The Polycyclic Polyprenylated Acylphloroglucinol Antibiotic PPAP 23 Targets the Membrane and Iron Metabolism in Staphylococcus aureus

Recently, a series of endo-type B polycyclic polyprenylated acylphloroglucinols (PPAP) derivatives with high antimicrobial activities were chemically synthesized. One of the derivatives, PPAP 23, which showed high antimicrobial activity and low cytotoxicity, was chosen for further investigation of its bactericidal profiles and mode of action. PPAP 23 showed a better efficacy in killing methicillin resistant Staphylococcus aureus (MRSA) and decreasing the metabolic activity of 5-day-old biofilm cells than vancomycin. Moreover, S. aureus did not appear to develop resistance against PPAP 23. The antimicrobial mechanism of PPAP 23 was investigated by RNA-seq combined with phenotypic and biochemical approaches. RNA-seq suggested that PPAP 23 signaled iron overload to the bacterial cells because genes involved in iron transport were downregulated and iron storage gene was upregulated by PPAP 23. PPAP 23 affected the membrane integrity but did not induce pore formation; it inhibited bacterial respiration. PPAP 23 preferentially inhibited Fe–S cluster enzymes; it has a mild iron chelating activity and supplementation of exogenous iron attenuated its antimicrobial activity. PPAP 23 was more effective in inhibiting the growth of S. aureus under iron-restricted condition. The crystal structure of a benzylated analog of PPAP 23 showed a highly defined octahedral coordination of three PPAP ligands around a Fe (3+) core. This suggests that PPAPs are generally capable of iron chelation and are able to form defined stable complexes. PPAP 23 was found to induce reactive oxygen species (ROS) and oxidative stress. Fluorescence microscopic analysis showed that PPAP 23 caused an enlargement of the bacterial cells, perturbed the membrane, and dislocated the nucleoid. Taken together, we postulate that PPAP 23 interacts with the cytoplasmic membrane with its hydrophobic pocket and interferes with the iron metabolism to exert its antimicrobial activity in Staphylococcus aureus

Proteomic analysis of early responsive resistance proteins of wheat (Triticum aestivum) to yellow rust (Puccinia striiformis f. sp tritici) using ProteomeLab PF2D

WOS: 000317715600004Wheat (Triticum aestivum L.) yellow rust, caused by Puccinia striiformis f. sp. tritici (Pst), is one of the most destructive diseases of wheat worldwide. To clarify the molecular details and components of the resistance response in wheat offers further possibilities to combat yellow rust. In this study, differentially regulated early response proteins in wheat leaves infected by Pst isolates were investigated by proteomic approaches. Total proteins extracts from leaves harvested at 24 hour post inoculation (hpi) were separated by two dimensional liquid chromatography system, ProteomeLab PF2D. Following PF2D analysis, six hundred and thirty-seven protein peaks were compared one by one between protein patterns obtained from pathogen-and mock-inoculated leaf tissue. Among those differentially expressed 33 proteins were identified in Pst-infected plants as compared with mock-inoculated controls by nanoLC-ESI-MS/MS. Six proteins were exhibited homology to fungal proteins. Two fungal proteins, including E3 ubiquitin protein ligase and Ubiquitin-like protein, are important members of ubiquitin-proteasome system which the importance of the its proteolytic function in regulating the virulence of pathogenic fungi has just been realized recently. Other identified 27 proteins were host proteins in response to Pst and classified in five groups based on their roles in diverse biological processes. The results indicated that identified defence related proteins such as pathogene related protein 1 and 4 (PR1, PR4), Glutathione S transferase (GST) are major component for systemic acquired resistance (SAR) which is one of the strong disease resistance form in plants and appears within several days following the initial pathogen attack.TUBITAK, COST programmeTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [109T293]This study was supported by TUBITAK, COST programme 109T293 project. We thank Kadir Akan, Ayse Yildiz and Lutfi Cetin for their help during plant inoculation and sampling. We also thank Abdulmecit Gokce and Yavuz Ozturk for their technical support for PF2D as well as Konca Bulut and Rahmi Buyukkeskin for their experimental assistance

Proteomic investigation of compatible interaction between wheat and yellow rust

WOS: 000209805600356

Molecular Characterization of Measles Viruses in Turkey (2010-2011): First Report of Genotype D9 Involved in an Outbreak in 2011

Bakkaloglu, Zekiye/0000-0001-9137-016XWOS: 000325153400010PubMed: 23959542Genetic characterization of measles viruses (MVs) combined with acquisition of epidemiologic information is essential for measles surveillance programs used in determining transmission pathways. This study describes the molecular characterization of 26 MV strains (3 from 2010, 23 from 2011) obtained from urine or throat swabs harvested from patients in Turkey. MV RNA samples (n=26) were subjected to sequence analysis of 450 nucleotides comprising the most variable C-terminal region of the nucleoprotein (N) gene. Phylogenetic analysis revealed 20 strains from 2011 belonged to genotype D9, 3 to D4, 2 strains from 2010 to genotype D4 and 1 to genotype B3. This study represents the first report describing the involvement of MV genotype D9 in an outbreak in Turkey. The sequence of the majority of genotype D9 strains was identical to those identified in Russia, Malaysia, Japan, and the UK. Despite lack of sufficient epidemiologic information, the presence of variants observed following phylogenetic analysis suggested that exposure to genotype D9 might have occurred due to importation more than once. Phylogenetic analysis of five genotype D4 strains revealed the presence of four variants. Epidemiological information and phylogenetic analysis suggested that three genotype D4 strains and one genotype B3 strain were associated with importation. This study suggests the presence of pockets of unimmunized individuals making Turkey susceptible to outbreaks. Continuing molecular surveillance of measles strains in Turkey is essential as a means of acquiring epidemiologic information to define viral transmission patterns and determine the effectiveness of measles vaccination programs designed to eliminate this virus. J. Med. Virol. 85:2128-2135, 2013. (c) 2013 Wiley Periodicals, Inc.Ministry of Health, Turkish National Public Health Agency, Adnan Saygun cad Ankara, TurkeyMinistry of Health - Turkey [55, 06100]Grant sponsor: Ministry of Health, Turkish National Public Health Agency, Adnan Saygun cad. No. 55, 06100 Ankara, Turke

