In vivo expression of Neisseria meningitidis proteins homologous to the Haemophilus influenzae Hap and Hia autotransporters

The genome sequences of Neisseria meningitidis serogroup B strain MC58 and serogroup A strain Z2491 were systematically searched for open reading frames (ORFs) encoding autotransporters. Eight ORFs were identified, six of which were present in both genomes, whereas two were specific for MC58. Among the identified ORFs was the gene encoding the known autotransporter IgA1 protease. The deduced amino acid sequences of the other identified ORFs were homologous to known autotransporters and found to contain an N-terminal signal sequence and a C-terminal domain that could constitute a beta-barrel in the outer membrane. The ORFs NMB1985 and NMB0992, encoding homologs of the Hap (for Haemophilus adhesion and penetration protein) and Hia (for Haemophilus influenzae adherence protein) autotransporters of H. influenzae, were cloned from serogroup B strain H44/76 and expressed in Escherichia coli. Western blots revealed that all sera of patients (n=14) and healthy carriers (n=3) tested contained antibodies against at least one of the recombinant proteins. These results indicate that both genes are widely distributed among N. meningitidis isolates and expressed during colonization and infection.     

Sequence analysis and relationships between meningococcal class 3 serotype proteins and other porins from pathogenic and non-pathogenic Neisseria species

The presence of highly conserved regions within previously determined porin gene sequences from Neisseria meningitidis and Neisseria gonorrhoeae permitted the construction of oligonucleotide primers for PCR amplification of other neisserial porin genes. Although two separate porin genes (porA and porB) are present in N. meningitidis only a single fragment, corresponding to porB, could be amplified from this species. The amplified porB genes from four different meningococcal serotypes, which express the class 3 outer membrane protein, were sequenced. Amplified fragments corresponding to porin genes from N. lactamica and N. sicca were also sequenced. In common with the known neisserial porins, models of the organisation of the predicted proteins indicated trans-membrane structures with eight surface exposed loops. In the meningococcal class 3 proteins the main regions of sequence variation, which must be responsible for serotype specificity, were located on loops 5 and 7. A phylogenetic analysis of the family of porins from the Neisseria confirmed the close relationship of the meningococcal class 3 protein with the gonococcal PIA protein, while the gonococcal PIB protein was shown to be closely related to the N. lactamica porin. The close relationship seen between porins of the pathogenic and non-pathogenic Neisseriae identified no obvious virulence-associated regions in the proteins, but did suggest that the current nomenclature for neisserial porin genes may need reviewing.     

Characterization of serogroup C meningococci isolated from 14 provinces of China during 1966-2005 using comparative genomic hybridization

Neisseria meningitidis is a major cause of bacterial meningitis and septicemia worldwide. In China, serogroup A strains were responsible for over 95% of the cases, while serogroup B strains were mainly the cause of localized outbreaks and sporadic cases. Before 2003, serogroup C strains were only recovered from a few sporadic cases. However, a sudden increase in the number of cases due to serogroup C strains occurred during 2003-2005 in Anhui Province, China. Many cases were found in other provinces at the same time. Multilocus sequence typing (MLST) results indicated that the unique sequence type 4821 clone meningococci, a new hyper-virulent lineage, was responsible for the serogroup C meningitis outbreaks. We have completed the project of sequencing the whole genome of the Chinese N. meningitidis serogroup C representative isolate 053442. We fabricated a whole-genome microarray of N. meningitidis isolate 053442 and analyzed the genome composition differences among 81 serogroup C isolates which were isolated from 14 provinces of China during 1966-2005. The comparative genomic hybridization (CGH) result shows that the genome compositions of nearly all serogroup C isolates are similar to that of 053442. The products of many absent open reading frames (ORFs) are conserved hypothetical proteins. The results will provide a valuable resource from which one can analyze the genome composition and genetic background of serogroup C meningococci in China.     

