Miriam Ender

Fitness Cost of SCCmec and Methicillin Resistance Levels in Staphylococcus aureus

ABSTRACT 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.

Fitness Cost of Staphylococcal Cassette Chromosome mec in Methicillin-Resistant Staphylococcus aureus by Way of Continuous Culture

ABSTRACT We examined the effect of introducing type I or IV staphylococcal cassette chromosome mec (SCCmec) elements on the growth yield of Staphylococcus aureus in glucose-limited continuous culture. Type I showed increased glucose consumption and ATP demand per gram of cells synthesized and decreased cell yield compared to those of the parent strain. In contrast, type IV SCCmec elements had no adverse energetic effect.

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

ABSTRACT 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.

Mosaic staphylococcal cassette chromosome mec containing two recombinase loci and a new mec complex, B2

ABSTRACT A novel staphylococcal cassette chromosome (SCC) mec from a clinical methicillin-resistant Staphylococcus aureus isolate (ST100/CC5) had a mosaic structure, composed of SCC DNA from several different backgrounds. It harbored two complete ccr loci and a new variant of mec complex B, with ΔmecR1 interrupted by the aminoglycoside resistance transposon Tn4001.

Protease-activated receptor-1 cleaved at R46 mediates cytoprotective effects.

Summary.  Background:  Activated protein C (aPC) mediates powerful cytoprotective effects through the protease‐activated receptor‐1 (PAR1) that translate into reduced harm in mouse injury models. However, it remains elusive how aPC‐activated PAR1 can mediate cytoprotective effects whereas thrombin activation does the opposite.

Variability in SCCmecN1 spreading among injection drug users in Zurich, Switzerland.

BackgroundAn extremely low level methicillin resistant Staphylococcus aureus (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 mec (SCCmec) element encoding a functional mecA gene and it produces PBP2a.ResultsThis clone carried a new 45.7-kb element, termed SCCmecN1, containing a class B mec complex (mecA- ΔmecR1::IS1272), a truncated Tn4003 harbouring the dfrA gene, and a fusB1 gene, conferring methicillin, trimethoprim and low level fusidic acid resistance, respectively. In addition to the two insertion site sequences (ISS) framing the SCCmec, a third ISS (ISS*) was identified within the element. SCCmecN1 also harboured two distinct ccrAB complexes belonging to the class 4 subtype, both of which were shown to be active and to be able to excise the SCCmecN1 or 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 SCCmec 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 SCCmec-chromosomal junction site. MRSA clones carrying the shorter SCCmec with the 6.4-kb deletion were usually ciprofloxacin resistant, while strains with the complete SCCmecN1 were co-trimoxazole resistant or had no additional resistances. This suggested that the genetic backbone of the host S. aureus, although identical by PFGE pattern, had at some stage diverged with one branch acquiring a sulfonomide resistance mutation and the other ciprofloxacin resistance.ConclusionThis description of the structure and variations of SCCmecN1 will allow for quicker and easier molecular detection of this clone and monitoring of its spread.

Transcriptional Profiling of XdrA, a New Regulator of spa Transcription in Staphylococcus aureus

Transcription of spa, encoding the virulence factor protein A in Staphylococcus aureus, is tightly controlled by a complex regulatory network, ensuring its temporal expression over growth and at appropriate stages of the infection process. Transcriptomic profiling of XdrA, a DNA-binding protein that is conserved in all S. aureus genomes and shares similarity with the XRE family of helix-turn-helix, antitoxin-like proteins, revealed it to be a previously unidentified activator of spa transcription. To assess how XdrA fits into the complex web of spa regulation, a series of regulatory mutants were constructed; consisting of single, double, triple, and quadruple mutants lacking XdrA and/or the three key regulators previously shown to influence spa transcription directly (SarS, SarA, and RNAIII). A series of lacZ reporter gene fusions containing nested deletions of the spa promoter identified regions influenced by XdrA and the other three regulators. XdrA had almost as strong an activating effect on spa as SarS and acted on the same spa operator regions as SarS, or closely overlapping regions. All data from microarrays, Northern and Western blot analyses, and reporter gene fusion experiments indicated that XdrA is a major activator of spa expression that appears to act directly on the spa promoter and not through previously characterized regulators.

A novel DNA-binding protein modulating methicillin resistance in Staphylococcus aureus

BackgroundMethicillin resistance in Staphylococcus aureus is conferred by the mecA-encoded penicillin-binding protein PBP2a. Additional genomic factors are also known to influence resistance levels in strain specific ways, although little is known about their contribution to resistance phenotypes in clinical isolates. Here we searched for novel proteins binding to the mec operator, in an attempt to identify new factor(s) controlling methicillin resistance phenotypes.ResultsAnalysis of proteins binding to a DNA fragment containing the mec operator region identified a novel, putative helix-turn-helix DNA-binding protein, SA1665. Nonpolar deletion of SA1665, in heterogeneously methicillin resistant S. aureus (MRSA) of different genetic backgrounds, increased methicillin resistance levels in a strain dependent manner. This phenotype could be fully complemented by reintroducing SA1665 in trans. Northern and Western blot analyses, however, revealed that SA1665 had no visible influence on mecA transcription or amounts of PBP2a produced.ConclusionSA1665 is a new chromosomal factor which influences methicillin resistance in MRSA. Although SA1665 bound to the mecA promoter region, it had no apparent influence on mecA transcription or translation, suggesting that this predicted DNA-binding protein modulates resistance indirectly, most likely through the control of other genomic factors which contribute to resistance.

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 M1.

Streptococcal SpeB Cleaved PAR-1 Suppresses ERK Phosphorylation and Blunts Thrombin-Induced Platelet Aggregation

Background The family of 4 related protease-activated receptors (PAR-1, 2, 3 & 4) expressed by mammalian cells allow to sense for and react to extracellular proteolytic activity. Since major human bacterial pathogens secret a wide array of protease(-s) we investigated whether they interfere with human PAR function. Methodology/Principal Findings Supernatants from cultures of major human bacterial pathogens were assayed for the presence of protease(-s) capable to cleave overexpressed human PAR-1, 2, 3 and 4 reporter constructs. Group A streptococcus (GAS) was found to secret a PAR-1-cleaving protease. Experiments involving genetical and pharmacological gain and loss of function identified streptococcal pyrogenic exotoxin B SpeB as the protease responsible. On the host’s side analysis of overexpressed PAR-1 carrying alanine substitutions and deletions showed the amino acid residue leucine44 on PAR-1’s extracellular N-terminus to be the only cleavage site. Complementary studies on endogenously expressed PAR-1 using PAR-1 blocking antibodies further supported our conclusion. Through PAR-1 cleavage SpeB efficiently blunted thrombin-induced induction of the ERK-pathway in endothelial cells and prevented platelets aggregation in response to thrombin. Conclusions/Significance Our results identify a novel function of the streptococcal virulence factor SpeB. By cleaving human PAR-1 at the N-terminal amino acid residue leucine44 SpeB rendered endothelial cells unresponsive to thrombin and prevented human platelets from thrombin-induced aggregation. These results suggest that by blunting PAR-1 signaling, SpeB modulates various innate host responses directed against invasive GAS potentially helping the invasive bacteria to escape. This may allow to tailor additional treatments in the future since upon invasion of the blood stream endothelial cells as well as platelets and mononuclear cells respond to PAR-1 agonists aiming to prevent further bacterial dissemination.