Biomedical subjects

M So

Publications and source records attributed to M So.

At least 37 records · Page 2Linked to original sources

PubMed1997

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.

Animals↗

PubMed1997

Attachment of piliated, Opa- and Opc- gonococci and meningococci to epithelial cells elicits cortical actin rearrangements and clustering of tyrosine-phosphorylated proteins.

Attachment of piliated Neisseria gonorrhoeae or Neisseria meningitidis cells to A431, Chang, HEC-1-B, or polarized T(84) cells triggers rearrangements of cortical microfilaments and the accumulation of phosphotyrosine-containing proteins at sites of bacterial contact. Actin stress fibers and the microtubule network remain unaltered in infected cells. The rearrangements reported here are triggered by piliated, Opa- and Opc- strains and also by nonpiliated gonococci (GC) that produce the invasion-associated OpaA protein. Thus, neisserial adhesion via either of at least two different adhesins can trigger cortical rearrangements. In contrast, these rearrangements are not triggered by nonadherent GC or meningococcal strains, by heat-killed or chloramphenicol-treated GC strains, or by Escherichia coli recombinants that adhere to cells via GC OpaA or Opal fusion proteins, suggesting that additional neisserial components are involved. Immunoblotting experiments did not detect consistent increases in the phosphorylation of specific proteins. Possible biological implications of these Neisseria-induced cortical rearrangements are discussed.

Actin Cytoskeleton↗

PubMed1997

The antifungal effect of lactoferrin and lysozyme on Candida krusei and Candida albicans.

Lactoferrin and lysozyme (muramidase) are non-immune defence factors present in various exocrine secretions, including saliva. Previous studies have shown that both proteins, either singly or in combination, are bactericidal in nature and their combined activity is synergistic. As little is known of their interactions with Candida species, 20 oral isolates of C. krusei and 5 isolates of C. albicans were studied for their susceptibility to human apo-lactoferrin and lysozyme, either singly or in combination, using an in vitro assay system. The two species exhibited significant interspecies differences in susceptibility to lactoferrin (p < 0.05), but not for lysozyme; C. krusei being more sensitive to lactoferrin (c 1.4 times) than C. albicans. Both species revealed significant intraspecies differences in their susceptibility to lysozyme (p < 0.05), but not for lactoferrin. No synergistic antifungal activity of the two proteins on either Candida species was noted. The results imply that the variable expression of the fungicidal activity of lactoferrin and lysozyme on Candida species may modulate the oral carriage of yeasts in a complex manner.

Antifungal Agents↗

PubMed1996

Peptide methionine sulfoxide reductase contributes to the maintenance of adhesins in three major pathogens.

Pathogenic bacteria rely on adhesins to bind to host tissues. Therefore, the maintenance of the functional properties of these extracellular macromolecules is essential for the pathogenicity of these microorganisms. We report that peptide methionine sulfoxide reductase (MsrA), a repair enzyme, contributes to the maintenance of adhesins in Streptococcus pneumoniae, Neisseria gonorrhoeae, and Escherichia coli. A screen of a library of pneumococcal mutants for loss of adherence uncovered a MsrA mutant with 75% reduced binding to GalNAcbeta1-4Gal containing eukaryotic cell receptors that are present on type II lung cells and vascular endothelial cells. Subsequently, it was shown that an E. coli msrA mutant displayed decreased type I fimbriae-mediated, mannose-dependent, agglutination of erythrocytes. Previous work [Taha, M. K., So, M., Seifert, H. S., Billyard, E. & Marchal, C. (1988) EMBO J. 7, 4367-4378] has shown that mutants with defects in the pilA-pilB locus from N. gonorrhoeae were altered in their production of type IV pili. We show that pneumococcal MsrA and gonococcal PilB expressed in E. coli have MsrA activity. Together these data suggest that MsrA is required for the proper expression or maintenance of functional adhesins on the surfaces of these three major pathogenic bacteria.

Adhesins, Bacterial↗

PubMed1996

Induction of SIV capsid-specific CTL and mucosal sIgA in mice immunized with a recombinant S. typhimurium aroA mutant.

