[show abstract][hide abstract] ABSTRACT: The type IV pilus of Neisseria meningitidis is the major factor for meningococcal adhesion to host cells. In this study, we showed that a mutant of N. meningitidis pilV, a minor pilin protein, internalized less efficiently to human endothelial and epithelial cells than the wild type strain. MALDI-TOF MS and ESI-MS/MS analyses showed that PilE, the major subunit of pili, was less glycosylated at its Serine 62 residue (Ser62) in ΔpilV mutant than in the pilV(+) strain, while phosphoglycerol at PilE Ser93 and phosphocholine at PilE Ser67 were not changed. Introduction of the pglL mutation, which results in complete loss of O-linked glycosylation from Ser62, slightly reduced N. meningitidis internalization into human brain microvasocular endothelial cells, while addition of the ΔpilV mutation greatly reduced N. meningitidis internalization. The accumulation of ezrin, which is part of the cytoskeleton ERM family, was observed with pilV(+), pglL(-) and pilES62A strains, but not with the ΔpilV mutant. These results suggested that, while N. meningitidis pilin originally had an adhesive activity that was less affected by minor pilin proteins, the invasive function evolved with incorporation of the PilV protein into the pili to promote the N. meningitidis internalization into human cells.
Infection and immunity 09/2012; · 4.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: Herein we present a case of Neisseria meningitidis-related sepsis and meningitis in a 60-year-old woman. The N. meningitidis strain was identified as serogroup B and sequence type (ST)-4893 by multilocus sequence typing (MLST). The patient in this case had visited France prior to development of symptoms. No meningococcal isolate belonging to ST-4893 has been identified in Japan previously, whereas an ST-4893 strain from France has been reported in the MLST database. These results strongly suggest that this case is likely to have been imported from France.
Japanese journal of infectious diseases. 01/2011; 64(1):61-2.
[show abstract][hide abstract] ABSTRACT: Meningococcal internalization into human cells is likely to be a consequence of meningococcal adhesion to human epithelial and endothelial cells. Here, we identified three transposon mutants of Neisseria meningitidis that were primarily defective in the internalization of human brain microvascular endothelial cells (HBMEC), with insertions occurring in the gltT (a sodium-independent L-glutamate transporter) gene or its neighboring gene, NMB1964 (unknown function). NMB1964 was tentatively named gltM in this study because of the presence of a mammalian cell entry (MCE)-related domain in the deduced amino acid sequences. The null ΔgltT-ΔgltM N. meningitidis mutant was also defective in the internalization into human umbilical vein endothelial cells and the human lung carcinoma epithelial cell line A549, and the defect was suppressed by transcomplementation of the mutants with gltT(+)-gltM(+) genes. The intracellular survival of the ΔgltT-ΔgltM mutant in HBMEC was not largely different from that of the wild-type strain under our experimental conditions. Introduction of a1-bp deletion and amber or ochre mutations in gltT-gltM genes resulted in the loss of efficient internalization into HBMEC. The defect in meningococcal internalization into HBMEC and L-glutamate uptake in the ΔgltT-ΔgltM mutant were suppressed only in strains expressing both GltT and GltM proteins. The efficiency of meningococcal invasion to HBMEC decreased under L-glutamate-depleted conditions. Furthermore, ezrin, a key membrane-cytoskeleton linker, accumulated beneath colonies of the gltT(+)-gltM(+) N. meningitidis strain but not of the ΔgltT-ΔgltM mutant. These findings suggest that l-glutamate influx via the GltT-GltM L-glutamate ABC transporter serves as a cue for N. meningitidis internalization into host cells.
