Investigation of rifampicin resistance mechanisms in Brucella abortus using MS-driven comparative proteomics

Mutations in the rpoB gene have already been shown to contribute to rifampicin resistance in many bacterial strains including Brucella species. Resistance against this antibiotic easily occurs and resistant strains have already been detected in human samples. We here present the first research project that combines proteomic, genomic, and microbiological analysis to investigate rifampicin resistance in an in vitro developed rifampicin resistant strain of Brucella abortus 2308. In silico analysis of the rpoB gene was performed and several antibiotics used in the therapy of Brucellosis were used for cross resistance testing. The proteomic profiles were examined and compared using MS-driven comparative proteomics. The resistant strain contained an already described mutation in the rpoB gene, V154F. A correlation between rifampicin resistance and reduced susceptibility on trimethoprim/sulfamethoxazole was detected by E-test and supported by the proteomics results. Using 12?836 MS/MS spectra we identified 6753 peptides corresponding to 456 proteins. The resistant strain presented 39 differentially regulated proteins most of which are involved in various metabolic pathways. Results from our research suggest that rifampicin resistance in Brucella mostly involves mutations in the rpoB gene, excitation of several metabolic processes, and perhaps the use of the already existing secretion mechanisms at a more efficient level.     

Exosomes as Biomarker Enriched Microvesicles: Characterization of Exosomal Proteins Derived from a Panel of Prostate Cell Lines with Distinct AR Phenotypes

Prostate cancer is the leading type of cancer diagnosed in men. In 2010, ∼217,730 new cases of prostate cancer were reported in the United States. Prompt diagnosis of the disease can substantially improve its clinical outcome. Improving capability for early detection, as well as developing new therapeutic targets in advanced disease are research priorities that will ultimately lead to better patient survival. Eukaryotic cells secrete proteins via distinct regulated mechanisms which are either ER/Golgi dependent or microvesicle mediated. The release of microvesicles has been shown to provide a novel mechanism for intercellular communication. Exosomes are nanometer sized cup-shaped membrane vesicles which are secreted from normal and cancerous cells. They are present in various biological fluids and are rich in characteristic proteins. Exosomes may thus have potential both in facilitating early diagnosis via less invasive procedures or be candidates for novel therapeutic approaches for castration resistance prostate cancer. Because exosomes have been shown previously to have a role in cell-cell communication in the local tumor microenvironment, conferring activation of numerous survival mechanisms, we characterized constitutive lipids, cholesterol and proteins from exosomes derived from six prostate cell lines and tracked their uptake in both cancerous and benign prostate cell lines respectively. Our comprehensive proteomic and lipidomic analysis of prostate derived exosomes could provide insight for future work on both biomarker and therapeutic targets for the treatment of prostate cancer.Prostate cancer (PCa)¹ is the leading type of cancer diagnosed in men. The American Cancer Society reported 217,730 new cases of PCa in the United States last year. Death from PCa follows its incidence profile closely as the third leading cause of cancer-related death in men (1). In the early stages, the disease is locally confined to the prostate and is hormone or androgen-dependent. It can be managed at this stage by surgical intervention or radiation treatment. However, over time (varying from months to years), many prostate cancers metastasize and, even with aggressive hormone deprivation therapy, progress to castration resistant prostate cancer (CRPC), which ultimately results in death. During early metastasis, a response to androgen deprivation therapy (ADT) is usually observed. Nonetheless, despite the reduction in androgen levels after ADT, androgen receptor (AR) remains active and contributes to CRPC progression (2–4).The routine screening test for PCa diagnosis in North America includes measurement of prostate specific antigen (PSA) in the blood, digital rectal examination and a prostate biopsy (5). PSA screening for PCa detection is controversial because certain activities can induce the production of PSA, unrelated to the presence of cancer (6). Consequently prostate biopsy, albeit an invasive procedure, remains the only definitive diagnostic test for PCa. There is an urgent current need, therefore, for the discovery of relevant biomarkers to replace the existing diagnostic tests for better and earlier detection of PCa (7).One possible source of biomarkers which could be used as part of a diagnostic test are exosomes. All cells produce and release exosomes, which are often found in different body fluids such as plasma (8), serum (9, 10) malignant ascites (11, 12) urine (13), amniotic fluid (14), bronchoalveolar lavage fluid (15, 16), and breast milk (17, 18). Recent studies suggest however that cancer cells produce exosomes, which may be differentiated from those derived from normal cells primarily based upon their cargo. Exosomes are cup-shaped (19) encapsulated by a bi-layer lipid membrane (20) with a membrane-bound compartment varying between 30–100 nm in size (19). As mentioned above, they are secreted from both normal cells and tumor cells (21) and although the underlying mechanism of exosome function is not fully understood it is known that exosomes are formed in the endosomal compartment of cells and are secreted upon fusion of multivesicular bodies (MVB) with the plasma membrane (21). The schematic cartoon in Fig. 1 depicts early endosome (EE) formation as a result of the invagination of specific regions of the plasma membrane. In addition, endocytotic cargo transported out of the cell is sorted from EE into intraluminal vesicles (ILV). Mechanisms involved in protein sorting into ILVs are still under investigation however there is evidence supporting the involvement of ubiquitin and endosomal sorting complex required for transport (ESCRT machinery) in this process. Finally, fusion of late endosome or MVB with plasma membrane releases ILVs into the extracellular matrix or the tissue microenvironment. Accumulating evidence suggests that induction of intracellular calcium (22–25), overexpression of Rab11 or citron kinase (26) as well as a reduction in membrane cholesterol, or inhibition of cholesterol biosynthesis (27), could stimulate the release of exosomes into the microenvironment.Open in a separate windowFig. 1.Mechanism involved in exosome formation and trafficking in the microenvironment.As shown in Fig. 1, once released, exosomes will interact with recipient target cells via different mechanisms such as fusion with the plasma membrane or adhesion to corresponding receptors on the plasma membrane (25).Although, the mechanisms underlying exosome formation and secretion is still under investigation, it is well-known that factors such as cell type, cell cycle, and stage of cancer, could affect the amount and composition of exosomes formed and secreted from various cells (19). It has been shown that exosomes are secreted in a multitude of cell types and though it is postulated that they are involved in membrane trafficking as communication vesicles, their relevance in cancer initiation and specifically prostate tumor growth and progression has yet to be determined (28–30). Studies on tumor-related microvesicles suggest that exosomes play a significant role in cell communication thus potentially influencing cancer progression via different mechanisms (31). Exosomes contain and protect the integrity of various proteins and an array of lipids, mRNA and miRNA which would otherwise be hydrolytically or enzymatically broken down if they existed as free soluble molecules in the extracellular microenvironment. The presence of differential exosomal protein markers involved in cancer progression combined with the presence of exosomes in accessible biological fluids highlights a potential role of exosomes as clinical biomarkers for PCa at diagnosis and progression (32, 33). Therefore isolation, purification and characterization of exosomes derived from different body fluids is an essential first step in identifying novel biomarkers from this source.In addition, exosomes may also present novel therapeutic strategies. If in fact implicated in cancer progression, exosomes present a new target set for development of novel therapeutics. Hence, a better understanding of the mechanisms involved in formation and secretion of exosomes for therapeutic targeting as well as investigating the relevance of the presence of different proteins in these membrane vesicles is required.Therefore the main purpose of the present study was to observe the release of exosomes by prostate cells, and determine characteristic differences between exosomes released by parent cells of different characteristic AR phenotypes. In order to answer this question, in addition to one nonmalignant cell line, we used five different PCa cell lines which contain/lack AR and were representative of different stages of PCa.We then confirmed the transfer of exosomes to target cells in culture using confocal microscopy of fluorescence labeled exosomes. We subsequently performed a comprehensive proteomic analysis of all six different prostate cell lines using mass spectrometry to understand differences between the protein profiles released via exosome externalization in different prostate cell lines. The final part of this study was to investigate the difference in broad classes of lipids and cholesterol as constituents of different prostate cell lines and their exosomes.Taken together the comprehensive characterization of exosomes derived from prostate cell lines which have distinct AR ±ve phenotypes, provides a basis for evaluating transfer of identified composite exosome proteins between different PCa cells as part of a recognized cell communication phenomenon. In addition this study forms a platform for future clinical validation research using exosomes as biomarkers for PCa diagnosis as well as potential therapeutic targets which could be important in the treatment of CRPC.     