We have developed a new expression system based on the E. coli groEL promoter. The suicide vector constructed (called APC vector) allows simultaneous attenuation of a Salmonella strain by disruption of the coding sequence for aroA and stable integration of a gene into the bacterial chromosome. High-level expression of antigen is achieved after Salmonella is taken up by macrophages, a major antigen processing cell of the host. The chloramphenicol acetyltransferase (CAT) and the simian immunodeficiency virus capsid (p27gag) genes were cloned downstream of the groEL promoter and expressed within S. typhimurium. By measuring CAT activity, we showed that the groEL promoter was up-regulated during infection of the J774 macrophage line. The immune response to SIV capsid was assessed in Balb/c mice given one oral dose of vaccine. A local mucosal secretory IgA response against SIV capsid was detected but no systemic antibody response to the same antigen. A systemic CTL response was detected as early as 28 days to as late as 70 days post-immunization. CTL activity was MHC restricted (H-2d) and was mediated by CD3+, CD8+, CD4- T-lymphocytes. These results indicate that with only one oral dose of recombinant Salmonella using the APC vector, a systemic CTL response and a mucosal secretory response against the SIV capsid antigen are elicited in a mouse model.

Animals↗

PubMed1996

Assembly and antigenicity of the Neisseria gonorrhoeae pilus mapped with antibodies.

The relationship between the sequence of Neisseria gonorrhoeae pilin and its quaternary assembly into pilus fibers was studied with a set of site-directed antibody probes and by mapping the specificities of antipilus antisera with peptides. Buried and exposed peptides in assembled pili were identified by competitive immunoassays and immunoelectron microscopy with polyclonal antibodies raised against 11 peptides spanning the pilin sequence. Pili did not compete significantly with pilin subunits for binding to antibodies against residues 13 to 31 (13-31) and 18-36. Pilus fibers competed well with pilin protein subunits for binding to antibodies raised against peptides 37-56, 58-78, 110-120, 115-127, 122-139, and 140-159 and competed weakly for antibodies against residues 79-93 and 94-108. Antibodies to sequence-conserved residues 37-56 and to semiconserved residues 94-108 preferentially bound pilus ends as shown by immunoelectron microscopy. The exposure of pilus regions to the immune system was tested by peptide mapping of antiserum specificities against sets of overlapping peptides representing all possible hexameric or octameric peptides from the N. gonorrhoeae MS11 pilin sequence. The immunogenicity of exposed peptides incorporating semiconserved residues 49-56 and 121-126 was revealed by strong, consistent antigenic reactivity to these regions measured in antipilus sera from rabbits, mice, and human and in sera from human volunteers with gonorrhea. The conservation and variation of antigenic responses among these three species clarify the relevance of immunological studies of other species to the human immune response against pathogens. Overall, our results explain the extreme conservation of the entire N-terminal one-third of the pilin protein by its dominant role in pilus assembly: hydrophobic residues 1-36 are implicated in buried lateral contacts, and polar residues 37-56 are implicated in longitudinal contacts within the pilus fiber.

Amino Acid Sequence↗

PubMed1996

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.

Amino Acid Sequence↗

PubMed1996

Traversal of a polarized epithelium by pathogenic Neisseriae: facilitation by type IV pili and maintenance of epithelial barrier function.

BACKGROUND: Gonococci (GC) and meningococci (MC) are gram-negative bacterial pathogens that infect human mucosal epithelia. We would like to understand the functions of specific bacterial components at each stage of mucosal colonization: adhesion, cell invasion, and traversal into subepithelial tissues. As no animal model of mucosal colonization by GC or MC is available, increasingly sophisticated in vitro approaches have been used to address these issues. MATERIALS AND METHODS: We adapted the polarized T84 human epithelial cell system to study GC and MC colonization. Epithelial barrier function was monitored by permeability to soluble tracers and with electrical resistance measurements. Polarized cells were used to assay bacterial traversal of the monolayers, and cells grown on plastic were used to assay adhesion and cell invasion. RESULTS: All pathogenic Neisseriae examined traversed the monolayers. The traversal times were species specific and identical to times established previously in organ culture studies. In contrast to experiments with some enteric pathogens, transmigration by GC and MC was not accompanied by disruption of the epithelial barrier. GC mutants lacking type IV pili were compromised in adhesion, invasion, and traversal of T84 cells. CONCLUSIONS: Experiments with polarized T84 cells mimic key features of organ culture infections and reveal additional aspects of neisserial infection. Epithelial barrier function can be retained during bacterial traversal. Experiments with a nonpiliated GC mutant and its wild-type parent indicated an unexpected role for pili in cell invasion. Our results are consistent with the hypothesis that bacterial adhesion, invasion, or both are rate-limiting for traversal across the epithelium.

