Alteration of epithelial cell transferrin-iron homeostasis by Neisseria meningitidis and Neisseria gonorrhoeae

Iron is an essential element for nearly all organisms. In mammals, iron is transported to body tissues by the serum glycoprotein transferrin. Transferrin-iron is internalized by binding to specific receptors followed by endocytosis. In vitro , Neisseria meningitidis and Neisseria gonorrhoeae can use iron from a variety of iron-containing compounds, including human transferrin. In vivo , transferrin is an important source of iron for N. gonorrhoeae : a mutant that is unable to bind and use transferrin-iron is unable to colonize the urethra of men or initiate disease at this site. As pathogenic Neisseria and its human host derive much of their iron from transferrin, we reasoned that a competition may exist between microbe and host epithelial cells for transferrin-iron at certain stages of infection. We therefore tested the hypothesis that N. meningitidis and N. gonorrhoeae may actively interfere with host transferrin-iron metabolism. We report that Neisseria-infected human epithelial cells have reduced levels of transferrin receptor messenger RNA and cycling transferrin receptors. The ability of infected cells to internalize transferrin receptor is also reduced. Finally, the relative distribution of surface and cycling transferrin receptors is altered in an infected cell. We conclude that Neisseria infection alters epithelial cell transferrin-iron homeostasis at multiple levels.     

Meningococcal adhesion suppresses proapoptotic gene expression and promotes expression of genes supporting early embryonic and cytoprotective signaling of human endothelial cells

Neisseria meningitidis colonizes the human nasopharynx and occasionally causes lethal or damaging septicemia and meningitis. Here, we examined the adherence-mediated signaling of meningococci to human cells by comparing gene expression profiles of human umbilical vein endothelial cells (HUVEC) infected by adherent wild-type, frpC-deficient mutant, or the nonadherent (DeltapilD) N. meningitidis. Pili-mediated adhesion of meningococci resulted in alterations of expression levels of human genes known to regulate apoptosis, cell proliferation, inflammatory response, adhesion and genes for signaling pathway proteins such as TGF-beta/Smad, Wnt/beta-catenin and Notch/Jagged. This reveals that adhering piliated meningocci manipulate host signaling pathways controlling cell proliferation while establishing a commensal relationship.     

PilC of Neisseria meningitidis is involved in class II pilus formation and restores pilus assembly, natural transformation competence and adherence to epithelial cells in PilC-deficient gonococci

Type 4 pili produced by the pathogenic Neisseria species constitute primary determinants for the adherence to host tissues. In addition to the major pilin subunit (PilE), neisserial pili contain the variable PilC proteins represented by two variant gene copies in most pathogenic Neisseria isolates. Based upon structural differences in the conserved regions of PilE, two pilus classes can be distinguished in Neisseria meningitidis . For class I pili found in both Neisseria gonorrhoeae and N. meningitidis , PilC proteins have been implicated in pilus assembly, natural transformation competence and adherence to epithelial cells. In this study, we used primers specific for the pilC2 gene of N. gonorrhoeae strain MS11 to amplify, by the polymerase chain reaction, and clone a homologous pilC gene from N. meningitidis strain A1493 which produces class II pili. This gene was sequenced and the deduced amino acid sequence showed 75.4% and 73.8% identity with the gonococcal PilC1 and PilC2, respectively. These values match the identity value of 74.1% calculated for the two N. gonorrhoeae MS11 PilC proteins, indicating a horizontal relationship between the N. gonorrhoeae and N. meningitidis pilC genes. We provide evidence that PilC functions in meningococcal class II pilus assembly and adherence. Furthermore, expression of the cloned N. meningitidis pilC gene in a gonococcal pilC1,2 mutant restores pilus assembly, adherence to ME-180 epithelial cells, and transformation competence to the wild-type level. Thus, PilC proteins exhibit indistinguishable functions in the context of class I and class II pili.     

