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1.
Antimicrob Agents Chemother ; 66(6): e0238321, 2022 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-35652645

RESUMO

Invasive meningococcal disease (IMD) due to serogroup Y Neisseria meningitidis (NmY) is rare in China; recently, an invasive NmY isolate, Nm512, was discovered in Shanghai with decreased susceptibility to penicillin (PenNS). Here, we investigated the epidemiology of NmY isolates in Shanghai and explored the potential commensal Neisseria lactamica donor of the PenNS NmY isolate. A total of 491 N. meningitidis and 724 commensal Neisseria spp. isolates were collected. Eleven NmY isolates were discovered from IMD (n = 1) and carriers (n = 10), including two PenNS isolates with five-key-mutation-harboring (F504L-A510V-I515V-H541N-I566V) penA genes. Five of the eight ST-175 complex (CC175) isolates had a genotype [Y:P1.5-1,2-2:F5-8:ST-175(CC175)] identical to that of the predominant invasive clone found in South Africa. Only one invasive NmY CC23 isolate (Nm512) was discovered; this isolate carried a novel PenNS penA832 allele, which was identified in commensal N. lactamica isolates locally. Recombination analysis and transformation of the penA allele highlighted that N. meningitidis Nm512 may acquire resistance from its commensal donor; this was supported by the similar distribution of transformation-required DNA uptake sequence variants and the highly cognate receptor ComP between N. meningitidis and N. lactamica. In 2,309 NmY CC23 genomes from the PubMLST database, isolates with key-mutation-harboring penA genes comprised 12% and have been increasing since the 1990s, accompanied by recruitment of the blaROB-1 and/or quinolone resistance allele. Moreover, penA22 was predominant among genomes without key mutations in penA. These results strongly suggest that Nm512 is a descendant of the penA22-harboring CC23 isolate from Europe and acquired its penicillin resistance locally from commensal N. lactamica species by natural transformation.


Assuntos
Infecções Meningocócicas , Neisseria lactamica , Neisseria meningitidis , China/epidemiologia , Humanos , Neisseria lactamica/genética , Neisseria meningitidis/genética , Neisseria meningitidis Sorogrupo Y , Resistência às Penicilinas/genética , Sorogrupo
2.
BMJ Open ; 12(5): e056081, 2022 05 18.
Artigo em Inglês | MEDLINE | ID: mdl-35584870

RESUMO

INTRODUCTION: Infant upper respiratory microbiota are derived partly from the maternal respiratory tract, and certain microbiota are associated with altered risk of infections and respiratory disease. Neisseria lactamica is a common pharyngeal commensal in young children and is associated with reduced carriage and invasive disease by Neisseria meningitidis. Nasal inoculation with N. lactamica safely and reproducibly reduces N. meningitidis colonisation in healthy adults. We propose nasal inoculation of pregnant women with N. lactamica, to establish if neonatal pharyngeal colonisation occurs after birth, and to characterise microbiome evolution in mother-infant pairs over 1 month post partum. METHODS AND ANALYSIS: 20 healthy pregnant women will receive nasal inoculation with N. lactamica (wild type strain Y92-1009) at 36-38 weeks gestation. Upper respiratory samples, as well as optional breastmilk, umbilical cord blood and infant venous blood samples, will be collected from mother-infant pairs over 1 month post partum. We will assess safety, N. lactamica colonisation (by targeted PCR) and longitudinal microevolution (by whole genome sequencing), and microbiome evolution (by 16S rRNA gene sequencing). ETHICS AND DISSEMINATION: This study has been approved by the London Central Research Ethics Committee (21/PR/0373). Findings will be published in peer-reviewed open-access journals as soon as possible. TRIAL REGISTRATION NUMBER: NCT04784845.


Assuntos
Microbiota , Neisseria lactamica , Neisseria meningitidis , Adulto , Criança , Pré-Escolar , Feminino , Humanos , Lactente , Recém-Nascido , Microbiota/genética , Mães , Neisseria lactamica/genética , Faringe , Projetos Piloto , Gravidez , RNA Ribossômico 16S
3.
Mol Cell ; 82(4): 852-867.e5, 2022 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-35051351

RESUMO

Leading CRISPR-Cas technologies employ Cas9 and Cas12 enzymes that generate RNA-guided dsDNA breaks. Yet, the most abundant microbial adaptive immune systems, Type I CRISPRs, are under-exploited for eukaryotic applications. Here, we report the adoption of a minimal CRISPR-Cas3 from Neisseria lactamica (Nla) type I-C system to create targeted large deletions in the human genome. RNP delivery of its processive Cas3 nuclease and target recognition complex Cascade can confer ∼95% editing efficiency. Unexpectedly, NlaCascade assembly in bacteria requires internal translation of a hidden component Cas11 from within the cas8 gene. Furthermore, expressing a separately encoded NlaCas11 is the key to enable plasmid- and mRNA-based editing in human cells. Finally, we demonstrate that supplying cas11 is a universal strategy to systematically implement divergent I-C, I-D, and I-B CRISPR-Cas3 editors with compact sizes, distinct PAM preferences, and guide orthogonality. These findings greatly expand our ability to engineer long-range genome edits.