Actins↗

PubMed1995

The Neisseria transcriptional regulator PilA has a GTPase activity.

The pilA gene of Neisseria gonorrhoeae encodes the response regulator of a two-component regulatory system that controls pilin gene expression. Examination of the primary sequence of PilA indicates that the protein contains at least two functional domains. The N-terminal region has a proposed helix-turn-helix motif thought to be involved in DNA binding. This region also contains the residues that are presumed to form the acidic pocket involved in phosphorylation by PilB, the sensor kinase of the system. The C terminus of the protein has extensive homology to the G (GTP-binding) domains of the eukaryotic signal recognition particle (SRP) 54-kDa protein and the alpha subunit of the SRP receptor, or docking protein. This homology also extends to similar regions of the bacterial SRP homologs Ffh and FtsY. Here, we demonstrate that purified PilA has significant GTPase activity, and that this activity has an absolute requirement for MgCl2 and is sensitive to KCl and low pH. We also show that PilA has a strict specificity for GTP, and that GTP hydrolysis follows first order kinetics, with a maximum velocity (Vmax) of 1900 pmol of Pi produced per min per mg of protein and a Km for GTP of 9.6 microM at 37 degrees C.

Bacterial Outer Membrane Proteins↗

PubMed1995

The Neisseria meningitidis haemoglobin receptor: its role in iron utilization and virulence.

The Neisseria meningitidis haemoglobin receptor gene, hmbR, was cloned by complementation in a porphyrin-requiring Escherichia coli mutant. hmbR encodes an 89.5 kDa outer membrane protein which shares amino acid homology with the TonB-dependent receptors of Gram-negative bacteria. HmbR had the highest similarity to Neisseria transferrin and lactoferrin receptors. The utilization of haemoglobin as an iron source required internalization of the haemin moiety by the cell. The mechanism of haemin internalization via the haemoglobin receptor was TonB-dependent in E. coli. A N. meningitidis hmbR mutant was unable to use haemoglobin but could still use haemin as a sole iron source. The existence of a second N. meningitidis receptor gene, specific for haemin, was shown by the isolation of cosmids which did not hybridize with the hmbR probe, but which were able to complement an E. coli hemA aroB mutant on haemin-supplemented plates. The N. meningitidis hmbR mutant was attenuated in an infant rat model for meningococcal infection, indicating that haemoglobin utilization is important for N. meningitidis virulence.

Aldehyde Oxidoreductases↗

PubMed1995

Interaction of pathogenic neisseriae with nonphagocytic cells.

The ability to interact with nonphagocytic cells is a crucial virulence attribute of the meningococcus and the genococcus. Like most bacterial pathogens, Neisseria meningitidis and Neisseria gonorrhoeae initiate infections by colonizing the mucosal epithelium, which serves as the site of entry. After this step, both bacteria cross the intact mucosal barrier. While N. gonorrhoeae is likely to remain in the subepithelial matrix, where it initiates an intense inflammatory reaction, N. meningitidis enters the bloodstream, and eventually the cerebrospinal fluid to cause meningitis. Both pathogens have evolved very similar mechanisms for interacting with host cells. Surface structures that influence bacterium-host interactions include pili, the meningococcal class 5 outer membrane proteins or the gonococcal opacity proteins, lipooligosaccharide, and the meningococcal capsule. This review examines what is known about the roles these structures play in bacterial adhesion and invasion, with special emphasis, on pilus-mediated adhesion. Finally, the importance of these structures in neisserial pathogenesis is discussed.

Amino Acid Sequence↗

PubMed1995

Interaction of the Neisseria gonorrhoeae PilA protein with the pilE promoter involves multiple sites on the DNA.

