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Chlamydia basic research society.

Report of the 1st biennial conference, Memphis 2003.

The Chlamydia basic research society was formed in 2002 with the objectives of:

• Encouraging basic research in chlamydial biology and pathogenesis

• Providing a forum for the exchange of information among scientists engaged in research on basic chlamydial biology and pathogenesis

• Promoting the development of young scientists in chlamydial research by encouraging their active participation in Society meetings and activities.

The Society's intention is to have a biennial scientific meeting held at a relatively low budget location so that young scientists are able to attend. On this basis the first meeting was hosted by the Fogelman conference centre of the University of Tennessee in Memphis, USA. The meeting was held from March 8 - 10, 2003. Thus this new conference series fits well between other established chlamydial conferences, such as the International Chlamydial Conference, held every 4 years (last held in Antalya, Turkey in June 2002) and the forthcoming conference of the European Chlamydia Research society, also held every 4 years ( & due to be held in Budapest in September 2004). Thus it is intended that a major chlamydial conference will be held every year.

Figures 1 - 4. The conference venue in Memphis.

The first conference of the Society was attended by some 220 delegates who listened to 53 oral papers and, if they were conscientious, scrutinised 61 posters. The oral presentations were split into 9 sessions which were: Genomics and Proteomics;  Gene Regulation and Signal Transduction; Chlamydial envelope and attachment; Chlamydial Protein Secretion; Persistence / Heat Shock Secretion; Host - Chlamydia interactions; Non - adaptive immune responses; Adaptive Immune responses and Cell culture / animal models & vaccination. 

From such a cornucopia of chlamydial research it is invidious and subjective to single out any particular papers for praise. Moreover some of the counterattractions of Memphis (see conclusions) meant that the writer was less than ideally diligent. This is therefore a necessarily arbitrary overview of the excellent papers which were presented. There may also be inaccuracies caused by deciphering partially legible notes written at speed during the presentation. [If I have misrepresented something or missed something important, or you would like to add something, please do contact the writer using the comments link at the bottom of the page].

The day one organiser, Ming Tan (Irvine) was able to put together an excellent program from the strong submitted research activity in the chlamydial genomics and proteomics field. His keynote introduction to the presentations and posters set the scene brilliantly. He also made an excellent choice of panellists able to stimulate productive discussion.

In the proteomics session, Brian VanDahl (Aarhus, Denmark) sought translated proteins of the putative C. pneumoniae type three secretion system. Much of this work is either published formally, or is viewable on the excellent chlamydial proteomics gels web site. Jyotika Sharma (San Antonio, Texas) was particularly interested in translated proteins likely to be interacting with host cells. Her strategy was to select from the chlamydial genomics databases open reading frames (ORFs) with the characteristics of encoding secreted or envelope proteins. She then prepared GST-fusion proteins of the selected ORFs, raised antibody to them, & localized the target protein by immunofluorescence microscopy. Chlamydial expression of the target protein and any post translational modification was then identified by Western Blot and / or radio immunoprecipation analysis (RIPA). Where indicated, half life of the chlamydial expressed protein was determined by pulse chase experiments. This was labour intensive work: so far ~300 ORFs cloned; ~200 GST fusions and ~130 antisera. They work hard in Guangming Zhong's lab!  Of the proteins so far localized, ~85% were detected inside the chlamydial inclusion, with  ~5% were associated with the inclusion membrane. 1-2% of proteins were in the host cell cytosol and ~10% had uncertain location (note, these percentages are not mutually exclusive). S35 met pulse chase and other experiments indicated that expression of the dnaJ chaperone protein peaks at 16 hr post infection and that it complexes with hsp70. Half life of the expressed protein was 6 - 8 hrs.

