ABSTRACT
The major outer membrane porin (PorB) expressed by Neisseria gonorrhoeae plays multiple roles during infection, in addition to its function as an outer membrane pore. We have generated a panel of mutants of N. gonorrhoeae strain FA1090 expressing a variety of mutant porB genes that all function as porins. We identified multiple regions of porin that are involved in its binding to the complement regulatory factors C4b-binding protein and factor H and confirmed that the ability to bind at least one factor is required for FA1090 to survive the bactericidal effects of human serum. We tested the ability of these mutants to inhibit both apoptosis and the oxidative burst in polymorphonuclear leukocytes but were unable to identify the porin domains required for either function. This study has identified nonessential porin domains and some potentially essential portions of the protein and has further expanded our understanding of the contribution of the porin domains required for complement regulation.
INTRODUCTION
Neisseria gonorrhoeae is the causative agent of the sexually transmitted infection gonorrhea, which typically causes uncomplicated urogenital infections but on rare occasions can also disseminate to mediate more severe disease phenotypes. This human-specific pathogen has evolved multiple mechanisms to counter both the innate and adaptive immune responses during infection, and some of these are mediated by its major outer membrane porin protein (PorB). PorB is a voltage-gated pore that mediates ion exchange between N. gonorrhoeae and the environment and is essential for bacterial viability, likely due to its ability to allow nutrients access to the periplasm (1, 2). Porin is found in the outer membrane as a homotrimeric β-pleated barrel; each monomer is 32 to 35 kDa and consists of 16 transmembrane-spanning segments and 8 extracellular loops (3, 4). N. gonorrhoeae strains contain a single porB gene in one of two allelic forms (P.IA or P.IB), and the two alleles have been associated with different biological phenotypes. P.IA-expressing strains tend to be associated with disseminated disease, whereas P.IB-expressing isolates typically cause localized urogenital infections (5, 6). There is ∼80% nucleotide sequence similarity between P.IA and P.IB, and the majority of the variation between alleles and between different isolates expressing the same allele occurs within the extracellular loops (7, 8).
In addition to its function as an outer membrane pore, PorB also appears to perform multiple functions contributing to N. gonorrhoeae pathogenesis. It can induce or inhibit apoptotic signaling in eukaryotic cells (9–13), it contributes to serum resistance via interactions with regulators of classical and alternative complement (14–19), it mediates epithelial cell invasion under low-phosphate conditions (20–22), and it can affect the generation of reactive oxygen species (ROS) by innate immune cells (23, 24). The related organism Neisseria meningitidis also possesses a porB gene which shares 60 to 70% amino acid sequence homology with gonococcal porB. Like the gonococcus, meningococcal PorB also inhibits apoptosis induction and can modulate immune responses via binding to and inducing signaling downstream of Toll-like receptor 2 (25–31).
Binding to factor H, the major regulator of the alternative complement pathway, can render Neisseria species resistant to killing by normal human serum (NHS), and N. gonorrhoeae strains expressing P.IA alleles can be serum resistant by virtue of direct P.IA binding to factor H. In contrast, P.IB-expressing strains require sialylation of lipooligosaccharide (LOS) for factor H binding to occur (14, 15). Factor H interactions with P.IA versus P.IB appear to be mediated by different PorB domains; extracellular loop 5 of P.IA can mediate interactions with factor H (14), and extracellular loops 1 and 2 appear to be required for factor H binding by P.IB (18). Serum resistance can also be mediated via binding to the classical complement regulatory protein C4b-binding protein (C4bp). Several surface components of N. gonorrhoeae are involved in interactions with C4bp, including LOS (32), type IV pili (33), and porin (16). Both P.IA and P.IB bind to C4bp, and again, different domains in each allele appear to mediate C4bp binding (extracellular loop 1 of P.IA and extracellular loops 5 and 7 of P.IB) (16). The significance of C4bp and factor H binding can be illustrated by experiments demonstrating that binding of either factor can rescue N. gonorrhoeae from killing by heterologous sera (17, 19).
In this study, we generated a variety of mutations in the PorB gene of strain FA1090 (which expresses the P.IB allele) in order to perform structure-function studies on the porin protein. Here we have further characterized determinants of serum resistance in FA1090 by identifying several regions within P.IB that are required for C4bp and/or factor H binding and correlated the binding of these complement regulatory factors with resistance to killing by human serum.
