RESUMEN
Two-component regulatory systems are involved in processes important for bacterial pathogenesis. The proposed misR/misS (or phoP/phoQ) system is one of four two-component systems of the obligate human pathogen Neisseria meningitidis. Inactivation of this system results in loss of phosphorylation of the lipooligosaccharide inner core and causes attenuation in a mouse model of meningococcal infection. MisR and the cytoplasmic domain of MisS were purified as His6 and maltose binding protein fusion proteins, respectively. The MisS fusion was shown to be autophosphorylated in the presence of ATP, and the phosphoryl group was subsequently transferred to MisR. The phosphotransfer reaction was halted with a MisR/D52A mutation, while a MisS/H246A mutation prevented autophosphorylation. Specific interaction of phosphorylated MisR (MisR approximately P) and MisR with the misR promoter was demonstrated by gel mobility shift assays, where MisR approximately P exhibited higher affinity than did the nonphosphorylated protein. The transcriptional start site of the misRS operon was mapped, and DNase I protection assays revealed that MisR interacted with a 15-bp region upstream of the transcriptional start site that shared no similarity to binding motifs of other two-component systems. Transcriptional reporter studies suggested that MisR phosphorylation is critical for the autoinduction of the misRS operon. Limited Mg2+ concentration failed to induce expression of the misRS operon, which is the only operon now proven to be under the direct control of the MisRS two-component system. Thus, these results indicate that the meningococcal MisRS system constitutes a functional signal transduction circuit and that both components are critical in the autoregulation of their expression.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/fisiología , Neisseria meningitidis/patogenicidad , Transducción de Señal/fisiología , Secuencia de Bases , Desoxirribonucleasa I , Regulación Bacteriana de la Expresión Génica , Homeostasis , Humanos , Datos de Secuencia Molecular , Neisseria meningitidis/genética , Neisseria meningitidis/fisiología , Plásmidos , Regiones Promotoras Genéticas , Alineación de Secuencia , Homología de Secuencia de Ácido NucleicoRESUMEN
In 2000, a large international outbreak of meningococcal disease caused by Neisseria meningitidis serogroup W-135 was identified among pilgrims returning from the Hajj in Saudi Arabia. To assess ongoing risk, we evaluated N. meningitidis carriage among US travelers to the 2001 Hajj. Of 25 N. meningitidis isolates obtained, 15 (60%) were nongroupable and 8 (32%) were serogroup W-135 when tested by standard slide-agglutination techniques. Two additional nongroupable isolates were characterized as serogroup W-135 when tested by polymerase chain reaction. Nine of 10 serogroup W-135 isolates were indistinguishable from the Hajj-2000 clone. None of the departing, but 9 (1.3%) of the returning, pilgrims carried serogroup W-135 (P=.01); all carriers reported previous vaccination. Carriage of N. meningitidis serogroup W-135 increased significantly in pilgrims returning from the Hajj. Although the risk of disease to pilgrims appears to be low, the risk of spread to others of this pathogenic strain remains a concern.
Asunto(s)
Portador Sano/epidemiología , Infecciones Meningocócicas/epidemiología , Neisseria meningitidis Serogrupo W-135/aislamiento & purificación , Viaje , Adulto , Anciano , Portador Sano/microbiología , Femenino , Humanos , Islamismo , Masculino , Infecciones Meningocócicas/microbiología , Persona de Mediana Edad , Neisseria meningitidis Serogrupo W-135/clasificación , Faringe/microbiología , Reacción en Cadena de la Polimerasa , Arabia Saudita , Serotipificación , Estados UnidosRESUMEN
The genetic basis for biosynthesis of the (alpha1-->4)-linked N-acetyl-D-glucosamine 1-phosphate capsule of Neisseria meningitidis serogroup X was defined. The biosynthesis gene cassette was a approximately 4.2-kb region located between ctrA of the capsule transport operon and galE, which encodes the UDP-glucose-4-epimerase. This location was identical to the locations of the biosynthesis cassettes in other meningococcal serogroups. Three open reading frames unique to meningococcus serogroup X were identified. Deletion-insertion mutation and colony immunoblotting confirmed that these three genes were essential for serogroup X capsule expression, and the genes were designated xcbA, xcbB, and xcbC (serogroup X capsule biosynthesis). Reverse transcriptase PCR indicated that the xcbABC genes form an operon and are cotranscribed divergently from ctrA. XcbA exhibited 52% amino acid similarity to SacB, the putative capsule polymerase of meningococcus serogroup A, suggesting that it plays a role as the serogroup X capsule polymerase. An IS1016 element was found within the intergenic region separating ctrA and xcbA in multiple strains, and this element did not interfere with capsule expression.
