Effect of siderophores on virulence of Neisseria gonorrhoeae.

The virulence of Neisseria gonorrhoeae for chicken embryos can be modified in a predictable manner by the addition of microbial siderophores to the inoculum. "Meningobactin" and "gonobactin," siderophores isolated from iron-limited cultures of meningococci and gonococci, respectively, enhance the virulence of the relatively avirulent colony type 3 (T3) organisms, but have essentially no effect on the virulence of T1 organisms. Both of these compounds were found previously to stimulate in vitro growth of the pathogenic Neisseria spp. under conditions made iron limiting by the addition of conalbumin, the transferrin counterpart of chickens. Similarly, ferrated schizokinen and arthrobactin, both dihydroxamate siderophores which stimulated growth in iron-limited conditions in vitro, also enhanced virulence of T3 organisms, whereas desferrioxamine B mesylate (Desferal), a trihydroxamate previously shown to be inhibitory in vitro, decreased the virulence of the T1 colony form. This was due to the iron-binding function of the molecule, as the iron-saturated form, ferrioxamine B mesylate, did not affect virulence. An additional trihydroxamate siderophore, ferrichrome A, which was inactive on Neisseria spp. in either the deferri- or ferrated forms in vitro, likewise did not affect virulence in the chicken embryo model. The neisserial siderophores were more effective than the other microbial siderophores in enhancing virulence of T3 gonococci. The results add to the evidence that the ability to acquire iron is an important determinant of virulence.     

Pathogenesis and Immunology of Experimental Gonococcal Infection: Virulence of Colony Types of Neisseria gonorrhoeae for Chicken Embryos

The virulence of gonococcal strains and colony types was evaluated in embryonated hen eggs of various ages inoculated by different routes. Striking differences in virulence of colony types were revealed by intravenous inoculation of 11-day embryos. T1 and T2 colony types were found to have high virulence for embryos, whereas T3 and T4 colony types were relatively avirulent. These observations are in accord with previous studies in volunteers. The differences in virulence were not related to differences in toxicity of killed gonococci, sonic lysates, or to the susceptibility of gonococci to cidal effects of chicken embryo blood. Rather, they appeared to involve differences in clearance of gonococci from the blood stream and subsequent multiplication of the virulent colony types. Infection with virulent colony types appears to be primarily bacteremic in this model. Preliminary experiments indicated that chicken embryos may be protected against the lethal infection by prior treatment of the inoculum with normal and immune rabbit serum. This protective effect was not associated with bactericidal activity. The chicken embryo model is potentially useful as a means of investigating attributes of virulence of gonococci and factors in immunity against gonorrhea.     

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.     

The growth-promoting effect of bacterial iron for serum-exposed bacteria

Bacterial ability to obtain iron in bovine serum or in media containing transferrin (Tr) or conalbumin (Ca) was investigated by using serum-resistant (virulent) and serum-sensitive (avirulent) strains of Escherichia coli and Salmonella typhimurium. Bacteria growing in bovine serum enriched with radioactive iron-saturated Tr or with radioactive iron-saturated enterobactin (E) did not acquire radioactive iron. It has been found that the passage of siderophore (Si)-iron complexes into bacteria is blocked in serum by Tr and in Ca-containing medium by Ca. The investigation of bacterial ability to take iron in synthetic media showed that bacteria take in Si-bound but no Tr-bound radioactive iron. In the absence of free iron, the growth of serum-exposed virulent bacteria was supported by their stored iron. Virulent bacteria passaged in medium void of usable iron became depleted in stored iron and did not grow in animal sera unless sera were enriched by the addition of exogenous iron. Experiments with serum-exposed avirulent bacteria showed that their growth in Si-enriched serum should not be attributed to the iron-providing activity of Si but to the stimulating effect of Si which facilitates the use of stored iron. As distinct from avirulent bacteria, virulent bacteria used stored iron without the stimulating activity of extracellular Si.     

Assmilation of iron by pathogenic Neisseria spp.

Iron assimilation by Neisseria meningitidis and Neisseria gonorrhoeae has been shown to be important to their growth and virulence. Iron acquisition in vitro was studied in an agar diffusion assay employing the iron-binding protein conalbumin. The ability of various iron compounds to alter the growth-inhibitory effect of conalbumin was investigated. On an equimolar iron basis, citrate-containing iron compounds were most effective; hemin was slightly less effective; ferrous sulfate and ferrous ammonium sulfate were even less effective; and the ferric compounds, ferric nitrate, ferric chloride, and ferric dextran (Imferon), were least effective. The results suggested that, as with Escherichia coli and certain other bacteria, the Neisseria spp. may utilize a citrate-mediated iron transport system. Microbial siderophores were also tested for their ability to relieve the growth-inhibitory effect of conalbumin. Two phenolate siderophores and Desferal enhanced growth inhibition in the deferrated form, but were inactive in the ferrated form. Several trihydroxamates of the ferrichrome family and coprogen were inactive in either the deferrated or ferrated forms. Of the 12 different siderophores tested, only the dihydroxamates (schizokinen, arthrobactin, and aerobactin) were stimulatory, but then only in the ferrated forms. Apparently, even though those siderophores could be utilized as specific iron transport agents by the Neisseria spp., they could not compete with conalbumin for iron under these assay conditions.     

