Contact stimulation of Tgl and type IV pili in Myxococcus xanthus

Myxococcus xanthus tgl mutants lack social motility and type IV pili but can be transiently stimulated to swarm and to make pili by contacting tgl+ cells. The absence of pili in tgl mutants is shown not to be due to the absence of pilin. The rate of pilus elongation after Tgl stimulation is shown to be similar to the rate of pilus elongation in wild-type cells, using a new more rapid assay for stimulation.     

Anti-Gal binds to pili of Neisseria meningitidis: the immunoglobulin A isotype blocks complement-mediated killing

alpha 1,3-Galactosyl antibodies (anti-Gal) are ubiquitous natural human serum and secretory polyclonal antibodies that bind to terminal galactose-alpha 1,3-galactose (alpha-galactosyl) residues. Serum immunoglobulin G (IgG) anti-Gal can block alternative complement pathway-mediated lysis of representative gram-negative enteric bacteria that bind it to lipopolysaccharide alpha-galactosyl structures, thereby promoting survival of such bacteria in the nonimmune host. We wanted to know whether anti-Gal also could bind to the lipooligosaccharides (LOS) of Neisseria meningitidis. To our surprise, we found that serum and secretory anti-Gal bound to pili but not to LOS of certain strains. This suggested the presence of an immunogenic pilus carbohydrate epitope. Mild periodate oxidation of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated outer membrane preparations from strains that bound anti-Gal followed by labeling of the neoaldehyde groups resulted in the labeling of bands that corresponded to pilin and LOS, confirming that pilin contains carbohydrate structures. A Bandeiraea simplicifolia lectin that also binds terminal alpha 1,3-galactosyl residues also bound to pilin. Serum IgG, IgA, and IgM anti-Gal as well as colostral secretory IgA anti-Gal bound to pilin, as judged by immunoblotting, and to the pili of intact piliated organisms, as judged by immunoelectron microscopy. Total serum anti-Gal (IgG, IgA, and IgM) and purified serum IgA1 anti-Gal, but not its purified IgG isotype, blocked complement-mediated lysis of a piliated meningococcal strain that bound anti-Gal to its pili. Colostral anti-Gal secretory IgA blocked killing of the same strain. Thus, anti-Gal IgA may promote disease when it binds to the pili of N. meningitidis strains.     

A simple carbohydrate fermentation test for identification of the pathogenic Neisseria

The carbohydrate fermentation test in cystine-Trypticase agar-tubed medium was compared with the Minitek system with carbohydrate-impregnated paper disks in Müeller-Hinton broth for identification of Neisseria gonorrhoeae and N. meningitidis. There was 100% agreement between the methods for confirmation of N. meningitidis. The paper disk method confirmed 98% of the N. gonorrhoeae isolates; the cystine-Trypticase agar method confirmed 96%. Reactions with the paper disk method could be read in 4 h.     

The phosphorylcholine epitope undergoes phase variation on a 43-kilodalton protein in Pseudomonas aeruginosa and on pili of Neisseria meningitidis and Neisseria gonorrhoeae

Phosphorylcholine (ChoP) is a component of the teichoic acids of Streptococcus pneumoniae and has been recently identified on the lipopolysaccharide of Haemophilus influenzae, also a major pathogen of the human respiratory tract. Other gram-negative pathogens that frequently infect the human respiratory tract were surveyed for the presence of the ChoP epitope as indicated by binding to monoclonal antibodies (MAbs) recognizing this structure. The ChoP epitope was found on a 43-kDa protein on all clinical isolates of Pseudomonas aeruginosa examined and on several class I and II pili of Neisseria meningitidis. The specificity of the anti-ChoP MAb was demonstrated by the inhibition of binding in the presence of ChoP but not structural analogs. As in the case of H. influenzae, the expression of this epitope was phase variable on these species. In P. aeruginosa, this epitope was expressed at detectable levels only at lower growth temperatures. Expression of the ChoP epitope on piliated neisseriae displayed phase variation, both linked to pilus expression and independently of fully piliated bacteria.     