Patrik Bavoil described the host range of 3 "Chlamydia" [sic] bacteriophages. [Comment: These bacteriophages are in fact restricted to the genus Chlamydophila not Chlamydia, which in my view is one of the best indications that the new taxonomy was right to draw a genus level distinction between these two taxa]. See: chlamydial phages. Chlamydiaphages are of interest because they may be molecular tools for probing the chlamydial surface and they might form the basis of systems for the eventual manipulation of the chlamydial genome. All chlamydiaphages have the so-called IN5 loop on the VP1 structural protein. This loop is thought to form a trimeric protrusion at each of the 3 fold axes of symmetry for recognition of host (chlamydial) receptors. Patrik reported that phiCPG1 (Chlamydophila caviae) and PhiAR39 (Chlamydophila pneumoniae) can also infect Chlamydophila abortus. [Conversely Everson et al 2002 showed that C. abortus phage Chp2 can infect C. felis and C. pecorum but not C. pneumoniae]. This raised the possibility that Chlamydophila species may have multiple phage receptor sites. Patrik reported that phage-infected chlamydiae undergo partial developmental arrest, with decreased expression of the late genes CopN and OmcB.

This site has consistently emphasized the potential importance to our developing knowledge of the Chlamydiales of the environmental chlamydiae [hence chlamydiae.com not chlamydia.com].  Matthias Horn (ex Munich now Vienna) [who reviewed Parachlamydia for this site], gave an exciting preview of the final stages of the Parachlamydia UWE25 genome sequencing project. This is particularly important as the first Chlamydiales sequence outside the Chlamydiaceae and one which may be expected to throw some insight on Chlamydiales evolution. The Parachlamydia genome at 2,429,974 nucleotides, is substantially larger than that of the Chlamydia or Chlamydophila species. Indeed it is the second largest known genome for an intracellular bacterial pathogen. This suggests that, if the normal rules of genomic degradation apply, the Parachlamydia may be closer to the Moulder last common ancestor than the Chlamydiaceae. 

Roughly 80% of the Parachlamydia genome is coding sequence, with some 1845 open reading frames of average length 1,059 nucleotides. The 35% G+C ratio is substantially lower than the 40 - 42% of the Chlamydiaceae, but some 44% of the open reading frames had clear homologues among the Chlamydiaceae. Put another way, 45% of open reading frames had highest homology to the Proteobacteria, 13% to the Cyanobacteria and a remarkable 10% to plant genes. Although two thirds of the unique open reading frames could not be assigned, in the remainder there appeared to be substantial remnants of mobile genetic elements including translocases. Some 50 open reading frames included leucine rich repeat proteins associated with transposed regions or inverted repeats. The cell envelope apparently lacked a MOMP (omp1) homologue, POMP motifs or a complete pathway for peptidoglycan biosynthesis. However they have an ADP translocase, an F-type ATPase, a complete TCA cycle and an additional proton-translocating NADH facility. Thus Parachlamydia appear to be more independent of the host cell than the Chlamydiaceae. A type III secretion operon was present but distributed throughout the chromosome rather than localised to pathogenicity islands. CPAF was also present, together with putative inclusion proteins. This genome on preliminary evaluation is already sufficiently different to the Chlamydiaceae to suggest it is going to be very interesting indeed.

Tim Read of TIGR compared the newly completed Chlamydophila caviae (GPIC) genome sequence with other Chlamydiaceae sequences. There were 155 C. caviae unique genes and 943 genes resembling other chlamydial genes. C. caviae had the most complete tryptophan metabolism of any of the Chlamydiaceae, whereas C. pneumoniae had very little. Comparison of the C. caviae and C. pneumoniae sequences showed a break down in synteny in the plasticity zone / terminator region, suggesting that lateral gene transfer might play an important role in chlamydial evolution [but compare contrary view presented by Rick Stephens at Antalya]. 

In the area of gene regulation and signal transduction, Chris Schaumberg (Irvine) reviewed the putative chlamydial promoter structures at the -35 and -10 positions for sigma 66 [See also Timm's Antalya presentation]. Using as a model the E. coli promoter which had an optimum of TTGACa, nucleotide substitutions were made at each of the 6 positions in an in vitro transcription translation system based on dnaK to define the optimum chlamydial promoter, which was TTGACA. At the -10 position the E. coli promoter was TATTAT but the chlamydial promoter was TAAGAT. A suboptimal -35 promoter in chlamydiae correlates with a novel positive cis-acting spacer AT region thought to compensate for the suboptimal promoter sequence. Anne-Marie Douglas (Memphis) was interested in the possible regulation of the three known chlamydial sigma factors. Sigma 66 is generally concerned with housekeeping genes, whereas sigma 28 and sigma 54 are alternative sigma factors which might be regulated by anti (eg RsbW) or even anti anti (eg RsbV1/2) sigma factors! She found that RsbV1/2 and RSbW were expressed throughout the developmental cycle. Recombinant rRsbW bound to recombinant sigma 28 and phosphorylated recombinant RsbV2. She therefore believed that Rsb proteins probably regulate sigma 28 activity but there may be alternative temporal regulators of sigma activity.