Rapid Evolution of the Sequences and Gene Repertoires of Secreted Proteins in Bacteria

Proteins secreted to the extracellular environment or to the periphery of the cell envelope, the secretome, play essential roles in foraging, antagonistic and mutualistic interactions. We hypothesize that arms races, genetic conflicts and varying selective pressures should lead to the rapid change of sequences and gene repertoires of the secretome. The analysis of 42 bacterial pan-genomes shows that secreted, and especially extracellular proteins, are predominantly encoded in the accessory genome, i.e. among genes not ubiquitous within the clade. Genes encoding outer membrane proteins might engage more frequently in intra-chromosomal gene conversion because they are more often in multi-genic families. The gene sequences encoding the secretome evolve faster than the rest of the genome and in particular at non-synonymous positions. Cell wall proteins in Firmicutes evolve particularly fast when compared with outer membrane proteins of Proteobacteria. Virulence factors are over-represented in the secretome, notably in outer membrane proteins, but cell localization explains more of the variance in substitution rates and gene repertoires than sequence homology to known virulence factors. Accordingly, the repertoires and sequences of the genes encoding the secretome change fast in the clades of obligatory and facultative pathogens and also in the clades of mutualists and free-living bacteria. Our study shows that cell localization shapes genome evolution. In agreement with our hypothesis, the repertoires and the sequences of genes encoding secreted proteins evolve fast. The particularly rapid change of extracellular proteins suggests that these public goods are key players in bacterial adaptation.     

Antigenic cross-reactivity between outer membrane proteins of Neisseria meningitidis and commensal Neisseria species

Two mouse sera against outer membrane proteins from a pathogenic Neisseria meningitidis strain and a commensal N. lactamica strain and two human sera from patients recovering from meningococcal meningitis were used to identify antigens common to pathogenic and commensal Neisseria species. Two major antigens of 55 kDa and 32 kDa, present in all N. meningitidis and N. lactamica strains tested, were demonstrable with all the sera used; the 55-kDa protein was iron-regulated. Demonstration of other common antigens was dependent on the serum used: a 65-kDa antigen was visualised with the human and the mouse anti-N. lactamica sera; a 37-kDa antigen identified as the meningococcal ferric binding protein (FbpA) was only detected with the mouse sera, and two antigens of 83 kDa and 15 kDa were only shown with the mouse anti-N. meningitidis serum. The results demonstrate the existence of several outer membrane antigens common to N. lactamica and N. meningitidis strains, in agreement with the hypothesis that natural immunity against meningitis is partially acquired through colonisation by commensal species, and open new perspectives for the design of vaccine formulations and the development of strategies for vaccination against meningitis.     

Identification of acquired DNA in Neisseria lactamica

Anomalous DNA (aDNA) in prokaryotic genomes, identified by its aberrant nucleotide composition, generally represents horizontally acquired DNA. Previous studies showed that frequent DNA transfer occurs between commensal Neisseriae and Neisseria meningitidis. Currently, it is unknown whether aDNA regions are also transferred between these species. The genome of Neisseria lactamica strain 892586 was assessed by a strategy that enables the selective isolation of aDNA, using endonucleases with recognition sites that are overrepresented in aDNA. Of eight regions with aDNA, five displayed similarity to virulence-associated meningococcal sequences. Of three aDNA fragments with limited or no similarity to neisserial sequences, one encodes a novel putative autotransporter/adhesin. The remaining two fragments are adjacent in the N. lactamica genome, and encode a novel putative ATPase/subtilisin-like protease operon. A similar operon is present in the genomes of different respiratory tract pathogens. The identification of aDNA from N. lactamica with similarity to meningococcal aDNA shows that genetic exchange between the Neisseriae is not limited to the neisserial core genome. The discovery of aDNA in N. lactamica similar to a locus in other pathogens substantially expands the neisserial gene pool.     

Identification of a 13-kDa protein associated with the polyhydroxyalkanoic acid granules from Acinetobacter spp.

Abstract Transferrin-binding proteins from Neisseria meningitidis vary among different isolates. We have identified and studied a hypervariable region adjacent to the carboxyl-end of the transferrin-binding domain of the Tbp2 molecule. The tbp2 genes from six strains of N. meningitidis were cloned and sequenced in this particular region. Sequence analysis of these regions along with five other sequences available from pathogenic Neisseria showed a common organisation of seven highly variable nucleotide stretches interspersed with six conserved nucleotide stretches. The variable regions correlated with the location of immunoreactive epitopes in polyclonal antisera raised to transferrin-binding proteins identified by peptide pin technology. Sequence analysis suggested a mosaic-like organisation of the tbp2 genes. Taken together, these data suggest that the antigenic variation in this part of the protein may result from a strong host immune pressure.     

