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Query: UNIPROT:P06889 (Mol)
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Integration host factor (IHF) is a small, heterodimeric DNA-binding protein with pleiotropic function. IHF was purified to apparent homogeneity from Neisseria gonorrhoeae. Gel-retardation assays demonstrated binding of IHF to the pilE promoter region. The IHF-binding site was identified by DNase I protection assays and mapped proximal to three previously defined pilE promoters. Removal of the putative IHF-binding domain from pilE promoter DNA negated retardation of the DNA fragment when assessed by gel-shift analysis. Kleinschmidt electron microscopy showed pronounced kinking of pilE promoter DNA following incubation with IHF. Isogenic N. gonorrhoeae strains were constructed that contained either a wild-type pilE locus or a deleted pilE locus where the IHF-binding domain was removed. Primer-extension analysis and Northern blotting of total gonococcal RNA showed that in the absence of IHF binding at the pilE promoter, transcription was reduced 10-fold. Together, these data indicate that IHF is a transcriptional co-activator of pilE.
Mol Microbiol 1997 Feb
PMID:Integration host factor is a transcriptional cofactor of pilE in Neisseria gonorrhoeae. 915 37

Studies of gonococcal pilus biogenesis are fundamental to understanding organelle structure/function relationships and identifying new approaches to controlling disease. This area of research is also relevant to elucidating the basic mechanisms of outer membrane translocation of macromolecules, which requires components highly related to those involved in type IV pilus expression. Previous studies have shown that products of several ancillary pil genes are required for organelle biogenesis but of these only PilQ, a member of the GspD protein family, is a component of the outer membrane. DNA sequencing of the region upstream of pilQ revealed the presence of two open reading frames (ORFs) whose deduced polypeptides shared significant identities with proteins required for pilus expression in Pseudomonas aeruginosa and Pseudomonas syringae, the genes for which are arrayed upstream of a gene encoding a PilQ homologue. Gonococcal mutants bearing transposon insertions in these ORFs were non-piliated and failed to express pilus-associated phenotypes, and the corresponding genes were designated PilO and pilP. The piliation defects in the mutants could not be ascribed to polarity on distal pilQ expression as shown by direct measurement of PilQ antigen in those backgrounds and the use of a novel technique to create tandem duplications in the gonococcus (Gc) genome. As predicted by the presence of a consensus lipoprotein signal sequence, PilP expressed in both Escherichia coli and Gc could be labelled with [3H]-palmitic acid. PilP- as well as PilQ- mutants shed PilC, a protein which facilitates pilus assembly and is implicated in epithelial cell adherence, in a soluble form. Combined with the finding that levels of multimerized PiIQ were greatly reduced in PilP- mutants, the results suggest that PilP is required for PilQ function and that PilQ and PilC may interact during the terminal stages of pilus biogenesis. The findings also support the hypothesis that the Gc PilQ multimer corresponds to a physiologically relevant form of the protein required for pilus biogenesis.
Mol Microbiol 1997 Feb
PMID:PilP, a pilus biogenesis lipoprotein in Neisseria gonorrhoeae, affects expression of PilQ as a high-molecular-mass multimer. 915 38

A recombinant plasmid capable of restoring UV resistance to an Escherichia coli uvrA mutant was isolated from a genomic library of Neisseria gonorrhoeae. Sequence analysis revealed an open reading frame whose deduced amino acid sequence displayed significant similarity to those of the UvrA proteins of other bacterial species. A second open reading frame (ORF259) was identified upstream from, and in the opposite orientation to the gonococcal uvrA gene. Transcriptional fusions between portions of the gonococcal uvrA upstream region and a reporter gene were used to localise promoter activity in both E. coli and N. gonorrhoeae. The transcriptional starting points of uvrA and ORF259 were mapped in E. coli by primer extension analysis, and corresponding sigma70 promoters were identified. The arrangement of the uvrA-ORF259 intergenic region is similar to that of the gonococcal recA-aroD intergenic region. Both contain inverted copies of the 10 bp neisserial DNA uptake sequence situated between divergently transcribed genes. However, there is no evidence that either the uptake sequence or the proximity of the promoters influences expression of these genes.
Mol Gen Genet 1997 May 20
PMID:Cloning, nucleotide sequence and transcriptional analysis of the uvrA gene from Neisseria gonorrhoeae. 919 6

