Neisseria gonorrhoeae Heme Biosynthetic Mutants Utilize Heme and Hemoglobin as a Heme Source but Fail To Grow within Epithelial Cells

Many bacterial pathogens, including pathogenic neisseriae, can use heme as an iron source for growth. To study heme utilization by Neisseria gonorrhoeae, two heme biosynthetic mutants were constructed, one with a mutation in hemH (the gene encoding ferrochelatase) and one with a mutation in hemA (the gene encoding γ-glutamyl tRNA reductase). The hemH mutant failed to grow without an exogenous supply of heme or hemoglobin, whereas the hemA mutant failed to grow unless heme, hemoglobin, or heme precursors were present. Growth of the mutants with hemoglobin required expression of the hemoglobin receptor (HpuAB) and was TonB dependent. However, growth with heme required neither HpuAB nor TonB. An fbpA mutant grew normally when either heme or hemoglobin was present in the medium. The heme biosynthetic mutants showed reduced intracellular survival, compared to the parent strain, within A-431 endocervical epithelial cell cultures. These studies demonstrate that in addition to synthesizing their own heme, N. gonorrhoeae strains are able to internalize and utilize exogenous heme independently of FbpA but appear unable to obtain heme from within epithelial cells for growth.     

Identification and purification of a hemoglobin-binding outer membrane protein from Neisseria gonorrhoeae.

The majority of in vitro-grown Neisseria gonorrhoeae strains were unable to use hemoglobin as the sole source of iron for growth (Hgb-), but a minor population was able to do so (Hgb+). The ability of Hgb+ gonococci to utilize hemoglobin as the iron source was associated with the expression of an iron-repressible 89-kDa hemoglobin-binding protein in the outer membrane. The N-terminal amino acid sequence of this protein revealed amino acids, from positions 2 to 16, identical to those of HpuB, an 85 kDa iron-regulated hemoglobin-haptoglobin utilization outer membrane protein of Neisseria meningitidis. Isogenic mutants constructed by allelic replacement with a meningococcal hpu::mini-Tn3erm construct no longer expressed the 89-kDa protein. Mutants could not utilize hemoglobin to support growth but still grew on heme. Thus, the gonococcal HpuB homolog is a functional hemoglobin receptor and is essential for growth with hemoglobin.     

Binding and accumulation of hemin in Neisseria gonorrhoeae.

The ability to utilize hemin and hemin-containing compounds for nutritional iron (Fe) uptake has been documented for several pathogenic bacteria. Neisseria gonorrhoeae can utilize free hemin as a source of Fe for growth; however, little is known concerning the mechanisms involved in hemin transport. In this study we have characterized the binding and accumulation of hemin by N. gonorrhoeae and defined the specificity of the gonococcal hemin receptor. N. gonorrhoeae F62 was grown in a chemically defined medium containing the iron chelator Desferal, and hemin transport was initiated by the addition of [59Fe]hemin (4.0 or 8.0 microM; specific activity, 7.0 Ci/mol). 59Fe uptake from radiolabeled hemin by N. gonorrhoeae was energy dependent, and 59Fe was shown to accumulate in the cell at a constant rate during logarithmic growth. However, we observed a decrease in the uptake of 59Fe from radiolabeled hemin when inorganic iron was present in the growth medium. Binding of 59Fe from radiolabeled hemin was inhibited by the addition of either cold hemin, hematoporphyrin, or hemoglobin, but not by ferric citrate. Although [14C]hemin was found to support the growth of N. gonorrhoeae, we did not detect the uptake of 14C from radiolabeled hemin. Extraction of the gonococcal periplasmic ferric binding protein (Fbp) from cultures grown with [59Fe]hemin indicated that a majority of the 59Fe was associated with the Fbp. Taken together, the results presented here indicate that hemin binds to a gonococcal outer membrane receptor through the protoporphyrin portion of the molecule and that following binding, iron is removed and transported into the cell, where it is associated with the gonococcal periplasmic ferric binding protein, Fbp.     

A pleiotropic iron-uptake mutant of Neisseria meningitidis lacks a 70-kilodalton iron-regulated protein.