PilA is the putative DNA-binding component of a two-component system that regulates transcription of the pilin expression locus (pilE) of Neisseria gonorrhoeae. Here we report the purification of the PilA protein and characterization of its DNA-binding activity. PilA was overproduced in Escherichia coli with an isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible expression vector. Cell extracts were prepared by sonication and fractionated by anion-exchange chromotography, followed by dye affinity chromatography with Cibacron Blue. Proteins were eluted by using a gradient of KCl, and PilA-containing fractions were identified by immunoblot analysis with a polyclonal anti-PilA antiserum. Purified PilA was judged to be > 90% pure, as determined by Coomassie blue staining and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. PilA purified in this manner was used to develop a gel retardation assay with a 301-bp fragment containing the pilE promoter (PpilE) and upstream sequences as a probe. A fragment of similar size containing the E. coli aroH promoter was used as a negative control. Competition experiments using a 100- to 1,000-fold excess of unlabelled DNA fragments confirmed the specificity of PilA binding to the pilE promoter. To localize the PilA binding site within the 301-bp PpilE fragment, stepwise deletions were generated by PCR and the fragments were examined in the gel shift assay. The results of these experiments show that there are two regions upstream of PpilE that are required for binding by PilA. Taken together, these data indicate that while PilA binds specifically to the upstream region of the pilE gene, this interaction is complex and likely involves multiple regions of this DNA sequence.

Bacterial Proteins↗

PubMed1995

Relationship between the cell surface hydrophobicity and adherence of Candida krusei and Candida albicans to epithelial and denture acrylic surfaces.

C. krusei is an emerging pathogen, especially in immunocompromised hosts, and is implicated, together with Candida albicans, as an etiological agent of oral candidoses. As the cell surface hydrophobicity of these yeasts appears to be important in the pathogenesis of superficial candidoses, 20 oral isolates of C. krusei and 5 oral isolates of C. albicans were investigated using a biphasic (hydrocarbon/aqueous) separation hydrophobicity assay. All the C. krusei isolates demonstrated significantly greater hydrophobicity than the C. albicans isolates (p < 0.001). Further, there were significant intraspecies differences in cell surface hydrophobicity amongst C. krusei isolates. When hydrophobicity and adhesion to surfaces were compared using data from a previous study, there was a positive correlation between the cell surface hydrophobicity and adhesion of C. krusei isolates to HeLa surfaces (r = 0.53, p < 0.05), but not to acrylic surfaces. The current data, while confirming the inter- and intraspecies differences in cell surface hydrophobicity of Candida species, indicate that this attribute together with other cell surface features may determine the hierarchy of virulence amongst the different Candida species.

Candida↗

PubMed1994

Roles of pilin and PilC in adhesion of Neisseria meningitidis to human epithelial and endothelial cells.

Pili and pilin antigenic variation play important roles in adhesion of Neisseria meningitidis (MC) to human epithelial and endothelial cells. We recently identified one pilin variant that confers high adhesiveness of MC to human epithelial cells in culture. However, other factor(s) also play a role in MC adhesiveness, since some nonadhesive variants of MC strain 8013 are piliated and produce the same pilin variant as adhesive derivatives. PilC1 and PilC2, high molecular weight outer membrane proteins in Neisseria gonorrhoeae, are proposed to play roles in pilus assembly. Strain 8013 also contains pilC1 and pilC2; their products function in a similar if not identical manner in pilus biogenesis. PilC1 has an additional function in that it also modulates adhesiveness of strain 8013.

Bacterial Adhesion↗

PubMed1994

Adhesion and colonisation of Candida krusei on host surfaces.