MATERIALS AND METHODS
Generating mutations in the FA1090 porB gene.To generate a construct in which to introduce mutations into FA1090 PorB, the porB gene and sequences from the downstream intergenic region (IGR) were cloned into the pBluescript vector. The region between porB nucleotide 1015 and 500 nucleotides downstream of the porB stop codon (the downstream IGR) was first amplified from FA1090 genomic DNA using KOD Hot Start DNA polymerase (Novagen), with a silent C-to-T mutation at porB nucleotide 1017 being incorporated into the primer to create an NheI site at the 5′ end and a SacI site being appended to the downstream end. This PCR product was cloned into pCR Blunt (Invitrogen) and then subcloned into pBluescript SK using NheI and SacI. Targeted insertion of a kanamycin resistance cassette (Kanr) into the downstream IGR was performed to provide a selectable marker, but this construct failed to yield viable FA1090 transformants, suggesting that we targeted an essential region. Random insertion of Kanr into the downstream IGR was then done using an EZ-Tn5 <Kan-2> insertion kit (Epicentre), after which the pool of constructs was transformed into FA1090 via spot transformation (34, 35). Transformants without growth defects were selected on gonococcal medium base (GCB) agar containing 50 μg/ml kanamycin, and the location of the Kanr insertions in a subset of clones was determined by PCR amplification and sequencing of part of the downstream region. A clone containing the Kanr inserted 232 nucleotides downstream from the FA1090 porB stop codon was then reamplified and cloned into pBluescript using the same procedure described above. The porB gene from nucleotides 50 to 1020 was then amplified from FA1090 genomic DNA, the silent C-to-T mutation at porB nucleotide 1017 was incorporated into the downstream primer, and an EcoRI site was incorporated into the upstream primer. This PCR product was cloned into pCR Blunt and then subcloned into pBluescript next to the downstream IGR using EcoRI and NheI. Introduction of the C-to-T transition into the porB gene had no effect on growth or colony morphology (data not shown).
To generate mutations in the context of the porB gene in pBluescript, several approaches were used. Site-directed mutagenesis was used to delete or mutate the eight extracellular loops, as well as to change residues conserved in pathogenic Neisseria to those found in commensal Neisseria. Amplification of the porB gene for site-directed mutagenesis was performed using a QuikChange II XL site-directed mutagenesis kit (Stratagene) according to the manufacturer's specifications (primer sequences are available upon request). The appropriate recombinants were identified by nucleotide sequencing. Random mutagenesis of the entire porB gene or of smaller regions of porB was performed using a GeneMorph II EZClone domain mutagenesis kit (Stratagene) according to the manufacturer's specifications. Briefly, the porB gene/domain was amplified under mutagenic PCR conditions to generate a pool of megaprimers containing random mutations. Megaprimers were then used to amplify the original pBluescript plasmid containing porB. The PCR products were digested with DpnI for 2 h at 37°C and then transformed into XL10-Gold ultracompetent cells (Stratagene). Plasmid DNA containing a random pool of porB mutations was purified from the resulting pool of colonies and was used in subsequent reactions for transformation into FA1090.
FA1090 growth and transformations.N. gonorrhoeae strains were cultured on GCB (Difco) agar plus Kellogg's supplements (36) and were typically grown at 37°C with 5% CO2 for 18 to 20 h. All strains expressing mutations in porB were generated in the background of a piliated FA1090 strain expressing multiple Opa proteins. The mutations were introduced into the FA1090 chromosome at the porB locus (NGO1812, between truA [NGO1811] and oxyR [NGO1813]) by spot transformation, as described previously (34, 35), and recombinants were selected for by plating transformants on GCB agar containing 50 μl/ml kanamycin. The presence of mutations was screened for by amplifying the region of interest from the chromosome and nucleotide sequencing.
For C4bp binding, factor H binding, and serum sensitivity assays, FA1090 strains expressing wild-type porin or P.IB mutations were grown overnight on GCB agar, suspended in gonococcal liquid medium containing Kellogg's supplements and 0.042% Na2HCO3 at an optical density at 550 nm of 0.16, and grown for 3 to 4 h at 37°C. For factor H binding and some serum sensitivity assays, the liquid medium was additionally supplemented with 2 μg/ml CMP–N-acetylneuraminic acid (CMP-NANA; Sigma).
C4bp- and factor H-binding assays.The C4bp- and factor H-binding assays were adapted from references 14 and 16. Gonococci (108) were fixed with 4% paraformaldehyde for 10 min and washed in Hanks balanced salt solution (HBSS; Gibco) with 0.15 mM CaCl2 and 1 mM MgCl2 (HBSS2+). Fixed bacteria were incubated with 10% NHS (Atlanta Biologicals) for 20 min at 37°C and washed with HBSS2+. Bacteria were also incubated with 4 μg purified factor H (Complement Technology, Inc.) under the same conditions in separate experiments to verify factor H-binding phenotypes. The purified factor H was assayed to be >97% pure by SDS-PAGE and should not contain significant amounts of the alternatively spliced form of factor H (factor H-like protein 1), which can also be detected by the anti-factor H antibody used for flow cytometry (see below) (37). In order to detect binding of complement regulatory proteins, bacteria were stained with either 1 μg anti-C4bp (A215; Quidel) or 1 μg anti-factor H (OX24; Santa Cruz) in a volume of 100 μl HBSS2+ plus 1% bovine serum albumin (BSA) for 20 min at room temperature, washed with HBSS2+, and then incubated with a fluorescein-conjugated goat anti-mouse secondary antibody (1:100 dilution in HBSS2+ plus 1% BSA; 115-095-146; Jackson). Fluorescence was assessed by flow cytometry (FACSCalibur; BD Biosciences).
