Resistance of neisseria meningitidis to the toxic effects of heme iron and other hydrophobic agents requires expression of ght.

Several genetic systems that allow the use of iron-protoporphyrin IX (heme) have been described for the pathogenic bacterium Neisseria meningitidis. However, many questions about the process of heme acquisition and utilization remain to be answered. To isolate and analyze unidentified genes that play a role in heme iron uptake and utilization, a Himar1 transposon mutant library was screened in N. meningitidis serogroup A strain IR4162. One locus identified by transposon mutagenesis conferred protection against heme toxicity. A mutant with a deletion in a gene termed ght (gene of hydrophobic agent tolerance) within this locus was susceptible to heme and other hydrophobic agents compared to the parental strain. Transcriptional analysis indicated that ght is cotranscribed with an upstream open reading frame NMA2149. Uncharacterized orthologues of ght were identified in many other gram-negative bacteria. We present genetic evidence for the importance of ght in resistance to hydrophobic agents and its potential role in interaction with other hydrophobic agent resistance mechanisms within N. meningitidis.

Identification of Neisseria meningitidis genetic loci involved in the modulation of phase variation frequencies.

It has been proposed that increased phase variation frequencies in Neisseria meningitidis augment transmissibility and invasiveness. A Himar1 mariner transposon mutant library was constructed in serogroup A N. meningitidis and screened for clones with increased phase variation frequencies. Insertions increasing the frequency of slippage events within mononucleotide repeat tracts were identified in three known phase variation-modulating genes (mutS, mutL, and uvrD), as well as six additional loci (pilP, fbpA, fbpB, NMA1233, and two intergenic regions). The implications of these insertion mutations are discussed.

Biofilm formation by Neisseria meningitidis.

Biofilm formation by the human pathogen Neisseria meningitidis was analyzed. Biofilm-forming meningococcal strains were identified and quantitated by crystal violet staining. Laser scanning confocal microscopy of the meningococcal biofilm revealed variable layers up to 90 microm in thickness. A total of 39 meningococcal isolates were studied; 23 were nasopharyngeal-carriage isolates, and 16 were invasive-disease isolates. Thirty percent of carriage isolates and 12.5% of invasive-disease isolates formed biofilms proficiently on a polystyrene surface. Generally, the strains that formed biofilms showed high-level cell surface hydrophobicity, characteristic of strains lacking a capsule. The inhibitory role of capsule in biofilm formation was further confirmed by comparing the biofilm-forming capabilities of a serogroup B wild-type strain of a disease-associated isolate to those of its capsule-deficient mutant (ctrA). Some strains of meningococci form biofilms, and this process is likely important in menigococcal colonization.

Natural transformation and phase variation modulation in Neisseria meningitidis.

Neisseria meningitidis has evolved the ability to control the expression-state of numerous genes by phase variation. It has been proposed that the process aids this human pathogen in coping with the diversity of microenvironments and host immune systems. Therefore, increased frequencies of phase variation may augment the organism's adaptability and virulence. In this study, we found that DNA derived from various neisserial co-colonizers of the human nasopharynx increased N. meningitidis switching frequencies, indicating that heterologous neisserial DNA modulates phase variation in a transformation-dependent manner. In order to determine whether the effect of heterologous DNA was specific to the Hb receptor, HmbR, we constructed a Universal Rates of Switching cassette (UROS). With this cassette, we demonstrated that heterologous DNA positively affects phase variation throughout the meningococcal genome, as UROS phase variation frequencies were also increased in the presence of neisserial DNA. Overexpressing components of the neisserial mismatch repair system partially alleviated DNA-induced changes in phase variation frequencies, thus implicating mismatch repair titration as a cause of these transformation-dependent increases in switching. The DNA-dependent effect on phase variation was transient and may serve as a mechanism for meningococcal genetic variability that avoids the fitness costs encountered by global mutators.

Iron transport systems in Neisseria meningitidis.

Acquisition of iron and iron complexes has long been recognized as a major determinant in the pathogenesis of Neisseria meningitidis. In this review, high-affinity iron uptake systems, which allow meningococci to utilize the human host proteins transferrin, lactoferrin, hemoglobin, and haptoglobin-hemoglobin as sources of essential iron, are described. Classic features of bacterial iron transport systems, such as regulation by the iron-responsive repressor Fur and TonB-dependent transport activity, are discussed, as well as more specific features of meningococcal iron transport. Our current understanding of how N. meningitidis acquires iron from the human host and the vaccine potentials of various components of these iron transport systems are also reviewed.

Epidemiology, hypermutation, within-host evolution and the virulence of Neisseria meningitidis.