Expression of multiple pili by Legionella pneumophila: identification and characterization of a type IV pilin gene and its role in adherence to mammalian and protozoan cells

Legionella pneumophila expresses pili of variable lengths, either long (0.8 to 1.5 microm) or short (0.1 to 0.6 microm), that can be observed by transmission electron microscopy. We have identified a gene in L. pneumophila with homology to the type IV pilin genes (pilEL). An insertion mutation was constructed in pilEL and introduced into the L. pneumophila wild-type strain by allelic exchange. The pilin mutant is defective for expression of long pili. Reintroduction of the pilin locus on a cosmid vector restores expression of the long pili. The L. pneumophila pilEL mutant exhibited approximately a 50% decrease in adherence to human epithelial cells (HeLa and WI-26 cells), macrophages (U937 cells), and Acanthamoeba polyphaga but had a wild-type phenotype for intracellular replication within these cells. Southern hybridization analysis showed that the pilEL locus is present in L. pneumophila serogroups 1 through 13 but is variable in 16 other Legionella species. The presence of a type IV pilin gene and its expression by L. pneumophila may provide an advantage for colonization of lung tissues during Legionnaires' disease and invasion of amoebas in the environment.     

Suppression of an absolute defect in type IV pilus biogenesis by loss-of-function mutations in pilT, a twitching motility gene in Neisseria gonorrhoeae

Type IV pili of Neisseria gonorrhoeae, the Gram-negative etiologic agent of gonorrhea, facilitate colonization of the human host. Gonococcal PilT, a protein belonging to a large family of molecules sharing a highly conserved nucleotide binding domain motif, has been shown to be dispensable for organelle biogenesis but essential for twitching motility and competence for genetic transformation. Here, we show that the defect in pilus biogenesis resulting from mutations in the pilC gene, encoding a putative pilus-associated adhesin for human tissue, can be suppressed by the absence of functional PilT. These data conclusively demonstrate that PilT influences the Type IV pilus biogenesis pathway and strongly suggest that organelle expression is a dynamic process. In addition, these findings imply that PilT antagonizes the process of organelle biogenesis and provide the basis for a model for how the counteractive roles of PilT and PilC might relate mechanistically to the phenomenon of twitching motility.     

Serotonin enhances the production of type IV collagen by human mesangial cells

BACKGROUND: The plasma concentration of 5-hydroxytryptamine (5-HT) in diabetic patients is higher than that in normal subjects. Since recent reports have demonstrated the presence of 5-HT2A receptor in glomerular mesangial cells, it is possible that 5-HT may be involved in the development of diabetic nephropathy through the 5-HT2A receptor in mesangial cells. Because expansion of the glomerular mesangial lesion is a characteristic feature of diabetic nephropathy, we examined the effect of 5-HT on the production of type IV collagen by human mesangial cells. METHODS: Human mesangial cells were incubated with 5-HT with or without 5-HT receptor antagonists, protein kinase C (PKC) inhibitor or transforming growth factor-beta (TGF-beta) antibody. Type IV collagen mRNA and protein concentration in medium were measured by Northern blot analysis and enzyme-linked immunosorbent assay (ELISA), respectively. TGF-beta mRNA and bioactivity in the medium were measured by Northern blot analysis and bioassay using mink lung epithelial cells, respectively. RESULTS: 5-HT stimulated the production of type IV collagen by human mesangial cells, which was inhibited by ketanserin and sarpogrelate hydrochloride, 5-HT2A receptor antagonists, but not by ondansetron, a 5-HT3 receptor antagonist. 5-HT increased the bioactivities of both active and total TGF-beta. However, the 5-HT-enhanced production of type IV collagen was completely inhibited by an anti-TGF-beta antibody. Furthermore, a PKC inhibitor, calphostin C, inhibited the 5-HT-induced increase in type IV collagen secretion, and the activity of membrane PKC was increased by 5-HT. Phorbol ester activated type IV collagen production as well as active and total TGF-beta. Calphostin C completely inhibited the 5-HT-enhanced activity of active TGF-beta, but did not inhibit exogenous TGF-beta-induced increase in type IV collagen secretion. CONCLUSIONS: Our results suggest that 5-HT-enhanced production of type IV collagen by human mesangial cells is mediated by activation of PKC and subsequent increase in active TGF-beta activity.     