Serum sensitivity assays.Serum sensitivity assays were adapted from a previous study (38). The parental FA1090 strain and strains expressing mutations in P.IB were cultured as described above in gonococcal liquid medium with supplements and 2 μg/ml CMP-NANA. Bacterial cells (105) were incubated with 10% NHS in HBSS2+ in a total volume of 200 μl, and 50 μl of the bacterial suspension was immediately removed for serial dilution and plating onto GCB agar plates to determine viable bacterial counts. The remaining 150 μl was incubated at 37°C for 30 min, after which bacteria were serially diluted and plated onto GCB plates. The percent survival of each strain in NHS was determined by calculating the ratio of the viable bacterial counts at 30 min relative to the viable counts at time zero. As a control, serum was treated at 56°C for 30 min, and bacterial survival was assessed in the presence of heat-inactivated serum.
Inhibition of apoptosis and ROS production.The ability of FA1090 porB mutants to inhibit apoptosis and ROS production was determined using HL-60 cells differentiated down the granulocytic pathway as previously described (39). HL-60 cells were differentiated in 0.7% dimethylformamide (Sigma) for a period of 5 days, after which cells were infected with the parental FA1090 strain or porB mutants. For the apoptosis inhibition assays, differentiated HL-60 cells were infected at a multiplicity of infection (MOI) of 50 for 3 h, followed by treatment of cells with 1 μM staurosporine for 3 h to induce apoptosis. Cells were washed with phosphate-buffered saline (PBS) before being treated with 50 μl cell lysis buffer (BD Pharmingen) to harvest cell lysates. To measure caspase-3 activity in the lysates, 5 μl reconstituted caspase-3 substrate (N-acetyl–Asp–Glu—Val–Asp–7-amino-4-methylcoumarin [Ac-DEVD-AMC]; BD Pharmingen) at a concentration of 1 mg/ml was incubated with assay buffer and 25 μl cell lysates for 60 min at 37°C. 7-Amino-4-methylcoumarin (AMC) fluorescence was measured using an excitation wavelength of 380 nm, an emission wavelength of 440 nm, and a SpectraMax M5 plate reader (Molecular Devices). Luminol-dependent chemiluminescence (LDCL) assays were performed to examine ROS production by infected HL-60 cells (39). Assays were carried out in a total volume of 0.2 ml PBS supplemented with 0.9 mM CaCl2, 0.5 mM MgCl2, and 7.5 mM glucose (PBSG) in black-bottom 96-well plates (Nunc). HL-60 cells were resuspended at a concentration of 4 × 107 cells/ml, and 106 cells were seeded in the presence of 100 μM luminol. The cells were then stimulated with phorbol 12-myristate 13-acetate (PMA) and N. gonorrhoeae that had been grown for 4 to 5 h in gonococcal liquid medium with supplements (see above). Following stimulation, LDCL was measured every 2 min over a total period of 60 min at 37°C using a plate reader.
RESULTS
Generation of FA1090 strains expressing mutant porin proteins.The major outer membrane porin (PorB) of pathogenic Neisseria species has been implicated in a variety of biological functions. In order to identify residues within PorB that participate in these various functions, we constructed a set of N. gonorrhoeae FA1090 strains expressing mutations in the P.IB allele. To generate the mutations within the porB gene, a plasmid containing the parental porB sequence next to a selectable marker was constructed (Fig. 1A). The majority of the porB signal sequence was removed from the construct, as introduction of mutations in the signal sequence could potentially prevent proper localization of the protein in the outer membrane and because Escherichia coli expressing the full-length porB gene does not grow well. A random transposon insertion (kanamycin resistance) that had no effect on bacterial viability or colony morphology was isolated in the downstream intergenic region of porB. Mutations in porB were then introduced in the context of the plasmid construct (see below) and transformed into FA1090 by selecting for kanamycin resistance carried on the transposon and screening for cotransformation of mutations via sequencing. The linkage frequencies of cotransformed mutations ranged from 0.1 to greater than 0.7 (data not shown).
