Search

Upregulation of the developmental Wnt/planar cell polarity pathway is observed in many cancers and is associated with cancer development at early and late stages. We recently showed that PRICKLE1 and VANGL2, two core Wnt/PCP components, are overexpressed in triple negative breast cancer and associated with poor prognosis. PRICKLE1 is a cytoplasmic protein phosphorylated by the poorly described serine/threonine kinase MINK1 which triggers its localization at the plasma membrane, a key step for its function. Knockdown experiments have demonstrated that MINK1 and PRICKLE1 contribute to TNBC cell motility and spreading in vitro and in vivo. However, the identity of MINK1 substrates and the role of MINK1 enzymatic activity in this process have not yet been addressed issues.We carried out a phosphoproteomic strategy and identified novel MINK1 substrates including LL5β. LL5β is a membrane scaffold molecule that anchors microtubules at the cell cortex through its association with the plus-end MT proteins CLASPs to trigger focal adhesion disassembly. LL5β is a prominent member of the MINK1-PRICKLE1 protein complex and is directly phosphorylated by MINK1 that promotes its interaction with CLASP. Using a kinase inhibitor, we demonstrate that the enzymatic activity of MINK1 is involved in the protein complex assembly and localization, and cell migration. Analysis of gene expression data show that the concomitant up-regulation of PRICKLE1 and LL5β mRNA levels encoding MINK1 substrates is associated with a poor metastasis-free survival for TNBC patients. Altogether, our results suggest that MINK1 may represent a potential target in TNBC.

The developmental Wnt/planar cell polarity (PCP) pathway is the most recently described branch of the Wnt signaling pathways and is strongly implicated in cancer development at early and late stages (PMID: 28718442 and PMID: 30952630). Upregulation of the Wnt/PCP components is observed in many cancers and is associated with cancer progression. However, how their molecular functions are regulated remains an open question. Recent data from our laboratory showed that Prickle1 and Vangl2, two core Wnt/PCP components, are overexpressed in Triple Negative Breast Cancers (TNBC) and associated with poor prognosis (PMID: 27184734 and 26754771). Prickle1 is a cytoplasmic protein phosphorylated by the serine/threonine kinase Mink1, which triggers Prickle1 localization at the plasma membrane and regulates its activity (PMID: 22037766). Activation of this axis contributes to breast cancer cell motility and metastatic spreading in vitro and in vivo (PMID: 27184734). To extend our knowledge of the Mink1 substrates, we carried out a phosphoproteomic strategy and identified LL5β. LL5β is a membrane scaffold molecule that anchors microtubules (MTs) at the cell cortex through its association with CLASP, a plus-end MT protein, to trigger focal adhesion disassembly. We found that LL5β is a prominent member of the Prickle1-associated protein complex and harbors a consensus motif for Mink1 phosphorylation. At the molecular level, we demonstrated a two-step phosphorylation cascade carried out by Mink1 that phosphorylates sequentially Prickle1 to mediate its association with LL5β, then LL5β to promote its interaction with CLASP. Finally, analysis of gene expression data suggests that the concomitant up-regulation of Prickle1 and LL5β mRNA levels is associated with a poor metastasis-free survival for TNBC patients. In an effort to counteract the Mink1-Prickle1-LL5β prometastatic pathway, we selected a Mink1 inhibitor that recapitulates all the phenotypes observed following the knockdown of Mink1 expression. This chemical compound is currently under investigation in preclinical assays using xenografted TNBC cell lines and patient-derived xenografts in nude mice. Citation Format: Avais Daulat, Monica Silveira Wagner, Malgorzata Kowalczewska, Pascal Finetti, Rémy Castellano, Stéphane Audebert, François Bertucci, Luc Camoin, Jean-Paul Borg. The serine-threonine kinase Mink1 as a potential therapeutic target in triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2290.

