Untersuchung zur antibakteriellen Wirkung und zum Biosynthese-Gencluster des Peptidantibiotikum Feglymycin

Feglymycin ist ein aus Streptomyces sp. DSM 11171 isoliertes, lineares 13mer-Peptid, das zu einem hohen Anteil aus den nicht-proteinogenen Aminosäuren Hpg (4-Hydroxyphenylglycine) und Dpg (3,5-Dihydroxyphenylglycine) besteht. Zudem besitzt es eine interessante, alternierende Abfolge von D- und L- Aminosäuren und strukturelle Ähnlichkeiten mit den Glycopeptiden der Vancomycin-Gruppe von Antibiotika und den Glycodepsipeptid-Antibiotika Ramoplanin und Enduracidin. Außerdem besitzt Feglymycin eine interessante Bioaktivität. Es wirkt in vivo antibakteriell gegen MRSA-Stämme (multi-resistente Staphylococcus aureus Stämme) und inhibitiert in vitro die Replikation von HIV-Viren im Zellkulturtest. Aufgrund seiner molekularen Masse und strukturellen Ähnlichkeit mit bekannten Zellwandbiosynthese-Inhibitoren wie z.B. Vancomycin und Ramoplanin, wurde auch Feglymycin als Zellwandbiosynthese-Inhibitor getestet. In diesen Tests zeigte Feglymycin keinen Effekt auf die membran-gebundenen zweite und die dritte Stufe der Peptidoglycanbiosynthese. Jedoch deuteten die Experimente auf eine Inhibition der früheren Biosyntheseschritte hin. Ziel dieser Arbeit war es, die antibakterielle Wirkung von Feglymycin auf die bakterielle Zellwandbiosynthese im Detail zu untersuchen und das biologische Target zu identifizieren. Zusätzlich wurde das Feglymycin Biosynthese-Gencluster untersucht. In LC-MS „one-pot assays“ wurde die Wirkung von Feglymycin auf die isolierten E. coli-Enzyme MurA-F getestet. Hierbei konnte reproduzierbar gezeigt werden, das Feglymycin die Enzyme MurA (Enopyruvyl-UDP-GlcNAc Synthase) und MurC (UDP-N-Acetyl-muramyl-L-alanin Ligase) inhibiert. In spektrophotometischen Assays mit den E. coli-Enzymen MurA und MurC konnte ein Ki-Wert von 0.33 +/- 0.04 μM für das MurC Enzym und ein Ki-Wert von 3.4 +/- 1.1 μM für das MurA Enzym bestimmt werden. Weitere Untersuchungen zeigten, dass Feglmycin auch die MurA (IC50 = 3.5 +/- 1.3 μM) und MurC (IC50 = 1.0 +/- 0.6 μM) Enzyme des gram-positiven Bakteriums Staphylococcus aureus inhibiert. Feglymycin zeigte dabei eine nicht-kompetitive Inhibition gegenüber der Bindung der Substrate des MurA Enzyms PEP (Phosphoenolpyruvat) und UDP-GlcNAc (UDP-N-Acetylglucosamin) und der Substrate des MurC Enzyms UDP-MurNAc (UDP-N-Acetylmuramat), ATP (Adenosintriphosphat) and L-Alanin. Feglmycin ist daher der erste Naturstoff der das MurC Enzym inhibiert. Zudem zeigt Feglymycin einen nicht-kompetitive Inhibitionstyp. Circulardichromismus (CD) Experimente mit den isolierten E. coli-Enzymen MurA und MurC und Feglymycin deuten einen möglichen allosterischen Effekt des Inhibitors auf die Enzyme an. Zusätzlich wurde die Feglmycinproduktion durch den Stamm Streptomyces sp. DSM 11171 und die Feglymycin-Detektion mittels LC-MS optimiert. Durch die Sequezierung des Genoms von Streptomyces sp. DSM 11171 konnte das Feglymycin Biosynthese-Gencluster identifiziert werden. Bei der Annotation des Genclusters zeigte sich, dass es sich bei Feglymycin um ein nicht-ribosomal synthetisiertes Peptid (NRPS) handelt dessen Biosynthese der Biosynthese der Glycopeptidantibiotika der Vancomycin-Gruppe von Antibiotika ähnelt. Zudem konnten im Streptomyces sp. DSM 11171 Genom weitere NRPS und Polyketidsynthase (PKS) Gencluster identifiziert und annotiert werden.Feglymycin is a linear 13-mer peptide produced by Streptomyces sp. DSM 11171 containing largely the non-proteinogenic Hpg (4-hydroxyphenylglycine) and the non-proteinogenic Dpg (3,5-dihydroxyphenylglycine) amino acids and an interesting alternation of D and L amino acids. It shows structural homogies to the glycopeptides of the vancomycin group of antibiotics and the glycodepsipeptide antibiotics ramoplanin and enduracidin. Feglymycin additionally shows an interesting biological activity. It possesses antibiotic activity against MRSA (multi-resistant Staphylococcus aureus) strains in vivo and inhibits syncytium formation in HIV infection in vitro. Due to its molecular mass and structural analogies to known inhibitors of the cell-wall biosynthesis, i.e. vancomycin and ramoplanin, feglymycin was tested as cell-wall biosynthesis inhibitor. In these tests feglymycin showed no effect on the membrane-bound second and third step of the peptidoglycan biosynthesis but the experiments indicated an inhibition of earlier biosynthetic steps. Aim of this work was to investigate the antibacterial activity of feglymycin on the bacterial cell-wall biosynthesis in more detail and to identify the biological target. Additionally the feglymycin biosynthesis gene cluster was investigated. Feglymycin was tested in a LC-MS one-pot assay against the isolated enzymes MurA-F from E. coli. Dereplication revealed that feglymycin specifically inhibits the enzymes MurA (enolpyruvyl-UDP-GlcNAc synthase) and MurC (UDP-N-acetyl-muramyl-L-alanine ligase). In in vitro assays with the enzymes MurA and MurC from gram-negative E. coli, a Ki value of 0.33 +/- 0.04 μM was determined for the MurC enzyme and a Ki value of 3.4 +/- 1.1 μM for the MurA enzyme. Further investigations showed that feglymycin also inhibits the MurA (IC50 = 3.5 +/- 1.3 μM) and MurC (IC50 = 1.0 +/- 0.6 μM) enzyme from gram-positive Staphylococcus aureus. The inhibition mode of feglymycin was found to be non-competitive with the binding of PEP (phosphoenolpyruvate) and UDP-GlcNAc (UDP-N-acetylglucosamine) in case of the MurA enzyme and non-competitive with binding of UDP-MurNAc (UDP-N-acetylmuramic acid), ATP (adenosine-triphosphate) and L-alanine in case of the MurC enzyme. Feglymycin is therefore the first natural compound found to inhibit the MurC enzyme showing a non-competitive inhibition type. Circular dichroism (CD) experiments with the isolated enzymes MurA and MurC from E. coli and feglymycin indicated a possible allosteric effect of feglymycin. Furthermore the feglymycin production by Streptomyces sp. DSM 11171 and feglymycin detection by LC-MS were optimized. Sequencing of the genome of Streptomyces sp. DSM 11171 allowed the idenfitication of the feglymycin biosynthesis gene cluster. Annotation of the gene cluster showed that feglymycin is a non-ribosomal synthesized peptide (NRPS) closely related to the glycopeptides of the vancomycin group of antibiotics. Additionally further NRPS and polyketide synthase (PKS) gene clusters were identified in the Streptomyces sp. DSM 11171 genome and annotated

