OUTLINE OF THE THESIS
In this work we first focused our interest on a better characterization of R. prowazekii adhesins thought to play a major role in adhesion and host cell invasion process. Two distinct adhesins, called Adr1 and Adr2, and which display a high sequence homology, were initially taken in consideration. However, for unexpected reasons, we failed to express the recombinant Adr1 (RP827) as a soluble protein. Only the rickettsial Adr2 encoded by RP828 was cloned, expressed and purified in amount sufficient for immunizations. The production of mAbs was achieved through the fusion of mouse myeloma cells and spleen cells from RickA-immunized mice. Both sensitivity and specificity of the mAbs anti-Adr2 were evaluated by western blot. Their efficiency to neutralize R. prowazekii entry into host cell was then investigated.
In the second work, we also generated selective mAbs to gain further insights into cell-to-cell spreading, another major event of rickettsia pathogenesis. More specifically, our aim was to localize RickA in R. conorii. While this protein was found able to promote actin polymerisation (Gouin et al., 2004), its role in rickettsia motility has been the subject of debates (Balraj et al., 2008; Kleba et al., 2010). Based on the lack of peptide signal, its localization as a membrane protein is for long questionnable. Immunofluorescence and immune electron microscopy are the strategies displayed to carefully examine this aspect.
In the last part of this work and based on the different potential rickettsial recombinant protein markers, we investigated the discrimination of infection between R. typhi and R. conorii by ELISA.
These works were described in the 3 publications presented below.
Rickettsia prowazekii is the etiologic agent of epidemic typhus and Brill-Zinsser disease (Bechah et al., 2008). This is a louse-borne human pathogen which has caused large outbreaks in situations where lack of hygiene and cold weather favour louse proliferation. Humans are exposed to R. prowazekii through direct contact with contaminated body louse feces. Rickettsia begins its life cycle in the human host by invading the endothelial cells via the process of induced phagocytosis. Then, it rapidy escapes from the phagosome into the host cytoplasm where it replicates and eventually causes the invaded cell to burst (Walker et al., 2007; Balraj et al., 2009).
Understanding the molecular mechanisms responsible for R. prowazekii virulence is an important challenge. Using two dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with high throughput matrix-assisted laser desorption/ionization time of fight (MALDI-TOF) the first proteome reference maps of both R. conorii and R. prowazekii were established (Renesto et al., 2005). This achievement in turn led to the identification of two putative rickettsial ligands recognized by endothelial cells and called Adr1 and Adr2 (Renesto et al., 2006).
Recognition of and binding to the host cell is a key step for pathogenesis. This is particularly true when considering the fact that these strictly intracellular bacteria must enter host cells to replicate and survive. Here, in order to get better knowledge about the rickettsial Adr2 adhesin, we produced mAbs directed against this protein. For this purpose, the recombinant Adr2 protein from R. prowazekii was cloned, expressed and purified to immunize mice. The capacity of the anti-Adr2 mAb to inhibit rickettsiae-induced cytotoxicity was also investigated.
Rickettsia prowazekii is the etiological agent of epidemic typhus. This bacterium is an obligate intracellular parasite that grows freely within the cytoplasm of its eukaryotic host cell rather than in phagosomes or phagolysosome . R. prowazekii can be isolated from shell vial cell cultures, which has replaced classic animal- and/or embryonated egg– based culture methods [2, 3]. The pathogen exhibits a slow generation time (8–12 h), undergoes steady multiplication and lyses the host cell by releasing hundreds of infectious bacteria . Understanding the mechanisms involved in this unique intracytoplasmic parasitism was the goal of current study.
Bacterial cell surface proteins are involved in host-parasite interactions and are targeted by the adaptive response of the host immune system . Adhesion is a key step for bacterial invasion of host tissues, and adhesins are bacterial surface proteins that recognize receptors on host cells. The expression of various genes during adhesion can activate the pathogenic process . Proteins as well as structural organelles on bacterial surface mediate adhesion. The bacterial components may be capsule, lipopolysaccharide, toxins and adhesins.
The R. japonica rOmpB autotransporter proteins function in rickettsial adherence to and invasion of Vero cells . These proteins belong to a large family of outer membrane proteins known as the surface cell antigen (Sca) family . Rickettsial entry into the host cell is mediated by the rOmpB protein, which binds to the host cell receptor Ku70, a component of the DNA dependent protein kinase . Cholesterol also acts as a membrane receptor for R. prowazekii binding [9-12]. The rOmpA protein is an immunodominant, surface-exposed autotransporter present only in the rickettsial spotted fever group [12, 13] and may be involved in the initial adhesion of R. rickettsii to the host cell . In the previous study, two putative rickettsial ligands recognized by host cell surface proteins were identified using high resolution 2D-PAGEcoupled with mass spectrometry . The results showed that one ligand corresponds to the C terminal extremity of rOmp B called β-peptide, the second one being a protein of unknown function encoded by RC1281 in R. conorii. RC1281 is located downstream of its paralog, RC1282 . Their orthologous genes in R. prowazekii are respectively RP827 and RP828 encoded proteins share striking homologies. They are respectively termed Adr1 and Adr2 for adhesion of Rickettsiae. Because of the presence of a signal peptide in Adr1 and Adr2 and their significant sequence homology with membrane proteins, they likely form a β barrel structure within the outer membrane, a location consistent with their putative function as adhesins. Adr1 and Adr2 are ubiquitously present within the Rickettsia genus and may play a critical role in their pathogenicity. However, the precise role of these proteins has not been investigated .
