Iron is essential to virtually all organisms, but poses problems of toxicity and poor solubility. Bacteria have evolved various mechanisms to counter the problems imposed by their iron dependence, allowing them to achieve effective iron homeostasis under a range of iron regimes. Highly efficient iron acquisition systems are used to scavenge iron from the environment under iron-restricted conditions. In many cases, this involves the secretion and internalisation of extracellular ferric chelators called siderophores. Ferrous iron can also be directly imported by the G protein-like transporter, FeoB. For pathogens, host–iron complexes (transferrin, lactoferrin, haem, haemoglobin) are directly used as iron sources. Bacterial iron storage proteins (ferritin, bacterioferritin) provide intracellular iron reserves for use when external supplies are restricted, and iron detoxification proteins (Dps) are employed to protect the chromosome from iron-induced free radical damage. There is evidence that bacteria control their iron requirements in response to iron availability by down-regulating the expression of iron proteins during iron-restricted growth. And finally, the expression of the iron homeostatic machinery is subject to iron-dependent global control ensuring that iron acquisition, storage and consumption are geared to iron availability and that intracellular levels of free iron do not reach toxic levels.

Bacterial iron homeostasis

▪ Abstract The ability of pathogens to obtain iron from transferrins, ferritin, hemoglobin, and other iron-containing proteins of their host is central to whether they live or die. To combat invading bacteria, animals go into an iron-withholding mode and also use a protein (Nramp1) to generate reactive oxygen species in an attempt to kill the pathogens. Some invading bacteria respond by producing specific iron chelators—siderophores—that remove the iron from the host sources. Other bacteria rely on direct contact with host iron proteins, either abstracting the iron at their surface or, as with heme, taking it up into the cytoplasm. The expression of a large number of genes (>40 in some cases) is directly controlled by the prevailing intracellular concentration of Fe(II) via its complexing to a regulatory protein (the Fur protein or equivalent). In this way, the biochemistry of the bacterial cell can accommodate the challenges from the host. Agents that interfere with bacterial iron metabolism may prove ex...

Iron metabolism in pathogenic bacteria.

Iron is an essential element for most organisms, including bacteria. The oxidized form is insoluble, and the reduced form is highly toxic for most macromolecules and, in biological systems, is generally sequestrated by iron- and heme-carrier proteins. Thus, despite its abundance on earth, there is practically no free iron available for bacteria whatever biotope they colonize. To fulfill their iron needs, bacteria have multiple iron acquisition systems, reflecting the diversity of their potential biotopes. The iron/heme acquisition systems in bacteria have one of two general mechanisms. The first involves direct contact between the bacterium and the exogenous iron/heme sources. The second mechanism relies on molecules (siderophores and hemophores) synthesized and released by bacteria into the extracellular medium; these molecules scavenge iron or heme from various sources. Recent genetic, biochemical, and crystallographic studies have allowed substantial progress in describing molecular mechanisms of siderophore and hemophore interactions with the outer membrane receptors, transport through the inner membrane, iron storage, and regulation of genes encoding biosynthesis and uptake proteins.

Bacterial Iron Sources: From Siderophores to Hemophores

https://www.cell.com/cell-host-microbe/pdf/S1931-3128(13)00152-2.pdf

Iron in Infection and Immunity

Neisseria gonorrhoeae is evolving into a superbug with resistance to previously and currently recommended antimicrobials for treatment of gonorrhea, which is a major public health concern globally. Given the global nature of gonorrhea, the high rate of usage of antimicrobials, suboptimal control and monitoring of antimicrobial resistance (AMR) and treatment failures, slow update of treatment guidelines in most geographical settings, and the extraordinary capacity of the gonococci to develop and retain AMR, it is likely that the global problem of gonococcal AMR will worsen in the foreseeable future and that the severe complications of gonorrhea will emerge as a silent epidemic. By understanding the evolution, emergence, and spread of AMR in N. gonorrhoeae, including its molecular and phenotypic mechanisms, resistance to antimicrobials used clinically can be anticipated, future methods for genetic testing for AMR might permit region-specific and tailor-made antimicrobial therapy, and the design of novel antimicrobials to circumvent the resistance problems can be undertaken more rationally. This review focuses on the history and evolution of gonorrhea treatment regimens and emerging resistance to them, on genetic and phenotypic determinants of gonococcal resistance to previously and currently recommended antimicrobials, including biological costs or benefits; and on crucial actions and future advances necessary to detect and treat resistant gonococcal strains and, ultimately, retain gonorrhea as a treatable infection.