Assuntos
Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/genética , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Deleção de Genes , Edição de Genes , Genoma Humano , Neisseria lactamica/genética , Proteínas de Bactérias/metabolismo , Proteínas Associadas a CRISPR/metabolismo , Células HEK293 , Células HeLa , Humanos , Neisseria lactamica/enzimologia , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo
4.
Methods Mol Biol ; 2414: 387-404, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34784048

RESUMO

Neisseria lactamica is a nonpathogenic commensal of the human upper respiratory tract that has been associated with protection against N. meningitidis colonization and disease. We have previously utilized the N. lactamica controlled human infection model to investigate the protective effect of N. lactamica colonization on N. meningitidis colonization, the nature of cross-reactive immune responses mounted toward N. meningitidis following N. lactamica colonization, and the microevolution of N. lactamica over a 5-month colonization period. More recently, we have assessed the possibility of utilizing genetically modified strains of N. lactamica to enable use of the commensal as a vehicle for prolonged exposure of the nasopharynx of humans to antigens of interest, expressed in carried organisms. A controlled infection with N. lactamica expressing the meningococcal antigen NadA has been executed and the results demonstrate that this strategy is effective at generating immune responses to the target antigen. Throughout this chapter, we outline in a step-by-step manner the methodologies utilized when performing controlled human infection with N. lactamica including procedures relating to: (1) the dilution of N. lactamica stock vials to derive intranasal inocula, (2) the delivery of intranasal inocula to human volunteers, (3) the determination of N. lactamica colonization status following intranasal inoculation using oropharyngeal swabbing and nasal wash sampling, (4) the microbiological procedures utilized to identify N. lactamica colonization among study volunteers, and (5) the identification of N. lactamica colonies as strain Y92-1009 using polymerase chain reaction.


Assuntos
Neisseria lactamica , Antígenos , Reações Cruzadas , Humanos , Nasofaringe , Neisseria meningitidis , Infecções por Neisseriaceae
5.
Sci Transl Med ; 13(601)2021 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-34233953

RESUMO

The human nasopharynx contains a stable microbial ecosystem of commensal and potentially pathogenic bacteria, which can elicit protective primary and secondary immune responses. Experimental intranasal infection of human adults with the commensal Neisseria lactamica produced safe, sustained pharyngeal colonization. This has potential utility as a vehicle for sustained release of antigen to the human mucosa, but commensals in general are thought to be immunologically tolerated. Here, we show that engineered N. lactamica, chromosomally transformed to express a heterologous vaccine antigen, safely induces systemic, antigen-specific immune responses during carriage in humans. When the N. lactamica expressing the meningococcal antigen Neisseria Adhesin A (NadA) was inoculated intranasally into human volunteers, all colonized participants carried the bacteria asymptomatically for at least 28 days, with most (86%) still carrying the bacteria at 90 days. Compared to an otherwise isogenic but phenotypically wild-type strain, colonization with NadA-expressing N. lactamica generated NadA-specific immunoglobulin G (IgG)- and IgA-secreting plasma cells within 14 days of colonization and NadA-specific IgG memory B cells within 28 days of colonization. NadA-specific IgG memory B cells were detected in peripheral blood of colonized participants for at least 90 days. Over the same period, there was seroconversion against NadA and generation of serum bactericidal antibody activity against a NadA-expressing meningococcus. The controlled infection was safe, and there was no transmission to adult bedroom sharers during the 90-day period. Genetically modified N. lactamica could therefore be used to generate beneficial immune responses to heterologous antigens during sustained pharyngeal carriage.