We isolated an iron-uptake mutant of Neisseria meningitidis M986-NCV-1 that was severely limited in the ability to use several sources of iron in the form of Fe3+. This mutant, FAM11, grew poorly or not at all with human transferrin (TF) or lactoferrin (LF) as the sole iron source in a defined medium but grew as well as wild-type meningococci with hemin or hemoglobin. Uptake of 55Fe bound to TF, LF, dicitrate complexes, aerobactin, or nitrilotriacetate was reduced to 0 to 4% of the wild-type level. FAM11 did not produce an iron-repressible outer membrane protein (FeRP) of 70 kilodaltons (kDa) found in membranes of iron-stressed M986-NCV-1. Western blot (immunoblot) analysis using rabbit antiserum against this protein revealed that at least 17 of 18 meningococcal and 10 of 14 gonococcal strains produced an FeRP of ca. 70 kDa. The 70-kDa FeRP was shown to be surface exposed by radioimmunoprecipitation with human immune sera. These data suggest that the 70-kDa FeRP is somehow involved in Fe uptake from TF and LF. However, we were unable to transform the iron-uptake phenotype from FAM11 into wild-type meningococci to confirm this. Revertants of FAM11 that grew with TF and LF did not regain the ability to make the 70-kDa FeRP but also did not completely regain the Fe-uptake phenotype of M986-NCV-1.     

Characterization of the hgbA locus encoding a hemoglobin receptor from Haemophilus ducreyi.

Haemophilus ducreyi can bind hemoglobin and use it as a source of heme, for which it has an obligate requirement. We previously identified and purified HgbA, a hemoglobin-binding outer membrane protein from H. ducreyi. In this report, we describe the molecular cloning, expression, DNA sequence, and mutagenesis of the structural gene for HgbA, hgbA. H. ducreyi and recombinant Escherichia coli expressing hgbA bound [125I]hemoglobin, establishing HgbA as a receptor. Insertions or deletions in the cloned hgbA gene abolished expression of HgbA and hemoglobin binding in E. coli. Mutagenesis of H. ducreyi by allelic exchange of insertions into hgbA abolished its ability to bind [125I]hemoglobin or utilize hemoglobin as a source of heme. The deduced protein sequence was similar to those of the TonB-dependent family of outer membrane receptors. The most similar member was HutA (heme receptor) from Vibrio cholerae. Tbp1 and Lbp1 (transferrin and lactoferrin receptors, respectively, from pathogenic Neisseria spp.) also showed very significant homology. Thus, by characterizing the hgbA locus, this work elucidates a potentially important role of HgbA in obtaining heme and/or iron from the host.     

CD46-independent binding of neisserial type IV pili and the major pilus adhesin, PilC, to human epithelial cells

Neisseria gonorrhoeae is a gram-negative bacterial pathogen which infects the human mucosal epithelium. An early critical event in neisserial infection is the type IV pilus-mediated adherence to the host cell. The PilC protein, located on the pilus tip, has earlier been identified as the major pilus adhesin. Previous studies suggested that the cell surface protein CD46 is a pilus receptor for Neisseria. We investigated the role of CD46 in pilus-mediated gonococcal infection of epithelial cells. Differences in binding efficiencies of piliated gonococci as well as purified pilus adhesin PilC2 on human epithelial cell lines did not correlate to the level of surface-expressed CD46. Additionally, no binding of piliated gonococci or PilC2 protein was observed on CD46-transfected CHO and MDCK cells. Furthermore, specific down-regulation of CD46 expression in human epithelial cell lines by RNA interference did not alter the binding efficiency of piliated gonococci or purified PilC2 protein, although other CD46-dependent processes, such as measles virus infection and C3b cleavage, were significantly reduced. These data support the notion that pilus-mediated gonococcal infection of epithelial cells can occur in a CD46-independent manner, thus questioning the function of CD46 as an essential pilus receptor for pathogenic neisseriae.     

Ability of Neisseria gonorrhoeae, Neisseria meningitidis, and commensal Neisseria species to obtain iron from lactoferrin.

The ability of 107 Neisseria isolates to compete for iron with human lactoferrin (LF) was examined. Each of 15 meningococci, 53% of 59 selected gonococci, and 24% of 33 commensal Neisseria could use LF-bound iron for growth. Isolates which could not obtain iron from LF were growth inhibited when sufficient LF was added to defined agar medium to bind available free iron. No difference was observed in the ability of colony type 1 and colony type 4 gonococci of the same strain to compete with LF for iron. LF was growth inhibitory for 50% of 22 disseminated disease isolates (DGI strains) and 51% of 35 local urogenital disease isolates (UGI strains). Only 14% of gonococcal isolates requiring arginine, hypoxanthine, and uracil for growth were able to compete with LF for iron, whereas 87% of all other gonococcal isolates could do so (P less than 0.005). Ability to obtain iron from LF does not appear to be required for survival of Neisseria on mucosal surfaces, nor essential for invasion of the bloodstream by gonococci. However, ability to utilize LF as a source of iron may contribute to differences in pathogenicity among certain gonococcal isolates.     