Identification and characterization of specific sequences encoding pathogenicity associated proteins in the genome of commensal Neisseria species

Abstract The distribution of distinct sequences in pathogenic and commensal Neisseria species was investigated systematically by dot blot analysis. Probes representing the genes of Rmp, pilin and IgA1 protease were found to hybridize exclusively to the chromosomal DNA of the pathogenic species, Neisseria gonorrhoeae and/or Neisseria meningitidis . In contrast, specific sequences for the genes of the porin protein Por and the opacity protein (Opa) were also detected in a panel of commensal Neisseria species such as N. lactamica, N. subflava, N, flava, N. mucosa and N. sicca . Using opa -specific oligonucleotides as probes in chromosomal blots, the genomes of the commensal Neisseria species show a totally reduced repertoire of cross-hybridizing loci compared to the complex opa gene family of N. gonorrhoeae . DNA sequence analysis of one opa -related gene derived from N. flava and N. sicca , respectively, revealed a large degree of homology with previously described gonococcal and meningococcal genes e.g., a typical repetitive sequence in the leader peptide and the distribution of the hypervariable and conserved regions. This observation, together with the finding, that the gene is constitutively transcribed, leads to the assumption that some of the commensal Neisseria species may have the potential for the expression of a protein harboring similar functions as the Opa proteins in pathogenic Neisseriae .     

Signal peptide-dependent protein transport in Bacillus subtilis: a genome-based survey of the secretome.

One of the most salient features of Bacillus subtilis and related bacilli is their natural capacity to secrete a variety of proteins into their environment, frequently to high concentrations. This has led to the commercial exploitation of bacilli as major "cell factories" for secreted enzymes. The recent sequencing of the genome of B. subtilis has provided major new impulse for analysis of the molecular mechanisms underlying protein secretion by this organism. Most importantly, the genome sequence has allowed predictions about the composition of the secretome, which includes both the pathways for protein transport and the secreted proteins. The present survey of the secretome describes four distinct pathways for protein export from the cytoplasm and approximately 300 proteins with the potential to be exported. By far the largest number of exported proteins are predicted to follow the major "Sec" pathway for protein secretion. In contrast, the twin-arginine translocation "Tat" pathway, a type IV prepilin-like export pathway for competence development, and ATP-binding cassette transporters can be regarded as "special-purpose" pathways, through which only a few proteins are transported. The properties of distinct classes of amino-terminal signal peptides, directing proteins into the various protein transport pathways, as well as the major components of each pathway are discussed. The predictions and comparisons in this review pinpoint important differences as well as similarities between protein transport systems in B. subtilis and other well-studied organisms, such as Escherichia coli and the yeast Saccharomyces cerevisiae. Thus, they may serve as a lead for future research and applications.     

Direct selection and phage display of a Gram-positive secretome

Surface, secreted and transmembrane protein-encoding open reading frames, collectively the secretome, can be identified in bacterial genome sequences using bioinformatics. However, functional analysis of translated secretomes is possible only if many secretome proteins are expressed and purified individually. We have now developed and applied a phage display system for direct selection, identification, expression and purification of bacterial secretome proteins.     

Secretomes of medically important fungi reflect morphological and phylogenetic diversity