Assuntos
Vacinas Meningocócicas , Neisseria lactamica , Adulto , Anticorpos Antibacterianos , Antígenos Heterófilos , Ecossistema , Humanos , Memória Imunológica
6.
J Infect Chemother ; 27(1): 65-69, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32873462

RESUMO

INTRODUCTION: Neisseria lactamica is a commensal bacterium of the upper respiratory tract in humans and is closely related to Neisseria meningitidis. N. lactamica colonization may contribute to preventing N. meningitidis colonization and invasive meningococcal disease. However, the transference of antimicrobial resistance genes from N. lactamica to N. meningitidis has been reported. METHODS: In this study, we aimed to identify N. lactamica using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and performed multilocus sequence typing of seven N. lactamica strains isolated from Japanese children. We also analyzed the antimicrobial susceptibility of these strains and the mutations in their antimicrobial resistance genes (penA, gyrA, and parC). RESULTS: All the N. lactamica strains could be identified using MALDI-TOF MS. All strains were of different sequence types (STs), including five new STs. Five strains had intermediate susceptibility, two were resistant to ampicillin, and all had five out of the five known PBP2 mutations. Six strains were resistant to levofloxacin. Among the quinolone-resistant strains, three had GyrA mutations, and three had both ParC and GyrA mutations. CONCLUSIONS: N. lactamica STs may vary in Japanese children, and penicillin- and quinolone-resistant strains may be prevalent. We should pay attention not only to the drug resistance of N. meningitidis but also to the drug susceptibility of N. lactamica whose drug-resistance genes may transfer to N. meningitidis.


Assuntos
Infecções Meningocócicas , Neisseria lactamica , Neisseria meningitidis , Criança , Humanos , Japão/epidemiologia , Neisseria lactamica/genética , Neisseria meningitidis/genética , Sistema Respiratório
7.
Genome Biol Evol ; 12(2): 3938-3950, 2020 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-32031617

RESUMO

Neisseria spp. possess four genogroups of filamentous prophages, termed Nf1 to 4. A filamentous bacteriophage from the Nf1 genogroup termed meningococcal disease-associated phage (MDA φ) is associated with clonal complexes of Neisseria meningitidis that cause invasive meningococcal disease. Recently, we recovered an isolate of Neisseria gonorrhoeae (ExNg63) from a rare case of gonococcal meningitis, and found that it possessed a region with 90% similarity to Nf1 prophages, specifically, the meningococcal MDA φ. This led to the hypothesis that the Nf1 prophage may be more widely distributed amongst the genus Neisseria. An analysis of 92 reference genomes revealed the presence of intact Nf1 prophages in the commensal species, Neisseria lactamica and Neisseria cinerea in addition to the pathogen N. gonorrhoeae. In N. gonorrhoeae, Nf1 prophages had a restricted distribution but were present in all representatives of MLST ST1918. Of the 160 phage integration sites identified, only one common insertion site was found between one isolate of N. gonorrhoeae and N. meningitidis. There was an absence of any obvious conservation of the receptor for prophage entry, PilE, suggesting that the phage may have been obtained by natural transformation. An examination of the restriction modification systems and mutated mismatch repair systems with prophage presence suggested that there was no obvious preference for these hosts. A timed phylogeny inferred that N. meningitidis was the donor of the Nf1 prophages in N. lactamica and N. gonorrhoeae. Further work is required to determine whether Nf1 prophages are active and can act as accessory colonization factors in these species.


Assuntos
Infecções Meningocócicas/virologia , Neisseria/virologia , Prófagos/genética , Transferência Genética Horizontal/genética , Transferência Genética Horizontal/fisiologia , Inovirus/genética , Neisseria cinerea/virologia , Neisseria gonorrhoeae/virologia , Neisseria lactamica/virologia , Filogenia
8.
J Infect ; 80(1): 61-68, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31586462

RESUMO

OBJECTIVE: Neisseria lactamica has an important influence on carriage and antimicrobial susceptibility of N. meningitidis, a major pathogen of septicemia and meningitis. In China, quinolone resistance is highly prevalent in N. meningitidis but unknown in N. lactamica. This study investigates the carriage rate, sequence type, and ciprofloxacin resistance of N. lactamica in children in China. METHODS: During 2014-2016, throat swabs were collected from 2,239 children in Shanghai. The ciprofloxacin minimum inhibitory concentrations of the isolates were determined by the agar dilution method. RESULTS: The overall carriage rate of N. lactamica was higher (8.9%) than that of N. meningitidis (0.9%) and peaked at two years (37.1%). The resistance frequency of N. lactamica to ciprofloxacin was 98.5% (197/200). There were 65 sequence types (STs). Clonal complex (cc) 640 (45.5%) dominated, while ST-14031 was predominant (37%, 74/200). All isolates possessed a GyrA mutation; 17 isolates (8.5%) harbored additionally a ParC mutation. Assigned to 39 different alleles, the gyrA sequences from these N. lactamica isolates formed an N. lactamica cluster, which also included eight alleles from N. meningitidis. CONCLUSION: The N. lactamica isolates in China showed distinct characteristics with lower genetic diversity and a much higher prevalence of quinolone resistance than in other countries.


