Differential expression of Pmp10 in cell culture infected with Chlamydia pneumoniae CWL029

The complete genome of Chlamydia pneumoniae contains a total of 21 genes encoding polymorphic membrane proteins (Pmp). From this large Pmp family three genes, pmp8, pmp10 and pmp11, were cloned and antibodies against recombinant full-length Pmp proteins were produced. Indirect immunofluorescence microscopy of HEp-2 cells infected with C. pneumoniae CWL029 was performed with the Pmp antibodies in combination with a Chlamydia-specific anti-lipopolysaccharide (LPS) antibody. This double staining technique clearly showed that expression of Pmp10 was differential. Additional double staining with monoclonal antibodies to the surface of C. pneumoniae elementary bodies and the anti-LPS antibody resulted in identification of seven monoclonal antibodies that reacted identically to the Pmp10 antibody indicating that Pmp10 is an immunodominant protein. Finally, the molecular mechanism responsible for differential expression is suggested to be variation in the guanine residues in the polyG tract of pmp10.     

A secondary structure motif predictive of protein localization to the chlamydial inclusion membrane

Chlamydiae are obligate intracellular pathogens that spend their entire growth phase sequestered in a membrane-bound vacuole called an inclusion. A set of chlamydial proteins, labelled Inc proteins, has been identified in the inclusion membrane (IM). The predicted IncA, IncB and IncC amino acid sequences share very limited similarity, but a common hydrophobicity motif is present within each Inc protein. In an effort to identify a relatively complete catalogue of Chlamydia trachomatis proteins present in the IM of infected cells, we have screened the genome for open reading frames encoding this structural motif. Hydropathy plot analysis was used to screen each translated open reading frame in the C. trachomatis genome database. Forty-six candidate IM proteins (C-lncs) that satisfied the criteria of containing a bilobed hydrophobic domain of at least 50 amino acids were identified. The genome of Chlamydia pneumoniae encodes a larger collection of C-lnc proteins, and only approximately half of the C-lncs are encoded within both genomes. In order to confirm the hydropathy plot screening method as a valid predictor of C-lncs, antisera and/or monoclonal antibodies were prepared against six of the C. trachomatis C-lncs. Immunofluorescence microscopy of C. trachomatis-infected cells probed with these antibodies showed that five out of six C-lncs are present in the chlamydial IM. Antisera were also produced against C. pneumoniae p186, a protein sharing identity with Chlamydia psittaci lncA and carrying a similar bilobed hydrophobic domain. These antisera labelled the inclusion membrane in C. pneumoniae infected cells, confirming that proteins sharing the unique secondary structural characteristic also localize to the inclusion membrane of C. pneumoniae. Sera from patients with high-titre antibodies to C. trachomatis were examined for reactivity with each tested C-lnc protein. Three out of six tested C-lncs were recognized by a majority of these patient sera. Collectively, these studies identify and characterize novel proteins localized to the chlamydial IM and demonstrate the existence of a potential secondary structural targeting motif for localization of chlamydial proteins to this unique intracellular environment.     

Genomic screening for Chlamydophila pneumoniae-specific antigens using serum samples from patients with primary infection

Chlamydophila pneumoniae, an obligate intracellular human pathogen, causes respiratory tract infections. The most common techniques used for the serological diagnosis of C.?pneumoniae infections are microimmunofluorescence tests and commercial serological ELISA tests; these are based on the detection of antibodies against whole chlamydial elementary bodies and lipopolysaccharide/outer membrane protein, respectively. Identification of more specific and highly immunodominant antigens is essential for the development of new serodiagnostic assays. To identify novel specific antigens from C.?pneumoniae, we screened 455 genes with unknown function in the genome of C.?pneumoniae J138. Extracts of Saccharomyces cerevisiae cells expressing GFP-tagged C.?pneumoniae proteins were subjected to Western blot analysis using serum samples from C.?pneumoniae-infected patients as the primary antibodies. From this comprehensive analysis, 58 clones expressing C.?pneumoniae open reading frames, including hypothetical proteins, were identified as antigens. These results have provided useful information for the development of new serological tools for the diagnosis for C.?pneumoniae infections and for the development of vaccines in future.     

