Influence of iron-limited and replete continuous culture on the physiology and virulence of Neisseria gonorrhoeae

Neisseria gonorrhoeae strains P9-2 (PenS) and KW2 (PenR) were grown in chemostats of nonferrous design at constant growth rate, pH and dissolved oxygen tension. Iron limitation (micromax 0.1 h-1) was imposed by omitting iron salts from the defined medium and titrating increasing concentrations of the non-metabolizable iron chelators ovotransferrin and Desferal, to progressively decrease the growth yield. Metabolic activity during iron limitation was very high, with a qGlc which was 2- or 11-fold greater than during cystine- or glucose-limited growth, respectively. More aspartate and isoleucine were metabolized during cystine-limited growth, while more glutamate, proline and serine were metabolized during glucose- or iron-limited growth. Significant concentrations of alanine or valine were excreted during cystine- or glucose-limited growth, respectively. Iron-limited growth of an initial inoculum of non-piliated, transparent colony-forming (P-O-) gonococci resulted in the selection of 100% piliated bacteria. Initial inocula of P+O- gonococci retained this phenotype for over 100 generations. Iron-limited gonococci were extremely virulent in the guinea-pig subcutaneous chamber model and inocula of even 12 bacteria grew rapidly and persisted. By contrast, cystine-limited (iron-replete) gonococci retained piliation but did not survive in the chambers. Transition from iron-limited to glucose-limited growth resulted in marked loss of piliation but the bacteria remained virulent. Loss of virulence did not correlate with susceptibility to killing by normal human serum, nor with changes in the content or composition of lipooligosaccharide, which contained 2.9, 3.7, 4.3 and 4.8 kDa moieties. Additional proteins were detectable in Sarkosyl-purified outer membranes of iron-limited gonococci but several proteins with molecular masses similar to those described in the literature for iron-restricted gonococci were detectable in cystine- or glucose-limited bacteria.     

Competition between Neisseria gonorrhoeae and Staphylococcus epidermidis during iron-limited or replete continuous culture

Neisseria gonorrhoeae strain P9-2 was grown in iron-limited or replete continuous culture at a dilution rate of 0.05 h-1, in the presence and absence of Staphylococcus epidermidis. Gonococci maintained expression of pili (P+) and the transparent colony phenotype in pure culture during transitions of iron- and cystine-limited growth. They competed well with staphylococci during iron-limited co-culture and comprised greater than 95% of the population. Transition to cystine-limited growth allowed the staphylococcus to predominate but the gonococcus did not wash out. Furthermore, the gonococcal opaque colony phenotype (O+), indicating synthesis of outer membrane proteins II, was now expressed. Restoration of iron limitation returned the co-culture to its original composition but with P+O+ gonococci dominating. These results suggest that environments might exist in Man where gonococci can compete successfully with normal indigenous bacteria during infection.     

Oxygen uptake kinetics of Pseudomonas chlororaphis grown in glucose- or glutamate-limited continuous cultures

Oxygen uptake and glucose and glutamate oxidation kinetics of the heterotrophic bacterium Pseudomonas chlororaphis grown in glucose- or glutamate-limited cultures under oxygen-saturating or oxygen-limiting conditions were determined. K _m values for oxygen were 1.4– 5.6 μM. Only in the case of glucose were significantly lower K _m values and enhanced specific oxygen affinity (V _max/K _m) per cell found under oxygen-limiting conditions. Both K _m and specific affinity values for glucose and glutamate oxidation were apparently affected by oxygen concentration, although a statistically significant enhancement of the oxidation kinetics was found only for glutamate. The kinetic data found for P. chlororaphis support the conclusion that the outcome of competition for oxygen with Nitrosomonas europaea in the rhizosphere of oxygen-releasing macrophytes will primarily be determined by oxidation kinetics of the electron donor instead of the oxygen uptake kinetics of the respective organisms. Received: 20 September 1996 / Accepted: 5 February 1997     

Lethal activity of miconazole in Neisseria gonorrhoeae grown in pure and mixed cultures

Abstract Low concentrations (e.g. 2 × 10⁻⁶ M) of an imidazole derivative anti-fungal agent, miconazole, were lethal for the Gram-negative, facultative aerobic pathogen Neisseria gonorrhoeae grown either alone or in mixed culture with the yeast Candida albicans . Electron microscopic observation of Neisseria cells exposed to miconazole showed the presence of blebs in the outer wall and areas of separation between the wall and the cytoplasmic membrane. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of cell lysates did not reveal differences in major outer membrane proteins between the treated and the untreated cells of any one strain. Imidazole derivatives are frequently used in the treatment of candidiasis. Our in vitro results show that low concentrations of one of them, miconazole, can be bactericidal for N. gonorrhoeae , a bacterium that can colonise sites of the human body where Candida is often found.     

