The Role of Melanocortin‐1 Receptor Polymorphism in Skin Cancer Risk Phenotypes

We have examined melanocortin‐1 receptor (MC1R) variant allele frequencies in the general population and in a collection of adolescent dizygotic and monozygotic twins to determine statistical associations of pigmentation phenotypes with increased skin cancer risk. This included hair and skin color, freckling, mole count and sun exposed skin reflectance. Nine variants were studied and designated as either strong R (OR = 63; 95% CI 32–140) or weak r (OR = 5; 95% CI 3–11) red hair alleles. Penetrance of each MC1R variant allele was consistent with an allelic model where effects were multiplicative for red hair but additive for skin reflectance. To assess the interaction of the brown eye color gene BEY2/OCA2 on the phenotypic effects of variant MC1R alleles we imputed OCA2 genotype in the twin collection. A modifying effect of OCA2 on MC1R variant alleles was seen on constitutive skin color, freckling and mole count. In order to study the individual effects of these variants on pigmentation phenotype we have established a series of human primary melanocyte strains genotyped for the MC1R receptor. These include strains which are MC1R wild‐type consensus, variant heterozygotes, and homozygotes for strong R alleles Arg151Cys and Arg160Trp. Ultrastructural analysis demonstrated that only consensus strains contained stage III and IV melanosomes in their terminal dendrites whereas Arg151Cys and Arg160Trp homozygous strains contained only immature stage I and II melanosomes. Such genetic association studies combined with the functional analysis of MC1R variant alleles in melanocytic cells should provide a link in understanding the association between pigmentary phototypes and skin cancer risk.     

Polyclonal Antisera To Distinguish Strains and Form Variants of Photorhabdus (Xenorhabdus) luminescens

In this study antisera against Photorhabdus luminescens strains were prepared for the first time. P. luminescens is a bacterial symbiont of entomopathogenic nematodes belonging to the genus Heterorhabditis. To characterize P. luminescens strains and form variants, we produced polyclonal antisera against P. luminescens PE (obtained from nematode strain NLH-E87.3) and against the primary and secondary forms of P. luminescens PSH (obtained from nematode strain DH-SH1). In double-diffusion tests all form variants of strain PE reacted with the antiserum against the primary form, but each variant produced a different diffusion pattern. The primary and secondary forms of strain PSH were also serologically different. Antiserum 9226 reacted with almost all P. luminescens strains tested, but it reacted differently with each strain in the double-diffusion test, showing that the strains were serologically different. The specificity of the antisera was increased by cross-absorption. After cross-absorption the antiserum against the strain PSH primary or secondary form was specific for that form and did not react with the other form. Using the cross-absorbed antisera in immunofluorescence cell-staining tests, we could distinguish primary and secondary form cells in a mixed strain PSH culture.     

A comparison of two strains of the anaerobic ciliate Trimyema compressum

Two strains of the anaerobic ciliate Trimyema compressum, isolated from different habitats, were compared. The cytoplasm of the ciliates contained hydrogenosome-like microbodies and methanogenic bacteria; the latter were lost during continued cultivation. In addition both strains harbored a non-methanogenic endosymbiont, which was lost in strain K. The ciliates lacked cytochromes, cytochrome oxidase and catalase but contained superoxide dismutase. Hydrogenase activity could be demonstrated only in strain N. In monoxenic culture strain K needed sterols as growth factors. The cells of both strains reacted similarly with respect to oxygen tolerance (up to 0.5 mg O₂/l), inhibition of growth by cyanide and azide, and resistance to antimycin A. Only cells of strain N showed growth inhibition by chloramphenicol. It is concluded that Trimyema compressum is an anaerobic, microaerotolerant organism, its microbodies show more resemblance to hydrogenosomes than to mitochondria.     