Aloe Emodin Reduces Phthiodiolone Dimycocerosate Potentiating Vancomycin Susceptibility on Mycobacteria

Treatment of tuberculosis still represent a major public health issue. The emergence of multi-and extensively-drug resistant (MDR and XDR) Mycobacterium tuberculosis clinical strains further pinpoint the urgent need for new anti-tuberculous drugs. We previously showed that vancomycin can target mycobacteria lacking cell wall integrity, especially those lacking related phthiocerol and phthiodolone dimycocerosates, PDIM A and PDIM B, respectively. As aloe emodin was previously hypothesized to be able to target the synthesis of mycobacterial cell wall lipids, we tested its ability to potentiate glycopeptides antimycobacterial activity. The aloe emodin with the vancomycin induced a combination effect beyond simple addition, close to synergism, at a concentration lower to reported IC₅₀ cytotoxic value, on M. bovis BCG and on H37Rv M. tuberculosis. Interestingly, out of six MDR and pre-XDR clinical strains, one showed a strong synergic susceptibility to the drug combination. Mycobacterial cell wall lipid analyses highlighted a selective reduction of PDIM B by aloe emodin.     

Genomic characterization of four novel bacteriophages infecting the clinical pathogen Klebsiella pneumoniae

Bacteriophages are an invaluable source of novel genetic diversity. Sequencing of phage genomes can reveal new proteins with potential uses as biotechnological and medical tools, and help unravel the diversity of biological mechanisms employed by phages to take over the host during viral infection. Aiming to expand the available collection of phage genomes, we have isolated, sequenced, and assembled the genome sequences of four phages that infect the clinical pathogen Klebsiella pneumoniae: vB_KpnP_FBKp16, vB_KpnP_FBKp27, vB_KpnM_FBKp34, and Jumbo phage vB_KpnM_FBKp24. The four phages show very low (0–13%) identity to genomic phage sequences deposited in the GenBank database. Three of the four phages encode tRNAs and have a GC content very dissimilar to that of the host. Importantly, the genome sequences of the phages reveal potentially novel DNA packaging mechanisms as well as distinct clades of tubulin spindle and nucleus shell proteins that some phages use to compartmentalize viral replication. Overall, this study contributes to uncovering previously unknown virus diversity, and provides novel candidates for phage therapy applications against antibiotic-resistant K. pneumoniae infections.