Ferric Enterobactin Binding and Utilization by Neisseria gonorrhoeae

FetA, formerly designated FrpB, an iron-regulated, 76-kDa neisserial outer membrane protein, shows sequence homology to the TonB-dependent family of receptors that transport iron into gram-negative bacteria. Although FetA is commonly expressed by most neisserial strains and is a potential vaccine candidate for both Neisseria gonorrhoeae and Neisseria meningitidis, its function in cell physiology was previously undefined. We now report that FetA functions as an enterobactin receptor. N. gonorrhoeae FA1090 utilized ferric enterobactin as the sole iron source when supplied with ferric enterobactin at approximately 10 μM, but growth stimulation was abolished when an omega (Ω) cassette was inserted within fetA or when tonB was insertionally interrupted. FA1090 FetA specifically bound 59Fe-enterobactin, with a Kd of approximately 5 μM. Monoclonal antibodies raised against the Escherichia coli enterobactin receptor, FepA, recognized FetA in Western blots, and amino acid sequence comparisons revealed that residues previously implicated in ferric enterobactin binding by FepA were partially conserved in FetA. An open reading frame downstream of fetA, designated fetB, predicted a protein with sequence similarity to the family of periplasmic binding proteins necessary for transporting siderophores through the periplasmic space of gram-negative bacteria. An Ω insertion within fetB abolished ferric enterobactin utilization without causing a loss of ferric enterobactin binding. These data show that FetA is a functional homolog of FepA that binds ferric enterobactin and may be part of a system responsible for transporting the siderophore into the cell

Ferric Enterobactin Binding and Utilization by Neisseria gonorrhoeae

FetA, formerly designated FrpB, an iron-regulated, 76-kDa neisserial outer membrane protein, shows sequence homology to the TonB-dependent family of receptors that transport iron into gram-negative bacteria. Although FetA is commonly expressed by most neisserial strains and is a potential vaccine candidate for both Neisseria gonorrhoeae and Neisseria meningitidis, its function in cell physiology was previously undefined. We now report that FetA functions as an enterobactin receptor. N. gonorrhoeae FA1090 utilized ferric enterobactin as the sole iron source when supplied with ferric enterobactin at approximately 10 μM, but growth stimulation was abolished when an omega (Ω) cassette was inserted within fetA or when tonB was insertionally interrupted. FA1090 FetA specifically bound 59Fe-enterobactin, with a Kd of approximately 5 μM. Monoclonal antibodies raised against the Escherichia coli enterobactin receptor, FepA, recognized FetA in Western blots, and amino acid sequence comparisons revealed that residues previously implicated in ferric enterobactin binding by FepA were partially conserved in FetA. An open reading frame downstream of fetA, designated fetB, predicted a protein with sequence similarity to the family of periplasmic binding proteins necessary for transporting siderophores through the periplasmic space of gram-negative bacteria. An Ω insertion within fetB abolished ferric enterobactin utilization without causing a loss of ferric enterobactin binding. These data show that FetA is a functional homolog of FepA that binds ferric enterobactin and may be part of a system responsible for transporting the siderophore into the cell

Defining the cellular precursors to human breast cancer

Human breast cancers are broadly classified based on their gene-expression profiles into luminal- and basal-type tumors. These two major tumor subtypes express markers corresponding to the major differentiation states of epithelial cells in the breast: luminal (EpCAM+) and basal/myoepithelial (CD10+). However, there are also rare types of breast cancers, such as metaplastic carcinomas, where tumor cells exhibit features of alternate cell types that no longer resemble breast epithelium. Until now, it has been difficult to identify the cell type(s) in the human breast that gives rise to these various forms of breast cancer. Here we report that transformation of EpCAM+ epithelial cells results in the formation of common forms of human breast cancer, including estrogen receptor-positive and estrogen receptor-negative tumors with luminal and basal-like characteristics, respectively, whereas transformation of CD10+ cells results in the development of rare metaplastic tumors reminiscent of the claudin-low subtype. We also demonstrate the existence of CD10+ breast cells with metaplastic traits that can give rise to skin and epidermal tissues. Furthermore, we show that the development of metaplastic breast cancer is attributable, in part, to the transformation of these metaplastic breast epithelial cells. These findings identify normal cellular precursors to human breast cancers and reveal the existence of a population of cells with epidermal progenitor activity within adult human breast tissues

Mapping the cellular and molecular heterogeneity of normal and malignant breast tissues and cultured cell lines