Isolation of Nontoxigenic Variants Associated with Enhanced Sporulation and Alteration in the Cell Wall from Clostridium botulinum Type A 190L by Treatment with Detergents

Nontoxigenic variants were isolated from Clostridium botulinum type A strain 190L after treatment with detergents such as deoxycholate, sodium dodecyl sulfate, Tween 80 and Brij-58. Deoxycholate was most effective for obtaining the variants. The variants exhibited a markedly increased frequency of sporulation compared with the oligosporogenic parent strain. The cell wall of the parent strain was composed of an outer layer and an inner layer, whereas that of the variants lost the outer layer. After treatment with mitomycin C the parent strain was subjected to lysis and produced bacteriophages with a hexagonal head and a contractible tail, while the nontoxigenic variants did not yield bacteriophages or phage-like structures. There appears to be a close relationship among the toxigenic and sporogenic properties, formation of the outer cell wall layer and lysogeny.     

Functional implications of the expression of PilC proteins in meningococci

Multiple forms of PilC were found in Neisseria meningitidis (Nm) strains isolated from the oropharynx, blood or cerebrospinal fluid expressing either Class I or Class II pili. PilC expression was observed less frequently in case as opposed to carrier isolates. Moreover, PilC and pili were not always co-expressed. Several heavily piliated strains had no detectable PilC protein as determined by Western blotting using an antiserum previously used to detect such proteins in adhesive variants (Nassif et al., 1994). Serogroup B strain MC58 produced large numbers of pili, but expressed barely detectable amounts of PilC. A clonal variant of this strain with increased expression of PilC concurrently exhibited increased adherence to Chang conjunctival epithelial cells and human umbilical vein endothelial cells (Huvecs), but with more rapid binding to the former. No alteration in pilin sequence occurred in this variant, suggesting the involvement of PilC in increased adhesion. A Pil^- backswitcher isolated from the hyper-adherent variant was PilC⁺ but was non-adherent, indicating that any PilC adherence function requires pilus expression. Parental variant (low PilC) produced pili in bundles that were easily detached from the bacterial surface and were frequently associated with Huvec surfaces after bacteria had been sheared off, but pili infrequently replaced bacteria during infection with the PilC-expressing variant. The hyper-adherent variant, which appeared to produce morphologically distinct pilus bundles, was able to withstand considerable shearing force and remained firmly attached to Huvecs. This raises the possibility that the observed hyper-adherence may arise from better anchorage of pili to the bacterial surface in addition to increased adhesion to some host cell surfaces.     

Antigenic population changes of Leptospira biflexa strains grown under the selective pressure of factorial antibodies

Serovars jequitaia and tororò of Leptospira biflexa were cultured in the presence of homologous factor serum containing factorial antibodies (FcAbs) to their major antigens. After 39 serial passages they were then re-tested to determine whether their major antigens had remained unchanged. It was found that each parent strain had been replaced by an antigenic variant. The disappearance of each parent strain and its replacement by an antigenic variant was attributed to the selective conditions imposed by FcAbs. The antigenic variants behaved like true mutants. They lacked the major serovar antigens of the parent strains and had acquired some major antigens similar to those of two different serovars, one of which belonged to the same serogroup as the parent strain and the other to a different serogroup. A comparison of the major antigens of the parent strains with those of their antigenic variants indicated that factorial antibodies may be used selectively to obtain antigenic variants with a predefined pattern of major antigens.     

A Comparative Study of the Sugar Composition of O-antigenic Lipopolysaccharides Isolated from Vibrio alginolyticus and Vibrio parahaemolyticus