Modulatory effect of iron on the pathogenesis of Pseudomonas aeruginosa mouse corneal infections.

The iron concentration of the culture medium used to prepare the inocula influenced the pathogenesis of mouse corneal infections by Pseudomonas aeruginosa. When the parental strain PAO1 was cultured in high-iron medium (5 micrograms of Fe per ml), it was less virulent than when it was cultured in low-iron medium (0.05 microgram of Fe per ml). The iron concentration of the growth medium had no effect on the virulence of a P. aeruginosa mutant which was resistant to the iron regulation of toxin A yields (PAO-toxFeR-18). A severely defective iron transport mutant, PAO-toxFeR-10, was avirulent regardless of the iron concentration of the growth medium. These studies indicate that both iron acquisition and iron regulation of toxin production are important factors in the determination of P. aeruginosa virulence.     

Virulence of transparent and opaque colony types of Neisseria gonorrhoeae for the genital tract of mice

The virulence of transparent (Tr) and opaque (Op) colony types of Neisseria gonorrhoeae in the genital tract of female mice was evaluated at two stages of oestrous. Isogenic pairs of Tr and Op variants were isolated from N. gonorrhoeae strain 57-120. Both variants exhibited a T2 morphology, but only the Op variant possessed protein II (P.II) in outer-membrane fractions. When administered by intravaginal inoculation Op gonococci were highly infective only for mice in late pro-oestrous, whereas Tr gonococci were virulent for mice at both late pro-oestrous and dioestrous. Gonococci recovered from the uterus were of both Tr and Op phenotypes in equal proportions when mice were infected at dioestrous with Tr cells. In contrast, greater than 90% of recovered colonies were of Op phenotype when mice were infected at late pro-oestrous with either Op or Tr cells. These results indicate that the virulence of gonococci for the genital tract of female mice differs from that for the chicken embryo. Furthermore, gonococcal survival in the female genital tract might be attributable to phase variation from Tr to Op phenotypes.     

Detection and differentiation of iron-responsive avirulent mutants on Congo red agar.

Agar medium containing Congo red dye differentiates virulent and avirulent colonies of Shigella, Vibrio cholerae, Escherichia coli, and Neisseria meningitidis. Like virulent plague bacilli, wild-type cells of these species absorb the dye and produce red colonies. Mutants or colonial variants have been isolated that fail to absorb the dye and produce colorless colonies. These mutants exhibit reduced virulence in the chicken embryo model, but their virulence is enhanced by supplementation with iron. Of those species tested, only Neisseria gonorrhoeae isolates failed to grow in the presence of this dye. Inhibition of growth by Congo red may thus provide a simple means for differentiating gonococci from other Neisseria.     

Inhibition of the growth of Neisseria meningitidis by reduced ferritin and other iron-binding agents.

Serogroups of N. meningitidis were characterized as virulent or avirulent according to their capacity to establish meningococcal infection in mice. An agar plate diffusion technique demonstrated that iron had a definite growth-supporting role for both of these meningococcal types. The avirulent strains could use ionic or chelated iron as well as the virulent strains. Iron-reversible growth inhibition occurred to the same extent for both bacterial types in the presence of the synthetic iron-chelating agents Desferal and ethylenediamine-di-orthohydroxy phenylacetic acid. A difference in response was demonstrated for these bacterial types when grown in the presence of various iron-binding proteins from animal body fluids and tissues. The growth of the avirulent strain was inhibited to a greater degree by egg white conalbumin. The humoral iron-binding protein transferrin showed a significant inhibitory capacity only when used in conjunction with bicarbonate. Under conditions of increased iron saturation of this protein, the avirulent strain was inhibited to the furthest extent. In the presence of ferritin, the cellular iron-binding protein, which had been reduced, inhibition of the growth of either strain type did not occur on iron-poor media (less than 5 micrograms/100 ml). However, with the incorporation of iron into the media, the inhibitory effect of the protein became evident. As the concentration of iron increased, the inhibition increased to a certain level and subsequently declined. A substantial difference in the ability of the avirulent type to grow in the presence of reduced horse spleen ferritin was observed. For this microorganism, a correlation appears to exist between the capacity to grow by utilizing the available iron in the presence of reduced ferritin and the ability to establish infection. The host protein ferritin, in the reduced state, apart from simply being a storage protein for iron, can prevent the growth of a procaryotic organism. Our experiments suggest a role for ferritin in the prevention of emningococcal disease. A cehmotherapeutic potential for Desferal is also implied.     