First, our attention was to characterize the role of these two adhesins Adr1 and Adr2 in rickettsial entry mechanisms to the host cell. However, we failed expression of recombinant protein Adr1 (RP827) and only the rickettsial gene Adr2 (RP828) could be cloned, expressed and purified in the amounts sufficient for mice immunizations. We produced monoclonal antibody (mAb) anti-Adr2 which was used to determine the neutralizing effect of R. prowazekii entry into host cell.
Distribution of Adr1 and Adr2 within bacterial species
The sequence similarities of the putative adhesins Adr1 and Adr2 for all studied rickettsial species are shown in Suppl. M1. Adr1 and Adr2 are conserved across all rickettsial species, and the highest sequence similarity was found between R. sibirica and R. africae Adr1 (98%) and between R. sibirica and R. rickettsii Adr2 (99%). The similarity between Adr1 and Adr2 sequences was about 40% among all rickettsial species. When comparing the rickettsial ORFs (Open Reading Frame) coding for Adr1 and Adr2 against the NCBI database, using the blastP software, we found that these proteins have homologs in other bacterial species (more than 30% amino acid sequence identity) (Fig. 1). These homologs were found predominantly among the α-proteobacteria, but were also identified in γ-proteobacteria such as Escherichia spp. and Salmonella spp. (Fig. 1).
Identification of the rickettsial adhesins using the overlay assay
To identify proteins expressed on the surface of R. prowazekii, an overlay assay was used. As illustrated in Fig. 2, this technique allows for the localization and identification of the rickettsial adhesins. Adr1 (RP827) and Adr2 (RP828) have a theoretical molecular weight of 23 kDa and 26 kDa, respectively. To further characterize the adhesins, the separation of the protein was carried out in 2D-PAGE and detected by silver staining. Following silver staining (Fig. 2A), intensely stained protein spots were excised from the gel, and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) was used for identification and analysis. A comparison of the 2D-gel/MALDI-TOF MS analysis and the overlay assay demonstrated that the spots identified in both methods were the presumed adhesions in R. prowazekii, RP827 and RP828. Interestingly, we have missed identification of RP827 in Rprowazekii and homolog of RP828 in R. conorii . We identified RC1281 which shares sequence homology with RP827 and sca5 (β-peptide) respectively in R. conorii. Only RP828 and β-peptide were identified in R. prowazekii. Moreover, we identified immunoreactive spot m1 which corresponds to prohibitin-2 (Mus musculus), but failed identification of other 2 immunoreactive spots m2, m3. Thus, this work completes and confirms previous results . 2.2.1. Cloning, expression and purification of rickettsial adhesins Initially, two R. prowazekii genes encoding Adr1 and Adr2 were selected for cloning and expression experiments by using Gateway technology (Invitrogen, Carlsbad, CA, USA). However, despite using 2 different constructions (with N terminal Histag -DsbC and Histag-thioredoxine fusion), we have successfully attempted the expression of only one protein of R. prowazekii. Thus, Adr2 was purified in soluble form in sufficient yield by a Nickel affinity chromatography (suppl. M2) for further experiments. In the case of two different Adr1constructions the cloning was successful, but expression of recombinant fusion proteins (Dsbc- Adr1, trx-Adr1 and trx-Adr2) in E. coli Rosetta (DE3) pLysS strain failed. The migration profile of the recombinant fusion protein is shown in Fig. 3A (Coomassie staining) showing DsbC-Adr2 fusion protein about 55 kDa, which corresponds to 26 kDa Adr2 protein in fusion with DsbC (28.4 kDa). The identity of recombinant protein Adr2 was confirmed by western blot using an anti-His antibody (Fig. 3B) and by MALDI-TOF MS, respectively.
The genes encoding: groEL and RP059 were subsequently cloned according to manufacturer’s instructions (Gateway Cloning Technology/Invitrogen Life Technologies). Then, expression of clones containing an N-His6 tag plus a fusion protein thioredoxin (TRX)  that enhances expression of the fusion partner [17, 18] was performed as described below. The identity of these proteins was checked by MALDI-TOF as described for Adr2. Purified recombinant proteins were used to generate mAbs included as controls in 137 neutralization assay (see 4).
Production of monoclonal antibodies against Adr2
Monoclonal antibodies (mAbs) were generated against the recombinant R. prowazekii Adr2. The antigenic profile of the recombinant protein was analyzed using western blots and silver staining. In a western blot, the monoclonal antibody obtained from immunized mice with R. prowazekii Adr2, recognized proteins at the positions corresponding to the theoretical location of Adr2 (Fig. 4A). The corresponding silver-stained spot was identified by MALDI-TOF MS (Fig. 4B) as Adr2 protein.