Proteome profiling of the compatible interaction between wheat and stripe rust

WOS: 000379860300018Over the last decade, comparative molecular profiling studies between compatible and incompatible plant-pathogen interactions have shown that susceptible response of the host to a pathogen requires factors that promote disease development. In this study, we examined proteome profiles during a compatible interaction between wheat and stripe rust. A 2D-LC system (ProteomeLab PF2D) was used for protein separation and to compare the proteome from infected and control samples. More than 700 protein peaks at each time point were compared between pathogen- and mock-inoculated samples. Selected proteins, with significant differences in abundance were identified by nanoLC-ESI- MS/MS and generated spectra were searched against the wheat protein databases from UniProt, and NCBI and the Puccinia database from The Broad Institute. In total, the identified proteins comprised of 62 % wheat and 38 % Pst proteins. All identified proteins were searched by bioinformatics-based algorithms to detect their subcellular localization and signal peptide motifs which have the potential to catch the candidate effector proteins. The wheat proteins were classified based on their function. Although a compatible interaction, many wheat proteins, such as antioxidants, PRs and cold-responsive proteins, are implicated in defense and stress tolerance. On the pathogen side, 64 proteins were identified, and included some important pathogenicity proteins that can play role in pathogen virulence and suppress the host defense. In addition, we discovered that nine proteins have a signal sequence and three of the hypothetical fungal proteins, PGTG_11681T0, PGTG_07231T0 and CBH50687.1, have been tentatively identified as candidate effectors.TUBITAKTurkiye Bilimsel ve Teknolojik Arastirma Kurumu (TUBITAK) [109 T293]This study was supported by TUBITAK, COST programme 109 T293 project. We thank Kadir Akan, Aysc Yildiz and Lutfi Cetin for their help during plant inoculation and sampling. We also thank Abdulmecit Gokce and Yavuz Ozturk for their technical support for PF2D as well as Konca Bulut and Rahmi Buyukkeskin for their experimental assistance

Evidence for heterogeneity in response to treatment in mammary tumors of dogs as happens in humans

Tumors are formed by various clones developed over a long time. This gives rise to a heterogeneous nature. This heterogeneity is the hardest challenge in the treatment of cancers because it is the main reason for drug resistance. This is a well-known fact in human cancer. Therefore, we have reasoned that if the tumor heterogeneity in canine mammary gland tumors (CMGTs) could be shown by an ex vivo assay, which will be used first time in veterinary oncology practice, this could be used further in clinics. To achieve this, twenty-six patients were included in the study. Tumor tissues were obtained from animals during routine surgery. Tumor cells were isolated and seeded ex vivo. The cells were exposed to anticancer drugs that are clinically used. Seven days after the treatment, chemosensitivity has luminometrically been assayed by ATP-tumor chemosensitivity assay (ATP-TCA). It has clearly been shown that all the tumor tissues have responded to treatment differently, implying that heterogeneity exists in mammary tumors. There has also been found that there was a weak to moderate statistically significant correlation between tumor size and drug index. However, there has been no correlation between drug index and metastasis to lymph nodes. Hyperplasic areas had relatively higher PCNA values. The results of our study demonstrate the heterogeneity in responses to in vitro drugs. Clinical trials based on test results and follow-up studies with large numbers of animals are needed to prove that such chemotherapeutic activity assessment tests can be clinically useful in predicting drug responses in CMGTs

Comparison of the anti-cancer activity of 5-aminolevulinic acid-mediated photodynamic therapy after continuous wave and pulse irradiation in different histological types of canine mammary sarcoma tumors

Canine mammary sarcoma tumors (CMST) are the most aggressive tumors with poor prognosis in dogs. Due to inadequate treatment options for CMST, recent studies have focused on alternative treatment strategies. We previously determined the optimized protocol of 5-ALA-based photodynamic therapy (PDT) in canine liposarcoma. However, its molecular mechanisms in the treatment of different histological types of CMST remain unclear.In this context, we, for the first time, assessed 5-aminolevulinic acid (5-ALA)-PDT-mediated anti-cancer activity and its molecular mechanism after continuous wave (CW) and pulse radiation (PR) on three different histological types (liposarcoma, chondrosarcoma, and osteosarcoma) of CMST cells by WST-1, Annexin V, ROS, acridine orange/propidium iodide staining, RT-PCR, and western blot analysis.Our findings showed that 5-ALA/PDT significantly suppressed the proliferation of CMST cells (p < 0.01) and induced apoptosis via increased ROS level and overexpression of Caspase-9 and Caspase-3 mRNA and cleaved protein levels in especially liposarcoma and chondrosarcoma cells following CW and PR irradiation at 9 J/cm(2). However, the response of CMST cells to 5-ALA was different upon CW and PR irradiation due to differences in their origin.Collectively, our findings provided the first evidence that 5-ALA-based PDT could be used as an alternative treatment strategy, especially liposarcoma and chondrosarcoma. However, further in vitro and in vivo studies are required to elucidate the underlying molecular mechanism of the efficacy of 5-ALA in CMST cells at the molecular level