Generation of FA1090 strains expressing mutated P.IB proteins and locations of mutated residues. (A) Schematic of the plasmid construct used to generate mutations. The FA1090 porB gene (minus the first 49 nucleotides) flanked by restriction sites was amplified and cloned next to 500 nucleotides of the porB downstream IGR, which contained a kanamycin resistance cassette (Kanr) inserted 232 nucleotides downstream of the porB-coding region. The orientation of Kanr is shown with an arrow. A silent mutation at nucleotide 1017 of the gene was engineered to create an NheI site to facilitate cloning. Transformation of FA1090 with the plasmid construct resulted in recombination at the porB locus (NGO1812) in the chromosome. (B) Localization of the recovered mutations in the context of the FA1090 P.IB protein sequence. The boxed residues contain the signal sequence, with the arrow indicating where the plasmid construct begins. The underlined residues are the predicted extracellular loops, inferred via sequence alignment with a meningococcal PorB (43). Residues shaded in gray were successfully mutated in this study.
A panel of FA1090 strains expressing a variety of mutations in the porB gene was generated using several different methods (Table 1; Fig. 1B). Site-directed mutagenesis was utilized to generate mutations in the eight P.IB extracellular loops. Deletions were attempted for loops 1, 2, 3, 4, 5, and 7, but only recombinant FA1090 expressing porins in which loops 4 and 5 were individually deleted could be recovered. Loops 1, 2, 3, and 7 were mutated via alanine-scanning mutagenesis, and several loop alanine-scanning mutants were recovered (Table 1, extracellular loop alanine-scanning mutants). Loops 6 and 8 are predicted to be shorter (∼6 amino acids each) and were subjected to alanine-scanning mutagenesis; however, only recombinants expressing mutations in loop 6 were isolated. Most of the loop domains were therefore successfully mutated, with the exception of loop domains at the extreme N and C termini of the protein.
FA1090 strains expressing mutant P.IB proteins generated and utilized in this study
Since some of the described functions of PorB appear to be specific for porins from pathogenic Neisseria species (2, 11, 13, 22), we also wanted to examine the potential functions of domains within porin that are unique to pathogenic Neisseria. Several regions in FA1090 PorB were targeted for mutagenesis by aligning porin protein sequences from both pathogenic and commensal Neisseria species and identifying regions that are conserved in pathogenic Neisseria strains but divergent in commensal neisserial or E. coli porins. These regions in FA1090 P.IB were mutated by site-directed mutagenesis to the respective residues found in porin from Neisseria mucosa (strain NCTC 10777-M3) (4) (Table 1, site-directed mutants). In addition to the domains listed, an attempt was also made to mutate P.IB amino acids 224 to 226 (224EKL226 to 224KTG226), but we were unsuccessful in recovering viable transformants.
Lastly, a group of P.IB mutants was generated by random PCR-based mutagenesis either of the entire porin gene or of smaller 150- to 200-nucleotide domains within the gene (Table 1, random mutants). Attempts to introduce random mutations across the entire porB gene yielded a low frequency of FA1090 recombinants expressing mutations (<20% of clones with at least 1 mutation), probably because multiple mutations occurring along the entirety of the gene would be more likely to yield a protein that cannot form a functional pore. Random mutagenesis of smaller regions gave increased frequencies of mutants (between 3 and 42% clones with at least 1 mutation), but the mutagenesis was not efficient enough to perform an efficient saturating screen.
Each of the strains expressing mutated P.IB proteins grew normally on GCB plates and had a normal colony morphology, showing that these mutations did not alter essential porin functions. Additionally, all of the strains expressed levels of P.IB comparable to those expressed by the parent strain, as assessed by Western blotting, confirming that the mutations did not alter porin expression (data not shown). Overall, we deleted or mutated 102 out of the 348 amino acids in FA1090 P.IB and showed that most of the extracellular loops and several residues in the transmembrane regions are dispensable for pore function (Fig. 1B).
Identification of P.IB domains required for C4bp binding.PorB has been shown to mediate resistance to complement-mediated killing via binding to the complement regulatory factors C4bp and factor H (14–19). Previous studies identified strain FA1090 as being able to bind C4bp and identified extracellular loop 1 of P.IA and extracellular loops 5 and 7 of P.IB as being involved in mediating the binding of C4bp to porin (16). To determine whether additional regions of P.IB might play a role in C4bp binding, the ability of each P.IB mutant strain to bind to C4bp was assessed. FA1090 strains grown in liquid culture for 3 h were fixed, incubated with 10% normal human serum, and stained with an anti-C4bp antibody and a fluorescent secondary antibody. Levels of C4bp binding were determined by flow cytometry (Fig. 2). FA1090 expressing wild-type P.IB bound to the C4bp present in serum, as did a subset of strains expressing mutant P.IB genes (represented by strain SDM 65-71 in Fig. 2). A smaller subset of P.IB mutant strains showed a lack of binding to C4bp (represented by strains ΔLoop4 and Loop6 254-259 in Fig. 2), and one mutant showed an intermediate binding phenotype (RM760 #22; Fig. 2). The results of C4bp binding for the strains tested are summarized in Table 2. These data show that residues in extracellular loops 4 through 7, as well as transmembrane residues adjacent to loop 6, play a role in mediating C4bp binding to FA1090 P.IB.