Genetic transformation of pilation and virulence into Neisseria gonorrhoeae T4.

Genetic transformation of nonpilated strains of Neisserai gonorrhoeae to pilated forms is described. The transformants displayed phenotypic T1 and T2 colonial morphology on agar and possessed pili visualized by electron microscopy. When T1 or T2 transformant cells were injected into 11-day-old chicken embryos, they exhibited virulence characteristics only slightly less than the parental donor strains, though the parental recipient strains were avirulent. Competence was maximal in the late log phase of growth, and the frequency of transformation of clonal T4s to pilation and virulence approached 2%. DNA extracted from transformants could be used to transform other T4 cells. In the course of this work, a shift to a novel colonial type, designated T2-T3 wrinkled, was observed as a consequence of growth of T4 in presence of enzymatic digests of either DNA or RNA, nucleases or individual deoxy- or ribonucleosides. In sharp distinction to the parental T4, these novel organisms were very pilated; however, they were only minimally virulent. Various nucleic acid analogs could neither induce nor inhibit this population shift. Additionally, DNA extracted from this T2-T3 wrinkled variant could be used to transform genetically both T1 and T4 gonococci to the new morphology.     

Cultivation of Neisseria gonorrhoeae in an L-929 cell culture

N. gonorrhoeae strain b has been found to be capable of retaining its viability in medium 199 with 10% of inactivated cattle serum added and in monolayer cell culture L-929 in the above medium. The characteristics obtained in the present investigation permit simulating the mixed association of gonococci and chlamydiae in the culture system used in this work.     

Physiology and metabolism of pathogenic Neisseria: partial characterization of the respiratory chain of Neisseria gonorrhoeae.

The cell membrane-associated respiratory electron transport chain of Neisseria gonorrhoeae was examined using electron paramagnetic spectroscopy (EPR) at liquid helium temperatures and optical spectroscopy at liquid nitrogen and room temperatures. EPR spectra of dithionite-reduced particles indicated the presence of centers N-1 and N-3 in the site I region of the respiratory chain, whereas reduction with succinate revealed the existence of center S-1 from the succinate cytochrome c reductase segment. Free radical(s) resembling that due to falvin semiquinone were observed with both reductants. Low temperature (77 K) optical difference spectra indicated the presence of cytochromes with alpha band maxima at 549, 557, and 562. Bands at 567, 535, and 417 nm, characteristic of the CO compound of cytochrome o, were also identified. Cytochromes a1 and a3 were not detected; however, a broad but weak absorbance with an alpha band maximun at 600 nm and a Soret shoulder at 440 nm was observed. Hence the respiratory chain of N. gonorrhoeae appears to contain several nonheme iron centers, cytochrome c, two b cytochromes, with cytochrome o which probably serves as the terminal oxidase.     

Effect of growth rate on ethanol production byThermoanaerobacter ethanolicus in glucose- or xylose-limited continuous culture

Summary Growth ofT. ethanolicus in fermentors was limited by either glucose and the growth rate was varied. Under xylose-limitation, lower ethanol yields occurred at higher growth rates.Regression analysis showed that for growth on either sugar, ethanol yield was more dependent upon length of cultivation than upon growth rate. As well, a high degree of inter-experimental trial variation was found, though a yield of 0.44 g/g of ethanol from xylose was repeatedly achieved.     

Physiology and metabolism of pathogenic neisseria: tricarboxylic acid cycle activity in Neisseria gonorrhoeae.