Growth of Neisseria gonorrhoeae in the female mouse genital tract does not require the gonococcal transferrin or hemoglobin receptors and may be enhanced by commensal lactobacilli

Neisseria gonorrhoeae is capable of utilizing a variety of iron sources in vitro, including human transferrin, human lactoferrin, hemoglobin, hemoglobin-haptoglobin complexes, heme, and heterologous siderophores. Transferrin has been implicated as a critical iron store for N. gonorrhoeae in the human male urethra. The demonstration that gonococci can infect the lower genital tracts of estradiol-treated BALB/c mice in the absence of human transferrin, however, suggests that other usable iron sources are present in the murine genital tract. Here we demonstrate that gonococcal transferrin and hemoglobin receptor mutants are not attenuated in mice, thereby ruling out transferrin and hemoglobin as essential for murine infection. An increased frequency of phase variants with the hemoglobin receptor "on" (Hg(+)) occurred in ca. 50% of infected mice; this increase was temporally associated with an influx of neutrophils and detectable levels of hemoglobin in the vagina, suggesting that the presence of hemoglobin in inflammatory exudates selects for Hg(+) phase variants during infection. We also demonstrate that commensal lactobacilli support the growth of N. gonorrhoeae in vitro unless an iron chelator is added to the medium. We hypothesize that commensal lactobacilli may enhance growth of gonococci in vivo by promoting the solubilization of iron on mucosal surfaces through the production of metabolic intermediates. Finally, transferrin-binding lipoprotein (TbpB) was detected on gonococci in vaginal smears, suggesting that although gonococci replicate within the genital tracts of mice, they may be sufficiently iron-stressed to express iron-repressible proteins. In summary, these studies support the potential role of nontransferrin, nonhemoglobin iron sources during gonococcal infection of the female genital tract.     

Ability of Neisseria gonorrhoeae, Neisseria meningitidis, and commensal Neisseria species to obtain iron from transferrin and iron compounds.

The ability of Neisseria species to use iron compounds and to compete with iron-binding proteins was examined with deferrated defined medium and the iron chelator deferoxamine. All Neisseria species were able to assimilate a variety of ferric and ferrous iron salts. They were not able to efficiently solubilize an inorganic iron salt such as ferric nitrate, but were able to use iron chelated by citrate, oxalacetate, pyrophosphate, or nitrilotriacetate. Each of the 95 Neisseria isolates examined was able to use hemin as a sole source of iron, and most, but not all, of the isolates were able to obtain iron from hemoglobin. Heated human serum stimulated growth of all gonococci, meningococci, and some commensal Neisseria species in iron-deficient medium. All gonococci and meningococci were able to scavenge iron from 25% saturated transferrin, whereas most commensal organisms were inhibited by this iron-binding protein. The ability to compete with transferrin was specific, since partially saturated conalbumin was bacteriostatic for all Neisseria species. Although the pathogenic Neisseria species were able to compete more efficiently with transferrin for iron than were the nonpathogenic Neisseria species, no correlation was observed between the virulence of different strains or colony types of gonococci and the ability to scavenge iron in vitro from transferrin or other chelators.     

Passively released heme from hemoglobin and myoglobin is a potential source of nutrient iron for Bordetella bronchiseptica