A comparative study of the sugar composition of O-antigenic lipopolysaccharides (LPS) isolated from Vibrio alginolyticus and those from V. parahaemolyticus was carried out. 3-Deoxy-d-mannooctulosonic acid, 2-keto-3-deoxy octonate (KDO), a regular sugar constituent of gram-negative bacterial LPS, was totally absent from LPS of all V. alginolyticus strains examined as it was from those of V. parahaemolyticus. Furthermore, a KDO-like thiobarbituric acid test-positive substance, identical with that of either V. parahaemolyticus 07 or 012, was also found in LPS from three strains, 505–78, 905–78, and 1013–79 (designated tentatively as group I), out of the five strains of V. alginolyticus tested. LPS from the members of group I contained, as component sugars, glucose, galactose, l-glycero-d-manno-heptose, glucosamine, galactosamine, the KDO-like substance, and an unidentified amino sugar P1. Thus, LPS of the members of group I possessed a similar sugar composition which is similar to that of LPS from either V. parahaemolyticus 07 or 012. LPS of strain 1027–79, one of the other two strains (designated tentatively as gorup II), contained as component sugars, glucose, l-glycero-d-mannoheptose, glucosamine, galactosamine, and the other unidentified amino sugar P2, while LPS of strain 53–79, the other member of group II, contained galactose as an additional component. The results indicate that LPS of strain 1027–79 has a sugar composition similar to that of V. parahaemolyticus 09 LPS.     

The role of melanocortin-1 receptor polymorphism in skin cancer risk phenotypes

We have examined melanocortin-1 receptor (MC1R) variant allele frequencies in the general population and in a collection of adolescent dizygotic and monozygotic twins to determine statistical associations of pigmentation phenotypes with increased skin cancer risk. This included hair and skin color, freckling, mole count and sun exposed skin reflectance. Nine variants were studied and designated as either strong R (OR = 63; 95% CI 32-140) or weak r (OR = 5; 95% CI 3-11) red hair alleles. Penetrance of each MC1R variant allele was consistent with an allelic model where effects were multiplicative for red hair but additive for skin reflectance. To assess the interaction of the brown eye color gene BEY2/OCA2 on the phenotypic effects of variant MC1R alleles we imputed OCA2 genotype in the twin collection. A modifying effect of OCA2 on MC1R variant alleles was seen on constitutive skin color, freckling and mole count. In order to study the individual effects of these variants on pigmentation phenotype we have established a series of human primary melanocyte strains genotyped for the MC1R receptor. These include strains which are MC1R wild-type consensus, variant heterozygotes, and homozygotes for strong R alleles Arg151Cys and Arg160Trp. Ultrastructural analysis demonstrated that only consensus strains contained stage III and IV melanosomes in their terminal dendrites whereas Arg151Cys and Arg160Trp homozygous strains contained only immature stage I and II melanosomes. Such genetic association studies combined with the functional analysis of MC1R variant alleles in melanocytic cells should provide a link in understanding the association between pigmentary phototypes and skin cancer risk.     

Conversion of biological and immunological properties during a process of decapsulation in a strain of Staphylococcus hyicus

Repeated subculture at 42 degrees C of Staphylococcus hyicus strain ST67P, which exhibits streaming-type growth in a soft-agar medium, yielded three variants, ST67L, ST67S and ST67C, which had different colonial morphologies; small compact colonies possessing long and short tails and perfect compact colonies. The parent strain and ST67L respectively gave strong and weak positive intensity when stained by rabbit antisera prepared by capsular type I and II strains of Staph. epidermidis conjugated with fluorescein isothiocyanate. Variant ST67L gave a positive result with antiserum prepared by capsular type I strain and no staining was observed with variants ST67S and ST67C against these antisera preparations. Strain ST67C had the lowest virulence although no remarkable difference was shown between the parent strain and variants ST67L and ST67S. The cell volume index of the parent strain was 1.35, 2.43 and 3.71 times larger than those of ST67L, ST67S and ST67C, respectively. The converting activity of rabbit anti-ST67P strain serum absorbed by strain ST67C required four times more of the organisms than strain ST67P, changing the colonial morphology of the strain from diffuse to compact type by the addition of antiserum to soft agar medium. Positive coagulase and false positive clumping factor reaction were shown in variants ST67C, but no remarkable alteration was observed with 19 biochemical properties determined by a conventional identification kit. In ulta-thin sections of the parent strain labelled with rabbit anti-strain ST67P serum conjugated with ferritin, large capsule surrounded by ferritin granules were demonstrated by electron microscopy.(ABSTRACT TRUNCATED AT 250 WORDS)     

Relation of colonial morphologies in soft agar to morphological and biological properties of the K-9 strain of Klebsiella pneumoniae and its variants.