Infection and immunity 10/2010; 79(1):380-92. · 4.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: In the current study, we investigated the activity of lipopolysaccharide (LPS) purified from Yersinia pestis grown at either 27 degrees C or 37 degrees C (termed LPS-27 and LPS-37, respectively). LPS-27 containing hexa-acylated lipid A, similar to the LPS present in usual gram-negative bacteria, stimulated an inflammatory response in human U937 cells through Toll-like receptor 4 (TLR4). LPS-37, which did not contain hexa-acylated lipid A, exhibited strong antagonistic activity to the TLR4-mediated inflammatory response. The phagocytic activity in the cells was not affected by LPS-37. To estimate the activity of LPS in its bacterial binding form, formalin-killed bacteria (FKB) were prepared from Y. pestis cells grown at 27 degrees C or 37 degrees C (termed FKB-27 and FKB-37, respectively). FKB-27 strongly stimulated the inflammatory response. This activity was suppressed in the presence of an anti-TLR4 antibody but not an anti-TLR2 antibody. In addition, this activity was almost completely suppressed by LPS-37, indicating that the activity of FKB-27 is predominantly derived from the LPS-27 bacterial binding form. In contrast, FKB-37 showed no antagonistic activity. The results arising from the current study indicate that Y. pestis causes infection in humans without stimulating the TLR4-based defense system via bacterial binding of LPS-37, even when bacterial free LPS-37 is not released to suppress the defense system. This is in contrast to the findings for bacteria that possess agonistic LPS types, which are easily recognized by the defense system via the bacterial binding forms.
[show abstract][hide abstract] ABSTRACT: The lipooligosaccharide (LOS) of Neisseria meningitidis can be decorated with phosphoethanolamine (PEA) at the 4' position of lipid A and at the O-3 and O-6 positions of the inner core of the heptose II residue. The biological role of PEA modification in N. meningitidis remains unclear. During the course of our studies to elucidate the pathogenicity of the ST-2032 (invasive) meningococcal clonal group, disruption of lptA, the gene that encodes the PEA transferase for 4' lipid A, led to a approximately 10-fold decrease in N. meningitidis adhesion to four kinds of human endothelial and epithelial cell lines at an multiplicity of infection of 5,000. Complementation of the lptA gene in a Delta lptA mutant restored wild-type adherence. By matrix-assisted laser desorption ionization-time-of-flight mass spectrometry analysis, PEA was lost from the lipid A of the Delta lptA mutant compared to that of the wild-type strain. The effect of LptA on meningococcal adhesion was independent of other adhesins such as pili, Opc, Opa, and PilC but was inhibited by the presence of capsule. These results indicate that modification of LOS with PEA by LptA enhances meningococcal adhesion to human endothelial and epithelial cells in unencapsulated N. meningitidis.
Infection and immunity 10/2008; 76(12):5777-89. · 4.21 Impact Factor
[show abstract][hide abstract] ABSTRACT: The Japan-specific sequence type (ST) clones, as well as several major epidemic-prone clones such as ST-32, have been identified previously among Neisseria meningitidis isolates in Japan. In this study, the infectious properties of various ST clones, including the two common Japan-specific ones, were examined and compared by in vitro infection assays using human endothelial and epithelial cell lines. The known invasive clones, as well as the Japan-specific ST-2032 strains that were frequently isolated from patients, exhibited high infectious abilities in adherence and invasion. In contrast, the Japan-specific ST-2046 and ST-198 strains, both of which were frequently isolated from carriers in Japan, were less efficient in adherence and invasion. The expression of the bacterial surface molecules such as pilin, Opc, Opa and PilC, and the lipooligosaccharide structure, did not differ between disease-associated and carrier-associated isolates. These results suggest that in vitro infection assays may discriminate between disease-associated (patient-dominant) and carrier-associated (carrier-dominant) meningococcal ST clones. The ST-2032 clone showed the highest infectious activity in vitro, suggesting that it may possess some unidentified factors necessary for the infectious ability that were not present in the ST-2046 clone with the lowest infectious ability.
[show abstract][hide abstract] ABSTRACT: We report a case of meningococcemia without meningitis, which is a rare infectious disease in Japan. A 32-year-old woman was referred to our hospital with fever and joint pain. Her clinical presentation and the results of laboratory examination on admission suggested viral infection. However, her condition rapidly progressed to septic shock with fulminans purpura. Blood culture grew Neisseria meningitidis. She received antimicrobial therapy and underwent localized therapy for skin lesions. Meningococcal infection should be considered in patients who have fever along with skin rash or petechiae even when there are no signs of meningitis. In this report, we also review case reports of meningococcemia without meningitis in Japan.