Studies on the Virulence of Neisseria gonorrhoeae I. Relation of Colonial Morphology and Resistance to Phagocytosis by Polymorphonuclear Leukocytes

Colonial varieties of Neisseria gonorrhoeae that are associated with virulence, types 1 and 2, were more resistant to phagocytosis by rabbit exudative polymorphonuclear leukocytes than colonial types of lesser virulence, types 3 and 4. Type 1 bacteria were resistant and type 4 gonococci were susceptible to phagocytosis by human polymorphonuclear leukocytes. Recent local type 1 isolates were similar in resistance to type 1 organisms of a standard laboratory strain (F62). Living and Formalin-treated, heat-killed, type 1 gonococci were equally resistant to phagocytosis. The antiphagocytic property of virulent colonial types was independent of leukotoxic action. Phagocytosis of both type 1 and type 4 gonococci by rabbit and human leukocytes was bactericidal. Rabbit leukocytes were superior to human leukocytes in killing gonococci. The results suggest that N. gonorrhoeae has virulence properties similar to those of extracellular bacterial pathogens, i.e., virulence is associated with antiphagocytic properties.     

Effects of iron and culture filtrates on killing of Neisseria gonorrhoeae by normal human serum.

Neisseria gonorrhoeae GC9, both colony types T2 and T4, were killed by normal human serum, although populations of colony type T4 were more susceptible. Ferric ammonium citrate prevented the killing of populations of both T2 and T4 colony types. Other iron compounds tested showed no protective effect, nor did ammonium citrate or the divalent cations magnesium or calcium. A filtrate from cultures of an N. gonorrhoeae strain grown in a liquid defined medium showed a similar protective effect in the serum assay. The filtrate appeared to chelate iron, as measured by decreased ability of iron-free transferin to bind iron in the presence of the filtrate. However, the two effects did not appear to be related. Neither ferric ammonium citrate nor the culture filtrate sufficiently inactivated complement to account for protection.     

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.     

Role of urinary solutes in natural immunity to gonorrhea.

Natural resistance of the male urethra to gonococci has not been explained by classical immune mechanisms but could result from antibacterial properties of urine. Accordingly, we measured survival in midmorning urine of 10(7) F-62 T2 gonococci per ml by serial dilutions and plate counts. Fifteen killer urines from eight people all killed greater than 3 logs (average, 5.3), and 13 of 15 were sterilized. Fourteen nonkiller (inhibitor) urines from seven subjects allowed no growth. Killer urines were more acidic (pH 5.4 versus 6.4) and more concentrated (861 versus 717 mosmol/kg) than nonkillers. Upon addition of hydrogen ion, urea, and sodium chloride to urines and broth, pH proved to be the major killing factor, but urea and NaCl were also bactericidal. Susceptibility to urine bactericidal power did not vary with colony type (T2 versus T4) or strain (F-62 versus two fresh isolates). Killing was rapid (0.5 to 3 h) and not bacteriolytic. Escherichia coli multiplied 10-fold in urines that inhibited growth of gonococci. Thus, the bacteriostatic effect of urine may explain why gonococci do not infect the bladder and kidney during gonorrhea. The bactericidal properties of urine may contribute to resistance against gonococcal urethritis.     

Role of exotoxin A and elastase in the pathogenicity of Pseudomonas aeruginosa strain PAO experimental mouse burn infection

We examined the virulence of Pseudomonas aeruginosa strain PAO and xcp (extracellular proteins deficient) and xch (extracellular proteins hyperproducing) mutants derived from strain PAO in an experimental mouse burn infection model. The results showed that xcp mutants, which produced little or no extracellular elastase and exotoxin A, were as virulent as their corresponding xcp+ strains. The xch mutants produced more elastase and exotoxin A than the wild type strain, however, they had significantly lower virulence, probably due to reduced ability of these strains to take up iron. Treatment of mice with ferric ammonium citrate had no effect on the wild type strain but enhanced mortality in mice challenged with xch mutants. Neither elastase nor exotoxin A seem to play any role in burn infections with P. aeruginosa strain PAO. However, ability for iron uptake is an important virulence factor.     