Tricarboyxlic acid cycle activity was examined in Neisseria gonorrhoeae CS-7. The catabolism of glucose in N. gonorrheae by a combination of the Entner-Doudoroff and pentose phosphate pathways resulted in the accumulation of acetate, which was not further catabolized until the glucose was depleted or growth became limiting. Radiorespirometric studies revealed that the label in the 1 position of acetate was converted to CO2 at twice the rate of the label in the 2 position, indicating the presence of a tricarboxylic acid cycle. Growth on glucose markedly reduced the levels of all tricarboxylic acid cycle enzymes except citrate synthase (EC 4.1.3.7). Extracts of glucose-grown cells contained detectable levels of all tricarboxylic acid cycle enzymes except aconitase (EC 4.2.1.3), isocitrate dehydrogenase (EC 1.1.1.42), and a pyridine nucleotide-dependent malate dehydrogenase (EC 1.1.1.37). Extracts of cells capable of oxidizing acetate lacked only the pyridine nucleotide-dependent malate dehydrogenase. In lieu of this enzyem, a particulate pyridine nucleotide-independent malate oxidase (EC 1.1.3.3) was present. This enzyme required flavin adenine dinucleotide for activity and appeared to be associated with the electron transport chain. Radiorespirometric studies utilizing labeled glutamate demonstrated that a portion of the tricarboxylic acid cycle functioned during glucose catabolism. In spite of the presence of all tricarboxylic acid cycle enzymes, N. gonorrhoeae CS-7 was unable to grow in medium supplemented with cycle intermediates.     

Progress of O-acetylation and cross-linking of peptidoglycan in Neisseria gonorrhoeae grown in the presence of penicillin

The synthesis, cross-linking and O-acetylation of gonococcal peptidoglycan in growing cells were studied by following incorporation of radioactive glucosamine and separation of the SDS-insoluble peptidoglycan into uncross-linked (monomer) and cross-linked (dimer and oligomer) fragments. Cultures to which penicillin or piperacillin at concentrations near the minimum growth inhibitory concentration (MIC) had been added 20 min before the radioactive label were compared with controls. The beta-lactams affected the early stage of cross-linking (up to 3 min) but had no effect thereafter. The deficit of cross-linking, however, did not recover. The O-acetylation, particularly of the monomer fraction, was decreased by beta-lactams even at concentrations that had no effect on culture optical density, viable counts or overall peptidoglycan synthesis. These effects on O-acetylation occurred mainly after the first 3 min of incorporation, rather than before. O-Acetylation of the oligomer fraction was also followed. Here penicillin led to increased levels of O-acetylation during the early stages of incorporation but the final value was never exceeded; indeed at higher drug concentrations the later stages of O-acetylation of oligomers were inhibited (e.g. almost completely at 2.5 X MIC).     

Mitogenic activity of Neisseria gonorrhoeae surface antigens in mouse splenic lymphocyte culture.

The mitogenic effects of Neisseria gonorrhoeae endotoxin, fractionated envelope componenents, and intact cells were examined on unsensitized mouse splenic lymphocytes in vitro. The stimulatory effect of these substances was measured by increased [3H]thymidine incorporation in spleen cell cultures. Intact cells, purified lipopolysaccharide (LPS), and cell envelope preparations were highly stimulatory and the stimulation index was dose dependent. Fractionated components of the envelope demonstrated variable stimulation when tested at identical LPS concentrations, reflecting the mitogenic activity of the protein moieties. The stimulatory dose responses for purified N. gonorrhoeae and Escherichia coli LPS were compared and mitogenicity was higher with gonococcal LPS at all concentrations tested. Alkaline detoxification or succinylation of N. gonorrhoeae LPS results in loss of ability to induce blast transformation. The mitogenicity of cell-surface components of N. gonorrhoeae is discussed in terms of LPS and protein content.     

Variations in surface protein composition associated with virulence properties in opacity types of Neisseria gonorrhoeae.

The biological properties of a series of opacity variants of Neisseria gonorrhoeae P9 have been examined. A novel protein, designated protein IId* (mol. wt 28 850), was identified within the set of heat-modifiable surface proteins previously reported. All variants producing extra outer membrane proteins were less sensitive to the bactericidal action of serum than the prototype transparent strain, with protein IIa* (mol. wt 28 500) being associated with increased resistance. The production of a different protein, protein II* (mol. wt 29 000), was correlated with resistance to low molecular weight antimicrobial agents (penicillin, fusidic acid, Cu2+, Zn2+). Increased adhesion to human buccal epithelial cells was demonstrated in all variants that produced extra surface proteins. These variants did not show increased binding to hexyl- and phenyl-substituted Sepharose gels suggesting that hydrophobic interaction was not responsible for their cohesive properties. The prototype strain lacking additional proteins demonstrated the greatest binding to erythrocytes, indicating that adhesion to buccal cells and red blood cells is mediated by different mechanisms. One variant producing protein IIa* showed increased association with leukocytes, whereas another producing protein IIb* showed decreased association with leukocytes. These results show that the heat-modifiable surface proteins are important virulence attributes of the gonococcus: this must be considered in the selection of strains for vaccine trials.     