Secretome represents a main target for understanding the mechanisms of fungal adaptation. In the present study, we focus on the secretomes of fungi associated with infections in humans and other mammals in order to explore relationships between the diverse morphological and phylogenetic groups. Almost all the mammalian pathogenic fungi analyzed have secretome sizes smaller than 1000 proteins and, secreted proteins comprise between 5% and 10% of the total proteome. As expected, the correlation pattern between the secretome size and the total proteome was similar to that described in previous secretome studies of fungi. With regard to the morphological groups, minimum secretome sizes of less than 250 secreted proteins and low values for the fraction of secreted proteins are shown in mammalian pathogenic fungi with reduced proteomes such as microsporidia, atypical fungi and some species of yeasts and yeast-like fungi (Malassezia). On the other hand, filamentous fungi have significantly more secreted proteins and the highest numbers are present in species of filamentous fungi that also are plant or insect pathogens (Fusarium verticilloides, Fusarium oxysporum and Basidiobolus meristosporus). With respect to phylogeny, there are also variations in secretome size across fungal subphyla: Microsporidia, Taphrinomycotina, Ustilagomycotina and Saccharomycotina contain small secretomes; whereas larger secretomes are found in Agaricomycotina, Pezizomycotina, Mucoromycotina and Entomophthoromycotina. Finally, principal component analysis (PCA) was conducted on the complete secretomes. The PCA results revealed that, in general, secretomes of fungi belonging to the same morphological group or subphyla cluster together. In conclusion, our results point out that in medically important fungi there is a relationship between the secretome and the morphological group or phylogenetic classification.     

Determination of the number of tuf genes in Chlamydia trachomatis and Neisseria gonorrhoeae

Restriction endonuclease fragments of DNA from Neisseria gonorrhoeae and Chlamydia trachomatis (mouse pneumonitis biovar) were hybridized to probes from the N-terminal and C-terminal portions of the Escherichia coli tufA gene. In common with other Gram-negative bacteria, the genome of N. gonorrhoeae was found to contain two homologous sequences (presumptive tuf genes). The C. trachomatis genome contained a single tuf sequence.     

植物病原真菌分泌蛋白质组学研究进展

分泌蛋白质组是指在特定时间和特定条件下,由组织或细胞等分泌的全部蛋白质。在病原真菌与植物的相互作用过程中,病原真菌会分泌大量的蛋白质和代谢产物,在病原真菌对植物的侵入、定殖和扩展等致病过程中起着重要作用。本文主要介绍了分泌蛋白质在植物病原真菌致病性中的作用、重要植物病原真菌分泌蛋白质组的研究进展、及植物病原真菌分泌蛋白质组的生物信息学预测分析等,对于全面了解植物病原真菌的致病机理具有重要意义。     

稻瘟病菌含信号肽小蛋白的计算机分析

结合计算机技术和生物信息学的方法,采用组合的信号肽分析软件SignalPv3.0、TargetPv1.1、Big-PIpredictor、TMHMMv2.0和SecretomeP对已公布的1486个稻瘟菌(magnaporthegrisea)小蛋白基因的N-端氨基酸序列进行信号肽分析,同时系统分析了信号肽的类型及结构。分析结果表明,在1486个稻瘟病菌小蛋白中,119个具有N-端信号肽的典型分泌蛋白。其中116个具有分泌型信号肽,1个具RR-motif型信号肽,2个具信号肽酶II型信号肽。在稻瘟病菌基因组中,分泌型小蛋白的序列是高度趋异的,仅出现少数氨基酸组成完全一致的信号肽,为进一步确认具有相同信号肽的分泌蛋白是否具有同源性,分别用BLAST2SEQUENCES对具有相同信号肽的分泌蛋白进行了序列对比。结果表明,具有相同信号肽的分泌蛋白同源性非常高。同时还采用Sublocv1.0对1486个小蛋白的亚细胞位置进行了预测,结果显示小蛋白的可能功能场所包括细胞质、细胞外、线立体和细胞核,功能场所位于细胞核的小蛋白是最多的。     

Analysis of Neisseria lactamica antigens putatively implicated in acquisition of natural immunity to Neisseria meningitidis

Sera from healthy human volunteers, patients convalescent from meningococcal meningitis, and mice immunized with outer membrane proteins from Neisseria meningitidis and Neisseria lactamica strains were used to analyze and identify antigens cross-reactive to both neisserial species. All classes of meningococcal proteins except class 1 (PorA) and class 5 cross-reacted with N. lactamica proteins and two other proteins of 65 and 55 kDa (an iron-regulated protein). Results obtained with the mouse sera demonstrate that cross-reactive antibodies can be elicited by either N. meningitidis or N. lactamica. These results support the suggestion that N. lactamica contributes to the development of natural immunity against N. meningitidis during the first years of life. The use of vaccines containing proteins other than PorA could interfere in colonization of mucosal surfaces by N. lactamica, hampering the natural mechanisms of immunity acquisition in humans. Only convalescent sera reacted with the 55 and 65 kDa proteins, which suggests that they might be relevant for pathogenicity.     

