Antimicrobial activity of ceftaroline against methicillin-resistant Staphylococcus aureus (MRSA) isolates collected in 2013-2014 at the Geneva University Hospitals.

Ceftaroline is a broad-spectrum antibiotic with activity against methicillin-resistant Staphylococcus aureus (MRSA) strains. Ceftaroline susceptibility of an MRSA set archived between 1994 and 2003 in the Geneva University Hospitals detected a high percentage (66 %) of ceftaroline resistance in clonotypes ST228 and ST247 and correlated with mutations in PBP2a. The ceftaroline mechanism of action is based on the inhibition of PBP2a; thus, the identification of PBP2a mutations of recently circulating clonotypes in our institution was investigated. We analyzed ceftaroline susceptibility in MRSA isolates (2013 and 2014) and established that resistant strains correlated with PBP2a mutations and specific clonotypes. Ninety-six MRSA strains were analyzed from independent patients and were isolated from blood cultures (23 %), deep infections (38.5 %), and superficial (skin or wound) infections (38.5 %). This sample showed a ceftaroline minimum inhibitory concentration (MIC) range between 0.25 and 2 µg/ml and disk diameters ranging from 10 to 30 mm, with a majority of strains showing diameters ≥20 mm. Based on the European Committee on Antimicrobial Susceptibility Testing (EUCAST) breakpoints, 76 % (73/96) of isolates showed susceptibility to ceftaroline. Nevertheless, we still observed 24 % (23/96) of resistant isolates (MIC = 2 µg/ml). All resistant isolates were assigned to clonotype ST228 and carried the N146K mutation in PBP2a. Only two ST228 isolates showed ceftaroline susceptibility. The decreasing percentage of ceftaroline-resistant isolates in our hospital can be explained by the decline of ST228 clonotype circulating in our hospital since 2008. We present evidence that ceftaroline is active against recent MRSA strains from our hospital; however, the presence of PBP2a variants in particular clonotypes may affect ceftaroline efficacy.

The djlA gene acts synergistically with dnaJ in promoting Escherichia coli growth.

The DnaK chaperone of Escherichia coli is known to interact with the J domains of DnaJ, CbpA, and DjlA. By constructing multiple mutants, we found that the djlA gene was essential for bacterial growth above 37 degrees C in the absence of dnaJ. This essentiality depended upon the J domain of DjlA but not upon the normal membrane location of DjlA.

Mitochondria recycle Ca(2+) to the endoplasmic reticulum and prevent the depletion of neighboring endoplasmic reticulum regions.

To study Ca(2+) fluxes between mitochondria and the endoplasmic reticulum (ER), we used "cameleon" indicators targeted to the cytosol, the ER lumen, and the mitochondrial matrix. High affinity mitochondrial probes saturated in approximately 20% of mitochondria during histamine stimulation of HeLa cells, whereas a low affinity probe reported averaged peak values of 106 +/- 5 microm, indicating that Ca(2+) transients reach high levels in a fraction of mitochondria. In concurrent ER measurements, [Ca(2+)](ER) averaged 371 +/- 21 microm at rest and decreased to 133 +/- 14 microm and 59 +/- 5 microm upon stimulation with histamine and thapsigargin, respectively, indicating that substantial ER refilling occur during agonist stimulation. A larger ER depletion was observed when mitochondrial Ca(2+) uptake was prevented by oligomycin and rotenone or when Ca(2+) efflux from mitochondria was blocked by CGP 37157, indicating that some of the Ca(2+) taken up by mitochondria is re-used for ER refilling. Accordingly, ER regions close to mitochondria released less Ca(2+) than ER regions lacking mitochondria. The ER heterogeneity was abolished by thapsigargin, oligomycin/rotenone, or CGP 37157, indicating that mitochondrial Ca(2+) uptake locally modulate ER refilling. These observations indicate that some mitochondria are very close to the sites of Ca(2+) release and recycle a substantial portion of the captured Ca(2+) back to vicinal ER domains. The distance between the two organelles thus determines both the amplitude of mitochondrial Ca(2+) signals and the filling state of neighboring ER regions.