Identification of high- and low-virulent strains of Chlamydia pneumoniae by their characterization in a mouse pneumonia model

Contradicting reports exist about the pathogenicity of Chlamydia pneumoniae and the severity of the respiratory disease they cause. This study aimed to clarify, in mice, our hypothesis that marked differences in virulence of well-defined C. pneumoniae strains might exist for lung infections. C57BL/6J mice were intranasally infected with equal amounts of five different, identically prepared laboratory strains of C. pneumoniae . Based on the clinical score, weight, histopathological score, the granulocyte marker-enzyme myeloperoxidase, and the amount of Chlamydiae in the lung tissue, the C. pneumoniae isolates exhibited clear differences in overall growth characteristics or clearance, and pathological potential. Thus, we could identify chlamydial strains (Kajaani-K6 and CWL-029), where mice became seriously ill, as well as a relatively low-virulent isolate (TWAR-183). Cytokine profiles also varied drastically between the five strains in extent and kinetic. Our results indicate that C. pneumoniae isolates differ markedly with regard to their interaction with the host and their pathological potential. This might also be true for the infection in humans. Because the genomic diversity of C. pneumoniae is rather small, more subtle genomic deviations account most likely for the apparent functional differences. Our results will be useful to identify additional virulence factors in the future.     

Sequence analysis of a group of low molecular-weight plasmids carrying multiple IS903 elements flanking a kanamycin resistance aph gene in Salmonella enterica serovars

A group of low molecular-weight ColE1-like plasmids carrying the aph sequence type aph(ii) from three different Salmonella serovars were sequenced. These plasmids carry two or more copies of IS903 elements, with up to 21bp sequence differences to one another, two of which flank the aph gene. This group of plasmids did not appear to carry any known mobilization genes and instead carry three open reading frames encoding hypothetical proteins of unknown function possibly organized in an operon. The plasmid replication region (RNA I/II--rom) of this plasmid group showed extensive homology to that of pKPN2 plasmid of Klebsiella pneumoniae and pCol-let plasmid of Escherichia coli. Three of the four plasmids had identical sequences, and the fourth had an extra copy of IS903 with target duplication, suggesting a recent divergence in the different Salmonella serovars from a common ancestor.     

Identification of a gene encoding an immuno-reactive membrane protein from Campylobacter jejuni

A gene encoding a putative membrane protein has been identified from Campylobacter jejuni NCTC 11168 following an immuno-screen of a lambda ZAP II genomic DNA library with antiserum raised against glycine-extractable proteins. The nucleotide sequence of the entire genomic insert revealed six open reading frames, all but one of which have sequence homologues in the complete genome sequence of Helicobacter pylori. The gene encoding the immuno-reactive protein was further identified by independent expression of these reading frames in Escherichia coli. The gene encodes an integral membrane protein, expression of which in E. coli results in a profound filamentous phenotype.     

Characterisation of a novel repetitive DNA sequence from Mycobacterium bovis

We report characterisation of three copies of a novel repeat sequence isolated from a Mycobacterium bovis genomic library. The repeat occurs within open reading frames, potentially encoding a conserved tandem array of a pentapeptide sequence with the consensus X-Gly-Asn-X-Gly. The tandem array is present up to five times in M. bovis and it is proposed that they may occur in a family of genes expressing functionally related proteins. We postulate that these proteins may play a role in binding of M. bovis to host cell receptors.     

Analysis of the first genome fragment from the marine sponge-associated, novel candidate phylum Poribacteria by environmental genomics

The novel candidate phylum Poribacteria is specifically associated with several marine demosponge genera. Because no representatives of Poribacteria have been cultivated, an environmental genomic approach was used to gain insights into genomic properties and possibly physiological/functional features of this elusive candidate division. In a large-insert library harbouring an estimated 1.1 Gb of microbial community DNA from Aplysina aerophoba, a Poribacteria-positive 16S rRNA gene locus was identified. Sequencing and sequence annotation of the 39 kb size insert revealed 27 open reading frames (ORFs) and two genes for stable RNAs. The fragment exhibited an overall G+C content of 50.5% and a coding density of 86.1%. The 16S rRNA gene was unlinked from a conventional rrn operon. Its flanking regions did not show any synteny to other 16S rRNA encoding loci from microorganisms with unlinked rrn operons. Two of the predicted hypothetical proteins were highly similar to homologues from Rhodopirellula baltica. Furthermore, a novel kind of molybdenum containing oxidoreductase was predicted as well as a series of eight ORFs encoding for unusual transporters, channel or pore forming proteins. This environmental genomics approach provides, for the first time, genomic and, by inference, functional information on the so far uncultivated, sponge-associated candidate division Poribacteria.     

