RESUMEN
In an effort to identify potential cytotoxins expressed by Neisseria gonorrhoeae, we have identified a locus that, when mutated in the gonococcus, results in a significant increase in toxicity of the strain to human fallopian tube organ cultures (HFTOC). This locus, gly1, contains two open reading frames (ORFs) which are likely cotranscribed. ORF1 encodes a polypeptide of 17.8 kDa with a signal sequence that is recognized and processed in Escherichia coli and N. gonorrhoeae. The 15.6-kDa processed polypeptide has been observed in membrane fractions and filtered spent media from cultures of E. coli expressing gly1 and in outer membrane preparations of wild-type N. gonorrhoeae. The gly1 locus is not essential for bacterial survival, and it does not play a detectable role in epithelial cell adhesion, invasion, or intracellular survival. However, a gly1 null mutant causes much more damage to fallopian tube tissues than its isogenic wild-type parent. A strain complemented in trans for the gly1 mutation showed a level of toxicity to HFTOC similar to the level elicited by the wild-type parent. Taken together, these results indicate an involvement of the gly1 locus in the toxicity of N. gonorrhoeae to human fallopian tubes.
Asunto(s)
Proteínas Bacterianas , Toxinas Bacterianas/genética , Citotoxinas/genética , Trompas Uterinas/microbiología , Neisseria gonorrhoeae/genética , Secuencia de Aminoácidos , Animales , Anticuerpos Antibacterianos/biosíntesis , Anticuerpos Antibacterianos/inmunología , Adhesión Bacteriana , Toxinas Bacterianas/toxicidad , Secuencia de Bases , Línea Celular , Técnicas de Cultivo , Citotoxinas/toxicidad , ADN Bacteriano , Trompas Uterinas/patología , Femenino , Genes Bacterianos , Prueba de Complementación Genética , Humanos , Datos de Secuencia Molecular , Mutagénesis , Mutagénesis Insercional , Neisseria gonorrhoeae/fisiología , Sistemas de Lectura Abierta , Péptidos/inmunología , Conejos , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
The pilA gene of Neisseria gonorrhoeae was initially identified in a screen for transcriptional regulators of pilE, the expression locus for pilin, the major structural component of gonococcal pili. The predicted protein sequence for PilA has significant homology to two GTPases of the mammalian signal recognition particle (SRP), SRP54 and SRalpha. Homologs of SRP54 and SRalpha were subsequently identified in bacteria (Ffh and FtsY, respectively) and appear to form an SRP-like apparatus in prokaryotes. Of the two proteins, PilA is the most similar to FtsY (47% identical and 67% similar at the amino acid level). Like FtsY, PilA is essential for viability and hydrolyzes GTP. The similarities between PilA and the bacterial FtsY led us to ask whether PilA might function as the gonococcal FtsY. In this work, we show that overproduction of PilA in Escherichia coli leads to an accumulation of pre-beta-lactamase, similar to previous observations with other bacterial SRP components. Low-level expression of pilA in an ftsY conditional mutant can complement the ftsY mutation and restore normal growth to this strain under nonpermissive conditions. In addition, purified PilA can replace FtsY in an in vitro translocation assay using purified E. coli SRP components. A PilA mutant that is severely affected in its GTPase activity cannot replace FtsY in vivo or in vitro. However, overexpression of the GTPase mutant leads to the accumulation of pre-beta-lactamase, suggesting that the mutant protein may interact with the SRP apparatus to affect protein maturation. Taken together, these results show that the gonococcal PilA is an FtsY homolog and that the GTPase activity is necessary for its function.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Proteínas Fimbrias , GTP Fosfohidrolasas/genética , Neisseria gonorrhoeae/genética , Receptores Citoplasmáticos y Nucleares/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Clonación Molecular , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Precursores Enzimáticos/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismo , GTP Fosfohidrolasas/metabolismo , Genes Bacterianos , Genes Reguladores , Prueba de Complementación Genética , Mutagénesis Sitio-Dirigida , Neisseria gonorrhoeae/metabolismo , Proteínas Recombinantes/metabolismo , beta-Lactamasas/genéticaRESUMEN
PilA is the response regulator of a two-component regulatory system that controls a number of genes in the pathogenic Neisseria. Previous work has shown that Neisseria gonorrhoeae (GC) PilA binds DNA and also hydrolyzes GTP. Here, we report the cloning, sequencing, purification, and biochemical characterization of PilA from N. meningitidis (MC) strain 8013. MC pilA is 94% identical to GC pilA at the nucleotide level. Of the 78 nucleotide changes, 52 are silent, while 26 result in a total of 20 amino acid changes. Additionally, the MC homolog has a 4-amino acid insertion between the putative DNA-binding and GTP-binding domains. Purified MC PilA binds to the same DNA fragment we have previously shown to be bound by GC PilA specifically and also hydrolyzes GTP. The K(m) of MC PilA for GTP is 8.6 microM, similar to that determined for the GC protein. However, the maximum velocity (Vmax) is approximately 35-fold greater than the GC PilA activity. Additionally, the nucleotide specificity of MC PilA differs from that of GC PilA. While GC PilA hydrolyzes only GTP, MC PilA hydrolyzes GTP and ATP equally well, and CTP and UTP also compete for this activity.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Fimbrias , Neisseria meningitidis/química , Adenosina Trifosfatasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Clonación Molecular , Proteínas de Unión al ADN/análisis , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Inhibidores Enzimáticos/farmacología , Escherichia coli/genética , GTP Fosfohidrolasas/metabolismo , Regulación Bacteriana de la Expresión Génica , Guanosina Trifosfato/metabolismo , Cinética , Datos de Secuencia Molecular , Neisseria gonorrhoeae/química , Nucleótidos/metabolismo , Nucleótidos/farmacología , Proteínas Recombinantes/aislamiento & purificación , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
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.