Environmental Modulation of Oral Treponeme Virulence in a Murine Model

This investigation examined the effects of environmental alteration on the virulence of the oral treponemes Treponema denticola and Treponema pectinovorum. The environmental effects were assessed by using a model of localized inflammatory abscesses in mice. In vitro growth of T. denticola and T. pectinovorum as a function of modification of the cysteine concentration significantly enhanced abscess formation and size. In contrast, growth of T. denticola or T. pectinovorum under iron-limiting conditions (e.g., dipyridyl chelation) had no effect on abscess induction in comparison to that when the strains were grown under normal iron conditions. In vivo modulation of the microenvironment at the focus of infection with Cytodex beads demonstrated that increasing the local inflammation had no effect on lesion induction or size. In vivo studies involved the determination of the effects of increased systemic iron availability (e.g., iron dextran or phenylhydrazine) on the induction, kinetics, and size of lesions. T. denticola induced significantly larger lesions in mice with iron pretreatment and demonstrated systemic manifestations of the infectious challenge and an accompanying spreading lesion with phenylhydrazine pretreatment (e.g., increases in circulating free hemoglobin). In contrast, T. pectinovorum virulence was minimally affected by this in vivo treatment to increase iron availability. T. denticola virulence, as evaluated by lesion size, was increased additively by in vivo iron availability, and cysteine modified growth of the microorganism. Additionally, galactosamine sensitized mice to a lethal outcome following infection with both T. denticola and T. pectinovorum, suggesting an endotoxin-like activity in these treponemes. These findings demonstrated the ability to modify the virulence capacity of T. denticola and T. pectinovorum by environmental conditions which can be evaluated by using in vivo murine models.     

Effects of Injected Iron and Siderophores on Infections in Normal and Immune Mice

The fate of virulent and avirulent strains of Salmonella typhimurium in untreated and iron-injected mice and in transferrin-containing media demonstrated a direct relationship between bacterial virulence and the ability of bacteria to acquire transferrin-bound iron. Effects of injected iron on the development of infections with virulent and avirulent bacterial strains were determined in normal and immune mice by determinations of bacterial numbers in tissue homogenates and the mortality of infected animals. Results showed that infected and iron-injected mice died much more rapidly and frequently from overwhelming infections than infected and saline-injected mice. The infection-promoting effect of iron varied with the degree of bacterial virulence; the more virulent the bacteria, the more helpful was iron for the development of lethal infections. Siderophores promoted lethal infections in mice infected with virulent but not with avirulent bacteria. Experiments with vaccinated animals showed that iron exerted a deleterious effect on acquired immunity. Immune mice infected with virulent bacteria and injected with iron developed lethal infections as rapidly and nearly as frequently as similarly treated normal mice. Siderophores did not promote the development of lethal infections in immune mice. The effectiveness of iron, but not of siderophores, to promote bacterial infections in vaccinated mice revealed that acquired immunity is dependent upon the activity of an iron-neutralizable antibacterial system.     

Interaction of Gonococci with Phagocytic Leukocytes from Men and Mice

The interaction of human and mouse phagocytic leukocytes with representative virulent (F62-T1) and avirulent (F62-T4, RD-5) strains of Neisseria gonorrhoeae was studied in vitro. Leukocyte monolayers were incubated with gonococci for 30 min at 37 C, washed repeatedly, reincubated with fresh medium, and sampled for viable bacteria at intervals. After the initial incubation period and washing, human leukocytes retained larger numbers of viable T1 than of T4. During the subsequent 120 min of incubation, the numbers of viable T1 remained approximately constant, whereas viable counts of T4 declined by about two-thirds. In contrast, mouse leukocytes under similar conditions destroyed 70% of both types of gonococci. When human bactericidal serum was applied to infected human leukocytes, it had no effect on T4 but inactivated over 50% of T1. It is concluded that T4 are phagocytized by human leukocytes and are thus exposed to internal digestion, but are protected from bactericidal serum. T1, on the other hand, either adhere to the surface of the leukocytes or remain located so that they are neither digested by the leukocytes nor protected from bactericidal serum.     

Reversion of Kellogg's colonial types of Neisseria gonorrhoeae in liquid medium.

Colonial type variation of gonococci is well known, but change from type 4 to type 4 to type 1 is rare except in vivo. By observing quantitatively subcultures from a new liquid medium it was possible to follow the day-to-day changes in the ratio of colonial types present. The results obtained showed that type 1 colonies could be derived from type 5 inocula even in unsupplemented media. In unsupplemented liquid medium, type 4 inocula did not revert to other types and indeed colonial type 4 appeared to be the final form of the organism before it died out. If, however, iron in the form of ferric citrate was added to the medium, reversion occurred and type 1 colonies rapidly came to predominate.     

