The serine/threonine kinase MINK1 directly regulates the function of promigratory proteins.

Upregulation of the developmental Wnt planar cell polarity (Wnt/PCP) pathway is observed in many cancers and is associated with cancer development. We have recently shown that PRICKLE1, a core Wnt/PCP pathway component, is a marker of poor prognosis in triple-negative breast cancer (TNBC). PRICKLE1 is phosphorylated by the serine/threonine kinase MINK1 and contributes to TNBC cell motility and invasiveness. However, the identity of the substrates of MINK1 and the role of MINK1 enzymatic activity in this process remain to be addressed. We used a phosphoproteomic strategy to identify MINK1 substrates, including LL5ß (also known as PHLDB2). LL5ß anchors microtubules at the cell cortex through its association with CLASP proteins to trigger focal adhesion disassembly. LL5ß is phosphorylated by MINK1, promoting its interaction with CLASP proteins. Using a kinase inhibitor, we demonstrate that the enzymatic activity of MINK1 is involved in PRICKLE1-LL5ß complex assembly and localization, as well as in cell migration. Analysis of gene expression data reveals that the concomitant upregulation of levels of mRNA encoding PRICKLE1 and LL5ß, which are MINK1 substrates, is associated with poor metastasis-free survival in TNBC patients. Taken together, our results suggest that MINK1 may represent a potential target for treatment of TNBC.

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.

Multi-omics Analysis Sheds Light on the Evolution and the Intracellular Lifestyle Strategies of Spotted Fever Group Rickettsia spp.

Arthropod-borne Rickettsia species are obligate intracellular bacteria which are pathogenic for humans. Within this genus, Rickettsia slovaca and Rickettsia conorii cause frequent and potentially severe infections, whereas Rickettsia raoultii and Rickettsia massiliae cause rare and milder infections. All four species belong to spotted fever group (SFG) rickettsiae. However, R. slovaca and R. raoultii cause scalp eschar and neck lymphadenopathy (SENLAT) and are mainly associated with Dermacentor ticks, whereas the other two species cause Mediterranean spotted fever (MSF) and are mainly transmitted by Rhipicephalus ticks. To identify the potential genes and protein profiles and to understand the evolutionary processes that could, comprehensively, relate to the differences in virulence and pathogenicity observed between these four species, we compared their genomes and proteomes. The virulent and milder agents displayed divergent phylogenomic evolution in two major clades, whereas either SENLAT or MSF disease suggests a discrete convergent evolution of one virulent and one milder agent, despite their distant genetic relatedness. Moreover, the two virulent species underwent strong reductive genomic evolution and protein structural variations, as well as a probable loss of plasmid(s), compared to the two milder species. However, an abundance of mobilome genes was observed only in the less pathogenic species. After infecting Xenopus laevis cells, the virulent agents displayed less up-regulated than down-regulated proteins, as well as less number of identified core proteins. Furthermore, their similar and distinct protein profiles did not contain some genes (e.g., ompA/B and rickA) known to be related to rickettsial adhesion, motility and/or virulence, but may include other putative virulence-, antivirulence-, and/or disease-related proteins. The identified evolutionary forces herein may have a strong impact on intracellular expressions and strategies in these rickettsiae, and that may contribute to the emergence of distinct virulence and diseases in humans. Thus, the current multi-omics data provide new insights into the evolution and fitness of SFG virulence and pathogenicity, and intracellular pathogenic bacteria.

Polysaccharide peptides from Coriolus versicolor exert differential immunomodulatory effects on blood lymphocytes and breast cancer cell line MCF-7 in vitro.

The protein-bound polysaccharides (PBP), isolated from Coriolus versicolor (CV) fungus, are considered as natural compounds with potential therapeutic applications. The immunopotentiating and antitumor activity of polysaccharopeptides has been previously examined, however similar findings could not be achieved. The source of PBP, variations in extraction process as well as environmental factors seems to affect the biological properties of these active CV components. Since further analysis are needed to draw more definite conclusion, the present study aimed to investigate the immunomodulatory properties of the PBP extract, isolated from commercially available capsules of C. versicolor. Our results revealed that the effect mediated by PBP extract depends on the target cells. We reported that the polysaccharopeptides induced a significant decrease in breast cancer MCF-7 cells growth, which was TNF-α-dependent phenomenon. Interestingly, the level of two others cytokines, IL-1ß and IL-6 was not affected. On the other hand, in this study we noticed that protein-bound polysaccharides extracted from CV significantly augmented the proliferative response of blood lymphocytes in a time-dependent manner, which was associated with IL-6 and IL-1ß mRNA upregulation. Moreover we found that the cells response to PBP stimuli might be inversely related to its concentration.

Polysaccharide peptide from Coriolus versicolor induces interleukin 6-related extension of endotoxin fever in rats.