Representative C4bp-binding phenotypes of FA1090 P.IB mutant strains. Bacterial strains were incubated with NHS, and C4bp binding was assessed by flow cytometry after staining fixed cells with an anti-C4bp primary antibody and a fluorescein isothiocyanate-conjugated secondary antibody. Results for unstained bacteria, as well as stained bacteria expressing wild-type P.IB, are shown as controls. Results for representative mutant strains are shown and are representative of those from at least 3 independent experiments.
Summary of C4bp- and factor H-binding data for FA1090 P.IB mutant strains
We observed that a subset of P.IB mutant strains showed reduced C4bp binding when they were swabbed directly from plates compared with that seen when they were first subject to a liquid growth step (data not shown). We postulated that a lack of C4bp binding by those strains might be due to the decreased viability of bacteria taken from plates. Consistent with this hypothesis, liquid-grown, heat-killed bacteria of strains showing differential binding exhibited decreased C4bp binding compared with live, liquid-grown bacteria. The parental FA1090 strain expressing wild-type P.IB showed no difference in C4bp binding when plate- versus liquid-grown bacteria were compared and also showed no decrease in C4bp binding after bacteria were heat killed (data not shown). It is unknown why some P.IB mutant strains show differential C4bp binding depending on the growth condition or viability of the bacterial cells, but it is possible that porin conformation or expression changes under different physiological conditions.
Identification of P.IB domains required for factor H binding.N. gonorrhoeae can also interact with the alternative complement regulatory protein factor H. Although N. gonorrhoeae strains expressing P.IA alleles of PorB can bind directly to factor H, strains expressing P.IB alleles bind only when their lipooligosaccharide (LOS) is modified by sialylation (18). The N-terminal extracellular loops 1 and 2 of P.IB (from strain F62) were previously shown to be necessary for mediation of binding to factor H but not sufficient to mediate binding to factor H (18). To determine whether the porin from FA1090 is also involved in factor H binding, the parental strain and each of the P.IB mutant strains was tested for the ability to bind factor H in the presence of LOS sialylation. Because NHS is a source of C3b, which can also interact with factor H when bound to bacteria (15), the factor H-binding assays were performed with both NHS and purified factor H. The bacteria were fixed, incubated with either 10% NHS or purified factor H, stained with an anti-factor H antibody and a fluorescent secondary antibody, and assessed for factor H binding by flow cytometry (Fig. 3). FA1090 expressing wild-type P.IB bound well to factor H in the presence of LOS sialylation, and a majority of strains expressing P.IB mutants showed no defect in factor H binding relative to the parental strain (represented by SDM 65-71 in Fig. 3A). Four strains (ΔLoop4, ΔLoop5, SDM 261-266, Loop7 290-295) showed a lack of binding to factor H when either human serum or purified factor H was used as the source for factor H (represented by ΔLoop4 in Fig. 3A). Five additional strains (Loop3 116-121, RM760a #40, SDM 248-250, Loop6 254-259, SDM 300-303) showed intermediate factor H binding with NHS (represented by Loop6 254-259 in Fig. 3A), and two of those strains (Loop3 116-121 and SDM 248-250, represented by Loop3 116-121 in Fig. 3B) exhibited further decreases in binding when purified factor H instead of NHS was used as the factor H source, suggesting that another component of NHS, such as C3b, may be facilitating factor H binding to these mutants. The results for all of the strains are summarized in Table 2. To explore the possibility that the lack of factor H binding was due to changes in LOS expression and a loss of LOS sialylation, LOS was purified from FA1090 expressing wild-type P.IB and the six strains expressing P.IB mutations exhibiting reduced factor H binding. Comparison of LOS expression by SDS-PAGE and silver staining showed that all strains expressed the same LOS species (data not shown). Since we know that the LOS expressed by the parental strain can be sialylated due to differential factor H binding in the presence and absence of CMP-NANA, it is likely that the LOS expressed by the P.IB mutant strains can also be sialylated. The regions found to be required for efficient P.IB-factor H interactions were mostly localized in the C-terminal half of the protein, suggesting that this region may constitute a binding domain.
