditions on polystyrene plates. The presence of a capsule is known to inhibit biofilm formation (Yi et al., 2004; Lappann et al., 2006), but α153 is a natural capsule null mutant. We also included the capsule-deficient reference strains HB-1 and BB-1, which were previously used in biofilm assays (Arenas et al., 2013a). After 1 h of incubation, α153 generated a biofilm mass intermediate between that of BB-1 and HB-1 (Figure 6A). Interestingly, deletion of autB drastically impaired biofilm formation to a level comparable with that of strain BB-1 (Figure 6A). This phenotype was complemented when either AutB1 or AutB2 was expressed in trans from plasmid (Figure 6A), demonstrating that both variants have a similar role.

N. meningitidis has two different strategies of biofilm formation, either dependent or independent of eDNA (Lappann et al., 2010). HB-1 and BB-1 follow the eDNA-dependent and -independent routes of biofilm formation, respectively (Arenas et al., 2013a). To investigate whether AutB-mediated biofilm formation is dependent on eDNA, biofilm formation of strain α153 and its autB mutant derivative was assessed in the presence of DNase I. This treatment considerably reduced biofilm mass in α153 to the level of the ΔautB mutant, but it did not impact on the residual biofilm formation of the mutant (Figure 6A, right panel). This data indicates that AutB-mediated biofilm formation is dependent on eDNA and suggests that the remaining biofilm formation in the autB mutant of α153 is eDNA independent. Presumably, AutB at the cell surface binds eDNA that acts as a glue facilitating interbacterial and bacterium-substratum interactions. Previously, the α-peptide of IgA protease and NHBA were reported to represent the main surface-exposed DNA-binding proteins involved in biofilm formation in strain HB-1 (Arenas et al., 2013a). Western blot analysis, however, revealed that the α-peptide of IgA protease is released into the medium in strain α153 (Figure 5A), whilst examination of the α153 genome sequence revealed that the nhbA gene is disrupted. Thus, probably, AutB is the main DNA-binding protein in α153 and, therefore, its inactivation has a drastic impact on biofilm formation. To investigate the DNA-binding capacities of AutB, we produced the entire passenger of AutB of strain HB-1 in E. coli. The protein formed inclusion bodies, which were purified, but we could not establish proper conditions to fold the protein in vitro.

To investigate the influence of AutB synthesis on biofilm formation in more detail, we used a flow-cell model. With this method, the flow in the nasopharynx or the bloodstream is mimicked, and biofilm development can continuously be monitored by microscopy. Biofilms formed by α153 were constituted of round aggregates of different sizes, which were surrounded by single cells (see representative pictures in Figure 6B and the development of the biofilm in Figure S3B). These aggregates fused into larger aggregates, which constituted the biomass of the biofilm (Figure S3B). These biofilm structures had a clearly different appearance than those reported previously for strains HB-1, BB-1, and α14 (Arenas et al., 2013a, 2015). Analysis of the α153ΔautB mutant did not reveal structured biofilms; instead, only few single cells or very small aggregates were randomly attached to the substratum (Figure 6B). COMSTAT analysis revealed a biomass and thickness significantly reduced as compared with the wild type (Figure 6C) in accordance with the results of the biofilm assays under static conditions (Figure 6A). Furthermore, biofilm roughness, also determined by COMSTAT, increased significantly. All phenotypic differences were complemented when AutB₁ was expressed in trans from plasmid (Figures 6B,C). These data confirm that AutB is required for biofilm formation and architecture and demonstrate its biological relevance.

To investigate whether the synthesis of AutB also affects the interaction of the bacteria with eukaryotic cells, we first determined the adherence of α153 and its autB mutant derivative to NCI-H292 cells by counting the number of cell-associated CFU 3 h after initiating infection. Although adherence of the mutant appeared slightly reduced (~25%), a defect that was complemented when AutB was expressed in trans (Figure 7A), this difference was not statistically significant.

Next, we studied the ability of the strains to cross epithelial cell layers, a process necessary for host invasion. NCI-H292 cells were grown for 6 days on a permeable support to generate cellular junctions, as verified by a trans-epithelial resistance of 30 ± 2 Ω × cm², in accordance with values previously reported (van Schilfgaarde et al., 1995). The cell layers were then infected with bacteria and the numbers of bacteria that reached the basal compartment after passage of the cell layers were determined between 6 and 12 h post infection by counting CFU. Cells of the autB mutant reached the basal compartment faster than those of the parental strain as demonstrated, for example, by the significantly higher numbers of autB mutant bacteria than of wild-type bacteria in the basal compartment 10 h after infection (Figure 7B), a phenotype that was complemented by expression of a functional copy of autB from pENAutB₁. Clones of cells derived from the wild type that reached the trans-epithelial compartment early after infection were collected from different experiments and further analyzed. To investigate whether AutB synthesis and, consequently, their biofilm-formation capacity was altered, we tested biofilm formation under static conditions. Interestingly, all clones had a reduced biofilm-forming capacity like the autB mutant (see Figure 7C for representatives). Sequencing of the AAGC region in all clones did not show alterations, suggesting that phase variation was not the origin of such defect. To test whether autB expression was lost we analyzed one clone by RT-PCR assays and found no detectable autB expression (Figure 7C). Sequencing of the complete autB gene and flanking regions, however, did not reveal an obvious reason for this down-regulation, suggesting these clones had acquired a mutation in a regulatory gene. Together, these data demonstrate that autB expression negatively affects bacterial epithelial transmigration and suggests that biofilm formation is inversely correlated with invasion.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.