Colonization by Bordetella bronchiseptica results in a variety of inflammatory respiratory infections, including canine kennel cough, porcine atrophic rhinitis, and a whooping cough-like disease in humans. For successful colonization, B. bronchiseptica must acquire iron (Fe) from the infected host. A vast amount of Fe within the host is sequestered within heme, a metalloporphyrin which is coordinately bound in hemoglobin and myoglobin. Utilization of hemoglobin and myoglobin as sources of nutrient Fe by B. bronchiseptica requires expression of BhuR, an outer membrane protein. We hypothesize that hemin is acquired by B. bronchiseptica in a BhuR-dependent manner after spontaneous loss of the metalloporphyrin from hemoglobin and/or myoglobin. Sequestration experiments demonstrated that direct contact with hemoglobin or myoglobin was not required to support growth of B. bronchiseptica in an Fe-limiting environment. Mutant myoglobins, each exhibiting a different affinity for heme, were employed to demonstrate that the rate of growth of B. bronchiseptica was directly correlated with the rate at which heme was lost from the hemoprotein. Finally, Escherichia coli cells expressing recombinant BhuR had the capacity to remove hemin from solution. Collectively, these experiments provided strong experimental support for the model that BhuR is a hemin receptor and B. bronchiseptica likely acquires heme during infection after passive loss of the metalloporphyrin from hemoglobin and/or myoglobin. These results also suggest that spontaneous hemin loss by hemoglobin and myoglobin may be a common mechanism by which many pathogenic bacteria acquire heme and heme-bound Fe.     

The iron-repressed, AraC-like regulator MpeR activates expression of fetA in Neisseria gonorrhoeae

Neisseria gonorrhoeae is an obligate human pathogen that causes the common sexually transmitted infection gonorrhea. Gonococcal infections cause significant morbidity, particularly among women, as the organism ascends to the upper reproductive tract, resulting in pelvic inflammatory disease, ectopic pregnancy, and infertility. In the last few years, antibiotic resistance rates have risen dramatically, leading to severe restriction of treatment options for gonococcal disease. Gonococcal infections do not elicit protective immunity, nor is there an effective vaccine to prevent the disease. Thus, further understanding of the expression, function, and regulation of surface antigens could lead to better treatment and prevention modalities in the future. In the current study, we determined that an iron-repressed regulator, MpeR, interacted specifically with the DNA sequence upstream of fetA and activated FetA expression. Interestingly, MpeR was previously shown to regulate the expression of gonococcal antimicrobial efflux systems. We confirmed that the outer membrane transporter FetA allows gonococcal strain FA1090 to utilize the xenosiderophore ferric enterobactin as an iron source. However, we further demonstrated that FetA has an extended range of substrates that encompasses other catecholate xenosiderophores, including ferric salmochelin and the dimers and trimers of dihydroxybenzoylserine. We demonstrated that fetA is part of an iron-repressed, MpeR-activated operon which putatively encodes other iron transport proteins. This is the first study to describe a regulatory linkage between antimicrobial efflux and iron transport in N. gonorrhoeae. The regulatory nidus that links these systems, MpeR, is expressed exclusively by pathogenic neisseriae and is therefore expected to be an important virulence factor.     

Presence of antibodies to the major anaerobically induced gonococcal outer membrane protein in sera from patients with gonococcal infections

Anaerobically grown Neisseria gonorrhoeae induces and represses the synthesis of outer membrane proteins. One of the anaerobically induced proteins, Pan 1, reacted strongly on Western blots with sera from patients with uncomplicated gonococcal infection, pelvic inflammatory disease, and disseminated gonococcal infection, but not with normal human serum. The pattern of reactivity of the sera against Pan 1 from several gonococcal strains suggested that the protein was antigenically heterogeneous, containing both common and unique epitopes. Staphylococcus aureus V8 protease digestion of Pan 1 from four gonococcal strains revealed the presence of common peptides, with one strain also containing some unique peptides and lacking others. The class of the antibody reactive with gonococcal outer membrane antigens was examined; anti-Pan 1 antibody was found to be IgG or IgM, but not IgA. The IgM antibody present reacted predominantly with Pan 1. These data indicate that the Pan 1 protein is expressed in vivo and strongly suggest that N. gonorrhoeae can grow anaerobically in vivo.     

Neisseria spp. and AIDS.

Neisseria meningitidis from various serogroups and two commensal neisseriae (N. sicca and N. perflava) were isolated from 15 patients at various stages of human immunodeficiency virus infection in this clinical and bacteriological study. The cases were grouped into the following three classes: (i) infections with an N. meningitidis strain of a serogroup known to be pathogenic (A, B, or C) and apparently independent of the human immunodeficiency virus infection, (ii) infections with a N. meningitidis strain of a serogroup which is normally either commensal or poorly pathogenic (serogroups Y, X, Z, and Z,29E), (iii) pulmonary and disseminated infections occurring in the course of the clinical evolutionary stage of AIDS, in two cases of which commensal neisseriae (N. sicca and N. perflava) were isolated from blood cultures.     

Protein sources of heme for Haemophilus influenzae.

