High Throughput Sequencing and Proteomics to Identify Immunogenic Proteins of a New Pathogen: The Dirty Genome Approach

Abstract

BackgroundWith the availability of new generation sequencing technologies, bacterial genome projects have undergone a major boost. Still, chromosome completion needs a costly and time-consuming gap closure, especially when containing highly repetitive elements. However, incomplete genome data may be sufficiently informative to derive the pursued information. For emerging pathogens, i.e. newly identified pathogens, lack of release of genome data during gap closure stage is clearly medically counterproductive.Methods/Principal FindingsWe thus investigated the feasibility of a dirty genome approach, i.e. the release of unfinished genome sequences to develop serological diagnostic tools. We showed that almost the whole genome sequence of the emerging pathogen Parachlamydia acanthamoebae was retrieved even with relatively short reads from Genome Sequencer 20 and Solexa. The bacterial proteome was analyzed to select immunogenic proteins, which were then expressed and used to elaborate the first steps of an ELISA.Conclusions/SignificanceThis work constitutes the proof of principle for a dirty genome approach, i.e. the use of unfinished genome sequences of pathogenic bacteria, coupled with proteomics to rapidly identify new immunogenic proteins useful to develop in the future specific diagnostic tests such as ELISA, immunohistochemistry and direct antigen detection. Although applied here to an emerging pathogen, this combined dirty genome sequencing/proteomic approach may be used for any pathogen for which better diagnostics are needed. These genome sequences may also be very useful to develop DNA based diagnostic tests. All these diagnostic tools will allow further evaluations of the pathogenic potential of this obligate intracellular bacterium.

Identification of Immunogenic Proteins of Waddlia chondrophila

“…In addition, W. chondrophila hypothetical protein #4 (Wim4), also identified in this work, is homolog to a P. acanthamoebae hypothetical protein (pah_c029o043), which was previously shown to be immunogenic [20].…”

“…Although, no homolog to MOMP could be identified in the P. acanthamoebae or in the P. amoebophila genomes, a putative cysteine-rich MOMP-family is present in the W. chondrophila genome [20], [24], [38]. However, no protein of this family was identified in the present work, most probably because we had, on purpose, chosen extraction and isolation conditions which should leave the putative waddlial disulphide linked matrix as an insoluble complex.…”

Metabolic Features of Protochlamydia amoebophila Elementary Bodies – A Link between Activity and Infectivity in Chlamydiae

“…Although environmental chlamydiae, including P. amoebophila , have a significantly larger genome size than the Chlamydiaceae , their metabolic potential is only slightly increased, and they are thus also strictly dependent on eukaryotic host cells [14], [16]–[19]. Despite being auxotrophic for most amino acids, cofactors, and nucleotides and being able to take up host-derived ATP, chlamydiae have at least partially maintained metabolic pathways devoted to carbon metabolism and energy generation [19]–[21].…”

Waddlia: An emerging pathogen and a model organism to study the biology of chlamydiae

“…This extended waddlial OmpA-like protein family may explain the large host diversity of W. chondrophila. Like other Chlamydiales, W. chondrophila also possesses clusters of genes encoding a T3SS for inoculation of bacterial effectors into the host cytoplasm [35], but do not possess a T4SS contrarily to Parachlamydia acanthamoebae and Protochlamydia amoebophila [40,41].…”