Pathogenic Neisseria hitchhike on the uropod of human neutrophils

Polymorphonuclear neutrophils (PMNs) are important components of the human innate immune system and are rapidly recruited at the site of bacterial infection. Despite the effective phagocytic activity of PMNs, Neisseria gonorrhoeae infections are characterized by high survival within PMNs. We reveal a novel type IV pilus-mediated adherence of pathogenic Neisseria to the uropod (the rear) of polarized PMNs. The direct pilus-uropod interaction was visualized by scanning electron microscopy and total internal reflection fluorescence (TIRF) microscopy. We showed that N. meningitidis adhesion to the PMN uropod depended on both pilus-associated proteins PilC1 and PilC2, while N. gonorrhoeae adhesion did not. Bacterial adhesion elicited accumulation of the complement regulator CD46, but not I-domain-containing integrins, beneath the adherent bacterial microcolony. Electrographs and live-cell imaging of PMNs suggested that bacterial adherence to the uropod is followed by internalization into PMNs via the uropod. We also present data showing that pathogenic Neisseria can hitchhike on PMNs to hide from their phagocytic activity as well as to facilitate the spread of the pathogen through the epithelial cell layer.     

The PilC adhesin of the Neisseria type IV pilus-binding specificities and new insights into the nature of the host cell receptor

Type IV pili of Neisseria gonorrhoeae and Neisseria meningitidis mediate the first contact to human mucosal epithelial cells, an interaction which is also critical for the interaction with vascular endothelial cells. The PilC proteins have been characterized as the principal pilus-associated adhesin. Here we show that PilC2 exhibits a defined cell and tissue tropism, as it binds to human epithelial and endothelial cell lines, but not to human T cells or fibroblasts. Piliated gonococci and PilC2 exhibit similar patterns of binding to human epithelial and endothelial cells, supporting the function of PilC as the key pilus adhesin. Although CD46 has previously been suggested to be a pilus receptor, several observations indicate that neisserial type IV pili and the pilus adhesin PilC2 interact with epithelial cells in a CD46 independent manner. Biochemical approaches were used to characterize the nature of host cell factors mediating binding of piliated gonococci and PilC2 protein. Our data indicate that the putative host cell receptor for gonococcal pili and the PilC2 pilus adhesin is a surface protein. Glycostructures were found to not be involved in binding. Moreover, we observed the uptake of purified PilC2 protein together with its receptor via receptor-mediated endocytosis and subsequent receptor re-exposure on the cell surface. Our data support the existence of a specific pilus receptor and provide intriguing information on the nature of the receptor.     

The pilus and porin of Neisseria gonorrhoeae cooperatively induce Ca(2+) transients in infected epithelial cells

Purified pili and porin from Neisseria quickly mobilize calcium (Ca(2+)) stores in monocytes and epithelial cells, ultimately influencing host cell viability as well as bacterial intracellular survival. Here, we examined the Ca(2+) transients induced in human epithelial cells during infection by live, piliated N. gonorrhoeae. Porin induced an influx of Ca(2+) from the extracellular medium less than 60 s post infection. The porin-induced transient is followed by a pilus-induced release of Ca(2+) from intracellular stores. The timing of these events is similar to that observed using purified proteins. Interestingly, the porin-induced Ca(2+) flux is required for the pilus-induced transient, indicating that the pilus-induced Ca(2+) release is, itself, Ca(2+) dependent. Several lines of evidence indicate that porin is present on pili. Moreover, pilus retraction strongly influences the porin- and pilus-induced Ca(2+) fluxes. These and other results strongly suggest that the pilus and porin cooperate to modulate calcium signalling in epithelial cells, and propose a model to explain how N. gonorrhoeae triggers Ca(2+) transients in the initial stages of pilus-mediated attachment.     

Pilus biogenesis and epithelial cell adherence of Neisseria gonorrhoeae pilC double knock-out mutants

The phase-variable PilC proteins of pathogenic Neisseria species have recently been implicated in both assembly and cellular adherence functions of the type 4 pili of these pathogens. We describe here the cloning of full-length pilC1 and pilC2 genes and the complete sequencing of the pilC2 gene of Neisseria gonorrhoeae MS11. Sequential inactivation of both genes by gene replacement in piliated (P⁺) variants of N. gonorrhoeae MS11 led initially to a non-piliated (P⁻) phenotype; however, spontaneous P⁺ variants could be derived from some pilC1,2 double mutants which produced morphologically intact pili. Purified pili from pilC1,2 mutants revealed no detectable PilC protein. Instead, a novel protein about 70 kDa in size appeared in the pili preparations of P⁺ mutants; this protein exhibited no immunological cross-reactivity with PilC1 or PilC2. We propose that this novel factor replaces the function of PilC in pilus biogenesis. Using isogenic N. gonorrhoeae strains which produce identical PilE (pilin) proteins we demonstrate that pili associated with the 70 kDa protein do not confer gonococcal adherence to human epithelial cells, in contrast to pili assembled in the presence of PilC1 or PilC2.     