Internal Medicine 02/2008; 47(17):1543-7. · 0.97 Impact Factor
[show abstract][hide abstract] ABSTRACT: We report the case of a patient with acute bronchitis caused by Neisseria meningitidis associated with HIV infection. A 36-year-old homosexual Japanese man being treated for HIV infection and reporting fever and a productive cough was found in laboratory findings to have elevated C-reactive protein but no leukocytosis. Chest radiography showed no infiltrates in either lung, but a sputum smear yielded large numbers of Gram-negative cocci, including some phagocytized by white blood cells. One day later N. meningitidis was reported to be the predominant isolate from sputum culture. The patient was diagnosed with acute bronchitis caused by N. meningitidis. The strain isolated from this patient was in serogroup B. The sequence type based on multilocus sequence typing was ST-5583, a subtype of ST-32. This strain is resistant to penicillin G in vitro, so we administered tosufloxacin at 600mg b.i.d., which showed excellent efficacy. Because of frequent sex with many men, homosexual contact was suspected as a possible route of infection. Meningococcal infections apart from meningitis rarely are surveyed epidemiologically in Japan, and the frequency of meningococcal infections in general is not clear. Vigilance is needed to identify trends in meningococcal infection, because N. meningitidis can cause acute bronchitis.
Kansenshogaku zasshi. The Journal of the Japanese Association for Infectious Diseases 12/2007; 81(6):731-5.
[show abstract][hide abstract] ABSTRACT: A real-time PCR assay with the cycling probe method was used to detect mutations at codons 83 and 87 in the DNA gyrase A subunit encoded by gyrA in Salmonella enterica serovar Typhi and Paratyphi A clinical isolates. The susceptibility estimated from the results of the gyrA mutation assay was consistent with that identified by the culture method using an E-test. This assay allows rapid screening of S. enterica serovar Typhi and Paratyphi A with reduced susceptibility to ciprofloxacin.
Microbiology and Immunology 02/2006; 50(9):707-11. · 1.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: Between September 2000 and March 2003 healthy subjects in 10 prefectures of Japan were investigated to identify carriers of Neisseria meningitidis. Twenty-five N. meningitidis strains were isolated from 5886 throat swab specimens collected from healthy persons, such as students, elderly, and foreigners. Of the 25 carriers, 9 were teenagers, 15 were in their twenties, and only one was in the fifties. The male-female ratio of the carriers was 17 to 8, showing male dominance. The serogroups of the 25 strains were B (9 strains), Y (4 strains) and non-groupable (12 strains). One of the strains was found to be deficient in gamma-glutamyl aminopeptidase activity, which is an identification marker for N. meningitidis.
Kansenshogaku zasshi. The Journal of the Japanese Association for Infectious Diseases 09/2005; 79(8):527-33.
[show abstract][hide abstract] ABSTRACT: It has been speculated that the gamma-glutamyl transpeptidase (ggt) gene is present only in Neisseria meningitidis and not among related species such as Neisseria gonorrhoeae and Neisseria lactamica, because N. meningitidis is the only bacterium with GGT activity. However, nucleotide sequences highly homologous to the meningococcal ggt gene were found in the genomes of N. gonorrhoeae isolates.
The gonococcal homologue (ggt gonococcal homologue; ggh) was analyzed. The nucleotide sequence of the ggh gene was approximately 95% identical to that of the meningococcal ggt gene. An open reading frame in the ggh gene was disrupted by an ochre mutation and frameshift mutations induced by a 7-base deletion, but the amino acid sequences deduced from the artificially corrected ggh nucleotide sequences were approximately 97% identical to that of the meningococcal ggt gene. The analyses of the sequences flanking the ggt and ggh genes revealed that both genes were localized in a common DNA region containing the fbp-ggt (or ggh)-glyA-opcA-dedA-abcZ gene cluster. The expression of the ggh RNA could be detected by dot blot, RT-PCR and primer extension analyses. Moreover, the truncated form of ggh-translational product was also found in some of the gonococcal isolates.
This study has shown that the gonococcal ggh gene is a pseudogene of the meningococcal ggt gene, which can also be designated as Psiggt. The gonococcal ggh (Psiggt) gene is the first identified bacterial pseudogene that is transcriptionally active but phenotypically silent.