International audience

The laboratory diagnosis of rickettsioses is based on serology (reference method), cell culture and/or molecular tools. However, the main drawback of serology is its incapacity to provide identification of Rickettsiae at the level of species. The aim of this study was to propose the versatile protein markers able to discriminate the patients with murine typhus from those with Mediterranean spotted fever. We have cloned and expressed 20 proteins of Rickettsia prowazekii and Rickettsia rickettsii, respectively, using the GATEWAY approach. These recombinant proteins were screened by ELISA with sera of infected patients with Rickettsia typhi and Rickettsia conorii, respectively. We identified several potential markers which allowed infection due to R. typhi to be discriminated from those due to R. conorii. However, the values of test-operating parameters were not sufficient for its 'routine' clinical use. Our diagnostic test requires further optimization for be applied as a point-of-care strategy in the management of patients with suspected cases of rickettsiosis.

The aim of this study was to identify candidate proteins for serodiagnostics of Q fever by monoclonal antibodies (MAbs), and to clone, express, and purify the selected proteins for use as antigens in ELISA. The reactivity of three MAbs to Coxiella burnetii (C. b.) Nine Mile strain and one MAb to Priscilla strain was tested using SDS-PAGE, 2-D gel electrophoresis, immunoblot analysis, and mass spectrometry. Three immunoreactive Q fever-specific proteins discriminated by MAbs, namely the CBU_0937 protein, outer membrane Com1 (CBU_1910) protein, and elongation factor Tu (CBU_0236) were identified. Successful PCR-amplification, cloning, expression, and purification of the recombinant proteins Com1 and CBU_0937 allowed their use for the screening of sera from patients with Q fever endocarditis (18) or acute Q fever (16) in ELISA. The recombinant protein CBU_0937 with unknown biological function proved to be a more applicable diagnostic tool for Q fever ELISA as compared to the Com1 protein.

Bartonella henselae is an emerging gram-negative facultative intracellular pathogen transmitted via Ctenocephalides felis (cat fleas) or cat scratches. Bartonellosis is present mainly in the form of cat scratch disease (CSD), bacillary angiomatosis and infective endocarditis (IE). The methods used to diagnose B. henselae rely on culturing, immunofluorescent assays and molecular techniques. The objective of the present study was to identify candidate proteins for the serodiagnosis of bartonellosis with the differential discrimination of both clinical scenarios: CSD and IE. For this, an immunoproteomic approach combined with 2-DE, immunoblotting and matrix-assisted laser desorption/ionization time-of-flight MS has been developed. Immunoproteomic profiles of sera collected from patients with CSD and IE were compared with those of blood donors. We identified several candidate proteins as phage-encoding Pap31 protein and an outer membrane protein of BH11510 that, in our view, might be useful for the serodiagnosis of bartonellosis.

The proteome of Tropheryma whipplei, the intracellular bacterium responsible for Whipple's disease (WD), was analyzed using two complementary approaches: 2-DE coupled with MALDI-TOF and SDS-PAGE with nanoLC-MS/MS. This strategy led to the identification of 206 proteins of 808 predicted ORFs, resolving some questions raised by the genomic sequence of this bacterium. We successfully identified antibiotic targets and proteins with predicted N-terminal signal sequences. Additionally, we identified a family of surface proteins (known as T. whipplei surface proteins (WiSPs)), which are encoded by a unique group of species-specific genes and serve as both coding regions and DNA repeats that promote genomic recombination. Comparison of the protein expression profiles of the intracellular facultative host-associated WD bacterium with other host-associated, intracellular obligate, and environmental bacteria revealed that T. whipplei shares a proteomic expression profile with other host-associated facultative intracellular bacteria. In summary, this study describes the global protein expression pattern of T. whipplei and reveals some specific features of the T. whipplei proteome.

