Helena Källström

Membrane cofactor protein (MCP or CD46) is a cellular pilus receptor for pathogenic Neisseria

Pili of Neisseria gonorrhoeae and Neisseria meningitidis mediate binding of the bacteria to human cell‐surface receptors. We found that purified pili bound to a 55‐ to 60‐kDa doublet band on SDS–PAGE of separated human epithelial cell extracts. This is a migration pattern typical of membrane cofactor protein (MCP or CD46). MCP is a widely distributed human complement regulatory protein. Attachment of the bacteria to epithelial cells was blocked by polyclonal and monoclonal antibodies directed against MCP, suggesting that this complement regulator is a receptor for piliated Neisseria. We proved this hypothesis by demonstrating that piliated, but not non‐piliated, gonococci bound to CHO cells transfected with human MCP‐cDNA. We also demonstrated a direct interaction between purified recombinant MCP and piliated Neisseria. Finally, recombinant MCP protein produced in E. coli inhibited attachment of the bacteria to target cells. Taken together, our data show that MCP is a human cell‐surface receptor for piliated pathogenic Neisseria.

Cell Signaling by the Type IV Pili of Pathogenic Neisseria

Neisseria gonorrhoeae andNeisseria meningitidis are Gram-negative bacterial pathogens that infect human mucosal epithelia. Type IV pilus-mediated adherence of these bacteria is a crucial early event for establishment of infection. In this work, we show that the type IV pili transduce a signal into the eucaryotic host cell. Purified adherent pili, but not pili from a low binding mutant, trigger an increase in the cytosolic free calcium ([Ca2+] i ) in target epithelial cells, a signal known to control many cellular responses. The [Ca2+] i increase was blocked by antibodies against CD46, a putative pilus receptor, suggesting a role for this protein in signal transduction. Pilus-mediated attachment was inhibited by depletion of host cell intracellular Ca2+ stores but not by removal of extracellular Ca2+. Further, kinase inhibition studies showed that pilus-mediated adherence is dependent on casein kinase II. In summary, these data reveal a novel function of the type IV pili, namely induction of signal transduction pathways in host cells.

Cdc25B cooperates with Cdc25A to induce mitosis but has a unique role in activating cyclin B1–Cdk1 at the centrosome

Cdc25 phosphatases are essential for the activation of mitotic cyclin–Cdks, but the precise roles of the three mammalian isoforms (A, B, and C) are unclear. Using RNA interference to reduce the expression of each Cdc25 isoform in HeLa and HEK293 cells, we observed that Cdc25A and -B are both needed for mitotic entry, whereas Cdc25C alone cannot induce mitosis. We found that the G2 delay caused by small interfering RNA to Cdc25A or -B was accompanied by reduced activities of both cyclin B1–Cdk1 and cyclin A–Cdk2 complexes and a delayed accumulation of cyclin B1 protein. Further, three-dimensional time-lapse microscopy and quantification of Cdk1 phosphorylation versus cyclin B1 levels in individual cells revealed that Cdc25A and -B exert specific functions in the initiation of mitosis: Cdc25A may play a role in chromatin condensation, whereas Cdc25B specifically activates cyclin B1–Cdk1 on centrosomes.

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.

Attachment of Neisseria gonorrhoeae to the cellular pilus receptor CD46: identification of domains important for bacterial adherence

Pili of Neisseria gonorrhoeae mediate binding of the bacteria to human host cells. Membrane cofactor protein (MCP or CD46), a human cell‐surface protein involved in regulation of complement activation, acts as a cellular pilus receptor. In this work, we examined which domains of CD46 mediate bacterial adherence. The CD46 expression was quantified and characterized in human epithelial cell lines. N. gonorrhoeae showed the highest adherence to ME180 cells, which have BC1 as the dominant phenotype. The BC isoforms of CD46 were expressed in all cell lines tested. The adherence was not enhanced by high expression of other isoforms, showing that the BC domain of CD46 is important in adherence of N. gonorrhoeae to human cells. To characterize the pilus‐binding site within the CD46 molecule, a set of CD46–BC1 deletion constructs were transfected into COS‐7 cells. Piliated N. gonorrhoeae attached well to CD46–BC1‐expressing COS‐7 cells. We show that the complement control protein repeat 3 (CCP‐3) and the serine–threonine–proline (STP)‐rich domain of CD46 are important for efficient adherence to host cells. Further, partial deletion of the cytoplasmic tail of CD46 results in low bacterial binding, indicating that the cytoplasmic tail takes part in the process of establishing a stable interaction between N. gonorrhoeae and host cells.

Characterisation of Cdc25B localisation and nuclear export during the cell cycle and in response to stress.