Candida krusei is an emerging pathogen, especially in immunocompromised hosts. As the adherence of this organism both to host epithelial surfaces and to catheter and prosthetic surfaces appears to be important in the pathogenesis of superficial as well as systemic candidoses, the adhesion of 20 oral isolates of C. krusei and five oral isolates of C. albicans was compared with the following substrates: cultured (HeLa) epithelial cells, buccal epithelial cells (BEC) from healthy adults and bone marrow transplant patients, and acrylic (polymethylmethacrylate) surfaces. Animal experiments in Sprague Dawley rats were also conducted to evaluate the relative oral carriage rate of the two Candida spp. C. krusei isolates adhered in far greater numbers to acrylic surfaces than to either of the cell surfaces. Significant intra-species differences in C. krusei adhesion for acrylic surfaces were noted between 74 (39%) of 190 pair comparisons in contrast to 18 (9.5%) of 190 with HeLa surfaces (p < 0.05). A positive correlation was also observed between the adhesion of C. krusei isolates to HeLa cells and acrylic surfaces. Five isolates of C. albicans showed very low adherence to HeLa surfaces when compared with BEC obtained from either healthy individuals or bone marrow transplant patients. The adherence of C. albicans to BEC from the healthy individuals was c. 12-fold greater than that of C. krusei, a figure similar to the relative murine oral carriage rate of the two Candida spp.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1994

The opaH locus of Neisseria gonorrhoeae MS11A is involved in epithelial cell invasion.

In order to produce a successful infection, Neisseria gonorrhoeae (GC) must attach to and invade mucosal epithelial cells. To identify GC gene products involved in this early interaction with host cells we constructed a gene bank derived from a clinical isolate of GC, and isolated a clone which had the capacity to adhere to the human endometrial adenocarcinoma tissue-culture line HEC-1-B. The cloned sequence was identified as a member of the opa gene family whose protein products have been associated with virulence. The GC chromosome contains numerous variant opa genes which, in MS11, are designated opaA-K. Previous work showed that expression of opaC confers a highly invasive phenotype upon strain MS11. When our cloned opa gene was mutated and returned to the GC MS11A chromosome by transformation and homologous recombination, we isolated one transformant that was significantly reduced in its invasive capacity. The locus mutated in this transformant was identified as opaH. Our results indicate that invasiveness of GC for human epithelial cells can be determined by more than one opa gene in strain MS11A.

Amino Acid Sequence↗

PubMed1993

Transcriptional control of gonococcal pilE expression: involvement of an alternate sigma factor.

The pilE gene encoding Neisseria gonorrhoeae (Gc) pilin contains two putative promoter sequences 5' to the transcription start point (tsp), a Pribnow box and an RpoN-binding consensus sequence. Sequence analysis shows that the nucleotide (nt) sequence of the pilE promoter region is completely conserved among eight different Gc isolates. Using a pilE::lacZ transcriptional fusion, we demonstrate that the RpoN sigma factor can function in Escherichia coli to increase pilE transcription when the NifA activator from Klebsiella is present in trans. In addition, over-production of the native pilin protein using RpoN and NifA is lethal to E. coli. Finally, we show that the RpoN sigma factor decreases the basal expression of pilE when an activator is not present. These results suggest that, in Gc, pilE transcription may be regulated by RpoN in conjunction with an activator and that sigma 70 can also act to direct transcription of pilE.

Bacterial Proteins↗

PubMed1993

Antigenic variation of pilin regulates adhesion of Neisseria meningitidis to human epithelial cells.

Pili have been shown to play an essential role in the adhesion of Neisseria meningitidis to epithelial cells. However, among piliated strains, both inter- and intrastrain variability exist with respect to their degree of adhesion to epithelial cells in vitro (Virji et al., 1992). This suggests that factors other than the presence of pili per se are involved in this process. The N. meningitidis pilin subunit undergoes extensive antigenic variation. Piliated low- and high-adhesive derivatives of the same N. meningitidis strain were selected and the nucleotide sequence of the pilin gene expressed in each was determined. The highly adhesive derivatives had the same pilin sequence. The alleles encoding the pilin subunit of the low-adhesive derivatives were completely different from the one found in the high-adhesive isolates. Using polyclonal antibodies raised against one hyperadhesive variant, it was confirmed that the low-adhesive piliated derivatives expressed pilin variants antigenically different from the highly adhesive strains. The role of antigenic variation in the adhesive process of N. meningitidis was confirmed by performing allelic exchanges of the pilE locus between low- and high-adhesive isolates. Antigenic variation has been considered a means by which virulent bacteria evade the host immune system. This work provides genetic proof that a bacterial pathogen, N. meningitidis, can use antigenic variation to modulate their degree of virulence.

Amino Acid Sequence↗