The type-4 pilus of Neisseria gonorrhoeae is a dominant surface antigen which facilitates adhesion to host target cells, an essential event in gonococcal infection. pilC2 encodes a 110-kDa protein involved in pilus assembly, pilus-mediated adherence to human epithelial cells in culture and natural competence for DNA transformation. Luciferase activity directed from a chromosomal pilC2::luxAB transcriptional fusion was reduced approximately 4-fold when cells were grown anaerobically. We observed a concomitant reduction in gonococcal piliation by electron microscopy and a reduction in the ability to adhere to ME-180 human epithelial cells when bacteria were grown in the absence of oxygen. Furthermore, we present evidence for growth-phase regulation of the gonococcal pilC2 gene in Escherichia coli, and show that all sequences necessary for growth-phase regulation are contained on a 121-bp pilC2 fragment. Expression from the minimal pilC2 fragment fused to lacZ in single-copy in E. coli was induced 2-fold when cells entered stationary phase. Surprisingly, induction does not require rpoS, the gene, which encodes the starvation-induced sigma factor RpoS. In summary, we have demonstrated that pilC2 is both positively and negatively regulated at the level of transcription. This regulation is most probably relevant to physiological conditions within the human host which influence gonococcal infections.
Mol Gen Genet 1997 Jul
PMID:Transcriptional regulation of pilC2 in Neisseria gonorrhoeae: response to oxygen availability and evidence for growth-phase regulation in Escherichia coli. 926 19

We have identified a gene encoding an autolysin (atlA) from Neisseria gonorrhoeae. The deduced amino acid sequence of AtlA shows significant similarity to the peptidoglycan degrading transglycosylases (endolysins) of bacteriophages lambda and P2, suggesting that the encoded protein also functions in peptidoglycan hydrolysis. An atlA mutant was identical to the wild-type strain in exponential growth rate, but demonstrated reduced lysis and peptidoglycan turnover in the stationary phase of growth. When transferred into a buffer solution, at a pH non-permissive for other gonococcal autolysins, an autolytic activity was detectable in the wild-type strain that was not present in the mutant. The most dramatic phenotype of the mutant occurred after extended time in stationary phase. After approximately 16h in stationary phase, both strains underwent an apparent replication event, after which the wild-type strain died rapidly whereas the atlA mutant survived considerably longer. Even after both the wild-type and mutant cells were dead, many of the mutant cells maintained intact morphology, whereas the wild-type cells were lysed. These results suggest that AtlA is a peptidoglycan transglycosylase related to bacteriophage endolysins and acts as an autolysin in the stationary phase.
Mol Microbiol 1997 Sep
PMID:A peptidoglycan hydrolase similar to bacteriophage endolysins acts as an autolysin in Neisseria gonorrhoeae. 936 15

The pathogenic Neisseria spp. are capable of iron utilization from host iron-binding proteins including transferrin and lactoferrin. Transferrin iron utilization is an energy-dependent, receptor-mediated event in which two identified transferrin-binding proteins participate. One of these proteins, TbpA, is homologous to the TonB-dependent family of outer membrane receptors that are required for high-affinity uptake of vitamin B12 and ferric siderophores. The 'TonB box' is a conserved domain near the amino-terminus of these proteins that has been implicated in interaction with TonB. Interaction between a periplasmic domain of TonB and the TonB box allows energy transduction to occur from the cytoplasmic membrane to the energy-dependent receptor in the outer membrane. We created a TonB box mutant of gonococcal TbpA and demonstrated that its binding and protease accessibility characteristics were indistinguishable from those of gonococcal Ton system mutants. The protease exposure of the second transferrin-binding protein, TbpB, was affected by the energization of TbpA, consistent with an interaction between these proteins. TbpB expressed by the de-energized mutants was readily accessible to protease, similar to TbpB expressed in the absence of TbpA. The de-energized mutants exhibited a marked decrease in transferrin diffusion rate, suggesting that receptor energization was necessary for ligand release. We propose a model to explain the observed Ton-dependent changes in the binding parameters and exposures of TbpA and TbpB.
Mol Microbiol 1997 Oct
PMID:Energy-dependent changes in the gonococcal transferrin receptor. 938 87

The aniA gene of Neisseria gonorrhoeae encodes an outer membrane lipoprotein which is strongly induced when gonococci are grown anaerobically in vitro in the presence of nitrite. Database searches with the amino acid sequence derived from the aniA structural gene revealed significant homologies to copper-containing nitrite reductases from several denitrifying bacteria. We constructed an insertional mutation in the aniA locus of strain MS11 by allelic replacement, to determine whether this locus was necessary for growth in oxygen-depleted environments, and to demonstrate that AniA was indeed a nitrite reductase. The mutant was severely impaired in its ability to grow micro-aerophilically in the presence of nitrite, and we observed a loss in viability over several hours of incubation. No measurable nitrite reductase activity was detected in the aniA mutant strain, and activity in the strain with a wild-type locus was inducible. Finally, we report investigations to determine whether AniA protein is involved in gonococcal pathogenesis.
Mol Gen Genet 1997 Nov
PMID:The Neisseria gonorrhoeae gene aniA encodes an inducible nitrite reductase. 941 36