Cdc25 phosphatases are essential regulators of the cell cycle. In mammalian cells, the Cdc25B isoform activates cyclin A- and cyclin B1-containing complexes and is necessary for entry into mitosis. In this report, we characterise the subcellular localisation of Cdc25B by immunofluorescence in combination with RNA interference to identify specific antibody staining. We find that endogenous Cdc25B is mainly nuclear, but a fraction resides in the cytoplasm during the G2 phase of the cell cycle. Cdc25B starts to appear in S-phase cells and accumulates until prophase, after which the protein disappears. We characterise a nuclear export sequence in the N-terminus of Cdc25B (amino acids 54-67) that, when mutated, greatly reduces the ability of Cdc25B to shuttle in a fluorescence loss in photobleaching assay. Mutation of the nuclear export sequence makes Cdc25B less efficient in inducing mitosis, suggesting that an important mitotic function of Cdc25B occurs in the cytoplasm. Furthermore, we find that when cells are exposed to cycloheximide or ultraviolet irradiation, Cdc25B partially translocates to the cytoplasm. The dependence of this translocation event on a functional nuclear export sequence, an intact serine 323 residue (a 14-3-3 binding site) and p38 mitogen-activated protein kinase activity indicates that the p38 pathway regulates Cdc25B localisation in different situations of cellular stress.

Neisseria meningitidis Undergoes PilC Phase Variation and PilE Sequence Variation during Invasive Disease

Neisseria meningitidis colonizes the upper respiratory tract (URT), enters the blood stream, and reaches the cerebrospinal fluid (CSF). In the present study, we show that bacteria isolated from the URT adhere better to human epithelial cells, compared with bacteria from blood or CSF, which suggests that important changes of virulence-associated proteins take place during bacterial dissemination. Phase variation in the pilus adhesin PilC and sequence variation in the pilus subunit PilE occurred among strains from 1 patient. Changes were not found in the invasion-associated opacity proteins or in lipooligosaccharides. PilC was frequently expressed in serogroup B strains and in URT strains but was often switched off in other serogroups and in CSF strains. Strains lacking PilC showed impaired adhesion to epithelial cells. These data argue that N. meningitidis undergoes PilC phase variation and PilE sequence variation during invasive disease.

The phase-variable pilus-associated protein PilC is commonly expressed in clinical isolates of Neisseria gonorrhoeae, and shows sequence variability among strains

PilC is a phase-variable protein associated with pilus-mediated adherence of pathogenic Neisseria to target cells. In this study, 24 strains of Neisseria gonorrhoeae with known epidemiological data were examined for expression of PilC. All strains produced PilC independently of serovar and site of isolation. To investigate whether the PilC protein is conserved or variable among gonococcal strains, the complete nucleotide sequence of pilC in four strains, isolated from either rectum, throat or blood, was determined. The deduced amino acid sequence in these strains differed from each other and from the two PilC proteins of N. gonorrhoeae MS11. These data demonstrate that PilC is commonly expressed, but the PilC sequence may vary among gonococcal strains.

Cholera toxin and extracellular Ca2+ induce adherence of non-piliated Neisseria: evidence for an important role of G-proteins and Rho in the bacteria-cell interaction.

In this study, we characterize the interaction between non‐piliated (P−) Neisseria gonorrhoeae and human epithelial cells. P− mutants lacking the pilus subunit protein PilE attach at low levels to cells. Although the binding may not lead to heavy inflammatory responses, the interaction between P−Neisseria and host cells most probably play a role in colonization and asymptomatic carriage of the pathogen. Here we show that the adherence of P−N. gonorrhoeae is blocked by GDP‐β‐S [guanosine 5′‐O‐(thio)diphosphate], a non‐hydrolyzable GTP analogue, and by C3 exotoxin, an inhibitor of the small G‐protein Rho. G‐protein activators such as cholera toxin, that activates Gs, and fluoroaluminate, a general G‐protein activator, induced bacterial adherence. Furthermore, increase of the extracellular free [Ca2+] dramatically enhanced adherence of non‐piliated Neisseria. The pharynx and the urogenital tract are natural entry sites of the pathogenic Neisseria species, and at both sites the epithelial cells can be exposed to wide variations in Ca2+ concentration. Taken together, these data show the importance of extracellular Ca2+ in the pathogenic Neisseria‐host interaction, and reveal a novel function of cholera toxin, namely induction of bacterial adherence.

Characterization of the region downstream of the pilus biogenesis gene pilC1 in Neisseria gonorrhoeae

The nucleotide sequence of a 3 kb region downstream of pilC1 in Neisseria gonorrhoeae MS11 was analyzed. This region contains two open reading frames, ORF1 and ORF2, and several repetitive DNA elements. ORF1 encodes an outer membrane protein that shows homology to orf98 of Pediococcus acidilactici. PCR with primers specific for ORF1 revealed that the gene is present in all gonococcal strains tested. The other open reading frame, ORF2, is highly homologous to the putative integral membrane protein HI1680 of Haemophilus influenzae.