Activation of type IV procollagenases by human tumor-associated trypsin-2

Increased production of proteinases, such as matrix metalloproteinases (MMPs), is a characteristic feature of malignant tumors. Some human cancers and cell lines derived from them also express trypsinogen, but the function of the extrapancreatic trypsin has remained unclear. In this study we cloned and sequenced trypsinogen-2 cDNA from human COLO 205 colon carcinoma cells and characterized the ability of the enzyme to activate latent human type IV procollagenases (proMMP-2 and proMMP-9). As shown by cloning and N-terminal amino acid sequencing, the amino acid sequence of tumor-associated trypsin-2 is identical to that of pancreatic trypsin-2. We found that both pancreatic trypsin-2 and tumor cell-derived trypsin-2 are efficient activators of proMMP-9 and are capable of activating proMMP-9 at a molar ratio of 1:1000, the lowest reported so far. Human trypsin-2 was a more efficient activator than widely used bovine trypsin and converted the 92-kDa proMMP-9 to a single 77-kDa product that was not fragmented further. The single peptide bond cleaved by trypsin-2 in proMMP-9 was Arg87-Phe88. The generation of the 77-kDa species coincided with the increase in specific activity of MMP-9. In contrast, trypsin-2 only partially activated proMMP-2. Trypsin-2 cleaved the Arg99-Lys100 peptide bond of proMMP-2 generating 62-65-kDa MMP-2 species. Trypsin-2-induced proMMP-2 and -9 conversions were inhibited by tumor-associated trypsin inhibitor added either prior to or during activation indicating that proMMPs were not activated autocatalytically. Trypsin-2 also activated proMMPs associated with tissue inhibitor of matrix metalloproteinases, the complexes of which are thought to be the major MMP forms in vivo. The ability of human tumor cell-derived trypsin-2 to activate latent MMPs suggests a role for trypsin-2 in initiating the proteinase cascade that mediates tumor invasion and metastasis formation.     

Role of glycosylation at Ser63 in production of soluble pilin in pathogenic Neisseria

Pilus-mediated adhesion is essential in the pathogenesis of Neisseria meningitidis (MC) and Neisseria gonorrhoeae (GC). Pili are assembled from a protein subunit called pilin. Pilin is a glycoprotein, and pilin antigenic variation has been shown to be responsible for intrastrain variability with respect to the degree of adhesion in both MC and GC. In MC, high-adhesion pilins are responsible for the formation of bundles of pili which bind bacteria and cause them to grow as colonies on infected monolayers. In this work, we selected MC and GC pilin variants responsible for high and low adhesiveness and introduced them into the other species. Our results demonstrated that a given pilin variant expressed an identical phenotype in either GC or MC with respect to bundling and adhesiveness to epithelial cells. However, the production of truncated soluble pilin (S pilin) was consistently more abundant in GC than in MC. In the latter species, the glycosylation of pilin at Ser63 was shown to be required for the production of a truncated monomer of S pilin. In order to determine whether the same was true for GC, we engineered various pilin derivatives with an altered Ser63 glycosylation site. The results of these experiments demonstrated that the production of S pilin in GC was indeed more abundant when pilin was posttranslationally modified at Ser63. However, nonglycosylated variants remained capable of producing large amounts of S pilin. These data demonstrated that for GC, unlike for MC, glycosylation at Ser63 is not required for S-pilin production, suggesting that the mechanisms leading to the production of S pilin in GC and MC are different.     

Transformation of tooth type induced by inhibition of BMP signaling

Mammalian dentitions are highly patterned, with different types of teeth positioned in different regions of the jaws. BMP4 is an early oral epithelial protein signal that directs odontogenic gene expression in mesenchyme cells of the developing mandibular arch. BMP4 was shown to inhibit expression of the homeobox gene Barx-1 and to restrict expression to the proximal, presumptive molar mesenchyme of mouse embryos at embryonic day 10. The inhibition of BMP signaling early in mandible development by the action of exogenous Noggin protein resulted in ectopic Barx-1 expression in the distal, presumptive incisor mesenchyme and a transformation of tooth identity from incisor to molar.     

Identification of potential ferric binding residues in the iron-binding protein of pathogenic Neisseria meningitidis through structure-based multiple sequence alignments

Toxic effects of hyperglycemia-induced advanced glycosylated end products (AGEs) may explain some vasculopathic complications of diabetes. Aminoguanidine, a known inhibitor of AGE formation, was administered by gavage to Sprague-Dawley streptozotocin-induced diabetic rats made azotemic by surgical reduction of renal mass. All rats became hyperglycemic. Renal ablation caused renal insufficiency, as evidenced by markedly reduced endogenous creatinine clearances at days 7 and 14. Aminoguanidine-treated rats had significantly (P < 0.04) superior survival to that of untreated azotemic diabetic rats. We infer from the extended life in a rat model of uremia in diabetic nephropathy that aminoguanidine may prove beneficial in human diabetes.     