[show abstract][hide abstract] ABSTRACT: Analysis of 182 Neisseria meningitidis strains isolated over the past 30 years in Japan by serogroup typing and multilocus sequence typing (MLST) was performed. The serogroups of the 182 Japanese isolates were B (103 isolates), Y (39), W135 (1) and non-groupable (39). By MLST analysis, 65 different sequence types (ST) were identified, 42 of which were not found in the MLST database as of January 2004 and seemed to be unique to Japan. Statistical analysis of the MLST results revealed that, although the Japanese isolates seemed to be genetically divergent, they were classified into six major clonal complexes and other minor complexes. Among these isolates, well-documented ST complexes found worldwide were present, such as ST-23 complex (49 isolates), ST-44 complex (41 isolates) and ST-32 complex (8 isolates). On the other hand, a new clonal complex designated ST-2046 complex (28 isolates), which has not been identified in other countries, was also found, suggesting that this clone was indigenous to Japan. Taken together, it was speculated that meningococcal isolates in Japan comprised heterogeneous clones, which were derived both from clones identified in other countries and clones unique to Japan.
Journal of Medical Microbiology 08/2004; 53(Pt 7):657-62. · 2.30 Impact Factor
[show abstract][hide abstract] ABSTRACT: We previously demonstrated that gamma-glutamyl aminopeptidase (also called gamma-glutamyl transpeptidase) (GGT) of Neisseria meningitidis is involved in the bacterial multiplication in cerebrospinal fluid. To further understand the function of meningococcal GGT, the biochemical properties were investigated in this study. The deduced amino acid sequence in N. meningitidis GGT was 37% identical to that of Escherichia coli GGT and that of Helicobacter pylori GGT, respectively, while a typical signal sequence was not found at the N-terminus of meningococcal GGT. Western blotting using rabbit antiserum against recombinant meningococcal GGT protein demonstrated that the meningococcal GGT is processed into two subunits in N. meningitidis at the conserved amino acid, threonine 427. The experiments on subcellular fractionation suggested that the majority of meningococcal GGT is associated with inner membrane facing to the cytoplasmic side. This cell localization might be unique for N. meningitidis compared to other GGTs.
[show abstract][hide abstract] ABSTRACT: The growth of a gamma-glutamyl aminopeptidase (GGT)-deficient Neisseria meningitidis strain was much slower than that of the parent strain in rat cerebrospinal fluid (CSF) and in a synthetic CSF-mimicking medium, and the growth failure was suppressed by the addition of cysteine. These results suggested that, in the environment of cysteine shortage, meningococcal GGT provided an advantage for meningococcal multiplication by supplying cysteine from environmental gamma-glutamyl-cysteinyl peptides.
Journal of Bacteriology 02/2004; 186(1):244-7. · 3.19 Impact Factor
[show abstract][hide abstract] ABSTRACT: Detection of gamma-glutamyl transpeptidase (GGT; ggt ) activity is one of the useful methods for a specific identification of Neisseria meningitidis. However, we previously happened to isolate a ggt -deficient N. meningitidis strain (NIID113) from a healthy carrier. In this study, in order to re-examine the reliability of the marker, we again investigated the GGT activity of 245 N. meningitidis human isolates and identified two other GGT-defective N. meningitidis isolates besides NIID113. The isolation frequency (1.2%) of ggt mutants among human isolates strongly confirmed the 98.8% reliability of GGT activity as the identification marker for N. meningitidis.
Microbiology and Immunology 02/2004; 48(6):485-7. · 1.55 Impact Factor
[show abstract][hide abstract] ABSTRACT: A multiplex polymerase chain reaction (PCR) assay was developed for the identification of Salmonella enterica serovar Typhimurium. Three sets of primers were designed for detecting O4, H:i, and H:1,2 antigen genes from the antigen-specific genes rfbJ, fliC, and fljB, respectively. These were evaluated in a multiplex PCR assay by using DNAs from S. enterica serovar Typhimurium, 15 other Salmonella serovars, and 8 non-Salmonella enteric pathogens. Multiplex PCR proved to be capable of identifying S. enterica serovar Typhimurium specifically and differentiating it from other Salmonella serovars in addition to non-Salmonella enteric pathogens. Thus, this multiplex PCR assay can be practically applied to the identification of S. enterica serovar Typhimurium.
Japanese journal of infectious diseases 09/2003; 56(4):151-5. · 1.51 Impact Factor