Representative factor H-binding phenotypes of FA1090 P.IB mutant strains. (A) Bacterial strains grown in the presence of 2 μg/ml CMP-NANA were incubated with purified factor H, and binding was assessed by flow cytometry after staining fixed cells with an anti-factor H primary antibody and a fluorescein isothiocyanate-conjugated secondary antibody. Results for unstained bacteria, as well as stained bacteria expressing wild-type P.IB, used as controls, are also shown. For the mutant P.IB-expressing strains, the levels of factor H binding are shown in solid black, and the levels of factor H binding of bacteria expressing wild-type P.IB are shown overlaid in gray. Results for representative mutant strains are shown and are representative of those from at least 3 independent experiments where bacterial strains were incubated with either NHS or purified factor H. (B) Certain P.IB mutant strains show differential binding to factor H after incubation with either 10% NHS or purified factor H (fH). Levels of factor H binding by the Loop3 116-121 mutant strain are shown in solid black, and those of factor H binding by the wild-type P.IB-expressing strain are overlaid in gray.
Serum resistance mediated by C4bp and factor H binding.Resistance to killing by serum is correlated with both C4bp and factor H binding (14, 16). We have identified strains expressing mutant porin proteins that (i) bind to both factor H and C4bp, (ii) bind to factor H but not C4bp, or (iii) bind to neither factor H nor C4bp (Table 2). We did not identify any mutant strains which bound to C4bp but not factor H when NHS was used as the source of factor H. Three strains representative of each binding phenotype were tested for resistance to killing by 10% NHS. The parental FA1090 and P.IB mutant strains were grown for 3 h in liquid culture in the presence of 2 μg/ml CMP-NANA, 105 CFU was incubated in 10% NHS at 37°C, and relative bacterial survival was assessed after 30 min (Fig. 4). Strains expressing P.IB mutants that still bound to both C4bp and factor H (SDM 65-71, Loop1 43-48, Loop2 83-88) exhibited complete resistance to serum-mediated killing, similar to the parental strain. Strains expressing P.IB mutations that bound to factor H but not C4bp (Loop6 254-259, RM598 #31, RM760a #40) also showed resistance to serum-mediated killing. Strains which bound to neither C4bp nor factor H (ΔLoop4, ΔLoop5, SDM 261-266) showed a 78% to 98.5% reduction in viability after incubation with serum. When serum was treated at 56°C for 30 min prior to incubation with bacteria, none of the strains tested showed reduced viability in the presence of heat-inactivated serum (data not shown). When the same P.IB mutant strains were grown in the absence of CMP-NANA, the strains that bound to only factor H (Loop6 254-259, RM598 #31, RM760a #40) then became sensitive to killing by serum (data not shown). These results suggest that the loss of C4bp binding is not sufficient to mediate the loss of serum resistance in this strain and that the loss of both C4bp and factor H binding correlates with the sensitivity of FA1090 to serum-mediated killing.
Serum sensitivity of FA1090 P.IB mutant strains that bind differentially to C4bp and factor H. FA1090 strains expressing different mutations in the porB gene were grown in the presence of 2 μg/ml CMP-NANA, and bacterial survival was assessed after incubation of 105 CFU in normal human serum for 30 min. The data are presented as percent survival relative to the number of bacteria at time zero for each strain and are representative of those from at least 6 independent experiments. *, P < 0.001 by Student's t test.
Porin's role in the inhibition of apoptosis and ROS production.The PorB proteins from both N. gonorrhoeae and N. meningitidis have been shown to affect apoptosis induction or inhibition in a wide variety of cell types (9–13, 25, 27). We and others have shown that N. gonorrhoeae inhibits apoptosis in polymorphonuclear leukocytes (PMNs) and PMN-like HL-60 cells (39, 40). In an attempt to define regions of PorB which might play a role in apoptosis inhibition in PMNs, each of the P.IB mutant strains was tested for its ability to inhibit staurosporine-induced apoptosis in HL-60 cells differentiated down the granulocytic pathway (39). However, we were unable to identify any strains exhibiting a lack of apoptosis inhibition, as all the mutants were able to inhibit apoptosis to the same extent as the parental FA1090 strain (data not shown).
Porin has also been shown to affect the production of ROS in phagocytic cells (23, 24), and N. gonorrhoeae actively inhibits the oxidative burst in PMNs and PMN-like HL-60 cells (39, 41). All of the P.IB mutant strains were also tested for their ability to inhibit phorbol 12-myristate 13-acetate (PMA)-induced ROS production in differentiated HL-60 cells, and all of the mutants were able to inhibit ROS production to the same extent as FA1090 expressing wild-type PorB (data not shown). Thus, we were unable to identify the PorB residues involved in inhibition of either apoptosis or the oxidative burst in granulocytic cells.