Assuntos
Neisseria lactamica , Neisseria meningitidis , Quinolonas , Criança , China/epidemiologia , Farmacorresistência Bacteriana , Humanos , Testes de Sensibilidade Microbiana , Neisseria meningitidis/genética , Prevalência , Quinolonas/farmacologia
9.
BMJ Open ; 9(4): e026544, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31048443

RESUMO

INTRODUCTION: Neisseria lactamica is a commensal organism found in the human nasopharynx and is closely related to the pathogen N. meningitidis (meningococcus). Carriage of N. lactamica is associated with reduced meningococcal carriage and disease. We summarise an ethically approved protocol for an experimental human challenge study using a genetically modified strain of N. lactamica that expresses the meningococcal antigen NadA. We aim to develop a model to study the role of specific bacterial antigens in nasopharyngeal carriage and immunity, to evaluate vaccines for their efficacy in preventing colonisation and to provide a proof of principle for the development of bacterial medicines. METHODS AND ANALYSIS: Healthy adult volunteers aged 18-45 years will receive an intranasal inoculation of either the NadA containing strain of N. lactamica or a genetically modified, but wild-type equivalent control strain. These challenge volunteers will be admitted for 4.5 days observation following inoculation and will then be discharged with strict infection control rules. Bedroom contacts of the challenge volunteers will also be enrolled as contact volunteers. Safety, colonisation, shedding, transmission and immunogenicity will be assessed over 90 days after which carriage will be terminated with antibiotic eradication therapy. ETHICS AND DISSEMINATION: This study has been approved by the Department for Environment, Food and Rural Affairs and South Central Oxford A Research Ethics Committee (reference: 18/SC/0133). Findings will be published in peer-reviewed open-access journals as soon as possible. TRIAL REGISTRATION NUMBER: NCT03630250; Pre-results.


Assuntos
Adesinas Bacterianas/biossíntese , Adesinas Bacterianas/imunologia , Antígenos/imunologia , Vacinas Meningocócicas/imunologia , Microrganismos Geneticamente Modificados , Neisseria lactamica/genética , Neisseria lactamica/metabolismo , Neisseria meningitidis/imunologia , Projetos de Pesquisa , Adolescente , Adulto , Pesquisa Biomédica , Humanos , Pessoa de Meia-Idade , Estudos Prospectivos , Adulto Jovem
10.
J Mol Biol ; 431(11): 2082-2094, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-30995450

RESUMO

Specificity engineering is challenging and particularly difficult for enzymes that have the catalytic machinery and specificity determinants in close proximity. Restriction endonucleases have been used as a paradigm for protein engineering, but successful cases are rare. Here, we present the results of a directed evolution approach to the engineering of a dimeric, blunt end cutting restriction enzyme NlaIV (GGN/NCC). Based on the remote similarity to EcoRV endonuclease, regions for random mutagenesis and in vitro evolution were chosen. The obtained variants cleaved target sites with an up to 100-fold kcat/KM preference for AT or TA (GGW/WCC) over GC or CG (GGS/SCC) in the central dinucleotide step, compared to the only ~17-fold preference of the wild-type enzyme. To understand the basis of the increased specificity, we determined the crystal structure of NlaIV. Despite the presence of DNA in the crystallization mix, the enzyme crystallized in the free form. We therefore constructed a computational model of the NlaIV-DNA complex. According to the model, the mutagenesis of the regions that were in the proximity of DNA did not lead to the desired specificity change, which was instead conveyed in an indirect manner by substitutions in the more distant regions.


Assuntos
Proteínas de Bactérias/química , Desoxirribonucleases de Sítio Específico do Tipo II/química , Neisseria lactamica/química , Proteínas de Bactérias/genética , Cristalografia por Raios X , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Neisseria lactamica/genética , Infecções por Neisseriaceae/microbiologia , Conformação Proteica , Especificidade por Substrato
11.
Nat Commun ; 9(1): 4753, 2018 11 12.
Artigo em Inglês | MEDLINE | ID: mdl-30420631

RESUMO

Neisseria lactamica is a harmless coloniser of the infant respiratory tract, and has a mutually-excluding relationship with the pathogen Neisseria meningitidis. Here we report controlled human infection with genomically-defined N. lactamica and subsequent bacterial microevolution during 26 weeks of colonisation. We find that most mutations that occur during nasopharyngeal carriage are transient indels within repetitive tracts of putative phase-variable loci associated with host-microbe interactions (pgl and lgt) and iron acquisition (fetA promotor and hpuA). Recurrent polymorphisms occurred in genes associated with energy metabolism (nuoN, rssA) and the CRISPR-associated cas1. A gene encoding a large hypothetical protein was often mutated in 27% of the subjects. In volunteers who were naturally co-colonised with meningococci, recombination altered allelic identity in N. lactamica to resemble meningococcal alleles, including loci associated with metabolism, outer membrane proteins and immune response activators. Our results suggest that phase variable genes are often mutated during carriage-associated microevolution.