Asunto(s)
Fimbrias Bacterianas/metabolismo , Neisseria/metabolismo , Actinas/análisis , Actinas/metabolismo , Adhesión Bacteriana/fisiología , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Impedancia Eléctrica , Epitelio/metabolismo , Humanos , Manitol/metabolismo , Microscopía Fluorescente , Neisseria/clasificación , Neisseria/patogenicidad , Técnicas de Cultivo de Órganos , PermeabilidadRESUMEN
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.
Asunto(s)
Adhesinas Bacterianas/biosíntesis , Adhesión Bacteriana/fisiología , Escherichia coli/enzimología , Neisseria gonorrhoeae/enzimología , Oxidorreductasas/metabolismo , Receptores de Superficie Celular , Receptores Acoplados a Proteínas G , Streptococcus pneumoniae/enzimología , Secuencia de Aminoácidos , Animales , Adhesión Bacteriana/genética , Secuencia de Bases , Secuencia de Carbohidratos , Cartilla de ADN , Disacáridos/química , Escherichia coli/genética , Escherichia coli/patogenicidad , Expresión Génica , Glicoconjugados , Cobayas , Pruebas de Hemaglutinación , Metionina Sulfóxido Reductasas , Datos de Secuencia Molecular , Neisseria gonorrhoeae/genética , Neisseria gonorrhoeae/patogenicidad , Oxidorreductasas/biosíntesis , Oxidorreductasas/química , Glicoproteínas de Membrana Plaquetaria/fisiología , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/patogenicidadRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , GTP Fosfohidrolasas/metabolismo , Regulación Bacteriana de la Expresión Génica , Neisseria gonorrhoeae/enzimología , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Escherichia coli/genética , Proteínas Fimbrias , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/aislamiento & purificación , Genes Bacterianos , Cinética , Matemática , Neisseria gonorrhoeae/genética , Pili Sexual , Ribonucleótidos/farmacologíaRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Fimbrias , Neisseria gonorrhoeae/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Bacterianas/aislamiento & purificación , Secuencia de Bases , Sitios de Unión/genética , Unión Competitiva , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Datos de Secuencia Molecular , Unión Proteica , Proteínas Recombinantes/metabolismo , Relación Estructura-ActividadRESUMEN
Tryptophan biosynthesis in Escherichia coli is regulated by the product of the trpR gene, the tryptophan (Trp) repressor. Trp aporepressor binds the corepressor, L-tryptophan, to form a holorepressor complex, which binds trp operator DNA tightly, and inhibits transcription of the tryptophan biosynthetic operon. The conservation of trp operator sequences among enteric Gram-negative bacteria suggests that trpR genes from other bacterial species can be cloned by complementation in E. coli. To clone trpR homologues, a deletion of the E. coli trpR gene, delta trpR504, was made on a plasmid by site-directed mutagenesis, then crossed onto the E. coli genome. Plasmid clones of the trpR genes of Enterobacter aerogenes and Enterobacter cloacae were isolated by complementation of the delta trpR504 allele, scored as the ability to repress beta-galactosidase synthesis from a prophage-borne trpE-lacZ gene fusion. The predicted amino acid sequences of four enteric TrpR proteins show differences, clustered on the backside of the folded repressor, opposite the DNA-binding helix-turn-helix substructures. These differences are predicted to have little effect on the interactions of the aporepressor with tryptophan, holorepressor with operator DNA, or tandemly bound holorepressor dimers with one another. Although there is some variation observed at the dimer interface, interactions predicted to stabilize the interface are conserved. The phylogenetic relationships revealed by the TrpR amino acid sequence alignment agree with the results of others.