DISCUSSION
Acquisition of serum resistance by pathogenic Neisseria species likely contributes significantly to bacterial pathogenesis, as it allows organisms to escape the bactericidal effects of complement. Complement is present in cervical mucus at levels approximating 10% of the level observed in serum (42) and is a potential source of antibacterial activity. Previous studies have shown that binding of N. gonorrhoeae to either C4bp or factor H via the outer membrane porin PorB can mediate serum resistance in certain strains. In this study, we generated a panel of N. gonorrhoeae strains expressing mutations in the porB gene to further map additional residues involved in binding of FA1090 P.IB to both C4bp and factor H and correlated the binding of both with serum resistance.
Our aim was to conduct structure-function studies of PorB by generating several strains expressing various mutations in the P.IB protein of N. gonorrhoeae strain FA1090 and using these mutants to examine several important biological phenotypes associated with PorB. Random mutagenesis was attempted with both the entire porB gene and smaller domains; however, the frequency of obtaining random mutants was too low to conduct a saturating screen. We also mutated the 8 extracellular loops, since previous studies suggested that at least a subset of the loops in P.IB is nonessential for bacterial viability (21) and that loop regions can be swapped between different PorB alleles (7, 14, 16, 18). We successfully deleted the majority of extracellular loops 4 and 5 but were unable to delete loops 1, 2, 3, and 7 (deletion of loops 6 and 8 was not attempted due to their short length). Alanine-scanning mutagenesis was performed on the remaining loops, such that 5 to 6 amino acids were mutated at once (Fig. 1B; Table 1). Most of our attempted mutations were successful; however, we were unable to recover recombinants expressing changes in extracellular loop 8 and the C-terminal half of loop 7, and we had difficulty constructing a plasmid containing mutations in the N-terminal half of loop 1. Our experience suggests that the majority of sequences in the PorB extracellular loops do not contribute in an essential way to the mediation of pore function but, instead, mediate interactions with other molecules. We have not attempted to combine mutations, but that possibility remains for future studies.
We also isolated a number of mutations in the transmembrane regions. The transmembrane domains are more highly conserved between different PorB alleles and across the porins expressed in different Neisseria species. While some of the mutations that we isolated were conservative, there were many nonconservative substitutions isolated in the transmembrane domains (Fig. 1B; Table 1). These results suggest that not all of the conserved residues found in the transmembrane domains are required for structure or porin function and raise the hypothesis that they play another unknown role.
We identified several regions that, when deleted or mutated, abrogated binding of either C4bp or factor H, or both. These regions were mapped onto a predicted structure of FA1090 P.IB and are shown in Fig. 5. Our results are consistent with those of previous studies showing that extracellular loops 5 and 7 are involved in C4bp binding, but those studies also suggested a lack of involvement for extracellular loops 4 and 6 (16). However, those studies made use of hybrid porins with loops derived from either N. gonorrhoeae strain MS11 (with a P.IB which binds C4bp) or N. gonorrhoeae strain F62 (with a P.IB which does not bind C4bp), whereas our loop mutations involved either deleting the majority of the loop or changing multiple sequential amino acids to alanines, which may account for the differences in the results. It is possible that structural or amino acid sequence similarities between the MS11 and F62 loops 4 and 6 were still able to support binding in the context of the hybrid molecules.
FA1090 P.IB structure and localization of residues involved in C4bp and factor H binding. The structure of the FA1090 P.IB monomer was predicted using the I-TASSER platform (44, 45) and visualized using the DeepView Swiss-PdbViewer (v. 4.1) (46). The Protein Data Bank accession numbers of the top four templates used by I-TASSER are 4AUI, 3A2S, 3VY8, and 3A2R. Extracellular loops 4 through 7 are labeled. (A) Regions of the protein involved in C4bp binding are labeled in red in the context of the predicted structure. (B) Regions of the protein involved in factor H binding are labeled in blue, and regions labeled in purple show an intermediate factor H-binding phenotype when mutated. Residues mutated in the RM760a #40 strain are not indicated, as the mutations are discontinuous and it is not obvious which residue(s) is critical for C4bp and/or factor H binding.
We also found that residues in the C terminus of P.IB were required for factor H binding (Fig. 5B), in contrast to previous studies suggesting that the N-terminal extracellular loops 1 and 2 are involved in PorB-factor H interactions when a different P.IB-expressing strain was used (18). We did not see a decrease in factor H binding when the C-terminal half of loop 1 was mutated, nor did we see a phenotype when the two halves of loop 2 were mutated separately. However, it is possible that mutagenesis of a larger domain is required to see an effect. We were also unable to generate a plasmid clone for testing a mutant with a mutation in the N-terminal half of loop 1. A hybrid porin expressing extracellular loops 3 through 8 of N. gonorrhoeae P.IB was previously not sufficient to mediate factor H binding (18); thus, it is likely that we have not identified all of the regions important for interactions with factor H.