PURPOSE: Polysaccharide peptide (PSP) extracted from the Coriolus versicolor mushroom is frequently suggested as an adjunct to the chemo- or radiotherapy in cancer patients. In a previous study we showed that PSP induced a tumour necrosis factor-α (TNF-α)-dependent anapyrexia-like response in rats. Thus, PSP appears to be a factor which modifies a number of pathophysiological responses. Because of this, PSP is suggested as a potential adjuvant for cancer therapy during which cancer patients frequently contract microbial infections accompanied by fever. The aim of the present study was to investigate whether or not PSP can modulate the course of the fever in response to an antigen such as lipopolysaccharide (LPS). MATERIALS AND METHODS: Body temperature (Tb) of male Wistar rats was measured by biotelemetry. PSP was injected intraperitoneally (i.p.) at a dose of 100 mg kg(-1), 2 h before LPS administration (50 µg kg(-1), i.p.). The levels of interleukin (IL)-6 and TNF-α in the plasma of rats were estimated 3 h and 14 h post-injection of PSP using a standard sandwich ELISA kit. RESULTS: We report that i.p. pre-injection of PSP 2 h before LPS administration expanded the duration of endotoxin fever in rats. This phenomenon was accompanied by a significant elevation of the blood IL-6 level of rats both 3 h and 14 h post-injection of PSP. Pre-treatment i.p. of the rats with anti-IL-6 antibody (30 µg/rat) prevented the PSP-induced prolongation of endotoxin fever. CONCLUSIONS: Based on these data, we conclude that PSP modifies the LPS-induced fever in IL-6-related fashion.

Analysis of the involvement of cytokines in allergy and breast cancer association.

AIM OF THE STUDY: The existence of a correlation between allergy disorders and cancer diseases has been confirmed by several epidemiological studies. Although the molecular mechanism involved in this phenomenon remains unknown, there are data indicating that certain cytokines, engaged in allergic processes, have antineoplastic activities. The aim of the present study was to explore the association between advanced breast cancer and allergic state on the molecular level. MATERIAL AND METHODS: We determined and compared the mRNA and protein expression of interleukin-1ß (IL-1ß), IL-4, IL-6, and interferon-γ (IFN-γ), cytokines known for antitumor properties, in the blood of advanced breast cancer patients and individuals with allergic diseases related to type 2 response. In addition, we performed an in vitro assay of reactivity of peripheral blood mononuclear cells after exogenous antigen stimulation. As a preliminary to molecular analysis we conducted a questionnaire study concerning the incidence of allergy among breast cancer patients and healthy subjects without malignancy. RESULTS: The results of the survey study revealed a negative relation between breast cancer and allergy prevalence. Subsequent molecular analysis, however, did not show statistically significant differences in cytokines mRNA and protein expression levels between allergic patients and those with malignancy. The in vitro reactivity test also did not reveal marked differences between IL-1ß, IL-4 and IL-6 production after PBMC triggering with exogenous antigen. CONCLUSIONS: We concluded that the studied cytokines (IL-1ß, IL-4, IL-6, and IFN-γ) are not engaged in breast cancer-allergy negative relation.

Cell extract-containing medium for culture of intracellular fastidious bacteria.

The culture of fastidious microorganisms is a critical step in infectious disease studies. As a proof-of-concept experiment, we evaluated an empirical medium containing eukaryotic cell extracts for its ability to support the growth of Coxiella burnetii. Here, we demonstrate the exponential growth of several bacterial strains, including the C. burnetii Nine Mile phase I and phase II strains, and C. burnetii isolates from humans and animals. Low-oxygen-tension conditions and the presence of small hydrophilic molecules and short peptides were critical for facilitating growth. Moreover, bacterial antigenicity was conserved, revealing the potential for this culture medium to be used in diagnostic tests and in the elaboration of vaccines against C. burnetii. We were also able to grow the majority of previously tested intracellular and fastidious bacterial species, including Tropheryma whipplei, Mycobacterium bovis, Leptospira spp., Borrelia spp., and most putative bioterrorism agents. However, we were unable to culture Rickettsia africae and Legionella spp. in this medium. The versatility of this medium should encourage its use as a replacement for the cell-based culture systems currently used for growing several facultative and putative intracellular bacterial species.

Axenic culture of fastidious and intracellular bacteria.

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.

Protein candidates for the serodiagnosis of rickettsioses.

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.

Protein candidates for Q fever serodiagnosis.

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.

Proteomics paves the way for Q fever diagnostics.

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.

Characterization of rickettsial adhesin Adr2 belonging to a new group of adhesins in α-proteobacteria.

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.

Identification of candidate proteins for the diagnosis of Bartonella henselae infections using an immunoproteomic approach.

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.

Global proteomic pattern of Tropheryma whipplei: a Whipple's disease bacterium.

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.

An immunoproteomic approach for identification of clinical biomarkers of Whipple's disease.

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.

Advances in Tropheryma whipplei research: the rush to find biomarkers for Whipple's disease.

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.

Production of monoclonal antibodies to Tropheryma whipplei and identification of recognized epitopes by two-dimensional electrophoresis and mass spectrometry.

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 of T. 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 of T. whipplei in 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 of T. whipplei in 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.

Identification of candidate antigen in Whipple's disease using a serological proteomic approach.

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.