Identification of rickettsial immunoreactive proteins using a proximity ligation assay Western blotting and the traditional immunoproteomic approach.

The closely related species Rickettsia conorii and R. africae are both etiological agents of rickettsiosis, a tick-borne serious infective disease. The laboratory diagnosis is based on serology, but remains not enough specific to provide the diagnosis at the species level. Here, we attempted to identify specific proteins that would enable the discrimination of R. africae sp from R. conorii sp infections. We screened 22 R. africae- and 24 R. conorii-infected sera at different course of infection using a traditional immunoproteomic approach. In parallel, we focused on the technical development of a "relatively new technique" named a proximity ligation assay coupled to two-dimensional Western blotting. The top range markers of R. africae early infection were rpoA, atpD, and acnA, ORF0029, R. africae active infection were rOmpB ß-peptide, OmpA, groEL and ORF1174, early R. conorii infection was prsA, RC0031, pepA, R. conorii active infection were ftsZ, cycM and rpoA. They are candidates for serodiagnosis of rickettsioses.

Structure of RC1339/APRc from Rickettsia conorii, a retropepsin-like aspartic protease.

The crystal structures of two constructs of RC1339/APRc from Rickettsia conorii, consisting of either residues 105-231 or 110-231 followed by a His tag, have been determined in three different crystal forms. As predicted, the fold of a monomer of APRc resembles one-half of the mandatory homodimer of retroviral pepsin-like aspartic proteases (retropepsins), but the quaternary structure of the dimer of APRc differs from that of the canonical retropepsins. The observed dimer is most likely an artifact of the expression and/or crystallization conditions since it cannot support the previously reported enzymatic activity of this bacterial aspartic protease. However, the fold of the core of each monomer is very closely related to the fold of retropepsins from a variety of retroviruses and to a single domain of pepsin-like eukaryotic enzymes, and may represent a putative common ancestor of monomeric and dimeric aspartic proteases.

Proteome analysis of Rickettsia conorii by two-dimensional gel electrophoresis coupled with mass spectrometry.

The availability of genome sequence offers the opportunity to further expand our knowledge about proteins expressed by Rickettsia conorii, strictly intracellular bacterium responsible for Mediterranean spotted fever. Using two-dimensional polyacrylamide gel electrophoresis combined with MALDI-TOF mass spectrometry, we established the first reference map of R. conorii proteome. This approach also allowed identification of GroEL as the major antigen recognized by rabbit serum and sera of infected patients. Altogether, this work opens the way to characterize the proteome of R. conorii, to compare protein profiles of different isolates or of bacteria maintained under different experimental conditions and to identify immunogenic proteins as potential vaccine targets.

Rickettsia conorii and R. prowazekii proteome analysis by 2DE-MS: a step toward functional analysis of rickettsial genomes.

In this work, we present a comparative two-dimensional (2D) PAGE analysis of Rickettsia conorii and Rickettsia prowazekii. This analysis reveals protein spots that were either unique to or common to both strains, some of them being identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

Protein coding palindromes are a unique but recurrent feature in Rickettsia.

Rickettsia are unique in inserting in-frame a number of palindromic sequences within protein coding regions. In this study, we extensively analyzed repeated sequences in the genome of Rickettsia conorii and examined their locations in regard to coding versus noncoding regions. We identified 656 interspersed repeated sequences classified into 10 distinct families. Of the 10 families, three palindromic sequence families showed clear cases of insertions into open reading frames (ORFs). The location of those in-frame insertions appears to be always compatible with the encoded protein three-dimensional (3-D) fold and function. We provide evidence for a progressive loss of the palindromic property over time after the insertions. This comprehensive study of Rickettsia repeats confirms and extends our previous observations and further indicates a significant role of selfish DNAs in the creation and modification of proteins.