DjlA is a third DnaK co-chaperone of Escherichia coli, and DjlA-mediated induction of colanic acid capsule requires DjlA-DnaK interaction.

DjlA is a 30-kDa type III membrane protein of Escherichia coli with the majority, including an extreme C-terminal putative J-domain, oriented toward the cytoplasm. No other regions of sequence similarity aside from the J-domain exist between DjlA and the known DnaK (Hsp70) co-chaperones DnaJ (Hsp40) and CbpA. In this study, we explored whether and to what extent DjlA possesses DnaK co-chaperone activity and under what conditions a DjlA-DnaK interaction could be important to the cell. We found that the DjlA J-domain can substitute fully for the J-domain of DnaJ using various in vivo functional complementation assays. In addition, the purified cytoplasmic fragment of DjlA was shown to be capable of stimulating DnaK ATPase in a manner indistinguishable from DnaJ, and, furthermore, DjlA could act as a DnaK co-chaperone in the reactivation of chemically denatured luciferase in vitro. DjlA expression in the cell is tightly controlled, and even its mild overexpression leads to induction of mucoid capsule. Previous analysis showed that DjlA-mediated induction of the wca capsule operon required the RcsC/RcsB two-component signaling system and that wca induction by DjlA was lost when cells contained mutations in either the dnaK or grpE gene. We now show using allele-specific genetic suppression analysis that DjlA must interact with DnaK for DjlA-mediated stimulation of capsule synthesis. Collectively, these results demonstrate that DjlA is a co-chaperone for DnaK and that this chaperone-co-chaperone pair is implicated directly, or indirectly, in the regulation of colanic acid capsule.

Selective inhibition of nuclear steroid receptor function by a protein from a human tumorigenic poxvirus.

The poxvirus molluscum contagiosum (MC) has a worldwide distribution and its prevalence is on the rise. Here we report that the MCV MC013L protein inhibits glucocorticoid and vitamin D, but not retinoid or estrogen, nuclear receptor transactivation. A direct interaction of MC013L with glucocorticoid and vitamin D receptor is supported by yeast two-hybrid, GST pull-down, and far Western blot analyses. Glucocorticoids act as potent inhibitors of keratinocyte proliferation, while vitamin D and retinoids promote and block terminal differentiation, respectively. Therefore, MC013L may promote efficient virus replication by blocking the differentiation of infected keratinocytes. MC013L may be the first member of a new class of poxvirus proteins that directly modulate nuclear receptor-mediated transcription.

Bcl-2 decreases the free Ca2+ concentration within the endoplasmic reticulum.

The antiapoptotic protein Bcl-2 localizes not only to mitochondria but also to the endoplasmic reticulum (ER). However, the function of Bcl-2 at the level of the ER is poorly understood. In this study, we have investigated the effects of Bcl-2 expression on Ca(2+) storage and release by the ER. The expression of Bcl-2 decreased the amount of Ca(2+) that could be released from intracellular stores, regardless of the mode of store depletion, the cell type, or the species from which Bcl-2 was derived. Bcl-2 also decreased cellular Ca(2+) store content in the presence of mitochondrial inhibitors, suggesting that its effects were not mediated through mitochondrial Ca(2+) uptake. Direct measurements with ER-targeted Ca(2+)-sensitive fluorescent "cameleon" proteins revealed that Bcl-2 decreased the free Ca(2+) concentration within the lumen of the ER, [Ca(2+)](ER). Analysis of the kinetics of Ca(2+) store depletion in response to the Ca(2+)-ATPase inhibitor thapsigargin revealed that Bcl-2 increased the permeability of the ER membrane. These results suggest that Bcl-2 decreases the free Ca(2+) concentration within the ER lumen by increasing the Ca(2+) permeability of the ER membrane. The increased ER Ca(2+) permeability conferred by Bcl-2 would be compatible with an ion channel function of Bcl-2 at the level of the ER membrane.

Recognition, targeting, and hydrolysis of the lambda O replication protein by the ClpP/ClpX protease.