Assuntos
Nasofaringe/microbiologia , Neisseria lactamica/crescimento & desenvolvimento , Infecções por Neisseriaceae/microbiologia , Portador Sadio , Contagem de Colônia Microbiana , Genes Bacterianos , Humanos , Mutação/genética , Taxa de Mutação , Neisseria lactamica/genética , Neisseria lactamica/isolamento & purificação , Recombinação Genética/genética
12.
Int J Med Microbiol ; 308(4): 454-458, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29605532

RESUMO

Neisseria lactamica is a nonpathogenic commensal bacterium that is potentially associated with the development of natural immunity against N. meningitidis. However, the genetic variation present in natural populations of N. lactamica has not been fully investigated. To better understand its epidemiology and genetic variation, we studied N. lactamica carriage in 1200 students aged 11-19 years old in Salvador, Brazil. The carriage prevalence was 4.5% (54/1200), with no statistical difference among sex and age, although we observed a trend towards higher carriage prevalence among 11-year-old individuals. Whole genome sequence analysis revealed a high genetic diversity among the isolates, with the presence of 32 different STs, 28 (87.5%) of which were new. A total of 21/50 (42%) isolates belonged to three different clonal complexes. While none of the isolates contained nadA or fHpb alleles, we detected 21 FetA variants, 20 NhbA variants and two variants of PorB. The data provide detailed information on circulating N. lactamica isolates in adolescents in Brazil and are complementary to studies in other countries.


Assuntos
Portador Sadio/epidemiologia , Portador Sadio/microbiologia , Infecções Meningocócicas/epidemiologia , Infecções Meningocócicas/microbiologia , Neisseria lactamica/genética , Adolescente , Alelos , Proteínas da Membrana Bacteriana Externa/genética , Brasil/epidemiologia , El Salvador/epidemiologia , Feminino , Genótipo , Humanos , Masculino , Epidemiologia Molecular , Neisseria lactamica/isolamento & purificação , Neisseria meningitidis/genética , Polimorfismo de Nucleotídeo Único , Porinas/genética , Estudantes , Sequenciamento Completo do Genoma , Adulto Jovem
13.
J Clin Microbiol ; 54(12): 2891-2899, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27629899

RESUMO

A carriage study was undertaken (n = 112) to ascertain the prevalence of Neisseria spp. following the eighth case of invasive meningococcal disease in young children (5 to 46 months) and members of a large extended indigenous ethnic minority Traveller family (n = 123), typically associated with high-occupancy living conditions. Nested multilocus sequence typing (MLST) was employed for case specimen extracts. Isolates were genome sequenced and then were assembled de novo and deposited into the Bacterial Isolate Genome Sequencing Database (BIGSdb). This facilitated an expanded MLST approach utilizing large numbers of loci for isolate characterization and discrimination. A rare sequence type, ST-6697, predominated in disease specimens and isolates that were carried (n = 8/14), persisting for at least 44 months, likely driven by the high population density of houses (n = 67/112) and trailers (n = 45/112). Carriage for Neisseria meningitidis (P < 0.05) and Neisseria lactamica (P < 0.002) (2-sided Fisher's exact test) was more likely in the smaller, more densely populated trailers. Meningococcal carriage was highest in 24- to 39-year-olds (45%, n = 9/20). Evidence of horizontal gene transfer (HGT) was observed in four individuals cocolonized by Neisseria lactamica and Neisseria meningitidis One HGT event resulted in the acquisition of 26 consecutive N. lactamica alleles. This study demonstrates how housing density can drive meningococcal transmission and carriage, which likely facilitated the persistence of ST-6697 and prolonged the outbreak. Whole-genome MLST effectively distinguished between highly similar outbreak strain isolates, including those isolated from person-to-person transmission, and also highlighted how a few HGT events can distort the true phylogenetic relationship between highly similar clonal isolates.


Assuntos
Surtos de Doenças , Infecções Meningocócicas/epidemiologia , Infecções Meningocócicas/transmissão , Neisseria lactamica/isolamento & purificação , Neisseria meningitidis Sorogrupo B/isolamento & purificação , Densidade Demográfica , Adolescente , Adulto , Criança , Pré-Escolar , Transferência Genética Horizontal/genética , Genoma Bacteriano/genética , Humanos , Lactente , Infecções Meningocócicas/microbiologia , Tipagem de Sequências Multilocus , Neisseria lactamica/genética , Neisseria meningitidis Sorogrupo B/genética , Adulto Jovem
14.
Fa Yi Xue Za Zhi ; 32(1): 1-6, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-27301077