Genome mining reveals a novel and promising NRPS gene cluster in #Xanthomonas albilineans#, #Xanthomonas oryzae# and #Xanthomonas translucens#

International audienceVarious bacteria use non-ribosomal peptide synthesis (NRPS) to produce peptides or other small molecules. These molecules exhibit broad structural diversity and display biological activities that range from adaptation to unfavorable environments, communication or competition with other microorganisms in their natural habitat, or even to action as virulence factors. Conserved features within the NRPS machinery allow the type, and sometimes even the structure, of the synthesized polypeptide to be predicted. Thus, bacterial genome mining via in siIico analyses of NRPS genes offers an attractive opportunity to uncover new bioactive non-ribosomally synthesized peptides. To date, the only known small molecule synthesized by NRPS in the genus Xanthomonas is albicidin produced by Xanthomonas albilineans, a xylem-invading pathogen that causes leaf scald-a lethal disease of sugarcane. In silica analysis of available genomic sequences of Xanthomonas strains led to the discovery of a novel NRPS gene cluster called META-B which doesn't resemble to any gene cluster de- scribed to date. This NRPS gene cluster occurs in (i) X. albilineans, (ii) two pathovars of Xanthomonas oryzae which are the causal agents of two agronomically important diseases of rice (bacterial leaf blight caused by X. oryzae pv. oryzae and bacterial leaf streak caused by X. oryzae pv. oryzicola), and (iii) Xanthomonas translucens , the causal agent of the bacterial leaf streak of wheat. Interestingly, the NRPS gene cluster META-B seems to be specific to strains of Xanthomonas associated with monocotyledonous plants, suggesting a putative involvement in plant-bacteria interactions. (Résumé d'auteur

Data from: Genome mining reveals the genus Xanthomonas to be a promising reservoir for new bioactive non-ribosomally synthesized peptides