大肠埃希氏杆菌UTI89分泌蛋白组的预测分析

目的：从大肠埃希氏杆菌UTI89基因组中筛选出全部潜在的分泌蛋白并进行初步研究。方法：使用SignalP3.0、TatP1.0、 SecretomeP2.0等蛋白分析软件对5211个ORF进行预测;对筛选出的信号肽及分泌蛋白的基本特征进行统计学分析;使用Blast 2 Sequences进行同源性分析。结果：共筛选出432个sec途径分泌蛋白,19个Tat途径分泌蛋白,386个非经典分泌蛋白;信号肽、分泌蛋白平均长度分别为25.5aa、282.8aa;信号肽中出现频率最高的3种氨基酸依次为L、A、S;仅有两个信号肽的氨基酸序列完全相同,相应的分泌蛋白高度同源。结论：大肠埃希氏杆菌UTI89基因组中有837个ORF可能编码分泌蛋白;分泌蛋白集中在500aa以下;组成信号肽的氨基酸相对保守,多数为疏水氨基酸;信号肽变异性较大,含相同信号肽的蛋白可能由同源基因编码。     

Genetic diversity of the iron-binding protein (Fbp) gene of the pathogenic and commensal Neisseria

Abstract The pathogenic Neisseria and most commensal Neisseria species produce an iron-binding protein (Fbp) when grown under iron-limited conditions. In the current study, we confirmed the presence of Fbp, as well as DNA sequences homologous to the gonococcal fbp , in strains of N. gonorrhoeae, N. meningitidis, N. cinerea, N. lactamica, N. subflava, N. kochii and N. polysaccharea . The fbp genes from these strains were amplified by the polymerase chain reaction, digested with Stu I or Rsa I, and the restriction patterns examined. The patterns for the gonococcal and meningococcal fbp were virtually identical; however, variations were observed in the fbp sequences of the commensal Neisseria species. N. flavescens, N. mucosa, N. sicca, N. ovis and Branhamella catarrhalis , did not produce Fbp as detected by sodium dodecyl sulfate-polyacrylamide gel electropheris and reactivity with an Fbp specific monoclonal antibody, nor did they hybridize to an fbp -specific DNA probe.     

Bioinformatic analysis of the Acinetobacter baumannii phage AB1 genome

As one of the pathogens of hospital-acquired infections, Acinetobacter baumannii poses great challenges to the public health. A. baumannii phage could be an effective way to fight multi-resistant A. baumannii. Here, we completed the whole genome sequencing of the complete genome of A. baumannii phage AB1, which consists of 45,159bp and is a double-stranded DNA molecule with an average GC content of 37.7%. The genome encodes one tRNA gene and 85 open reading frames (ORFs) and the average size of the ORF is 531bp in length. Among 85 ORFs, only 14 have been identified to share significant sequence similarities to the genes with known functions, while 28 are similar in sequence to the genes with function-unknown genes in the database and 43 ORFs are uniquely present in the phage AB1 genome. Fourteen function-assigned genes with putative functions include five phage structure proteins, an RNA polymerase, a big sub-unit and a small sub-unit of a terminase, a methylase and a recombinase and the proteins involved in DNA replication and so on. Multiple sequence alignment was conducted among those homologous proteins and the phylogenetic trees were reconstructed to analyze the evolutionary courses of these essential genes. From comparative genomics analysis, it turned out clearly that the frame of the phage genome mainly consisted of genes from Xanthomonas phages, Burkholderia ambifaria phages and Enterobacteria phages and while it comprises genes of its host A. baumannii only sporadically. The mosaic feature of the phage genome suggested that the horizontal gene transfer occurred among the phage genomes and between the phages and the host bacterium genomes. Analyzing the genome sequences of the phages should lay sound foundation to investigate how phages adapt to the environment and infect their hosts, and even help to facilitate the development of biological agents to deal with pathogenic bacteria.