Which contacts of patients with meningococcal disease carry the pathogenic strain of Neisseria meningitidis? A population based study

OBJECTIVES: To determine the prevalence of the pathogenic strain of Neisseria meningitidis in contacts of patients with meningococcal disease, and to determine which contact groups are likely to be carriers and warrant chemoprophylaxis. DESIGN: Population based study. SETTING: Norwegian county of Telemark. SUBJECTS: 1535 primary contacts of 48 patients with meningococcal disease, and 78 secondary contacts. INTERVENTIONS: Carriers of the pathogenic strain were treated with rifampicin. All household members and kissing contacts under 15 years of age were treated with oral penicillin. Contacts were taught to recognise the symptoms of meningococcal disease. RESULTS: In 27 of 48 cases investigated, contacts carrying the pathogenic strain of N meningitidis were found. A total of 42 such contacts were identified. Contacts were stratified into three classes according to the assumed closeness of contact with patients. In class 1 (household members and kissing contacts) the prevalence of the pathogenic strain was 12.4% (95% confidence interval 5.5% to 19.3%). In classes 2 and 3 the prevalence was 1.9% (0.9% to 3.4%) and 1.6% (0.14% to 3.1%). CONCLUSIONS: There is a high rate of carriage of the pathogenic strain of N meningitidis in patients' household members and kissing contacts, and this supports the practice of giving chemoprophylaxis to these contacts. The prevalence of carriage among other contacts is 2-3 times that found in the general population (0.7%); the benefits of chemoprophylaxis to these contacts may be marginal.     

Expression of type IV collagen in the developing human kidney

The distribution of alpha1-6 chains of type IV collagen (alpha1-6(IV)) in human fetal kidneys was examined by indirect immunofluorescence. By 11 weeks of gestation, alpha1, 2, 3, 4, and 6(IV) were already present, but alpha5(IV) appeared relatively late, at 21 weeks. Alpha1(IV) and alpha2(IV) were present in all basement membranes, alpha3(IV) and alpha4(IV) were restricted to the glomerular basement membrane and parts of the tubular basement membrane. Alpha5(IV) was distributed in the glomerular basement membrane, Bowman's capsule, and parts of the tubular basement membrane. Alpha6(IV) was present in the Bowman's capsule, parts of the tubular basement membrane, and occurred in parts of the glomerular basement membrane at the early capillary loop stage, but disappeared during the later capillary loop stage.     

Decreased type IV collagen expression by human decidual tissues in spontaneous abortion

To provide some insight into the etiology of spontaneous abortion, the expression of type IV collagen was investigated in human decidual tissues obtained after spontaneous abortion (n = 17) and normal pregnancy (n = 22). Indirect immunofluorescent staining was performed for type I, III, and IV collagen as well as laminin, and Northern blot analysis was conducted to assess the expression of messenger ribonucleic acid for the alpha 1(IV) chain. Immunohistochemical analysis did not reveal any significant differences between normal pregnancy and spontaneous abortion with respect to interstitial collagens (type I and III collagen) and laminin in the decidual tissue. However, although pericellular immunostaining for type IV collagen was recognized around the decidual cells in normal pregnancy, very weak or no staining was observed in spontaneous abortion. Northern blot analysis revealed that the decidual expression of messenger ribonucleic acid for the alpha 1(IV) chain was significantly reduced in spontaneous abortion compared to that in normal pregnancy (P < 0.001). These results suggest that type IV collagen might play an important role in the maintenance of pregnancy and that decreased expression of this collagen could be associated with spontaneous abortion.     

Biodegradation of cellulose acetate by Neisseria sicca

Bacteria capable of assimilating cellulose acetate, strains SB and SC, were isolated from soil on a medium containing cellulose acetate as a carbon source, and identified as Neisseria sicca. Both strains degraded cellulose acetate membrane filters (degree of substitution, DS, mixture of 2.8 and 2.0) and textiles (DS, 2.34) in a medium containing cellulose acetate (DS, 2.34) or its oligomer, but were not able to degrade these materials in a medium containing cellobiose octaacetate. Biodegradation of cellulose acetate (DS, 1.81 and 2.34) on the basis of biochemical oxygen demand reached 51 and 40% in the culture of N. sicca SB and 60 and 45% in the culture of N. sicca SC within 20 days. A decrease in the acetyl content of degraded cellulose acetate films and powder was confirmed by infrared and nuclear magnetic resonance analyses. After 10-day cultivation of N. sicca SB and SC, the number-average molecular weight of residual cellulose acetate decreased by 9 and 5%, respectively. Activities of enzymes that released acetic acid and produced reducing sugars from cellulose acetate were mainly present in the culture supernatant. Reactivity of enzymes for cellulose acetate (DS, 1.81) was higher than that for cellulose acetate (DS, 2.34).