During repeated subcultures of strain K-9 of Klebsiella pneumoniae, three variants, A, B, and C, were obtained showing different colonial morphologies in soft agar. The parent strain K-9 produced colonies that were large, smooth, and globular; variant A, wedged balloon forms somewhat elongate; variant B, rain-drop-like with some streaming; and variant C, small compact rough globules. These colonial forms in soft agar medium correlated well with the size of capsule determined by cell volume index and light microscopy. Organisms exhibiting large globular colonies did possess extra large capsules while strains producing compact-type colonies in soft agar were lacking capsules. Capsular size of the strains correlated well with mouse virulence, but most biochemical properties, including the amount of endotoxic substance, were the same in all strains.     

Antibiotic Resistant Group A Streptococci

A series of Streptococcus pyogenes strains, including strains isolated from patients, mutants which had acquired in vitro resistance to penicillin (Pc), mitomycin C (MC), tetracycline (TC) and chloramphenicol (CM), ultraviolet light induced α hemolytic mutants, as well as β hemolytic mutants (β mutants) derived from α hemolytic mutants (α mutants) were compared as to their antibiotic sensitivity, and physiological, biochemical and serological properties. To obtain β mutants from α mutants the following procedures were employed: (1) serial mouse passage, (2) serial serum-broth transfers, (3) cultivation in heat-killed cultures of parent strains, and (4) cultivation in broth containing bacterial DNA extracted from parent streptococcus cells. From the results obtained these strains could be divided into two major groups, each with two subgroups. Group 1 strains produce soluble hemolysins and are sensitive to Pc. Subgroup 1–1 strains are sensitive to other antibiotics too; subgroup 1–2 are resistant to certain antibiotics other than Pc, bacitracin and MC. Group 2 strains do not produce soluble hemolysins and resistant to Pc. Subgroup 2-1 strains are α hemolytic on horse blood agar and subgroup 2–2 are β hemolytic on the same medium. Pc resistance in group 2 strains was more than 100-fold higher than that of sensitive strains, and was accompanied by MC resistance, but to a lesser degree. Pc resistance in group 2 mutants could be induced by antibiotics other than Pc and also by ultraviolet irradiation. Although group 1 cells retained the characteristics of typical S. pyogenes, group 2 cells, both α and β hemolytic, lost most of the physiological, biochemical and serological properties of this species. The similarity of group 2 strains to group D or group N streptococcal strains in their general properties is discussed.     

Conversion of biological and immunological properties during a process of decapsulation in a strain of Staphylococcus hyicus

E. YOSHIDA, Y. ICHIMAN, M. SUGANUMA AND K. YOSHIDA. 1991. Repeated subculture at 42°C of Staphylococcus hyicus strain ST67P, which exhibits streaming-type growth in a soft-agar medium, yielded three variants, ST67L, ST67S and ST67C, which had different colonial morphologies; small compact colonies possessing long and short tails and perfect compact colonies. The parent strain and ST67L respectively gave strong and weak positive intensity when stained by rabbit antisera prepared by capsular type I and II strains of Staph. epidermidis conjugated with fluorescein isothiocyanate. Variant ST67L gave a positive result with antiserum prepared by capsular type I strain and no staining was observed with variants ST67S and ST67C against these antisera preparations. Strain ST67C had the lowest virulence although no remarkable difference was shown between the parent strain and variants ST67L and ST67S. The cell volume index of the parent strain was 1.35, 2.43 and 3.71 times larger than those of ST67L, ST67S and ST67C, respectively. The converting activity of rabbit anti-ST67P strain serum absorbed by strain ST67C required four times more of the organisms than strain ST67P, changing the colonial morphology of the strain from diffuse to compact type by the addition of antiserum to soft agar medium. Positive coagulase and false positive clumping factor reaction were shown in variants ST67C, but no remarkable alteration was observed with 19 biochemical properties determined by a conventional identification kit. In ulta-thin sections of the parent strain labelled with rabbit anti-strain ST67P serum conjugated with ferritin, large capsules surrounded by ferritin granules were demonstrated by electron microscopy. In variants ST67L and ST67S, but not ST67C medium size capsules surrounded by ferritin granules and only ferritin granules located around the cell wall, respectively, were observed.     

