Coiled-coil irregularities of the M1 protein structure promote M1-fibrinogen interaction and influence group A Streptococcus host cell interactions and virulence

Group A Streptococcus (GAS) is a human pathogen causing a wide range of mild to severe and life-threatening diseases. The GAS M1 protein is a major virulence factor promoting GAS invasiveness and resistance to host innate immune clearance. M1 displays an irregular coiled-coil structure, including the B-repeats that bind fibrinogen. Previously, we found that B-repeat stabilisation generates an idealised version of M1 (M1*) characterised by decreased fibrinogen binding in vitro. To extend these findings based on a soluble truncated version of M1, we now studied the importance of the B-repeat coiled-coil irregularities in full length M1 and M1* expressed in live GAS and tested whether the modulation of M1-fibrinogen interactions would open up novel therapeutic approaches. We found that altering either the M1 structure on the GAS cell surface or removing its target host protein fibrinogen blunted GAS virulence. GAS expressing M1* showed an impaired ability to adhere to and to invade human endothelial cells, was more readily killed by whole blood or neutrophils and most importantly was less virulent in a murine necrotising fasciitis model. M1-mediated virulence of wild-type GAS was strictly dependent on the presence and concentration of fibrinogen complementing our finding that M1-fibrinogen interactions are crucial for GAS virulence. Consistently blocking M1-fibrinogen interactions by fragment D reduced GAS virulence in vitro and in vivo. This supports our conclusion that M1-fibrinogen interactions are crucial for GAS virulence and that interference may open up novel complementary treatment options for GAS infections caused by the leading invasive GAS strain M

Creation of multiple nanodots by single ions

In the challenging search for tools that are able to modify surfaces on the nanometer scale, heavy ions with energies of several 10 MeV are becoming more and more attractive. In contrast to slow ions where nuclear stopping is important and the energy is dissipated into a large volume in the crystal, in the high energy regime the stopping is due to electronic excitations only. Because of the extremely local (< 1 nm) energy deposition with densities of up to 10E19 W/cm^2, nanoscaled hillocks can be created under normal incidence. Usually, each nanodot is due to the impact of a single ion and the dots are randomly distributed. We demonstrate that multiple periodically spaced dots separated by a few 10 nanometers can be created by a single ion if the sample is irradiated under grazing angles of incidence. By varying this angle the number of dots can be controlled.Comment: 12 pages, 6 figure

Discovery of microRNAs and other small RNAs in solid tumors

MicroRNAs (miRNAs) are ∼22-nt long, non-coding RNAs that regulate gene silencing. It is known that many human miRNAs are deregulated in numerous types of tumors. Here we report the sequencing of small RNAs (17–25 nt) from 23 breast, bladder, colon and lung tumor samples using high throughput sequencing. We identified 49 novel miRNA and miR-sized small RNAs. We further validated the expression of 10 novel small RNAs in 31 different types of blood, normal and tumor tissue samples using two independent platforms, namely microarray and RT–PCR. Some of the novel sequences show a large difference in expression between tumor and tumor-adjacent tissues, between different tumor stages, or between different tumor types. We also report the identification of novel small RNA classes in human: highly expressed small RNA derived from Y-RNA and endogenous siRNA. Finally, we identified dozens of new miRNA sequence variants that demonstrate the existence of miRNA-related SNP or post-transcriptional modifications. Our work extends the current knowledge of the tumor small RNA transcriptome and provides novel candidates for molecular biomarkers and drug targets

Variability in SCC<it>mec</it>_N1spreading among injection drug users in Zurich, Switzerland

<p>Abstract</p> <p>Background</p> <p>An extremely low level methicillin resistant <it>Staphylococcus aureus </it>(MRSA) belonging to ST45, circulates among intravenous drug users in the Zurich area. This clone can be misinterpreted as an MSSA by phenotypic oxacillin resistance tests, although it carries a staphylococcal cassette chromosome <it>mec </it>(SCC<it>mec</it>) element encoding a functional <it>mecA </it>gene and it produces PBP2a.</p> <p>Results</p> <p>This clone carried a new 45.7-kb element, termed SCC<it>mec</it>_N1, containing a class B <it>mec </it>complex (<it>mecA-</it>Δ<it>mecR1::IS1272</it>), a truncated Tn<it>4003 </it>harbouring the <it>dfrA </it>gene, and a <it>fusB1 </it>gene, conferring methicillin, trimethoprim and low level fusidic acid resistance, respectively. In addition to the two insertion site sequences (ISS) framing the SCC<it>mec</it>, a third ISS (ISS*) was identified within the element. SCC<it>mec</it>_N1also harboured two distinct <it>ccrAB </it>complexes belonging to the class 4 subtype, both of which were shown to be active and to be able to excise the SCC<it>mec</it>_N1or parts thereof. Slight variations in the SmaI-PFGE pattern of the clinical MRSA isolates belonging to this clone were traced back to differences in the sizes of the SCC<it>mec </it>J2 regions and/or to a 6.4-kb deletion extending from ISS* to the right end ISS. This latter deletion led to a variant right SCC<it>mec</it>-chromosomal junction site. MRSA clones carrying the shorter SCC<it>mec </it>with the 6.4-kb deletion were usually ciprofloxacin resistant, while strains with the complete SCC<it>mec</it>_N1were co-trimoxazole resistant or had no additional resistances. This suggested that the genetic backbone of the host <it>S. aureus</it>, although identical by PFGE pattern, had at some stage diverged with one branch acquiring a sulfonomide resistance mutation and the other ciprofloxacin resistance.</p> <p>Conclusion</p> <p>This description of the structure and variations of SCC<it>mec</it>_N1will allow for quicker and easier molecular detection of this clone and monitoring of its spread.</p