The ability of all 11 variable opacity (Opa) proteins encoded by Neisseria gonorrhoeae MS11 to interact directly with the five CD66 antigens was determined. Transfected HeLa cell lines expressing individual CD66 antigens were infected with recombinant N. gonorrhoeae and Escherichia coli strains expressing defined Opas. Based upon the ability of these bacteria to bind and invade and to isolate specifically CD66 antigens from detergent-soluble extracts of the corresponding cell lines, distinct specificity groups of Opa interaction with CD66 were seen. Defining these specificity groups allowed us to assign a specific function for CD66a in the Opa-mediated interaction of gonococci with two different target cell types, which are both known to co-express multiple CD66 antigens. The competence of individual Opas to interact with CD66a was strictly correlated with their ability to induce an oxidative response by polymorphonuclear neutrophils. The same Opa specificity was observed for the level of gonococcal binding to primary endothelial cells after stimulation with TNFalpha, which was shown to increase the expression of CD66a rather than CD66e. As CD66e alone is expressed on other target tissues of gonococcal pathogenicity, Opa variation probably contributes to the cell tropism displayed by gonococci.
Mol Microbiol 1997 Dec
PMID:Differential Opa specificities for CD66 receptors influence tissue interactions and cellular response to Neisseria gonorrhoeae. 942 34

Neisseria gonorrhoeae opacity-associated (Opa) proteins are a family of outer membrane proteins involved in gonococcal adherence to and invasion of human cells. We wanted to identify additional roles for Opa in the infectious process and used the yeast two-hybrid system to identify human epithelial cell proteins that interact with Opa proteins. Although this system has been used successfully to identify many types of interacting proteins, it has not been used to screen a human cell cDNA library for binding partners of a prokaryotic outer membrane protein. Therefore, we were also interested in exploring the versatility of the yeast two-hybrid system in identifying bacteria-host interactions. Using OpaP from strain F62SF as bait, we screened a HeLa cell cDNA library for Opa-interacting proteins (OIPs). We identified five different OIPs, designated OIP1-OIP5, two of which are homologous to human proteins--thyroid hormone receptor interacting protein (TRIP6) and pyruvate kinase isoenzyme M2 (PK). In the studies presented here, we investigated the interaction between Opa proteins and PK in more depth. Opa-PK interactions were confirmed by in vitro and in vivo assays independent of the yeast two-hybrid system. Escherichia coli expressing six different Opa proteins from gonococcal strain FA1090 all bound more PK than Opa-negative E. coli in in vitro binding assays. Using anti-PK antibody and fluorescence microscopy, we showed that human epithelial cell PK co-localizes with intracellular Opa+ gonococci and E. coli expressing Opa proteins. Using a mutant of N. gonorrhoeae unable to grow on pyruvate or lactate, it appears that intracellular pyruvate is essential for gonococcal growth and survival. These results suggest a novel mechanism in bacterial pathogenesis, i.e. the requirement for direct molecular interaction with a host metabolic enzyme (PK) for the acquisition of an essential intracellular carbon source and growth substrate (pyruvate). These results demonstrate that the yeast two-hybrid system is a valuable tool for identifying biologically relevant interactions between bacteria and host proteins, providing valuable leads for further investigations into novel mechanisms of bacterial pathogenesis.
Mol Microbiol 1998 Jan
PMID:Using the yeast two-hybrid system to identify human epithelial cell proteins that bind gonococcal Opa proteins: intracellular gonococci bind pyruvate kinase via their Opa proteins and require host pyruvate for growth. 946 65

Iron, an essential nutrient for most microorganisms, is sequestered by the host to decrease the concentration of iron available to bacterial pathogens. Neisseria gonorrhoeae, the causative agent of gonorrhoea, can acquire iron by direct interaction with human iron-binding proteins, including the serum glycoprotein, transferrin. Iron internalization from host transferrin requires the expression of a bacterial receptor, which specifically recognizes the human form of transferrin. Two gonococcal transferrin-binding proteins have been implicated in transferrin receptor function, TbpA and TbpB. We constructed a gonococcal transferrin receptor mutant without the introduction of additional antibiotic resistance markers and tested its ability to cause experimental urethritis in human male volunteers. The transferrin receptor mutant was incapable of initiating urethritis, although the same inoculum size of the wild-type parent strain, FA1090, causes urethritis in >90% of inoculated volunteers. To our knowledge, this is the first experimental demonstration that a bacterial iron acquisition system is an essential virulence factor for human infection.
Mol Microbiol 1998 Feb
PMID:The transferrin receptor expressed by gonococcal strain FA1090 is required for the experimental infection of human male volunteers. 948 72


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