Interactions of Neisseria gonorrhoeae with Human Neutrophils: Effects of Serum and Gonococcal Opacity on Phagocyte Killing and Chemiluminescence

Serum-sensitive strains of Neisseria gonorrhoeae were incubated with suspensions of normal or chronic granulomatous disease human neutrophils in the absence or presence of fresh or heat-inactivated human serum; phagocytosis, gonococcal viability, and chemiluminescence were measured. Nonpiliated opaque or transparent gonococci (colony types 3 and 4, respectively) were used for phagocytic bactericidal assays. In the presence of 2.0% fresh human serum, normal neutrophils killed >90% of types 3 and 4 gonococci by 135 min. Serum alone at this concentration was not bactericidal. In the absence of serum, type 4 gonococci were not killed, whereas type 3 gonococci were killed to the same degree as in the presence of serum. Interestingly, heat-inactivated normal serum slightly inhibited phagocytic killing of type 3 gonococci. Results almost identical to those above were obtained when 5% fresh human serum deficient in complement component 7 was substituted for 2% normal autologous serum. This indicated that the later components of complement were not involved in the observed results. To investigate the mechanisms responsible for the intracellular killing of the gonococci, we used neutrophils from patients with chronic granulomatous disease. These neutrophils are deficient in an activable NADPH oxidase and do not produce bactericidal oxygen products upon phagocytic stimulation. Neutrophils from two unrelated boys with chronic granulomatous disease killed type 3 and 4 gonococci to the same degree as did normal neutrophils. As with normal neutrophils, serum was needed for killing type 4 organisms. As expected, neutrophils from these patients showed absolutely no increased chemiluminescence in the presence of type 3 or 4 gonococci, with or without serum. The effects of serum on gonococcus-induced chemiluminescence by normal neutrophils was also investigated. For these studies, in addition to type 3 and 4 gonococci, we also used transparent colony types of lightly (type 1) and heavily (type 2) piliated organisms. Chemiluminescence induced by type 1, 2, or 3 gonococci (i.e., gonococci possessing either pili or opacity-associated proteins, but not both) was augmented only slightly by serum and then only at low ratios of gonococci to neutrophils. On the other hand, chemiluminescence induced by type 4 gonococci (i.e., gonococci possessing neither pili nor opacity-associated proteins) was substantially increased in the presence of serum. Stimulation of chemiluminescence by type 1, 2, 3, or 4 gonococci was dose dependent in the absence or presence of serum. Heat-killed type 3 gonococci induced chemiluminescence to the same degree as did viable organisms. Since the gonococci used in this research was strongly catalase positive, as are gonococci in general, and since it was killed by chronic granulomatous disease neutrophils, the results indicate that gonococci can be effectively killed within neutrophils, i.e., within phagolysosomes, by nonoxidative bactericidal mechanisms. Whereas type 3 gonococci were phagocytized and killed by neutrophils equally well with or without serum, serum was obligatory for phagocytic killing of type 4 gonococci, i.e., gonococci lacking opacity-associated proteins. In addition, either pili or opacity-associated proteins were apparently necessary for maximal stimulation of neutrophil chemiluminescence. The submaximal stimulation of chemiluminescence by gonococci lacking both pili and opacity-associated proteins, i.e., type 4 gonococci was augmented by low concentrations of nonimmune serum.     

Flavobacterium columnare colony types: Connection to adhesion and virulence?

Four different colony morphologies were produced by Flavobacterium columnare strains on Shieh agar plate cultures: rhizoid and flat (type 1), non-rhizoid and hard (type 2), round and soft (type 3), and irregularly shaped and soft (type 4). Colonies produced on AO agar differed from these to some extent. The colony types formed on Shieh agar were studied according to molecular characteristics [Amplified Fragment Length Polymorphism (AFLP), Automated Ribosomal Intergenic Spacer Analysis (ARISA), and whole cell protein SDS-PAGE profiles], virulence on rainbow trout fingerlings, and adhesion on polystyrene and fish gills. There were no molecular differences between colony types within one strain. Type 2 was the most adherent on polystyrene, but type 1 was the most virulent. Adhesion of F. columnare strains used in this study was not connected to virulence. From fish infected with colony type 1, three colony types (types 1, 2 and 4) were isolated. Contrary to previous studies, our results suggest that strong adhesion capacity may not be the main virulence factor of F. columnare. Colony morphology change might be caused by phase variation, and different colony types isolated from infected fish may indicate different roles of the colony morphologies in the infection process of columnaris disease.