Adam Wilson (Irvine) is interested in the regulation of chlamydial genes encoding heat shock proteins. In E. coli the acquisition of sigma 32 regulates the expression of heat shock protein, but in chlamydiae there is no sigma 32. In Bacillus subtillis, derepression of heat shock genes occurs by an operon / repressor model, in which HrcA binds to CIRCE. They had previously demonstrated that this mechanism is involved in dnaK regulation in chlamydiae [Wilson & Tan, 2002]; HrcA suppresses transcription of dnaK in a CIRCE-dependent manner. They reported that the groEL promoter has a CIRCE element but its affinity for HrcA was lower.

Nicole Grieshaber in Ted Hackstadt's laboratory wins the chlamydiae.com gold star for this conference !   It has long been known that chlamydiae have eukaryotic - type histone proteins which are expressed late in the chlamydial developmental cycle and which cause the condensation of the DNA nucleoid essential for elementary body development, with associated reduction in DNA transcription. Since the basic histone proteins have a very high affinity for DNA, the question arises how does the observed decondensation of chlamydial DNA occur at the differentiation of elementary bodies to reticulate bodies despite the continued expression of histone [see: developmental cycle]. She noted that a gene product of CT 804, a gene homologous to the enzyme encoded by ychB in E. coli involved in a non-mevalonate pathway of isoprenoid biosynthesis characteristic of some bacterial pathogens, protects chlamydial nucleoid ghosts prepared by zwittergent extraction from disruption by DNAse. Fosmidomycin, itself a novel herbicide and anti-malaria drug, inhibits this CT 804 rescue, presumably because isoprenoid intermediates downstream of CT 804 are not available. The molecule responsible [if I have copied it down correctly] is  the small molecule 2-C-Methyl-D-erythritol 2,4 cyclodiphosphate. It is unclear whether this compound is (i) directly competitive with DNA binding; (ii) whether it covalently modifies the histone; or (iii) whether it causes the condensed chromosome to unravel. A stunning piece of detective work among rare molecules.

In the final discussion, Christine Wu (Scripps Institute) presented a powerful liquid phase shotgun method of proteomic analysis using formic acid CNBr digestion, mass spectroscopy and bioinformatics. An impressive 384 unique EB components and 27 outer membrane (COMC) components have been identified together with other 100 phosphorylation sites that might be important for signalling and regulation. On MOMP, both phosphotyrosine and phosphoserine had been identified, with the phosphotyrosine apparently located on the MOMP adjacent to the periplasmic space while the phosphoserine was on the external surface of the protein in a suitable location for interaction with host cells. Phosphorylation sites such as these are likely to be important for signalling and regulation. For an example of this high-throughput technique see Florens et al., 2002.

In the genomics discussion it was generally agreed that there was substantial need for chlamydial genomic sequencing, particularly to understand the genomic basis of the different tissue tropisms and pathobiology of the C. trachomatis biovars. A considerable number of participants followed panellist Ian Clarke (Southampton) in commiserating on the difficulties of efforts to manipulate the genome of chlamydiae. It was suggested that www.chlamydiae.com might be a useful resource for sharing experiences in this area (under a suitable cloak of anonymity of course) as well as to provide a facility for updating chlamydial gene annotation. [These ideas are currently under discussion with TIGR].

[MEW] 27/03/2003

NEXT: Memphis 2 continued.

Reference

Everson J. S., Garner, S. A., Fane, B., Liu, B. L., Lambden, P. R. & Clarke, I. N. (2002). Biological Properties and Cell Tropism of Chp2, a Bacteriophage of the Obligate Intracellular Bacterium Chlamydophila abortus. Journal of Bacteriology 184, 2748 - 2754.

Florens, L., Washburn, M. P., Raine, J. D., Anthony, R. M., Grainger, M., Haynes, J. D. et al., (2002). A proteomic view of the Plasmodium falciparum life cycle. Nature 419, 520 - 526.