On the basis of the results obtained with the mutations that we were able to generate, there is considerable overlap between the residues involved in C4bp and factor H binding, as deletion/mutation of extracellular loops 4 and 5, part of loop 7, and sequences in the transmembrane region just downstream of loop 6 results in the loss of both C4bp and factor H binding. These strains showed a complete loss of resistance to killing by normal human serum (Fig. 4), consistent with previous observations that binding to these complement factors is required for N. gonorrhoeae serum resistance (14, 16). Strains which bound only to factor H and not C4bp were serum resistant when grown in the presence of CMP-NANA, suggesting that C4bp binding is not essential for serum resistance either when factor H binding can occur or when LOS is sialylated. We did identify P.IB mutant strains which bound to C4bp but not to purified factor H, but we could not examine serum resistance with those strains as they still showed intermediate factor H binding in the presence of NHS. In the absence of CMP-NANA, factor H-binding strains were sensitive to serum-mediated killing, as expected. Since all of the residues identified as being required for factor H binding are also required for C4bp binding in the presence of NHS, we also tested whether prebinding of factor H to PorB might interfere with subsequent C4bp binding. Prebinding of 2 μg purified factor H to the parental FA1090 strain did not result in a significant change in C4bp-binding levels, showing that the binding sites are not the same and that steric hindrance does not occur (data not shown).
We observed differential C4bp-binding phenotypes for some of our mutant strains when the bacteria were swabbed directly from plates rather than being grown in liquid broth for several hours. We saw similar phenotypes when the same strains were heat killed after growth in liquid broth (data not shown). Other mutant strains, as well as the parental strain expressing wild-type P.IB, did not exhibit differential binding under different culture conditions. The reasons for these differences are unclear, but our observations suggest that bacterial viability or different growth conditions might affect C4bp binding via either changes in porin conformation or changes in the properties of other molecules (e.g., LOS) that also play roles in C4bp interactions.
In addition to binding of complement regulatory proteins, we also tested all of the P.IB mutant strains for their abilities to inhibit staurosporine-induced apoptosis and the PMA-induced oxidative burst in differentiated HL-60 cells. There is abundant evidence to suggest that porin is able to modulate apoptotic signaling in a variety of cell types (9–13, 25, 27), but the specific domains in porin responsible for this phenotype have yet to be identified. Porin has also been shown to affect the oxidative burst, and recent results showing that N. gonorrhoeae actively inhibits the oxidative burst in PMNs and PMN-like cells (39, 41) led us to investigate whether porin might be playing a role. We were unable to isolate any mutations in PorB that resulted in a phenotype where the ability to inhibit apoptosis was lost and similarly did not identify any mutations which caused a defect in ROS inhibition. Our failure to identify residues involved in either of these processes could be due to several reasons, including the following: (i) the relevant domains were not mutated in this study, (ii) domains required for either or both phenotypes are also essential for pore function and bacterial viability, or (iii) porin does not play a significant role in either apoptosis inhibition or regulation of ROS production.
N. gonorrhoeae has evolved multiple mechanisms to evade the bactericidal effects of both the innate and adaptive immune responses during human infection. Many of these functions, including avoidance of the bactericidal effects of complement found in blood and at mucosal surfaces, are thought to be mediated at least in part via the major outer membrane porin elaborated by the organism. In this study, we have isolated several strains of N. gonorrhoeae expressing various mutations in the porin protein which could potentially be of utility for studying other biological functions of this important cell surface molecule. Our studies of the porin domains involved in regulating complement also lend further insight into the mechanisms used by N. gonorrhoeae to escape the innate immune response and survive in its host.
ACKNOWLEDGMENTS
Flow cytometry was performed at the Northwestern University Interdepartmental ImmunoBiology Flow Cytometry Core Facility. Traditional sequencing services were performed at the Northwestern University Genomics Core Facility. We thank Mark Anderson and Alice Château for critical reading and editing of the manuscript.
This work was supported by grants R01 AI044239 and R37 AI033493 to H.S.S. A.C. was partially supported by American Heart Association grant 10POST2550017 and The Wellcome Trust grant R24378/CN001.
FOOTNOTES
- Received 21 March 2013.
- Returned for modification 16 April 2013.
- Accepted 6 September 2013.
- Accepted manuscript posted online 16 September 2013.
- Copyright © 2013, American Society for Microbiology. All Rights Reserved.