The peptidoglycan of Neisseria gonorrhoeae, with or without O-acetyl groups, contains anhydro-muramyl residues

Muramidase digests of alkali-treated SDS-insoluble peptidoglycan from two strains of Neisseria gonorrhoeae were examined. Both strains contained disaccharide peptide monomers that had intramolecular 1,6-anhydro-muramyl ends. In contrast to strain 1L260, in which 50% of the monomer fraction is O-acetylated, the monomer fraction from strain RD5 was completely devoid of O-acetyl groups, as shown by HPLC. Penicillin decreased the O-acetylation of peptidoglycan but did not affect the proportion of anhydro-muramyl residues.     

Effect of anti-pilus antisera on virulence of variants of Neisseria gonorrhoeae for cultured epithelial cells

Variants of Neisseria gonorrhoeae P9 possessing a, gamma or delta pili were shown to vary in their toxicity and virulence for human epithelial cells. Studies with antisera raised against purified pili showed that attachment and virulence were reduced to a significant degree in the presence of antisera to homologous pili. Heterologous antisera, while capable of agglutinating whole organisms, were largely ineffective in reducing attachment and cytotoxicity. Using an enzyme-linked immunosorbent assay (ELISA) system, the cross-reactivity between pili and heterologous antisera was estimated to be no more than 10-20%.     

Opacity determinants of Neisseria gonorrhoeae: gene expression and chromosomal linkage to the gonococcal pilus gene

In N. gonorrhoeae, the expression of pilus and opacity (Op) proteins can be switched on and off and a single cell apparently has a whole repertoire of genes to express many serologically distinguishable protein types. We describe the isolation of several different Op genes and of nonexpressing gene equivalents, all derived from isogenic gonococcal variants. In the E. coli host, Op proteins identical with those made in the respective N. gonorrhoeae strain are produced. The Op genes map near the pilus expression locus. Genomic blotting experiments with an Op gene probe reveal complex hybridization patterns but little heterogeneity among the genes of Op variants. It appears that colonial variation involving the Op protein of N. gonorrhoeae is based on minor sequence alterations, in contrast to the pilus variation system, in which changes in the expression can be evoked by substantial genomic rearrangements.     

The surface properties of Neisseria gonorrhoeae: determinants of susceptibility to antibody complement killing.

Monovalent rabbit antisera were prepared to highly purified gonococcal lipopolysaccharide (LPS), to pili and to two major purified outer envelope proteins. All these antisera were free from significant specific IgM antibody and were standardized to 4 microgram specific IgG antibody per test, permitting accurate comparisons between the different gonococcal surface antigens as triggers of the complement-dependent bactericidal reaction. LPS was the most effective antigen at inducing a bactericidal response to homologous and heterologous gonococci, followed by the two individual outer envelope proteins. Pili were relatively ineffective. Strain P9 gonococci grown in vivo or which possessed a 'capsule' in vitro were more resistant to serum killing than the non-capsulated parent strain. One highly susceptible strain, F62, which was killed by complement in the absence of any LPS antibody, was able to directly activate complement by the alternative pathway.     

RecQ DNA helicase HRDC domains are critical determinants in Neisseria gonorrhoeae pilin antigenic variation and DNA repair

Neisseria gonorrhoeae (Gc), an obligate human bacterial pathogen, utilizes pilin antigenic variation to evade host immune defences. Antigenic variation is driven by recombination between expressed ( pilE ) and silent ( pilS ) copies of the pilin gene, which encodes the major structural component of the type IV pilus. We have investigated the role of the GcRecQ DNA helicase (GcRecQ) in this process. Whereas the vast majority of bacterial RecQ proteins encode a single 'Helicase and RNase D C-terminal' (HRDC) domain, GcRecQ encodes three tandem HRDC domains at its C-terminus. Gc mutants encoding versions of GcRecQ with either two or all three C-terminal HRDC domains removed are deficient in pilin variation and sensitized to UV light-induced DNA damage. Biochemical analysis of a GcRecQ protein variant lacking two HRDC domains, GcRecQΔHRDC2,3, shows it has decreased affinity for single-stranded and partial-duplex DNA and reduced unwinding activity on a synthetic Holliday junction substrate relative to full-length GcRecQ in the presence of Gc single-stranded DNA-binding protein (GcSSB). Our results demonstrate that the multiple HRDC domain architecture in GcRecQ is critical for structure-specific DNA binding and unwinding, and suggest that these features are central to GcRecQ's roles in Gc antigenic variation and DNA repair.