Although Haemophilus influenzae requires heme for growth, the source of heme during invasive infections is not known. We compared heme, lactoperoxidase, catalase, cytochrome c, myoglobin, and hemoglobin as sources of heme for growth in defined media. The minimum concentration of heme permitting unrestricted growth of strain E1a, an H. influenzae type b isolate from cerebrospinal fluid, was 0.02 micrograms/ml. Using molar equivalents of heme as lactoperoxidase, catalase, cytochrome c, myoglobin, and hemoglobin, we determined that myoglobin and hemoglobin permitted unrestricted growth at this concentration. To determine the ability of host defenses to sequester heme from H. influenzae, we used affinity chromatography to purify human haptoglobin and hemopexin, serum proteins which bind hemoglobin and heme. Plate assays revealed that 12 strains of H. influenzae acquired heme from hemoglobin, hemoglobin-haptoglobin, heme-hemopexin, and heme-albumin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of outer membrane proteins of strain E1a grown in heme-replete and heme-restricted conditions revealed a heme-repressible outer membrane protein with an apparent molecular mass of 38 kilodaltons. These results demonstrated that, unlike Escherichia coli, H. influenzae may acquire heme from hemoglobin-haptoglobin. H. influenzae also may acquire heme from hemopexin and albumin, which have not been previously investigated. The role of outer membrane proteins in the acquisition of heme is not yet clear.     

Cloning of a DNA fragment encoding a heme-repressible hemoglobin-binding outer membrane protein from Haemophilus influenzae.

Haemophilus influenzae is able to use hemoglobin as a sole source of heme, and heme-repressible hemoglobin binding to the cell surface has been demonstrated. Using an affinity purification methodology, a hemoglobin-binding protein of approximately 120 kDa was isolated from H. influenzae type b strain HI689 grown in heme-restricted but not in heme-replete conditions. The isolated protein was subjected to N-terminal amino acid sequencing, and the derived amino acid sequence was used to design corresponding oligonucleotides. The oligonucleotides were used to probe a Southern blot of EcoRI-digested HI689 genomic DNA. A hybridizing band of approximately 4.2 kb was successfully cloned into pUC19. Using a 1.9-kb internal BglII fragment of the 4.2-kb clone as a probe, hybridization was seen in both typeable and nontypeable H. influenzae but not in other bacterial species tested. Following partial nucleotide sequencing of the 4.2-kb insert, a putative open reading frame was subcloned into an expression vector. The host Escherichia coli strain in which the cloned fragment was expressed bound biotinylated human hemoglobin, whereas binding of hemoglobin was not detected in E. coli with the vector alone. In conclusion, we hypothesize that the DNA fragment encoding an approximately 120-kDa heme-repressible hemoglobin-binding protein mediates one step in the acquisition of hemoglobin by H. influenzae in vivo.     

Ability of Vibrio vulnificus to obtain iron from hemoglobin-haptoglobin complexes.

It has been suggested that the normal serum protein, haptoglobin (Hp), serves a bacteriostatic role by binding free hemoglobin (Hm), thus making heme iron unavailable for bacterial growth. Previous studies showed that, unlike Escherichia coli, Vibrio vulnificus was able to overcome this Hp-blocking effect. We report here a study on the iron-withholding property of the three major human Hp phenotypes, Hp 1, 2, and 2-1. Results of experiments with human serum showed that V. vulnificus C7184 was able to obtain iron from Hm bound to Hp types 1 and 2, but not that bound to Hp 2-1. E. coli 2395-80, on the other hand, was unable to overcome the blocking effect of any Hp phenotype. Using purified Hp 1, we also demonstrated that, although V. vulnificus was unable to grow in a deferrated medium without an additional iron source, it was able to grow with the addition of the Hm-Hp complex.     

Identification of pathogenic neisseriae by genetic transformation

The detection of pathogenic neisseriae by genetic transformation of a naturally occurring proline auxotroph of Neisseria gonorrhoeae strain F62 is described. Of 169 clinical isolates of N. gonorrhoeae, approximately 90% gave a positive transformation assay. Twelve clinical isolates of N. meningitidis and stock cultures of the various meningococcal serogroups also gave a positive result. However, the sensitivity of the assay was found to be approximately 1000-fold lower with N. meningitidis as test organism. Eleven other members of the family Neisseriaceae failed to transform the recipient organism. Although proline requirement did not appear to limit the value of the assay greatly, it probably was the main reason for negative results. The sensitivity of the assay and its ability to detect non-viable gonococci suggests that this method merits further investigation as a possible aid to diagnosis of gonococcal infection in special circumstances.     