Background: Various bacteria can use non-ribosomal peptide synthesis (NRPS) to produce peptides or other small molecules. Conserved features within the NRPS machinery allow the type, and sometimes even the structure, of the synthesized polypeptide to be predicted. Thus, bacterial genome mining via in silico analyses of NRPS genes offers an attractive opportunity to uncover new bioactive non-ribosomally synthesized peptides. Xanthomonas is a large genus of Gram-negative bacteria that cause disease in hundreds of plant species. To date, the only known small molecule synthesized by NRPS in this genus is albicidin produced by Xanthomonas albilineans. This study aims to estimate the biosynthetic potential of Xanthomonas spp. by in silico analyses of NRPS genes with unknown function recently identified in the sequenced genomes of X. albilineans and related species of Xanthomonas. Results: We performed in silico analyses of NRPS genes present in all published genome sequences of Xanthomonas spp., as well as in unpublished draft genome sequences of Xanthomonas oryzae pv. oryzae strain BAI3 and Xanthomonas spp. strain XaS3. These two latter strains, together with X. albilineans strain GPE PC73 and X. oryzae pv. oryzae strains X8-1A and X11-5A, possess novel NRPS gene clusters and share related NRPS-associated genes such as those required for the biosynthesis of non-proteinogenic amino acids or the secretion of peptides. In silico prediction of peptide structures according to NRPS architecture suggests eight different peptides, each specific to its producing strain. Interestingly, these eight peptides cannot be assigned to any known gene cluster or related to known compounds from natural product databases. PCR screening of a collection of 94 plant pathogenic bacteria indicates that these novel NRPS gene clusters are specific to the genus Xanthomonas and are also present in Xanthomonas translucens and X. oryzae pv. oryzicola. Further genome mining revealed other novel NRPS genes specific to X. oryzae pv. oryzicola or Xanthomonas sacchari. Conclusions: This study revealed the significant potential of the genus Xanthomonas to produce new non-ribosomally synthesized peptides. Interestingly, this biosynthetic potential seems to be specific to strains of Xanthomonas associated with monocotyledonous plants, suggesting a putative involvement of non-ribosomally synthesized peptides in plant-bacteria interactions

Genome mining reveals a novel and promising NRPS gene cluster in #Xanthomonas albilineans#, #Xanthomonas oryzae# and #Xanthomonas translucens#

International audienceVarious bacteria use non-ribosomal peptide synthesis (NRPS) to produce peptides or other small molecules. These molecules exhibit broad structural diversity and display biological activities that range from adaptation to unfavorable environments, communication or competition with other microorganisms in their natural habitat, or even to action as virulence factors. Conserved features within the NRPS machinery allow the type, and sometimes even the structure, of the synthesized polypeptide to be predicted. Thus, bacterial genome mining via in siIico analyses of NRPS genes offers an attractive opportunity to uncover new bioactive non-ribosomally synthesized peptides. To date, the only known small molecule synthesized by NRPS in the genus Xanthomonas is albicidin produced by Xanthomonas albilineans, a xylem-invading pathogen that causes leaf scald-a lethal disease of sugarcane. In silica analysis of available genomic sequences of Xanthomonas strains led to the discovery of a novel NRPS gene cluster called META-B which doesn't resemble to any gene cluster de- scribed to date. This NRPS gene cluster occurs in (i) X. albilineans, (ii) two pathovars of Xanthomonas oryzae which are the causal agents of two agronomically important diseases of rice (bacterial leaf blight caused by X. oryzae pv. oryzae and bacterial leaf streak caused by X. oryzae pv. oryzicola), and (iii) Xanthomonas translucens , the causal agent of the bacterial leaf streak of wheat. Interestingly, the NRPS gene cluster META-B seems to be specific to strains of Xanthomonas associated with monocotyledonous plants, suggesting a putative involvement in plant-bacteria interactions. (Résumé d'auteur

Mobile microscopy as a screening tool for oral cancer in India: A pilot study.

Oral cancer is the most common type of cancer among men in India and other countries in South Asia. Late diagnosis contributes significantly to this mortality, highlighting the need for effective and specific point-of-care diagnostic tools. The same regions with high prevalence of oral cancer have seen extensive growth in mobile phone infrastructure, which enables widespread access to telemedicine services. In this work, we describe the evaluation of an automated tablet-based mobile microscope as an adjunct for telemedicine-based oral cancer screening in India. Brush biopsy, a minimally invasive sampling technique was combined with a simplified staining protocol and a tablet-based mobile microscope to facilitate local collection of digital images and remote evaluation of the images by clinicians. The tablet-based mobile microscope (CellScope device) combines an iPad Mini with collection optics, LED illumination and Bluetooth-controlled motors to scan a slide specimen and capture high-resolution images of stained brush biopsy samples. Researchers at the Mazumdar Shaw Medical Foundation (MSMF) in Bangalore, India used the instrument to collect and send randomly selected images of each slide for telepathology review. Evaluation of the concordance between gold standard histology, conventional microscopy cytology, and remote pathologist review of the images was performed as part of a pilot study of mobile microscopy as a screening tool for oral cancer. Results indicated that the instrument successfully collected images of sufficient quality to enable remote diagnoses that show concordance with existing techniques. Further studies will evaluate the effectiveness of oral cancer screening with mobile microscopy by minimally trained technicians in low-resource settings

Tree of the amino acid sequences of C-domains of strains GPE PC73, XaS3, X11-5A, BAI3, and BLS256 together with C-domains identified by Rausch et al. as starter C-domains or as dual C/E-domains (Additional file 2).