The stability of toxigenicity in Clostridium botulinum types C and D.

Several type C and D strains of Clostridium botulinum, which had been converted to the toxgenic state by phages, were serially transferred through cooked meat medium with and without specific anti-phage serum. Most of the converted strains lost their toxigenicity even during transfer without antiserum, and the non-toxigenic variants that appeared were resistant to lysis and conversion by the original phage. However, in some combinations of phage and host bacteria toxigenicity was stable after ten transfers, though it showed a transient decrease, and the non-toxigenic variants that arose remained sensitive to lysis and conversion. When converted strains were transferred in medium containing anti-phage serum, toxigenicity was lost more rapidly than in the absence of serum and the non-toxigenic variants that appeared remained sensitive to lysis and conversion by the parent phage. Filtrates of the supernatants of culture fluids of strains transferred without anti-phase serum converted non-toxigenic strains to toxigenicity at varying rates.     

Induction of avirulent variants in Erwinia stewartii by incubation at supraoptimal temperatures.

High temperatures (37 degrees C) induced non-pigmented, and (or) small colony variants in some Erwinia stewartii strains. The former differed from the parent strain serologically and in having lost virulence to Zea mays. The small colony variants retained phytopathogenicity.     

Construction of mutants of Actinobacillus actinomycetemcomitans defective in serotype b-specific polysaccharide antigen by insertion of transposon Tn916.

Mutants of Actinobacillus actinomycetemcomitans strain Y4 defective in the capsular-like serotype b-specific polysaccharide antigen (SPA) were constructed by inserting the transposon Tn916. Southern blot analysis suggested that the transposon was inserted into a variety of different sites on the chromosome. Whole cells from two mutants (strains ST1 and ST2) lacked reactivity with a monoclonal antibody to SPA of A. actinomycetemcomitans Y4 (mAb S5) in enzyme-linked immunosorbent assay, but those from another nine mutants (e.g. strains ST3 and ST5) reacted very weakly with mAb S5. Immunodiffusion tests showed that mAb S5 or rabbit antiserum against whole cells of strain Y4 produced a fused precipitin band with purified SPA and autoclaved extract from strain Y4, but no precipitin band with autoclaved extracts from these four mutants. The hydrolysate of autoclaved extract from strain Y4 contained equal amounts of rhamnose and fucose, component sugars of SPA. The hydrolysates of autoclaved extracts from strains ST1 and ST2 contained a trace amount of rhamnose, but not fucose. Those of autoclaved extracts from strains ST3 and ST5 contained a trace amount of fucose, but not rhamnose. All of these SPA-defective mutants reacted with a mAb to lipopolysaccharide of strain Y4. The cell hydrophobicity of SPA-defective mutants was higher than that of the parent strain. These mutant clones will be useful for analysing the gene complex responsible for the synthesis of SPA of A. actinomycetemcomitans and the regulation of expression of the polysaccharide.     

