In this work we described the construction of a comprehensive and flexible gene collection of Chlamydia pneumoniae. This ORFeome consists of 869 ORFs maintained as homogeneous plasmid DNAs purified from single colonies plus 168 ORFs purified from separate ‘pools’ of entry clones. This coverage of 98.5% of all predicted Cpn ORFs is similar to the coverage reported for other bacterial ORFeomes
[10, 19–21]. Such ORFeomes offer a flexible resource with which to undertake functional genomic studies, including high-throughput screens
[22–24]. In our own laboratory we have successfully produced protein arrays/chips
 whereon thousands of proteins can be tested against other proteins or peptides. The described Cpn ORFeome is the basis for functional high-throughput analysis of this organism, providing a flexible tool for quick and efficient genome-wide examination and discovery.
We assessed the quality of the ORFeome by PCR and agarose gel-electrophoresis of all the entry clones obtained. Sequencing all positive single clones confirmed the identity of the entry clones. To minimize the occurrence of PCR-induced mutations, we optimized the PCR conditions, used a proofreading-capable DNA polymerase, and kept the number of amplification cycles low. Functional validation was done by recombinant protein production and PPI analysis using the Y2H system. According to our previous work
, we did not purify the first PCR products, because it does not minimize the possibility of primer dimers created by the secondary attachment primers. These primer dimers are the putative reason for the existence of samples for which no positive single clone could be found. In the present study we spot-tested 300 arbitrarily chosen first PCR products. Almost all products delivered a PCR band of the expected size. In addition, we examined PCR samples of negative candidates (no positive single clone) where the first-generated PCR product delivered, like the other samples examined, a strong PCR band. In most cases we could isolate positive candidates by investigating up to 20 clones of one reaction, affording a way to clone single genes but not hundreds of genes. We failed to produce PCR products for 15 ORFs. This could be due to possible errors in annotation of the CWL029 genome or, more likely, because of nucleotide differences between our Cpn isolate and the sequenced CWL029 genome or for technical reasons related to primer design and effectiveness specific to each missing ORF. The single entry clones are of higher quality than the pools of entry clones because they are homogeneous, and LR reactions performed subsequently with the pure clones will be more efficient. However, for reasons of economy and because of time constraints, it was not possible to screen every colony to obtain a pure isolate of the appropriate ORF-containing entry clone. However, we expect that additional positive single transformants are present in the liquid glycerol stocks. In future work, this version of the Cpn ORFeome may be extended by isolating more positive single clones.
Ninety-seven positive single clones (10% of the positive single clones) failed validation by DNA sequencing. Almost all of the affected genes harbor a mutation in the binding region of the primary forward primer, resulting in frameshift mutations. However, this type of problem could be solved by using primers of higher quality than the used Mass Spectrometry checked primers in this work. Another solution could be to re-order the 10% defective primers and re-clone the defective clones or to correct the mutations by using mutagenesis kits. Although these genes are not in frame with the commonly used Gateway® destination vectors, there is the possibility of transferring them to other vectors having the appropriate reading frame. Alternatively, there already exist reading-frame independent vectors suitable for Gateway® cloning
[15, 16]. The use of such vectors would increase the overall coverage of positive single clones.
The present work provides the first nearly complete ORFeome of Cpn. Production of recombinant proteins in the present system can be achieved with the well-known GST fusion tag. This tag could be used for further protein purification or affinity studies such as GST Pull-Down for validation of protein–protein interactions. We successfully produced fusion proteins of ten randomly chosen clones. ORFeome-wide protein chips already exist
[25–27] and could be applied to our Cpn collection for analyzing not only protein–protein interactions, but also the ability of proteins to bind to small molecules as well as other proteins. A special field in the case of Cpn would be the use of proteome chips in drug and drug target discovery, because such chips can provide a variety of systems for detecting drug–protein interactions among all proteins. The technique for proteome chips already exists in our working group, and was verified by finding a small epitope to a known antigen, another type of protein–peptide interaction
As a validation of our interaction screen, first we used FlhA, a Type-III secretion apparatus member. The Y2H screen identified the Type-III secretion chaperone sycE as an interaction partner. SycE is related to the class-I chaperones involved in binding of effector proteins during the secretion process. Since FlhA is located in the inner membrane and is involved in flagellar protein secretion, it is possible that SycE interacts with FlhA during the export of flagellar or other proteins. Supporting this finding, it was previously reported that another chaperon, FliJ, is also an interaction partner of FlhA
. We also identified the methyltransferase ada, which in E. coli is known to be involved in the repair of DNA damage induced by methylating agents. The link between ada and the Type-III secretion apparatus is unknown, but it is worth mentioning that chlamydia transports other methyltransferases to influence host gene expression via Type-III secretion
The second tested bait protein, FliF, is a member of the MS ring of the Type-III injectisome
. FliF is a previously described binding partner of FlhA
, but we could not detect this interaction. Although, the Y2H system is a powerful system to detect PPIs, nevertheless not all interactions can be detected within a single screen and further reasons could be loss of interaction due to the fusion with the yeast GAL4 protein domain, or the requirement that the interaction between bait and prey are obliged to happen in the nucleus of yeast cells. There are a couple of reasons leading to false-negatives in the Y2H system, a drawback actually existing in all PPI-methods. On the other hand, we identified FliN, a putative C-ring member
, as an interaction partner of FliF.
Our third bait protein was EUO, a chlamydial protein expressed early in the developmental cycle. EUO is able to bind to AT-rich sequences
 and also has been shown to have a histone H1-specific protease activity
. While the potential interaction partners of EUO are not known, we identified EUO itself, two hypothetical proteins, and the metabolic enzyme phosphocarrier protein ptsH as interacting partners. The exact roles of these interactions remain to be identified.
These screens were performed in a low-scale manner with a test Cpn protein set, but generation of an Cpn intrapathogen ‘interactome’ is conceivable through ORFeome-wide PPI screening by automated procedures. Likewise, a system-wide screen for host–pathogen PPIs could be conducted by screening against other available ORFeomes, e.g. the human ORFeome
, which would produce a detailed description of the host–pathogen interface at the molecular level, leading to improved understanding of Cpn pathogenesis in humans.