RESUMO

UNLABELLED: Abstract: OBJECTIVE: To investigate the bacterial succession on rat carcasses and to evaluate the use of bacterial succession for postmortem interval (PMI) estimation. METHODS: Adult female SD rat remains were placed in carton boxes. The bacterial colonization of circumocular skin, mouth and vagina was collected to be identified using culture-dependent biochemical methods. The changes in community composition were regularly documented. RESULTS: The bacterial succession in three habitats showed that Staphylococcus and Neisseria were predominated in early PMI, especially Staphylococcus aureus and Neisseria lactamica in 6 hours after death. Lactobacillus casei developed on the 3-4 days regularly, and kept stable at a certain level in late PMI. CONCLUSION: The involvement of normal and putrefactive bacteria in three body habitats of rat remains can be used for PMI estimation.


Assuntos
Medicina Legal/métodos , Neisseria lactamica , Mudanças Depois da Morte , Staphylococcus aureus , Animais , Autopsia , Cadáver , Morte , Ratos , Ratos Sprague-Dawley , Fatores de Tempo
15.
Vaccine ; 34(14): 1704-11, 2016 Mar 29.
Artigo em Inglês | MEDLINE | ID: mdl-26876441

RESUMO

BACKGROUND: Neisseria meningitidis are common colonizers of the human nasopharynx. In some circumstances, N. meningitidis becomes an opportunistic pathogen that invades tissues and causes meningitis. While a vaccine against a number of serogroups has been in effective use for many years, a vaccine against N. meningitidis group B has not yet been universally adopted. Bacterial heat shock protein complex (HSPC) vaccines comprise bacterial HSPs, purified with their chaperoned protein cargo. HSPC vaccines use the intrinsic adjuvant activity of their HSP, thought to act via Toll-like receptors (TLR), to induce an immune response against their cargo antigens. This study evaluated HSPC vaccines from N. meningitidis and the closely related commensal N. lactamica. RESULTS: The protein composition of N. lactamica and N. meningitidis HSPCs were similar. Using human HEK293 cells we found that both HSPCs can induce an innate immune response via activation of TLR2. However, stimulation of TLR2 or TLR4 deficient murine splenocytes revealed that HSPCs can activate an innate immune response via multiple receptors. Vaccination of wildtype mice with the Neisseria HSPC induced a strong antibody response and a Th1-restricted T helper response. However, vaccination of mice deficient in the major TLR adaptor protein, MyD88, revealed that while the Th1 response to Neisseria HSPC requires MyD88, these vaccines unexpectedly induced an antigen-specific antibody response via a MyD88-independent mechanism. CONCLUSIONS: N. lactamica and N. meningitidis HSPC vaccines both have potential utility for immunising against neisserial meningitis without the requirement for an exogenous adjuvant. The mode of action of these vaccines is highly complex, with HSPCs inducing immune responses via both MyD88-dependent and -independent mechanisms. In particular, these HSPC vaccines induced an antibody response without detectable T cell help.


Assuntos
Anticorpos Antibacterianos/sangue , Vacinas Bacterianas/imunologia , Proteínas de Choque Térmico/imunologia , Imunidade Inata , Neisseria meningitidis , Animais , Proteínas de Bactérias/imunologia , Citocinas/imunologia , Células HEK293 , Humanos , Imunidade Humoral , Imunoglobulina G/sangue , Meningite Meningocócica/prevenção & controle , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/imunologia , Neisseria lactamica , Proteoma , Baço/imunologia , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo
16.
Int J Med Microbiol ; 305(7): 729-35, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26321007

RESUMO

In meningococci, reduced penicillin susceptibility is associated with five specific mutations in the transpeptidase region of penicillin binding protein 2 (PBP2). We showed that the same set of mutations was present in 64 of 123 Neisseria lactamica strains obtained from a carriage study (MIC range: 0.125-2.0mg/L). The PBP2 encoding penA alleles in these strains were genetically similar to those found in intermediate resistant meningococci suggesting frequent interspecies genetic exchange. Fifty-six N. lactamica isolates with mostly lower penicillin MICs (range: 0.064-0.38mg/L) exhibited only three of the five mutations. The corresponding penA alleles were unique to N. lactamica and formed a distinct genetic clade. PenA alleles with no mutations on the other hand were unique to meningococci. Under penicillin selective pressure, genetic transformation of N. lactamica penA alleles in meningococci was only possible for alleles encoding five mutations, but not for those encoding three mutations; the transfer resulted in MICs comparable to those of meningococci harboring penA alleles that encoded PBP2 with five mutations, but considerably lower than those of the corresponding N. lactamica donor strains. Due to a transformation barrier the complete N. lactamica penA could not be transformed into N. meningitidis. In summary, penicillin MICs in N. lactamica were associated with the number of mutations in the transpeptidase region of PBP2. Evidence for interspecific genetic transfer was only observed for penA alleles associated with higher MICs, suggesting that alleles encoding only three mutations in the transpeptidase region are biologically not effective in N. meningitidis. Factors other than PBP2 seem to be responsible for the high levels of penicillin resistance in N. lactamica. A reduction of penicillin susceptibility in N. meningitidis by horizontal gene transfer from N. lactamica is unlikely to happen.