'Small' talk: Opa proteins as mediators of Neisseria-host-cell communication

Opa proteins are variable outer membrane proteins of Neisseria gonorrhoeae and Neisseria meningitidis that mediate tight interaction of these pathogens with human cells. They have emerged as a paradigm of a bacterial toolbox allowing recognition of different host receptors and orchestrating the cell type tropism displayed by pathogenic Neisseriae. Recent work has highlighted the molecular basis of Opa-protein-host-receptor interaction and has shed new light on the functional consequences of this interaction with regard to bacterial attachment, invasion, and responses elicited in particular host cells.     

The Neisseria type 2 IgA1 protease cleaves LAMP1 and promotes survival of bacteria within epithelial cells

Infection of human epithelial cells by Neisseria meningitidis (MC) and Neisseria gonorrhoeae (GC) increases the rate of degradation of LAMP1, a major integral membrane glycoprotein of late endosomes and lysosomes. Several lines of evidence indicate that the neisserial IgA1 protease is directly responsible for this LAMP1 degradation. LAMP1 contains an IgA1-like hinge region with potential cleavage sites for the neisserial type 1 and type 2 IgA1 proteases. Neisserial type 2 IgA1 protease cleaves purified LAMP1 in vitro . Unlike its wild-type isogenic parent, an iga ⁻ mutant of N. gonorrhoeae cannot affect LAMP1 turnover and its growth in epithelial cells is dramatically reduced. Thus, IgA1 protease cleavage of LAMP1 promotes intracellular survival of pathogenic Neisseria spp.     

PilT is required for PI(3,4,5)P3-mediated crosstalk between Neisseria gonorrhoeae and epithelial cells

The retractile type IV pilus participates in a number of fundamental bacterial processes, including motility, DNA transformation, fruiting body formation and attachment to host cells. Retraction of the N. gonorrhoeae type IV pilus requires a functional pilT. Retraction generates substantial force on its substrate (> 100 pN per retraction event), and it has been speculated that epithelial cells sense and respond to these forces during infection. We provide evidence that piliated, Opa non-expressing Neisseria gonorrhoeae activates the stress-responsive PI-3 kinase/Akt (PKB) pathway in human epithelial cells, and activation is enhanced by a functional pilT. PI-3 kinase inhibitors wortmannin and LY294002 reduce cell entry by 81% and 50%, respectively, illustrating the importance of this cascade in bacterial invasion. PI-3 kinase and its direct downstream effectors [PI(3,4,5)P3] and Akt are concentrated in the cell cortex beneath adherent bacteria, particularly at the periphery of the bacterial microcolonies. Furthermore, [PI(3,4,5)P3] is translocated to the outer leaflet of the plasma membrane. Finally, we show that [PI(3,4,5)P3] stimulates microcolony formation and upregulates pilT expression in vitro. We conclude that N. gonorrhoeae activation of PI-3 kinase triggers the host cell to produce a lipid second messenger that influences bacterial behaviour.     

HmbR outer membrane receptors of pathogenic Neisseria spp.: iron-regulated, hemoglobin-binding proteins with a high level of primary structure conservation.