Inhibition of R. prowazekii-induced cytotoxicity with anti-Adr2 monoclonal antibody
When R. prowazekii was pretreated for 20 min with increasing titers of anti-Adr2 monoclonal antibody and then added to L929 cells, cell cytotoxicity measured after 1h of incubation was 37% (dilution 1:100) and 40% (dilution 1:10), respectively. At the same time of sampling, inhibition of rickettsial entry assessed by rOmpB mAb was 53% (dilution 1:10) and 33% (dilution 1:100), respectively (Fig. 5). The % of inhibition assessed by negative specificity controls was about 5% for both mAbs: groEL and RP059. However, the greatest value of inhibition was obtained for 8h sample with the % of inhibition for Adr2 about 50% (dilution 1:10) and 43% (dilution 1:100). Indeed, the values obtained for rOmpB were 57% (dilution 1:10) and 43% (dilution 1:100). This inhibition was antibody concentration dependent for both Adr2 and rOmpB at 8h of incubation. Indeed, the % of inhibition was less than 10% for both controls: GroEL and RP059. We observe decreased % of inhibition for both rOmpB and Adr2 at the time of sampling 24h, 120h and 168h and ranging from 38% to 18% (Adr2, dilution 1:10), from 36% to 8% (Adr2, dilution 1:100) and 48% to 36% (rOmpB, dilution 1:10), 42% to 17% (rOmpBdilution 1:100), respectively. No significant variation was observed for GroEL and RP059, except at 24h time of sampling, the inhibition was 20% for groEL. The negative control consists on uninfected cells incubated with buffer only and showed noisy background of non specific cytotoxicity which ranged about 30%. In addition, Adr2 is sufficient to mediate R. prowazekii entry into the cell at early stage of mammalian cell infection.
In the present study, first, we selected in R. prowazekii genome the genes encoding for Adr1 (RP827) and Adr2 (RP828) based on previous work , sequenced and constructed the phylogenical tree showing the distribution of putative Adr1 and Adr2 within bacterial species including Rickettsiae, α- and γ-proteobacteria. Secondly, we identified in R. prowazekii proteome adhesins Adr1 and Adr2 and showed inhibition of R.prowazekii entry into the host cells by using monoclonal antibodies generated by mice immunization with recombinant fusion protein Adr2-Dsbc, rOmpB , as well as with recombinant proteins TRX-GroEL (RP626) and TRX-spo0J (RP059), respectively. All examined Rickettsia spp. share these both adhesins (Suppl. M1). Previous studies reported other adhesins differentially expressed in Rickettsia like the surface cell antigen (sca) family proteins and the outer membrane proteins, rOmpA and rOmpB [14, 20]. These genes have been used to study the phylogenetic relationships between Rickettsia spp. The Adr1 and Adr2 gene sequences show some heterogeneity between Rickettsia spp., in accordance with the four distinct rickettsial groups (e.g., the spotted fever group, the typhus group, R. canadensis and R. bellii). A highly resolved phylogenetic tree at the group level was constructed using the RP828 sequences (Fig. 1).We used overlay assays along with a proteomic approach to identify the adhesins . From a crude extract, proteins were separated using 2D-PAGE with 6–11 strips (Fig. 2), which allowed for better resolution of the protein than the previously optimized conditions . This approach allows for the localization and identification of the rickettsial adhesins using MALDI-TOF MS. Both RP827 and RP828 were detected. We observed the same pattern of results using the overlay assay, as seen in Fig. 2. Therefore, the protein identification was confirmed using both an overlay assay and western blot.
Table of contents :
II. An overview of antibodies as useful tools for diagnosis of the rickettsiosis and their contribution in exploration of rickettsial biology
II-2. Antibodies as tools in diagnosis of rickettsiosis
II-3. Antibodies as tools for physiopathological investigations
II-4. Monoclonal antibodies – Generalities and future prospects
OUTLINE OF THE THESIS
PREAMBLE ARTICLE 1
Characterization of rickettsial adhesin Adr2 belonging to a new group of adhesins in αproteobacteria Manohari Vellaiswamy, Malgorzata Kowalczewska, Vicky Merhej, Claude
Nappez, Renaud Vincentelli, Patricia Renesto, Didier Raoult Microbial Pathogenesis 50(5), p. 233-42, 2011
PREAMBLE ARTICLE 2
Transmission electron microscopy as a tool for exploring bacterial proteins: model of RickA in Rickettsia conorii Manohari Vellaiswamy, Bernard Campagna, Didier Raoult New Microbiologica, 34, p. 209-218, 2011
PREAMBLE ARTICLE 3
Protein candidates for the serodiagnosis of rickettsioses. Malgorzata Kowalczewska, Manohari Vellaiswamy, Claude Nappez, Renaud Vincentelli, Bernard La Scola and Didier Raoult
FEMS Immunology and Medical Microbiology, in revision
CONCLUSION AND PERSPECTIVES