Many so-called pathogenic bacteria such as Neisseria meningitidis, Haemophilus influenzae, Staphylococcus aureus and Streptococcus pneumoniae are far more likely to colonize and maintain populations in healthy individuals asymptomatically than to cause disease. Disease is a dead-end for these bacteria: virulence shortens the window of time during which transmission to new hosts can occur and the subpopulations of bacteria actually responsible for disease, like those in the blood or cerebral spinal fluid, are rarely transmitted to new hosts. Hence, the virulence factors underlying their occasional pathogenicity must evolve in response to selection for something other than making their hosts sick. What are those selective pressures? We address this general question of the evolution of virulence in the context of phase shifting in N. meningitidis, a mutational process that turns specific genes on and off, and, in particular, contingency loci that code for virulence determinants such as pili, lipopolysaccharides, capsular polysaccharides and outer membrane proteins. We use mathematical models of the epidemiology and the within-host infection dynamics of N. meningitidis to make the case that rapid phase shifting evolves as an adaptation for colonization of diverse hosts and that the virulence of this bacterium is an inadvertent consequence of short-sighted within-host evolution, which is exasperated by the increased mutation rates associated with phase shifting. We present evidence for and suggest experimental and retrospective tests of these hypotheses.

In vitro antimalarial activity of metalloporphyrins against Plasmodium falciparum.

The in vitro antimalarial activity against Plasmodium falciparum and heme polymerization were evaluated for ten metalloporphyrins: gallium protoporphyrin IX (GaPPIX), sodium salt of gallium protoporphyrin IX, silver protoporphyrin IX, palladium protoporphyrin IX, cobalt protoporphyrin IX, manganese protoporphyrin IX, tin protoporphyrin IX (SnPPIX), chromium protoporphyrin IX, gallium deuteroporphyrin IX (GaDPIX) and gallium hematoporphyrin IX. Metalloporphyrins inhibited parasite growth with 50% inhibitory concentrations (IC(50)) ranging from 15.5 microM to 190 microM. In trophozoite lysate-mediated heme polymerization assays, SnPPIX, GaPPIX and GaDPIX exerted potent inhibitory activity similar to that of artemisinin and chloroquine.

Development and evaluation of an improved mouse model of meningococcal colonization.

Studies of meningococcal pathogenesis have been severely restricted due to the absence of an adequate animal model. Given the significance of iron in meningococcal pathogenesis, we developed a model of Neisseria meningitidis colonization in outbred adult mice that included daily administration of iron dextran. While receiving iron, the animals were inoculated intranasally with the initial doses of bacterial suspension. Meningococci were recovered from the animals by nasopharyngeal washes. Approximately half of the animals inoculated with 10(7) CFU remained colonized 13 days after the initial bacterial inoculation. The model was further evaluated with genetically defined isogenic serogroup B mutant strains, and the colonization capabilities of the mutants were compared to that of the wild-type parent. A mutant that produces truncated lipooligosaccharide (KDO(2)-lipid A) and a mutant defective in capsule transport were dramatically impaired in colonization. A mutant defective in pilus transport (pilQ) showed moderately impaired colonization. The immunological aspect of the model was also evaluated by challenging mice after immunization with homologous whole-cell meningococci. The immunized mice were protected from colonization of the homologous strain. In this model, long-term meningococcal colonization was maintained, allowing us to study the effects of specific genetic mutation on colonization. In addition, this model allows investigation of the role of active immune response against meningococci.

Processing of heme and heme-containing proteins by bacteria.

An extensive amount of new knowledge on bacterial systems involved in heme processing has been accumulated in the last 10 years. We discuss common themes in heme transport across bacterial outer and inner membranes, emphasizing proteins and mechanisms involved. The processing of heme in the bacterial cytoplasm is extensively covered, and a new hypothesis about the fate of heme in the bacterial cell is presented. Auxiliary genes involved in heme utilization, i.e., TonB, proteases, proteins involved in heme storage and pigmentation, as well as genes involved in regulation of heme assimilation are reviewed.

Mutator clones of Neisseria meningitidis in epidemic serogroup A disease.

Serogroup A Neisseria meningitidis has repeatedly caused widespread epidemics of meningitis and septicemia throughout the 20th century. Recently, in a limited collection of strains, epidemic serogroup A isolates were found to have elevated mutation rates that was caused by defects in mismatch repair pathways. To ascertain the role of these mutators in the epidemic spread of this serogroup, the prevalence of hypermutability in a collection of 95 serogroup A N. meningitidis invasive isolates was determined. Overall mutability in Neisseriae can be described by measuring both missense mutation rates as well as phase variation frequencies of "contingency loci." Fifty-seven percent of serogroup A isolates possessed elevated mutability, which could be divided into two classes: intermediate and high level. Eleven of 20 high-level mutators, with phase variation rates >100-fold higher than wild-type isolates, were defective in mismatch repair. Ten of the 34 intermediate mutators possessing >10-fold increases in phase variation rates could be partially complemented by a wild-type mutL allele. A high prevalence of mutators in epidemic isolates indicates that hypermutability may play a major role in the transmission of this pathogen. The added diversity derived from increased phase variation rates may allow fixation of mutator alleles more frequently during epidemic spread.