Whipple's disease (WD) is a chronic multisystemic infection, caused by the bacterium Tropheryma whipplei. The main clinical presentations are classic WD (CWD) with histologic lesions in the gastrointestinal tract, endocarditis, and isolated neurologic infection. The current strategy for diagnosis remains invasive.The present study aimed to select the protein candidates for serological diagnosis of WD. The first step was to identify candidate proteins by an immunoproteomic approach combining 2-DE using a total extract of a T. whipplei, immunoblotting, and MS. The second step was to validate the discovered biomarkers using a recombinant protein-based ELISA. Serum samples from 18 patients with WD and from 54 control individuals were tested. A sugar ABC transporter, TWT328 (sensitivity (Se) 61%, specificity (Sp) 87%, positive predictive value (PPV) 61%, negative predictive value (NPV) 87%, and positive likelihood ratio (PLR) 4.69) was the best marker for development of serodiagnosis for CWD. We also obtained a reproducible immunoreactive protein pattern for patients with isolated neurological infection due to T. whipplei (Se 100%, Sp 93%, PPV 55.5%, NPV 100%, and PLR 13.51) as an encouraging step towards noninvasive diagnosis of this particular manifestation. Nine recombinant candidates have been successfully screened with serum samples. Results from these ELISA assays skewed with those obtained with immunoblots.

Tropheryma whipplei, the agent of Whipple's disease, is a gram-positive rod-shaped bacterium that belongs to the group of actinobacteria. In order to produce monoclonal antibodies (MAbs) against this bacterium, we inoculated mice with two different strains, Slow2 and Endo5. We produced 13 and 10 MAbs against Slow2 and Endo5, respectively. Nine of the Slow2 MAbs and seven of the Endo5 MAbs recognized a 58-kDa epitope. In addition, three other Endo5 MAbs detected a unique 84-kDa epitope. These MAbs were species specific, as they did not react with a selection of 22 different bacterial species, but they were not strain specific, as they did react with six other strains ofT. whipplei. Two-dimensional gel electrophoresis (2-DE) was combined with mass spectrometry (MS) to identify the 58-kDa and 84-kDa epitopes recognized by MAbs. After trypsin in-gel digestion of the spot, the 58-kDa protein was identified as an ATP synthase F1 complex beta chain, whereas the 84-kDa protein was identified as a polyribonucleotide nucleotidyltransferase by MS with matrix-assisted laser desorption ionization-time of flight. In an in vitro model, one of these MAbs allowed good detection ofT. whippleiin stool samples, contrary to a rabbit polyclonal antibody, which led to high fluorescent background. In the prospective studies, the produced MAb will be tested for detection ofT. whippleiin clinical samples, and the gene coding for identified 58-kDa and 84-kDa antigens will be tentatively cloned and then tested for its use in a diagnostic enzyme-linked immunosorbent assay for Whipple's disease.

The discriminatory diagnosis of Q fever remains difficult because of the unspecific clinical presentations of the disease. Additionally, the diagnosis is often delayed because serodiagnosis is not sensitive enough in the early stages of the disease when the immune response is not yet efficient. Similarly, the diagnosis of Q fever endocarditis can only be performed in approximately 35%, mainly via serology, which was a criterion postulated by Duke. Owing to the discriminatory diagnosis of Q fever and the high number of tests requested, we focused on expressing several proteins for ELISA studies with Coxiella burnetii-infected sera. Previously, we selected a list of 31 candidates [Sekeyova et al. (2009) Eur J Clin Microbiol Infect Dis 28: 287-295], of which we have successfully cloned and expressed 21. Finally, 15 recombinant proteins were prescreened with the sera of patients with acute Q fever and Q fever endocarditis, respectively. Sera from a control group were also screened. The nine most immunoreactive proteins from the first assay were tested with the sera from a larger group of patients. Our study identified CBU_0092 as the best marker of acute Q fever but failed to isolate a highly specific and sensitive marker of Q fever endocarditis.