Monoclonal antibody that recognizes an outer membrane antigen common to the pathogenic Neisseria species but not to most nonpathogenic Neisseria species.

A hybridoma derived from a mouse immunized with gonococcal outer membranes produced an antibody, designated H.8, that bound to all strains of Neisseria gonorrhoeae and N. meningitidis tested, and to N. lactamica and N. cinerea, but only rarely to other nonpathogenic Neisseria species. Studies with the gonococcal strain used in production of the antibody showed that the antibody bound to a surface-exposed, protease-sensitive, and heat-modifiable outer membrane antigen that we believe is distinct from previously described gonococcal outer membrane proteins.     

Involvement of HxuC outer membrane protein in utilization of hemoglobin by Haemophilus influenzae

Haemophilus influenzae can utilize different protein-bound forms of heme for growth in vitro. A previous study from this laboratory indicated that nontypeable Haemophilus influenzae (NTHI) strain N182 expressed three outer membrane proteins, designated HgbA, HgbB, and HgbC, that bound hemoglobin or hemoglobin-haptoglobin and were encoded by open reading frames (ORFs) that contained a CCAA nucleotide repeat. Testing of mutants expressing the HgbA, HgbB, and HgbC proteins individually revealed that expression of any one of these proteins was sufficient to allow wild-type growth with hemoglobin. In contrast, mutants that expressed only HgbA or HgbC grew significantly better with hemoglobin-haptoglobin than did a mutant expressing only HgbB. Construction of an isogenic hgbA hgbB hgbC mutant revealed that the absence of these three gene products did not affect the ability of NTHI N182 to utilize hemoglobin as a source of heme, although this mutant was severely impaired in its ability to utilize hemoglobin-haptoglobin. The introduction of a tonB mutation into this triple mutant eliminated its ability to utilize hemoglobin, indicating that the pathway for hemoglobin utilization in the absence of HgbA, HgbB, and HgbC involved a TonB-dependent process. Inactivation in this triple mutant of the hxuC gene, which encodes a predicted TonB-dependent outer membrane protein previously shown to be involved in the utilization of free heme, resulted in loss of the ability to utilize hemoglobin. The results of this study reinforce the redundant nature of the heme acquisition systems expressed by H. influenzae.     

Elaboration of a 3.6-kilodalton lipooligosaccharide, antibody against which is absent from human sera, is associated with serum resistance of Neisseria gonorrhoeae

Neisseria gonorrhoeae strains that resist lysis by normal human sera (NHS) do so, in part, because NHS contain immunoglobulin M (IgM) specific for lipooligosaccharide (LOS) antigens of serum-sensitive strains, but lack antibodies for LOS antigens that can serve as loci for immune lysis of serum-resistant (serr) strains. We used a monoclonal antibody (McAb), specific for an epitope within a 3.6-kilodalton (kDa) component of Neisseria meningitidis L8 LOS, that binds a 3.6-kDa gonococcal LOS component so that we could explore further serr gonococcal strains. The McAb bound to the LOS of 6 of 7 serr of strains but not to the LOS of 0 of 14 serum-sensitive and serum-intermediate gonococcal strains of diverse origin. We studied three serr strains further. Strain 7134 does not elaborate the 3.6-kDa LOS component and does not bind the McAb; strains WR220 and WR302 do elaborate the 3.6-kDa LOS component. The titer (log2) at which the McAb, diluted in NHS, lysed strain WR220 was 7.7; for WR302 it was 3.7, and for 7134 it was 0. Addition of McAb to NHS caused increased classical and alternative-pathway C3 deposition onto strain WR220, but only classical-pathway-activated C3 deposition onto strain WR302. The difference in lytic effectiveness of the McAb for the two strains, therefore, may result from differences in alternative-pathway augmentation of McAb-dependent classical-pathway activation on their surfaces. None of 40 randomly selected normal young adults had serum antibody that could compete with the McAb for binding to WR220 LOS in a solid-phase RIA. We conclude that the 3.6-kDa LOS component is commonly expressed by serr strains of N. gonorrhoeae and that antibody to it would be lytic if present in human serum, but that it is infrequently, if ever, present. As a result, strains elaborating this LOS are resistant to lysis by NHS.