Assuntos
Neisseria lactamica/efeitos dos fármacos , Neisseria meningitidis/efeitos dos fármacos , Resistência às Penicilinas , Proteínas de Ligação às Penicilinas/genética , Penicilinas/farmacologia , Polimorfismo Genético , Substituição de Aminoácidos , Transferência Genética Horizontal , Genótipo , Alemanha , Humanos , Testes de Sensibilidade Microbiana , Mutação de Sentido Incorreto , Neisseria lactamica/genética , Neisseria lactamica/isolamento & purificação , Neisseria meningitidis/genética , Neisseria meningitidis/isolamento & purificação , Infecções por Neisseriaceae/microbiologia , Transformação Bacteriana
17.
Clin Infect Dis ; 60(10): 1512-20, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25814628

RESUMO

BACKGROUND: Herd protection by meningococcal vaccines is conferred by population-level reduction of meningococcal nasopharyngeal colonization. Given the inverse epidemiological association between colonization by commensal Neisseria lactamica and meningococcal disease, we investigated whether controlled infection of human volunteers with N. lactamica prevents colonization by Neisseria meningitidis. METHODS: In a block-randomized human challenge study, 310 university students were inoculated with 10(4) colony-forming units of N. lactamica or were sham-inoculated, and carriage was monitored for 26 weeks, after which all participants were reinoculated with N. lactamica and resampled 2 weeks later. RESULTS: At baseline, natural N. meningitidis carriage in the control group was 22.4% (36/161), which increased to 33.6% (48/143) by week 26. Two weeks after inoculation of N. lactamica, 33.6% (48/143) of the challenge group became colonized with N. lactamica. In this group, meningococcal carriage reduced from 24.2% (36/149) at inoculation to 14.7% (21/143) 2 weeks after inoculation (-9.5%; P = .006). The inhibition of meningococcal carriage was only observed in carriers of N. lactamica, was due both to displacement of existing meningococci and to inhibition of new acquisition, and persisted over at least 16 weeks. Crossover inoculation of controls with N. lactamica replicated the result. Genome sequencing showed that inhibition affected multiple meningococcal sequence types. CONCLUSIONS: The inhibition of meningococcal carriage by N. lactamica is even more potent than after glycoconjugate meningococcal vaccination. Neisseria lactamica or its components could be a novel bacterial medicine to suppress meningococcal outbreaks. This observation explains the epidemiological observation of natural immunity conferred by carriage of N. lactamica. CLINICAL TRIALS REGISTRATION: NCT02249598.


Assuntos
Portador Sadio/microbiologia , Portador Sadio/prevenção & controle , Infecções Meningocócicas/microbiologia , Infecções Meningocócicas/prevenção & controle , Neisseria lactamica/crescimento & desenvolvimento , Neisseria meningitidis/isolamento & purificação , Probióticos/administração & dosagem , Adolescente , Adulto , Antibiose , Feminino , Humanos , Masculino , Estudos Prospectivos , Resultado do Tratamento , Adulto Jovem
18.
Gene ; 559(2): 177-83, 2015 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-25644076

RESUMO

Mitochondria are eukaryotic organelles which contain the own genetic material and evolved from free-living Eubacteria, namely hydrogen-producing Alphaproteobacteria. Since 1965, biologists provided, by research at molecular level, evidence for the prokaryotic origins of mitochondria. However, determining the precise origins of mitochondria is challenging due to inherent difficulties in phylogenetically reconstructing ancient evolutionary events. The use of new tools to evidence the prokaryotic origin of mitochondria could be useful to gain an insight into the bacterial endosymbiotic event that resulted in the permanent acquisition of bacteria, from the ancestral cell, that through time were transformed into mitochondria. Electron microscopy has shown that both proteobacterial and yeast cells during their growth in the presence of increasing amount of tellurite resulted in dose-dependent blackening of the culture due to elemental tellurium (Te(0)) that formed large deposits either along the proteobacterial membrane or along the yeast cell wall and mitochondria. Since the mitochondrial inner membrane composition is similar to that of proteobacterial membrane, in the present work we evidenced the black tellurium deposits on both, cell wall and mitochondria of ρ(+) and respiratory deficient ρ(-) mutants of yeast. A possible role of tellurite in studying the evolutionary origins of mitochondria will be discussed.