We have recently cloned and characterized the hemoglobin receptor gene from Neisseria meningitidis serogroup C. N. meningitidis cells expressing HmbR protein were able to bind biotinylated hemoglobin, and the binding was specifically inhibited by unlabeled hemoglobin and not heme. The HmbR-mediated hemoglobin binding activity of N. meningitidis cells was shown to be iron regulated. The presence of hemoglobin but not heme in the growth medium stimulated HmbR-mediated hemoglobin binding activity. The efficiency of utilization of different hemoglobins by the HmbR-expressing N. meningitidis cells was shown to be species specific; human hemoglobin was the best source of iron, followed by horse, rat, turkey, dog, mouse, and sheep hemoglobins, The phenotypic characterization of HmbR mutants of some clinical strains of N. meningitidis suggested the existence of two unrelated hemoglobin receptors. The HmbR-unrelated hemoglobin receptor was shown to be identical to Hpu, the hemoglobin-haptoglobin receptor of N. meningitidis. The Hpu-dependent hemoglobin utilization system was not able to distinguish between different sources of hemoglobin; all animal hemoglobins were utilized equally well. HmbR-like genes are also present in N. meningitidis serogroups A and B, Neisseria gonorrhoeae MS11 and FA19, Neisseria perflava, and Neisseria polysaccharea. The hemoglobin receptor genes from N. meningitidis serogroups A and B and N. gonorrhoeae MS11 were cloned, and their nucleotide sequences were determined. The nucleotide sequence identity ranged between 86.5% (for N. meningitidis serogroup B hmbR and MS11 hmbR) and 93.4% (for N. meningitidis serogroup B hmbR and N. meningitidis serogroup C hmbR). The deduced amino acid sequences of these neisserial hemoglobin receptors were also highly related, with overall 84.7% conserved amino acid residues. A stop codon was found in the hmbR gene of N. gonorrhoeae MS11. This strain was still able to use hemoglobin and hemoglobin-haptoglobin complexes as iron sources, indicating that some gonococci may express only the HmbR-independent hemoglobin utilization system.     

Networks and groups within the genus Neisseria: analysis of argF, recA, rho, and 16S rRNA sequences from human Neisseria species.

To understand the pattern of nucleotide sequence variation among bacteria that frequently exchange chromosomal genes, we analyzed sequences of the recA, argF, and rho genes, as well as part of the small-subunit (16S) rRNA gene, from about 50 isolates of human commensal Neisseria species and the pathogenic N. meningitidis and N. gonorrhoeae. Almost all isolates of these species could be assigned to five phylogenetic groups that are found for all genes examined and generally are supported by high bootstrap values. In contrast, the phylogenetic relationships among groups varied according to the gene analyzed with notable incongruences involving N. cinerea and N. lactamica. Further analysis using split decomposition showed that for each gene, including 16S rRNA, the patterns of sequence divergence within N. meningitidis and closely related species were inconsistent with a bifurcating treelike phylogeny and better represented by an interconnected network. These data indicate that the human commensal Neisseria species can be separated into discrete groups of related species but that the relationships both within and among these groups, including those reconstructed using 16S rRNA, have been distorted by interspecies recombination events.     

Neisseria gonorrhoeae infection induces altered amphiregulin processing and release

Adhesion of the human pathogen Neisseria gonorrhoeae has established effects on the host cell and evokes a variety of cellular events including growth factor activation. In the present study we report that infection with N. gonorrhoeae causes altered amphiregulin processing and release in human epithelial cells. Amphiregulin is a well-studied growth factor with functions in various cell processes and is upregulated in different forms cancer and proliferative diseases. The protein is prototypically cleaved on the cell surface in response to external stimuli. We demonstrate that upon infection, a massive upregulation of amphiregulin mRNA is seen. The protein changes its subcellular distribution and is also alternatively cleaved at the plasma membrane, which results in augmented release of an infection-specific 36 kDa amphiregulin product from the surface of human cervical epithelial cells. Further, using antibodies directed against different domains of the protein we could determine the impact of infection on pro-peptide processing. In summary, we present data showing that the infection of N. gonorrhoeae causes an alternative amphiregulin processing, subcellular distribution and release in human epithelial cervical cells that likely contribute to the predisposition cellular abnormalities and anti-apoptotic features of N. gonorrhoeae infections.     

PilC of pathogenic Neisseria is associated with the bacterial cell surface

Adherence of pathogenic Neisseria to target host cells is mediated by pili. PilC1 and PilC2 are two high-molecular-weight proteins involved in pilus assembly and cellular adherence functions of the pili. Inactivation of pilC1 or pilC2 in N. meningitidis resulted in clones that expressed the same number of pili as the parent, contained no alterations in pilE and showed no detectable differences in PilE glycosylation. However, the PilC2+ pilC1- mutant showed much reduced adherence to target cells, indicating that production of PilC1 is essential for pilus-mediated adherence. To study further the functional differences between the meningococcal pilC genes, we determined the complete nucleotide sequence of pilC1 and pilC2 of N. meningitidis. Alignment of six PilC sequences demonstrated that PilC is composed of both conserved and variable regions. By immunogold labelling of bacterial sections we showed that PilC is present in the membranes of both piliated and non-piliated bacteria. Further, we demonstrated that PilC is associated with the bacterial cell surface.