Fitness Cost of SCCmec and Methicillin Resistance Levels in Staphylococcus aureus

Transformation of a type I SCCmec element into Staphylococcus aureus yielded highly oxacillin-resistant transformants with a reduced growth rate. Faster-growing variants could again be selected at the cost of reduced resistance levels, demonstrating an inverse correlation between oxacillin resistance levels and growth rate

Selection vector for direct cloning of proof reading polymerase chain reaction products based on the lethal ccdB gene in Escherichia Coli

Introducing PCR products into plasmids vectors is key for molecular techniques. Ideally cloning vectors are easy to construct, modify and propagate, neither require advanced techniques nor special equipment or reagents and efficiently incorporate PCR products at close to zero empty vector background. We provide an easy to engineer self-made cloning vector, neither requiring sophisticated tools or techniques nor advanced cloning knowledge. Through recombination we obtained the pUC18ccdB vector, carrying the ccdB suicide gene within the pUC18 backbone. When SmaI cleaved (within the ccdB) vector was T4 ligated with small (0.2kbp) and intermediate (1.3 to 2.2kbp) blunt end PCR-products and transformed into E. coli, the amount of clones with incorporated PCR product was comparable to commercial PCR-cloning kits and at a close to zero PCR product negative background. In conclusion we present a simple, versatile and cheap approach to an efficient ‘home made’ PCR-cloning vector that allows integration of crude blunt end PCR products at close to zero background

Molecular Epidemiology of Methicillin-Resistant Staphylococcus aureus in Zürich, Switzerland (2003): Prevalence of Type IV SCCmec and a New SCCmec Element Associated with Isolates from Intravenous Drug Users

The majority of methicillin-resistant Staphylococcus aureus (MRSA) isolates, recovered in 2003 at the Department of Medical Microbiology in Zürich, Switzerland, belonged to major clones that are circulating worldwide. Staphylococcal cassette chromosome mec type IV (SCCmec-IV), harbored by half of the isolates, was found in sequence type 217 (ST217), which is an allelic variant of epidemic MRSA-15 (designated EMRSA-15), in a new local ST617 descending from clonal complex CC8 and in low-level oxacillin-resistant strains of multiple genetic lineages characteristic of community-onset MRSA. SCCmec-I, SCCmec-II, and SCCmec-III were in the minority, and four MRSA isolates had complex, rearranged SCCmec elements. A novel SCCmec-N1 of approximately 30 kb, associated with a dfrA gene and a ccr4-related recombinase complex, was identified in a large number of low-level oxacillin-resistant isolates, which descended from the successful clonal complex CC45 and are spreading among intraveneous drug users. In contrast, the SCCmec types of oxacillin-resistant coagulase-negative staphylococci (MRCNS) were of completely different composition. SCCmec type I (SCCmec-I) and SCCmec-II were more frequent than in the MRSA, while fewer contained SCCmec-IV. The other MRCNS displayed 11 different, complex patterns, suggesting frequent recombination between different SCCmec elements. With one ccr-negative exception, these strains amplified between one and three different ccr products, indicating either new varied complexes or multiple ccr loci. This suggests the presence of novel SCCmec types in MRCNS and no extensive interspecies SCCmec transfer between MRSA and MRCNS

GAS strains cleaving PAR-1 expressed functional SpeB.

<p>(<b>A</b>) 293T cells transiently expressing alkaline phosphatase tagged PAR-1 were incubated with overnight supernatants of well characterized clinical GAS isolates M1T1, M49 and new clinical GAS isolates (Cl1-8). Then AP-PAR-1 cleavage was assessed. Thrombin (IIa; 1nM) served as positive control. (<b>B</b>) Samples from (A) were analysed for proteolytic SpeB activity by evaluating Bz-Pro-Phe-Arg-Nan cleavage. Experiments were repeated at least 3 times with N=9, data presented as mean+/- SEM. </p