The tree was constructed using the maximum likelihood method and GTR as substitution model. Bootstrap percentages retrieved in 100 replications are shown at the main nodes. The scale bar (0.2) indicates the number of amino acid substitutions per site. C-domains belonging to the same clade as dual C/E-domains are in blue. C-domains belonging to the same clade as starter C-domains are in red. Putative starter C-domains of the loci META-A and META-C of strain GPE PC73, the contig G111 of strain XaS3 and the locus of strain BTAi similar to META-A and META-C are in green. C-domains Ax, Bx ad Cx correspond to C-domains of modules of the loci META-A, META-B and META-C of strain GPE PC73, respectively. C-domains Ox correspond to C-domains of modules of the locus META-B of strain BAI3. C-domains USxxx/x correspond to C-domains of modules of contigs of strain X11-5A. C-domains Gxxx/x correspond to C-domains of modules of contigs of strain XaS3. C-domains bradyx correspond to C-domains of the locus of Bradyrhizobium spp. strain BTAi similar to META-A and META-C (genes Bbta_6814, Bbta_6813, Bbta_6812). C-domains XOCx correspond to C-domains of the locus NRPS located in the same region as XaPPTase in strain BLS256. C-domains 0364 and 1145 correspond to C-domains of short NRPS genes XALc_0364 and XALc_1145 of strain GPE PC73, respectively. C-domain 0354XaS3 corresponds to the short NRPS gene of strain XaS3. C-domain Bbta4110 corresponds to the short NRPS gene of strain BTAi. C-domains identified by Rausch et al. [6] as starter C-domains were tagged “Starter1” to “Starter15” as follows: Starter1: Pseusyrin.NP_792633.1.m_1_leu Starter2: Pseusp.Q84BQ6.arfA_1_leu Starter3: Pseufluor.YP_259252.1.m_1_leu Starter4: Baciliche.YP_077640.1.lchAA_1_gln Starter5: Nocafarci.YP_117314.1.m_1_orn_lys_arg Starter6: Nocafarci.YP_119006.1.m_1_tyr Starter7: Nocafarci.YP_119328.1.m_1_ser Starter8: Nocafarci.YP_121279.1.m_1_ser Starter9: Strecoeli.NP_627443.1.m_1_ser Starter10: Strchrys.O68487.acmB_1_thr Starter11: Erwicarot.YP_049593.1.m_1_gln Starter12: Strprist.Q54959.snbC_1_thr Starter13: Bacisubti.NP_388230.1.srfAA_1_glu Starter14: Bacisubti.NP_389716.1.ppsA_1_glu Starter15: Baciliche.YP_090052.1.m_1_gln C-domains identified by Rausch et al. [6] as Dual C/E-domains were tagged “DualC/E1” to “DualC/E18” as follows: DualC/E1:Photlumin.NP_929905.1.m_9_thr_TO_val DualC/E2:Photlumin.NP_930489.1.m_2_val_TO_trp DualC/E3:Photlumin.NP_929905.1.m_6_bht_TO_trp DualC/E4:Bradjapon.NP_768748.1.m_3_ser_TO_phe DualC/E5:Chroviola.NP_902472.1.m_3_val_TO_ile_dual DualC/E6:Chroviola.NP_902472.1.m_1_thr_dual DualC/E7:Burkmalle.YP_106216.1.m_2_glu_TO_gly DualC/E8:Burkpseud.YP_111641.1.m_3_thr_TO_leu DualC/E9:Burkpseud.YP_111641.1.m_1_glu_gln DualC/E10:Pseusyrin.NP_792633.1.m_2_leu_TO_leu DualC/E11:Ralssolan.NP_522203.1.m_3_ser_TO_gly DualC/E12:Ralssolan.NP_522203.1.m_1_val DualC/E13:Pseufluor.YP_259253.1.m_4_leu_TO_ser DualC/E14:Pseufluor.YP_259253.1.m_2_thr_TO_ile DualC/E15:Pseusyrin.NP_792634.1.m_3_thr_TO_val DualC/E16:Pseusyrin.NP_792634.1.m_5_leu_TO_leu DualC/E17:Erwicarot.YP_049592.1.m_4_ser_TO_tyr_bht DualC/E18:Erwicarot.YP_049593.1.m_2_gln_TO_as