Asunto(s)
Cápsulas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , Neisseria meningitidis/metabolismo , Operón , Acetilglucosamina/metabolismo , Secuencia de Aminoácidos , Cápsulas Bacterianas/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Actividad Bactericida de la Sangre , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Humanos , Datos de Secuencia Molecular , Neisseria meningitidis/clasificación , Neisseria meningitidis/genética , Fosfatos/metabolismo , Análisis de Secuencia de ADN , Serotipificación , Factores de Transcripción/química , Factores de Transcripción/genética , UDPglucosa 4-Epimerasa/química , UDPglucosa 4-Epimerasa/genéticaRESUMEN
Neisseria meningitidis serogroup A capsular polysaccharide (CPS) is composed of a homopolymer of O-acetylated, alpha1-->6-linked ManNAc 1-phosphate that is distinct from the capsule structures of the other meningococcal disease-causing serogroups, B, C, Y, and W-135. The serogroup A capsule biosynthetic genetic cassette consists of four open reading frames, mynA-D (sacA-D), that are specific to serogroup A, but the functions of these genes have not been well characterized. mynC was found to encode an inner membrane-associated acetyltransferase that is responsible for the O-acetylation of the CPS of serogroup A. The wild-type CPS as revealed by 1H NMR had 60-70% O-acetylated ManNAc residues that contained acetyl groups at O-3, with some species acetylated at O-4 and at both O-3 and O-4. A non-polar mynC mutant generated by introducing an aphA-3 kanamycin resistance cassette produced CPS with no O-acetylation. A serogroup A capsule-specific monoclonal antibody was shown to recognize the wild-type O-acetylated CPS, but not the CPS of the mynC mutant, which lacked O-acetylation. MynC was C-terminally His-tagged and overexpressed in Escherichia coli to obtain the predicted approximately 26-kDa protein. The acetyltransferase activity of purified MynC was demonstrated in vitro using [14C]acetyl-CoA. MynC O-acetylated the O-acetylated CPS of the mynC mutant and further acetylated the wild-type CPS of serogroup A meningococci, but not the CPS of serogroup B or C meningococci. Genetic complementation of the mynC mutant confirmed the function of MynC as the serogroup A CPS O-3 and O-4 acetyltransferase. MynC represents a new subclass of O-acetyltransferases that utilize acetyl-CoA to decorate the D-mannosamine capsule of N. meningitidis serogroup A.
Asunto(s)
Acetiltransferasas/química , Neisseria meningitidis/metabolismo , Polisacáridos Bacterianos/química , Acetiltransferasas/metabolismo , Anticuerpos Monoclonales/química , Cápsulas Bacterianas/química , Membrana Celular/metabolismo , Cromatografía , Citosol/metabolismo , Bases de Datos como Asunto , Relación Dosis-Respuesta a Droga , Resistencia a Medicamentos , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/metabolismo , Prueba de Complementación Genética , Hexosaminas/química , Concentración de Iones de Hidrógeno , Immunoblotting , Kanamicina/farmacología , Espectroscopía de Resonancia Magnética , Modelos Genéticos , Mutación , Nitrofenoles/química , Reacción en Cadena de la Polimerasa , Polímeros , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Fracciones Subcelulares , Factores de TiempoRESUMEN
To resolve discrepancies in slide agglutination serotyping (SAST) results from state health departments and the Centers for Disease Control and Prevention (CDC), we characterized 141 of 751 invasive Haemophilus influenzae isolates that were identified in the United States from January 1998 to December 1999 through an active, laboratory-based, surveillance program coordinated by the CDC. We found discrepancies between the results of SAST performed at state health departments and those of PCR capsule typing performed at the CDC for 56 (40%) of the isolates characterized: 54 isolates that were identified as a particular serotype by SAST were shown to be unencapsulated by PCR, and two isolates that were reported as serotypes b and f were found to be serotypes f and e, respectively, by PCR. The laboratory error most likely to affect the perceived efficacy of the conjugate H. influenzae type b (Hib) vaccine was the misidentification of isolates as serotype b: of 40 isolates identified as serotype b by SAST, 27 (68%) did not contain the correlating capsule type genes. The frequency of errors fell substantially when standardized reagents and routine quality control of SAST were used during a study involving three laboratories. An overall 94% agreement between SAST and PCR results showed that slide agglutination could be a valid and reliable method for serotyping H. influenzae if the test was performed correctly, in accordance with standardized and recommended procedures. An ongoing prospective analysis of all H. influenzae surveillance isolates associated with invasive disease in children less than 5 years old will provide more accurate national figures for the burden of invasive disease caused by Hib and other H. influenzae serotypes.
Asunto(s)
Haemophilus influenzae/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Serotipificación/métodos , Aglutinación , ADN Bacteriano/análisis , Haemophilus influenzae/clasificación , Haemophilus influenzae/genética , Humanos , Estadística como AsuntoRESUMEN
In 2000, >400 cases of disease caused by Neisseria meningitidis serogroup W135 (MenW135), the largest MenW135 outbreak reported to date, occurred worldwide among Hajj pilgrims and their contacts. To elucidate the origin of the outbreak strains and to investigate their relatedness to major clonal groups, genotypic and phenotypic subtyping was performed on 26 MenW135 outbreak-associated isolates and 50 MenW135 isolates collected worldwide from 1970 through 2000. All outbreak-associated isolates were members of a single clone of the hypervirulent electrophoretic type (ET)-37 complex, designated the "(W)ET-37 clone"; 19 additional MenW135 strains were also members of this clone, and the remaining 31 MenW135 strains were clearly distinct. The 2000 MenW135 outbreak was not caused by emergence of a new MenW135 strain but rather by expansion of the (W)ET-37 clone that has been in circulation at least since 1970; the strains most closely related to those causing the 2000 outbreak have been isolated in Algeria, Mali, and The Gambia in the 1990s.