Phenotypic switching of Cryptococcus neoformans can influence the outcome of the human immune response

The human pathogenic fungus Cryptococcus neoformans exhibits the phenomenon of phenotypic switching, a process that generates variant colonies that can differ in morphology, virulence and other characteristics such as capsular glucuronoxylomannan (GXM) size and structure. A previous study established that mucoid colony (MC) variants of C. neoformans were more virulent and elicited a different inflammatory response than smooth colony (SM) variants. In this study, we investigated the interaction of cells from MC and SM variants and their respective GXMs with human T cells and monocytes. Specifically, we measured CD40, CD80 and CD86 expression, lymphoproliferation and interleukin (IL)-4, IL-10, interferon (IFN)-gamma and IL-12Rbeta2 expression in the presence and absence of variant cells and their GXMs. For some immune parameters, both MC and SM strains produced similar results, in particular no differences were observed in IL-4 induction. However, for other critical parameters, including CD86 expression, lymphoproliferation and IL-10 production, the MC variant had effects that can be expected to impair the immune response. Hence, a single C. neoformans strain can elicit several different immune responses depending on the colony type expressed, and this is unlikely to be accounted for by differences in phagocytosis only. The results provide a potential explanation for the higher virulence of the MC variant based on the concept that these cells inhibit the development of a vigorous immune response. Furthermore, the results suggest a mechanism by which phenotypic switching can generate variants able to evade the immune response.     

Characterization and infectivity of a spontaneous variant isolated fromFrankia sp. WEY 0131391

Summary A spontaneous variant, obtained from aFrankia isolate fromAlnus rubra nodules, was compared with the parent strain with regard to infectivity, nitrogenase activity, and electrophoretic and immunological profiles. Both the parent and the variant strain were equally effective in inducing nodulation in seedlings ofA. rubra. All inoculated plants had an active nitrogenase system as measured by the acetylene reduction assay. Electrophoresis of whole cell homogenates on SDS-polyacrylamide slab gels showed similar electrophoretic profiles; however, the variant strain also exhibited striking differences in protein patterns that distinguish it from the parent strain. Immunological analysis of the originalFrankia strain and its variant revealed shared antigens as well as immunologically distinct antigenic determinants in the two strains. The variant strain exhibits a distinct morphology and growth patterns which remain stable after many passages through culture.     

Antigenic differences among Campylobacter fetus S-layer proteins.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of S-layer proteins extracted from Campylobacter fetus strains by using acid glycine buffer showed that the predominant S-layer proteins of different strains had subunit molecular weights in the range of 90,000 to 140,000. Electron microscopy revealed oblique S-layer lattices with a spacing of approximately 5.6 nm (gamma = 75 degrees) on wild-type strains VC1, VC119, VC202, and VC203. Three variants of C. fetus VC119 producing a predominant S-layer subunit protein of different molecular weight (Mr) from that of the parent were also examined. Each variant produced an oblique lattice morphologically indistinguishable from that of the parent. Amino-terminal sequence analysis showed that the S-layer proteins of the VC119 parent and variants were identical up to residue 18 and that this sequence differed from but was related to the first 16 N-terminal residues shared by the S-layer proteins of the three other wild-type C. fetus isolates. Western immunoblot analysis with an antiserum prepared to the VC119 protein and an antiserum prepared to C. fetus 84-40 LP (Z. Pei, R. T. Ellison, R. V. Lewis, and M. J. Blaser, J. Biol. Chem. 263:6416-6420, 1988) showed that strains of C. fetus were capable of producing S-layer proteins with at least four different antigenic specificities. Immunoelectron microscopy with antiserum to the VC119 S-layer protein showed that C. fetus cultures contained cells with immunoreactive oblique S-layer lattices as well as cells with oblique S-layer lattices which did not bind antibody. This suggests that C. fetus S-layer proteins undergo antigenic variation. Thermal denaturation experiments indicated that the antigenicity conferred by the surface-exposed C. fetus S-layer epitopes was unusually resistant to heat, and the thermal stability appeared to be due to the highly organized lattice structure of the S. layer. Protease digestion of purified VC119 S-layer protein revealed a trypsin-, chymotrypsin-, and endoproteinase Glu-C-resistant domain with an apparent Mr of 110,000, which carried the majority of the epitopes of the S-layer protein, and a small enzyme-sensitive domain. The trypsin- and chymotrypsin-resistant polypeptides shared an overlapping sequence which differed from the N-terminal sequence of the intact S-layer protein.     