The culture of microorganisms has been the basis of microbiology. However, revolutionary tools such as metagenomics have made it possible to describe uncultivated bacteria, but several breakthroughs have occurred in culture leading to a revival of these techniques. In this review we focus on new applications that have successfully cultivated previously uncultivated bacteria. We also review the axenic cultivation of intracellular bacteria such as Tropheryma whipplei and Coxiella burnetii. These successes provide new tools for the design of axenic media for intracellular bacteria, such as Rickettsiae and Chlamydiae, or historically uncultivable pathogens, such as Mycobacterium leprae and Treponema pallidum. The future axenic culture of these microorganisms will facilitate antibiotic susceptibility testing and will provide insight into their microbial ecology and pathogenicity.

The laboratory diagnosis of rickettsioses is based on serology (reference method), cell culture and/or molecular tools. However, the main drawback of serology is its incapacity to provide identification of Rickettsiae at the level of species. The aim of this study was to propose the versatile protein markers able to discriminate the patients with murine typhus from those with Mediterranean spotted fever. We have cloned and expressed 20 proteins of Rickettsia prowazekii and Rickettsia rickettsii, respectively, using the GATEWAY approach. These recombinant proteins were screened by ELISA with sera of infected patients with Rickettsia typhi and Rickettsia conorii, respectively. We identified several potential markers which allowed infection due to R. typhi to be discriminated from those due to R. conorii. However, the values of test-operating parameters were not sufficient for its 'routine' clinical use. Our diagnostic test requires further optimization for be applied as a point-of-care strategy in the management of patients with suspected cases of rickettsiosis.

Background Rickettsia prowazekii is the etiological agent of epidemic typhus and is an obligate intracellular bacterium that grows as a parasite freely within the cytoplasm of a eukaryotic host cell. Previous studies have shown that rOmpA and rOmpB which belong to the family of rickettsial cell surface antigens are involved in vitro in the adhesion of Rickettsiae to epithelial cells. Recently, two putative rickettsial adhesins have been identified using high resolution 2D-PAGE coupled with mass spectrometry. In this study, we further characterize and describe the adhesin Adr2 from R. prowazekii . Methodology/Principal findings Using an overlay assay coupled with mass spectrometry two adhesins, Adr1 (RP827) and Adr2 (RP828), were identified from the R. prowazekii proteome Recombinant R. prowazekii Adr2 was expressed through fusion with Dsbc in Escherichia coli , purified and concentrated, thus allowing production of specific monoclonal antibodies, as confirmed by western blot assays. Finally, inhibition of rickettsiae-induced cytotoxicity with monoclonal anti-Adr2 antibody has showed a greatest impact on bacterial cell entry at 8 h post-infection (ca50%) and then decreased progressively to attempt 18% of inhibition at day 7. These, correlated to the inhibition of rickettsiae-induced cytotoxicity with monoclonal anti-rOmpB antibody. Thus, Adr2 is sufficient to mediate R. prowazekii entry into the cell at early stage of mammalian cell infection. Conclusions Our results suggest that R. prowazekii Adr2 could be the main actor promoting the entry of rickettsiae into the host cells. The present study opens the framework for future investigations for better understanding of the Adr2 -mediated mechanisms involved in adhesion/invasion or intracellular survival of R. prowazekii .

Bartonella henselae is an emerging gram-negative facultative intracellular pathogen transmitted via Ctenocephalides felis (cat fleas) or cat scratches. Bartonellosis is present mainly in the form of cat scratch disease (CSD), bacillary angiomatosis and infective endocarditis (IE). The methods used to diagnose B. henselae rely on culturing, immunofluorescent assays and molecular techniques. The objective of the present study was to identify candidate proteins for the serodiagnosis of bartonellosis with the differential discrimination of both clinical scenarios: CSD and IE. For this, an immunoproteomic approach combined with 2-DE, immunoblotting and matrix-assisted laser desorption/ionization time-of-flight MS has been developed. Immunoproteomic profiles of sera collected from patients with CSD and IE were compared with those of blood donors. We identified several candidate proteins as phage-encoding Pap31 protein and an outer membrane protein of BH11510 that, in our view, might be useful for the serodiagnosis of bartonellosis.