Antimicrobial Resistance in Neisseria gonorrhoeae in the 21st Century: Past, Evolution, and Future

Iron, an essential nutrient for most microorganisms, is sequestered by the host to decrease the concentration of iron available to bacterial pathogens. Neisseria gonorrhoeae, the causative agent of gonorrhoea, can acquire iron by direct interaction with human iron-binding proteins, including the serum glycoprotein, transferrin. Iron internalization from host transferrin requires the expression of a bacterial receptor, which specifically recognizes the human form of transferrin. Two gonococcal transferrin-binding proteins have been implicated in transferrin receptor function, TbpA and TbpB. We constructed a gonococcal transferrin receptor mutant without the introduction of additional antibiotic resistance markers and tested its ability to cause experimental urethritis in human male volunteers. The transferrin receptor mutant was incapable of initiating urethritis, although the same inoculum size of the wild-type parent strain, FA1090, causes urethritis in >90% of inoculated volunteers. To our knowledge, this is the first experimental demonstration that a bacterial iron acquisition system is an essential virulence factor for human infection.

The transferrin receptor expressed by gonococcal strain FA1090 is required for the experimental infection of human male volunteers

Pathogenic Neisseria species have been shown to scavenge iron from transferrin (Tf), although the mechanism is not yet fully understood. Two iron-repressible proteins that exhibit Tf-binding activity have been identified. This work describes the cloning and sequencing of tbpB, a 2.1-kb gene in N. gonorrhoeae that encodes Tbp2, an 85-kDa iron-repressible lipoprotein. Transcriptional interruption of tbpB had a strong polar effect on tbpA, the structural gene for Tbp1 that is located immediately downstream from tbpB. Such tbpB mutants did not express either Tbp2 or Tbp1, did not bind Tf to whole cells, did not grow on Tf plates, and did not take up iron from Tf. A mutant in which most of tbpB was deleted, presumably leaving tbpA under transcriptional control of the tbpB promoter, was constructed. This mutant did not express Tbp2 but expressed wild-type levels of Tbp1 and possessed the phenotype of reduced binding of Tf, decreased iron uptake from Tf, but normal growth on Tf plates. Mutants expressing Tbp2 and not Tbp1 bound less Tf, did not grow on Tf plates, and did not take up iron from Tf. These results suggest that tbpB and tbpA are polycistronic. Tbp2 apparently facilitates binding of Tf but is not essential for acquisition of iron from Tf under these in vitro conditions. Images

Gonococcal transferrin-binding protein 2 facilitates but is not essential for transferrin utilization.

All isolates of Neisseria gonorrhoeae express receptors that bind human transferrin (Tf). Although lactoferrin (Lf) is abundant on mucosa and in purulent exudates, many gonococci do not express an Lf receptor. The naturally occurring Lf receptor deletion mutant FA1090 (LbpB-LbpA-) is infectious, but a Tf receptor mutant of FA1090 is unable to infect male volunteers [Cornelissen, C.N., Kelley, M., Hobbs, M.M., Anderson, J.E., Cannon, J.G., Cohen, M.S., and Sparling, P.F. (1998) Mol Microbiol 27: 611-616]. Here, we report that expression of an Lf receptor in the absence of the Tf receptor was sufficient for infection, and that expression of both Lf and Tf receptors resulted in a competitive advantage over a strain that made only the Tf receptor in mixed infection of male volunteers. We confirmed that nearly 50% of clinical isolates do not make an Lf receptor. Surprisingly, about half of geographically diverse Lf - isolates representing many different auxotypes and porin serovars carried an identical lbpB lbpA deletion. Among Lf+ strains, all produced the integral outer membrane protein LbpA, but 70% did not express the lipoprotein LbpB. Thus, there are apparently selective pressures for expression of the Lf receptor in the male urethra that are balanced by others against expression of the Lf receptor in niches other than the male urethra.