Histidine pK(a) shifts and changes of tautomeric states induced by the binding of gallium-protoporphyrin IX in the hemophore HasA(SM).

The HasA(SM) hemophore, secreted by Serratia marcescens, binds free or hemoprotein bound heme with high affinity and delivers it to a specific outer membrane receptor, HasR. In HasA(SM), heme is held by two loops and coordinated to iron by two residues, His 32 and Tyr 75. A third residue His 83 was shown recently to play a crucial role in heme ligation. To address the mechanistic issues of the heme capture and release processes, the histidine protonation states were studied in both apo- and holo-forms of HasA(SM) in solution. Holo-HasA(SM) was formed with gallium-protoporphyrin IX (GaPPIX), giving rise to a diamagnetic protein. By use of heteronuclear correlation NMR spectroscopy, the imidazole side-chain (15)N and (1)H resonances of the six HasA(SM) histidines were assigned and their pKa values and predominant tautomeric states according to pH were determined. We show that protonation states of the heme pocket histidines can modulate the nucleophilic character of the two axial ligands and, consequently, control the heme binding. In particular, the essential role of the His 83 is emphasized according to its direct interaction with Tyr 75.

Replication of Neisseria meningitidis within epithelial cells requires TonB-dependent acquisition of host cell iron.

Neisseria meningitidis (meningococcus [MC]) is able to enter and replicate within epithelial cells. Iron, an essential nutrient for nearly all organisms, is an important determinant in the ability of MC to cause disease; however, its role in MC intracellular replication has not been investigated. We analyzed the growth of MC within the A431 human epithelial cell line and the dependence of this growth on iron uptake. We present evidence here that chelation of iron from infected tissue culture cells with Desferal strongly inhibited intracellular replication of wild-type (wt) MC. We also provide genetic evidence that iron must be acquired by MC from the host cell in order for it to replicate. An hmbR mutant that is unable to use hemoglobin iron and could not grow in tissue culture media without iron supplementation replicated more rapidly within epithelial cells than its wt parent strain. An fbpA mutant that is unable to utilize human transferrin iron or lactoferrin iron replicated normally within cells. In contrast, a tonB mutant could not replicate intracellularly unless infected cultures were supplemented with ferric nitrate. Taken together, these findings strongly suggest that MC intracellular replication requires TonB-dependent uptake of a novel host cell iron source.

Inducible, but not constitutive, resistance of gonococci to hydrophobic agents due to the MtrC-MtrD-MtrE efflux pump requires TonB-ExbB-ExbD proteins.

The MtrC-MtrD-MtrE efflux pump possessed by Neisseria gonorrhoeae is very similar to the MexA-MexB-OprM efflux pump of Pseudomonas aeruginosa. Because the antimicrobial resistance property afforded by the MexA-MexB-OprM efflux pump also requires the TonB protein, we asked whether a similar requirement exists for the gonococcal efflux pump. Unlike earlier studies with P. aeruginosa, we found that constitutive levels of gonococcal resistance to hydrophobic antimicrobial agents (i.e., Triton X-100 [TX-100]) did not require the TonB, ExbB, or ExbD protein. However, inducible levels of TX-100 resistance in gonococci had an absolute requirement for the TonB-ExbB-ExbD system, suggesting that such resistance in gonococci has an energy requirement above and beyond that required for constitutive pump activity.

Neisserial TonB-dependent outer-membrane proteins: detection, regulation and distribution of three putative candidates identified from the genome sequences.

Computer searches were carried out of the gonococcal and meningococcal genome databases for previously unknown members of the TonB-dependent family (Tdf) of outer-membrane receptor proteins. Seven putative non-contiguous genes were found and three of these (identified in gonococcal strain FA1090) were chosen for further study. Consensus motif analysis of the peptide sequences was consistent with the three genes encoding TonB-dependent receptors. In view of the five previously characterized TonB-dependent proteins of pathogenic neisseriae, the putative genes were labelled tdfF, tdfG and tdfH. TdfF had homology with the siderophore receptors FpvA of Pseudomonas aeruginosa and FhuE of Escherichia coli, whereas TdfG and TdfH had homology with the haemophore receptor HasR of Serratia marcescens. The aim of this project was to characterize these proteins and determine their expression, regulation, distribution and surface exposure. Strain surveys of iron-stressed commensal and pathogenic neisseriae revealed that TdfF is unlikely to be expressed, TdfG is expressed by gonococci only and that TdfH is expressed by both meningococci and gonococci. Expression of TdfH was unaffected by iron availability. Susceptibility of TdfH to cleavage by proteases in live gonococci was consistent with surface exposure of this protein. TdfH may function as a TonB-dependent receptor for a non-iron nutrient source. Furthermore, TdfH is worthy of future investigation as a potential meningococcal vaccine candidate as it is a highly conserved, widely distributed and surface-exposed outer-membrane protein.