Q fever is a worldwide zoonosis caused by Coxiella burnetii bacterium. Two clinical forms are present: acute Q fever and chronic disease, including endocarditis. Currently, the diagnosis of Q fever endocarditis is based on the detection of anti-phase I antibodies. The objective of the study was to identify candidate proteins for the serological diagnosis of endocarditis due to C. burnetii. The immunoreactivities of sera from 12 patients with C. burnetii infections, including the sera from patients with endocarditis and with the acute clinical form of Q fever, were compared with those of three control subjects who did not have Q fever. We identified 29 candidate antigenic proteins by mass spectrometry. Two proteins, arginine repressor and OmpH, were recognised exclusively by the sera of patients with Q fever endocarditis. These proteins are promising candidates for the development of serodiagnostic assays for Q fever endocarditis.

Whipple’s disease (WD) is a systemic chronic infection, caused by the Gram-positive bacterium Tropheryma whipplei. There are several clinical traits linked to WD: histological lesions in the GI tract in association with diverse clinical manifestations (classic WD), endocarditis with negative blood cultures, and isolated neurological infection. WD is rare, predominantly affects middle-aged men and is fatal without treatment. The most recent strategy for diagnosing WD uses the results of diastase-resistant periodic acid Schiff staining and PCR in parallel, both performed on involved organ/tissue biopsy (small intestine, cardiac valve and cerebrospinal fluid). The generation of rabbit polyclonal antibodies has enabled the detection of the bacterium in tissues by immunohistochemical staining. However, the diagnosis of WD remains an invasive procedure. The recent achievement of stable bacterial culture and sequencing of the T. whipplei genome has opened a framework for the development of a biomarker platform. Several studies in different fields have been performed, for example, transcriptomics, immunoproteomics and comparative proteomics. Biomarker candidates have been proposed for the development of less invasive procedures for diagnosing WD.

International audience; Whipple's disease (WD) is a chronic multisystemic infection, caused by Tropheryma whipplei, a Gram‐positive rod. Recently, a reliable method has been developed for cultivating T. whipplei in vitro. This together with the availability of complete genome sequence of T. whipplei prompted us to initiate proteome analysis of T. whipplei. The objective of the present study was to identify candidate proteins for serological diagnosis of WD. Immunoreactivities of sera collected from 18 patients with WD were compared with those of 24 control subjects who did not have WD. For this, we used 2‐DE, immunoblotting, and MS. In total, we identified 23 candidate antigenic proteins. These included a subset of six proteins, each of which was found significantly more frequently in cases as compared to their controls. The remaining 17 proteins were found exclusively in cases. The methods we used in the current study enabled us to identify candidate antigens that, in our view, might be useful for serological diagnosis of WD.

Q fever is a worldwide zoonosis caused by Coxiella burnetii. The disease most frequently manifests clinically as a self-limited febrile illness, as pneumonia (acute Q fever) or as a chronic illness that presents mainly as infective endocarditis. The extreme infectivity of the bacterium results in large outbreaks, and the recent outbreak in the Netherlands underlines its impact on public health. Recent studies on the bacterium have included genome sequencing, the investigation of host-bacterium interactions, the development of cellular and animal models of infection, and the comprehensive analysis of different clinical isolates by whole genome and proteomic approaches. Current approaches for diagnosing Q fever are based on serological methods and PCR techniques, but the diagnosis of early stage disease lacks specificity and sensitivity. Consequently, different platforms have been created to explore Q fever biomarkers. Several studies using a combination of proteomics and recombinant protein screening approaches have been undertaken for the development of diagnostics and vaccines. In this review, we highlight advances in the field of C. burnetii proteomics, focusing mainly on the contribution of these technologies to the development and improvement of Q fever diagnostics.