https://www.onlinelibrary.wiley.com/doi/pdf/10.1046/j.1365-2958.2003.03496.x

Opposing selective forces for expression of the gonococcal lactoferrin receptor.

Immune responses to the gonococcus after natural infection ordinarily result in little immunity to reinfection, due to antigenic variation of the gonococcus, and redirection or suppression of immune responses. Brinton and colleagues demonstrated that parenteral immunization of male human volunteers with a purified pilus vaccine gave partial protection against infection by the homologous strain. However, the vaccine failed in a clinical trial. Recent vaccine development efforts have focused on the female mouse model of genital gonococcal infection. Here we discuss the state of the field, including our unpublished data regarding efficacy in the mouse model of either viral replicon particle (VRP) vaccines, or outer membrane vesicle (OMV) vaccines. The OMV vaccines failed, despite excellent serum and mucosal antibody responses. Protection after a regimen consisting of a PorB-VRP prime plus recombinant PorB boost was correlated with apparent Th1, but not with antibody, responses. Protection probably was due to powerful adjuvant effects of the VRP vector. New tools including novel transgenic mice expressing human genes required for gonococcal infection should enable future research. Surrogates for immunity are needed. Increasing antimicrobial resistance trends among gonococci makes development of a vaccine more urgent.

/pdf/vaccines-for-gonorrhea-can-we-rise-to-the-challenge-39a1wx81ws.pdf

Vaccines for gonorrhea: can we rise to the challenge?

Summary Neisseria gonorrhoeae is able to utilize iron (Fe) from a variety of sources including transferrin (TF) and lactoferrin (LF). To gain insight into the molecular mechanisms used by gonococci to scavenge Fe from TF and LF, we cloned a 3.5 kb segment of wild-type DNA that repaired the defect in tlu mutants, which are unable to take up Fe from either TF or LF despite exhibiting apparently normal ligand binding to the receptor. Nucleotide sequence determination identified three open reading frames (ORFs), designated ORF1, ORF2, and ORF3, which were arranged in tandem. The deduced amino acid sequence of the 852 bp ORF1 encoded a 28 kDa protein that exhibited 26‐32% identity with TonB proteins of nine other bacteria. The 663 bp ORF2 predicted a 24 kDa protein and the 435 bp long ORF3 predicted a 15 kDa protein. These predicted protein sequences exhibited 32‐38% and 24‐36% identity, respectively, with ExbB and ExbD proteins of three other bacteria. Thus, the sequence comparison identified the ORF1, ORF2 and ORF3 as gonococcal homologues of the E. coli tonB, exbB and exbD genes. An insertional mutation in the tonB homologue resulted in the failure of gonococci to grow with TF, LF or human haemoglobin (HB) as sole Fe sources and in the inability to take up 55 Fe from TF and LF. The tonB mutation did not prevent the utilization of Fe from citrate (CT) or haemin (HM). Binding of TF, LF and HB to whole cells in a solid-phase binding assay was largely unaffected by the tonB mutation. We conclude that the pathways for utilization of Fe bound to TF, LF and HB but not to HM or CT were dependent on the TonB system.

James E. Anderson

Papers

The transferrin receptor expressed by gonococcal strain FA1090 is required for the experimental infection of human male volunteers

Gonococcal transferrin-binding protein 2 facilitates but is not essential for transferrin utilization.

Opposing selective forces for expression of the gonococcal lactoferrin receptor.

Vaccines for gonorrhea: can we rise to the challenge?

Cloning and functional characterization of Neisseria gonorrhoeae tonB, exbB and exbD genes