Pilus-facilitated adherence of Neisseria meningitidis to human epithelial and endothelial cells: modulation of adherence phenotype occurs concurrently with changes in primary amino acid sequence and the glycosylation status of pilin

Adherence of capsulate Neisseria meningitidis to endothelial and epithelial cells is facilitated in variants that express pili. Whereas piliated variants of N. meningitidis strain C311 adhered to endothelial cells in large numbers (<150 bacteria/cell), derivatives containing specific mutations that disrupt pilE encoding the pilin subunit were both non-piliated and failed to adhere to endothelial cells (<1 bacterium/ cell). In addition, meningococcal pili recognized human endothelial and epithelial cells but not cells originating from other animals. Variants of strain C311 were obtained that expressed pilins of reduced apparent M_r and exhibited a marked increase in adherence to epithelial cells. Structural analysis of pilins from two hyper-adherent variants and the parent strain were carried out by DNA sequencing of their pilE genes. Deduced molecular weights of pilins were considerably tower compared with their apparent M_r values on SDS-PAGE. Hyper-adherent pilins shared unique changes in sequence including substitution of Asn-113 for Asp-113 and changes from Asn-Asp-Thr-Asp to Thr-Asp-Ala-Lys at residues 127-130 in mature pilin. Asn residues 113 and 127 of‘parental’pilin both form part of the typical eukaryotic N-glycosylation motif Asn-X-Ser/Thr and could potentially be glycosylated post-translationally. The presence of carbohydrate on pilin was demonstrated and when pilins were deglycosylated, their migration on SDS-PAGE increased, supporting the notion that variable glycosylation accounts for discrepancies in apparent and deduced molecular weights. Functionally distinct pilins produced by two fully piliated variants of a second strain (MC58) differed only in that the putative glycosylation motif Asn-60-Asn-61-Thr-62 in an adherent variant was replaced with Asp-60-Asn-61-Ser-62 in a non-adherent variant. Fully adherent backswitchers obtained from the non-adherent variant always regained Asn-60 but retained Ser-62. We propose, therefore, that functional variations in N. meningitidis pili may be modulated in large part by primary amino acid sequence changes that ablate or create N-linked glycosylation sites on the pilin subunit.     

Variations in the expression of pili: the effect on adherence of Neisseria meningitidis to human epithelial and endothelial cells

The effect of variations in Neisseria meningitidis pili on bacterial interactions with three epithelial cell lines as well as human umbilical vein endothelial cells was studied using a panel of seven strains expressing Class I or Class II pili. Comparison of adherence of piliated and pilus-deficient variants of each strain to epithelial cells suggested that Class I pili may mediate bacterial adherence with all three epithelial cell lines. In contrast, Class II pili of the strains used did not increase bacterial adherence to Hep-2 larynx carcinoma cells, although an increase in adherence to Chang conjunctival and A549 lung carcinoma epithelial cells was observed in the Class II pili-expressing strains. In addition to these interclass functional variations, differences in adherence to epithelial cells were also observed among Class I and Class II strains. Functionally different pilin variants of one Class I strain, MC58, were obtained by single colony isolation. One piliated variant was identified which had concurrently lost the ability to adhere to both Chang and Hep-2 cells ('non-adherent' phenotype; adherence of less than 2 bacteria per cell). In addition, several adherent pilin variants were isolated from non-adherent Pil- and Pil+ bacteria by selection on Chang cells (adherence of 10-25 bacteria per cell). In contrast to epithelial cells, all variant pili, whether of Class I or Class II, adhered to endothelial cells in substantially larger numbers (greater than 50 bacteria per cell) and therefore implied the existence of distinct mechanisms in pilus-facilitated interactions of N. meningitidis with endothelial and epithelial cells.