Comparative genomics for identification of clone-specific sequence blocks in Streptococcus pneumoniae

The partial genome sequences of a serotype 3 and a serotype 2 pneumococcal strain were compared to the complete type 4 pneumococcal genome. Over 500000 and 150000 base pairs of the partial genome data, obtained from published patents, were analysed respectively. Global alignment showed that nearly the whole genome is highly conserved in accordance with data of multilocus sequence typing of housekeeping genes. The search for clone-specific genes revealed 17 new open reading frames in the type 3 strain, while no new open reading frame was detected in the type 2 strain. Allelic variation of genes was restricted by the use of crude sequence data, but still permitted identification of some new alleles and the observation that all surface proteins present in the partial genome data were highly conserved. In both strains we observed also a variety of chromosomal rearrangements and variations due to mobile genetic elements. All together, this comparative genomic approach gives a genome-based overview of strain relatedness and a prospective on what could be expected when sequencing other pneumococcal strains.     

柞蚕核型多角体病毒（AnpeNPV）三地理株的全基因组比较分析揭示其遗传多样性（英文）

【目的】柞蚕核型多角体病毒(Antheraea pernyi nucleopolyhedroviruses, AnpeNPV）能引起柞蚕脓病大面积暴发。尽管之前已完成了两株核型多角体病毒的基因组测序,但基于比较基因组的方法调查AnpeNPV间的遗传变异研究则相对较少。【方法】为了研究AnpeNPV不同地理株系间的遗传多样性,我们测序了1株从中国河南省分离的柞蚕NPV（AnpeNPV-H）基因组,并和之前从辽宁省分离的2株柞蚕NPV(AnpeNPV-L和AnpeNPV-Z)进行了比较基因组学分析。【结果】AnpeNPV-H的全基因组大小为125 605 bp,总共预测了146个开放阅读框（ORF）,包括95个功能注释蛋白和51个假定蛋白。我们发现这3株AnpeNPV间的基因组成非常相似。在这3个AnpeNPV株系间,有6个开放阅读框存在碱基变异而导致基因的变短。并鉴定超过200个单核苷酸多态性（single nucleotide variations, SNVs）,其中85%位于蛋白编码区。同时,odv-e 56, p94-like 和 egt 3个基因的非同义突变较高,表明这3个蛋白质编码基因可能经历了正向选择或纯化选择。我们发现在这3株AnpeNPV间一些基因的氨基酸发生变异,例如编码膜蛋白的基因、抑制凋亡和蜕皮的基因及与DNA复制相关的DNA聚合酶和解旋酶等基因。最后,展示了3株AnpeNPV间遗传重组的证据。【结论】本研究揭示了AnpeNPV种内水平的变异和株系内基因组的动态结构。     

Proteolytic cleavage of the Chlamydia pneumoniae major outer membrane protein in the absence of Pmp10

The genome of the obligate intracellular bacteria Chlamydia pneumoniae contains 21 genes encoding polymorphic membrane proteins (Pmp). While no function has yet been attributed to the Pmps, they may be involved in an antigenic variation of the Chlamydia surface. It has previously been demonstrated that Pmp10 is differentially expressed in the C. pneumoniae CWL029 isolate. To evaluate whether the absence of Pmp10 in the outer membrane causes further changes to the C. pneumoniae protein profile, we subcloned the CWL029 isolate and selected a clone with minimal Pmp10 expression. Subsequently, we compared the proteome of the CWL029 isolate with the proteome of the subcloned strain and identified a specific cleavage of the C-terminal part of the major outer membrane protein (MOMP), which occurred only in the absence of Pmp10. In contrast, when Pmp10 was expressed we predominantly observed full-length MOMP. No other proteins appeared to be regulated according to the presence or absence of Pmp10. These results suggest a close association between MOMP and Pmp10, where Pmp10 may protect the C-terminal part of MOMP from proteolytic cleavage.     

Complete Genome Sequence of a Chinese Isolate of Potato virus X and Analysis of Genetic Diversity

The complete genomic sequence of a Chinese Potato virus X isolate FX21 (PVX‐FX21) was determined from three overlapping cDNA clones. The genome of PVX‐FX21 is 6435 nucleotides in length excluding the poly(A) tail and contains five open reading frames (ORFs). Its entire genomic sequence shares 95.2–96.3% identities with Asian and European isolates, and 77.3–77.8% with American isolates. Phylogenetic analysis of the complete genomic sequence reveals two groups: the Eurasian group and the American group. PVX‐FX21 belongs to the Eurasian group and forms a separate sub‐branch with three Asian isolates. Similar analyses of the coat protein genes of 37 PVX isolates also reveal two major groups. All PVX isolates from Asia are clustered to group I, whereas isolates from Europe and America are clustered to both groups. Nucleotide sequence diversity analyses show that there is no geographical differentiation between PVX isolates and that constraint on the ORF encoding RNA‐dependent RNA polymerase is much higher than those on the other four ORFs.