Introduction: Normal and neoplastic breast tissues are comprised of heterogeneous populations of epithelial cells exhibiting various degrees of maturation and differentiation. While cultured cell lines have been derived from both normal and malignant tissues, it remains unclear to what extent they retain similar levels of differentiation and heterogeneity as that found within breast tissues. Methods: We used 12 reduction mammoplasty tissues, 15 primary breast cancer tissues, and 20 human breast epithelial cell lines (16 cancer lines, 4 normal lines) to perform flow cytometry for CD44, CD24, epithelial cell adhesion molecule (EpCAM), and CD49f expression, as well as immunohistochemistry, and in vivo tumor xenograft formation studies to extensively analyze the molecular and cellular characteristics of breast epithelial cell lineages. Results: Human breast tissues contain four distinguishable epithelial differentiation states (two luminal phenotypes and two basal phenotypes) that differ on the basis of CD24, EpCAM and CD49f expression. Primary human breast cancer tissues also contain these four cellular states, but in altered proportions compared to normal tissues. In contrast, cultured cancer cell lines are enriched for rare basal and mesenchymal epithelial phenotypes, which are normally present in small numbers within human tissues. Similarly, cultured normal human mammary epithelial cell lines are enriched for rare basal and mesenchymal phenotypes that represent a minor fraction of cells within reduction mammoplasty tissues. Furthermore, although normal human mammary epithelial cell lines exhibit features of bi-potent progenitor cells they are unable to differentiate into mature luminal breast epithelial cells under standard culture conditions. Conclusions: As a group breast cancer cell lines represent the heterogeneity of human breast tumors, but individually they exhibit increased lineage-restricted profiles that fall short of truly representing the intratumoral heterogeneity of individual breast tumors. Additionally, normal human mammary epithelial cell lines fail to retain much of the cellular diversity found in human breast tissues and are enriched for differentiation states that are a minority in breast tissues, although they do exhibit features of bi-potent basal progenitor cells. These findings suggest that collections of cell lines representing multiple cell types can be used to model the cellular heterogeneity of tissues

A Putative P-Type ATPase Required for Virulence and Resistance to Haem Toxicity in Listeria monocytogenes

Regulation of iron homeostasis in many pathogens is principally mediated by the ferric uptake regulator, Fur. Since acquisition of iron from the host is essential for the intracellular pathogen Listeria monocytogenes, we predicted the existence of Fur-regulated systems that support infection. We examined the contribution of nine Fur-regulated loci to the pathogenicity of L. monocytogenes in a murine model of infection. While mutating the majority of the genes failed to affect virulence, three mutants exhibited a significantly compromised virulence potential. Most striking was the role of the membrane protein we designate FrvA (Fur regulated virulence factor A; encoded by frvA [lmo0641]), which is absolutely required for the systemic phase of infection in mice and also for virulence in an alternative infection model, the Wax Moth Galleria mellonella. Further analysis of the ΔfrvA mutant revealed poor growth in iron deficient media and inhibition of growth by micromolar concentrations of haem or haemoglobin, a phenotype which may contribute to the attenuated growth of this mutant during infection. Uptake studies indicated that the ΔfrvA mutant is unaffected in the uptake of ferric citrate but demonstrates a significant increase in uptake of haem and haemin. The data suggest a potential role for FrvA as a haem exporter that functions, at least in part, to protect the cell against the potential toxicity of free haem

Effect of lipopolysaccharide structure on reactivity of antiporin monoclonal antibodies with the bacterial cell surface.

We studied the reactivity of 66 anti-Escherichia coli B/r porin monoclonal antibodies (MAbs) with several E. coli and Salmonella typhimurium strains. Western immunoblots showed complete immunological cross-reactivity between E. coli B/r and K-12; among 34 MAbs which recognized porin in immunoblots of denatured outer membranes of E. coli B/r, all reacted with OmpF in denatured outer membranes of E. coli K-12. Extensive reactivity, although less than that for strain B/r (31 of 34 MAbs), occurred for porin from a wild-type isolate, E. coli O8:K27. Only one of the MAbs reacted with porin in denatured outer membranes of S. typhimurium. Even with immunochemical amplification of the Western immunoblot technique, only six MAbs recognized S. typhimurium porin (OmpD), demonstrating that there is significant immunological divergence between the porins of these species. Antibody binding to the bacterial surface, which was analyzed by cytofluorimetry, was strongly influenced by lipopolysaccharide (LPS) structure. An intact O antigen, as in E. coli O8:K27, blocked adsorption of all 20 MAbs in the test panel. rfa+ E. coli K-12, without an O antigen but with an intact LPS core, bound seven MAbs. When assayed against a series of rfa E. coli K-12 mutants, the number of MAbs that recognized porin surface epitopes increased sequentially as the LPS core became shorter. A total of 17 MAbs bound porin in a deep rough rfaD strain. Similar results were obtained with S. typhimurium. None of the anti-E. coli B/r porin MAbs adsorbed to a smooth strain, but three antibodies recognized porin on deep rough (rfaF, rfaE) mutants. These data define six distinct porin surface epitopes that are shielded by LPS from reaction with antibodies