Assuntos
Telúrio/metabolismo , Evolução Biológica , DNA Mitocondrial/metabolismo , Escherichia coli/metabolismo , Escherichia coli/ultraestrutura , Halobacterium salinarum/metabolismo , Halobacterium salinarum/ultraestrutura , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/metabolismo , Neisseria lactamica/metabolismo , Neisseria lactamica/ultraestrutura , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Sphingomonas/metabolismo , Sphingomonas/ultraestrutura
19.
Mol Microbiol ; 93(2): 346-55, 2014 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-24910087

RESUMO

Neisseria meningitidis is an important human pathogen that is capable of killing within hours of infection. Its normal habitat is the nasopharynx of adult humans. Here we identify a genomic island (the prp gene cluster) in N. meningitidis that enables this species to utilize propionic acid as a supplementary carbon source during growth, particularly under nutrient poor growth conditions. The prp gene cluster encodes enzymes for a methylcitrate cycle. Novel aspects of the methylcitrate cycle in N. meningitidis include a propionate kinase which was purified and characterized, and a putative propionate transporter. This genomic island is absent from the close relative of N. meningitidis, the commensal Neisseria lactamica, which chiefly colonizes infants not adults. We reason that the possession of the prp genes provides a metabolic advantage to N. meningitidis in the adult oral cavity, which is rich in propionic acid-generating bacteria. Data from classical microbiological and sequence-based microbiome studies provide several lines of supporting evidence that N. meningitidis colonization is correlated with propionic acid generating bacteria, with a strong correlation between prp-containing Neisseria and propionic acid generating bacteria from the genus Porphyromonas, and that this may explain adolescent/adult colonization by N. meningitidis.


Assuntos
Regulação Bacteriana da Expressão Gênica , Ilhas Genômicas , Nasofaringe/microbiologia , Neisseria meningitidis/genética , Neisseria meningitidis/metabolismo , Propionatos/metabolismo , Adolescente , Adulto , Carbono/metabolismo , Feminino , Genoma Bacteriano , Humanos , Masculino , Microbiota , Família Multigênica , Neisseria lactamica/genética , Neisseria meningitidis/crescimento & desenvolvimento , Neisseria meningitidis/isolamento & purificação , Porphyromonas/metabolismo
20.
Clin Vaccine Immunol ; 20(9): 1360-9, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23803905

RESUMO

The poor immunogenicity of the meningococcal serogroup B (MenB) capsule has led to the development of vaccines targeting subcapsular antigens, in particular the immunodominant and diverse outer membrane porin, PorA. These vaccines are largely strain specific; however, they offer limited protection against the diverse MenB-associated diseases observed in many industrialized nations. To broaden the scope of its protection, the multicomponent vaccine (4CMenB) incorporates a PorA-containing outer membrane vesicle (OMV) alongside relatively conserved recombinant protein components, including factor H-binding protein (fHbp), Neisseria adhesin A (NadA), and neisserial heparin-binding antigen (NHBA). The expression of PorA is unique to meningococci (Neisseria meningitidis); however, many subcapsular antigens are shared with nonpathogenic members of the genus Neisseria that also inhabit the nasopharynx. These organisms may elicit cross-protective immunity against meningococci and/or occupy a niche that might otherwise accommodate pathogens. The potential for 4CMenB responses to impact such species (and vice versa) was investigated by determining the genetic distribution of the primary 4CMenB antigens among diverse members of the common childhood commensal, Neisseria lactamica. All the isolates possessed nhba but were devoid of fhbp and nadA. The nhba alleles were mainly distinct from but closely related to those observed among a representative panel of invasive MenB isolates from the same broad geographic region. We made similar findings for the immunogenic typing antigen, FetA, which constitutes a major part of the 4CMenB OMV. Thus, 4CMenB vaccine responses may impact or be impacted by nasopharyngeal carriage of commensal neisseriae. This highlights an area for further research and surveillance should the vaccine be routinely implemented.


Assuntos
Antígenos de Bactérias/imunologia , Vacinas Meningocócicas/imunologia , Neisseria lactamica/imunologia , Neisseria meningitidis Sorogrupo B/imunologia , Adolescente , Adulto , Antígenos de Bactérias/genética , Portador Sadio/imunologia , Portador Sadio/microbiologia , Criança , Pré-Escolar , Proteção Cruzada , Reações Cruzadas , Feminino , Humanos , Lactente , Masculino , Vacinas Meningocócicas/genética , Pessoa de Meia-Idade , Nasofaringe/microbiologia , Neisseria lactamica/genética , Neisseria meningitidis Sorogrupo B/genética , Adulto Jovem
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