It has previously been established that sequences at the C termini of polypeptide substrates are critical for efficient hydrolysis by the ClpP/ClpX ATP-dependent protease. We report for the bacteriophage lambda O replication protein, however, that N-terminal sequences play the most critical role in facilitating proteolysis by ClpP/ClpX. The N-terminal portion of lambda O is degraded at a rate comparable with that of wild type O protein, whereas the C-terminal domain of O is hydrolyzed at least 10-fold more slowly. Consistent with these results, deletion of the first 18 amino acids of lambda O blocks degradation of the N-terminal domain, whereas proteolysis of the O C-terminal domain is only slightly diminished as a result of deletion of the C-terminal 15 amino acids. We demonstrate that ClpX retains its capacity to bind to the N-terminal domain following removal of the first 18 amino acids of O. However, ClpX cannot efficiently promote the ATP-dependent binding of this truncated O polypeptide to ClpP, the catalytic subunit of the ClpP/ClpX protease. Based on our results with lambda O protein, we suggest that two distinct structural elements may be required in substrate polypeptides to enable efficient hydrolysis by the ClpP/ClpX protease: (i) a ClpX-binding site, which may be located remotely from substrate termini, and (ii) a proper N- or C-terminal sequence, whose exposure on the substrate surface may be induced by the binding of ClpX.

Molecular chaperones: How J domains turn on Hsp70s.

Molecular chaperones of the heat shock protein 70 (Hsp70) variety facilitate protein folding and assembly. They are assisted in this role by their Hsp40 partners, and recent studies have shed new light on how the 'J domains' of these 'cochaperones' activate substrate binding by Hsp70 molecules.

Mutational analysis of cysteine-string protein function in insulin exocytosis.

Cysteine-string proteins (Csps) are vesicle proteins involved in neurotransmission. They contain at least four domains: an N-terminal J-domain which can interact with the chaperone Hsc70, an adjacent linker region, the defining cysteine rich domain and a variable C terminus. As the relevance of these domains for the function of Csps in exocytosis is unknown, we have performed a mutational analysis of Csp domains using insulin release by large dense core vesicles (LDCVs) as a model of regulated exocytosis. All mutants were apparently palmitoylated and their subcellular distribution was similar to endogenous Csp. Point mutations within the highly conserved HPD motif of the J-domain abolished activation of Hsc70. However, these mutations altered the effect of Csp on exocytosis only after additional truncation of the extreme C terminus as found in the Csp splice variant Csp2. Furthermore, the strikingly conserved linker region adjacent to the J-domain was important for Csp function in exocytosis, but not for the activation of Hsc70 ATPase. The effects of Csp wild-type or mutants were preserved in permeabilized cells excluding an effect on transmembrane ion fluxes. These observations demonstrate a functional difference between the two isoforms and suggest a role for the J-domain co-chaperone function as well as for the newly defined linker region in LDCV exocytosis.

Cysteine-string proteins regulate exocytosis of insulin independent from transmembrane ion fluxes.

Cysteine-string proteins (Csps) are vesicle proteins involved in exocytosis of synaptic vesicles in Drosophila and modulation of presynaptic calcium influx. As both the contribution of calcium channel regulation to the role of Csp in exocytosis and a function of Csp outside the nervous system are unknown, we studied its function in endocrine exocytosis from large dense core vesicles (LDCVs) using insulin-secreting pancreatic beta-cells. Csps were expressed in primary and derived beta-cell lines on insulin-containing LDCVs. Suppression of Csp expression reduced not only depolarisation induced insulin release but also exocytosis in permeabilised cells directly stimulated by Ca2+. Thus, Csp is a secretory granule protein and is required for endocrine exocytosis independent of the modulation of transmembrane calcium fluxes.

The J-domain family and the recruitment of chaperone power.

The defining feature of the Hsp40 chaperone family is a approximately 70-amino-acid-residue signature, termed the J domain, that is necessary for orchestrating interactions with its Hsp70 chaperone partner(s). J-domain proteins play important regulatory roles as co-chaperones, recruiting Hsp70 partners and accelerating the ATP-hydrolysis step of the chaperone cycle. Certain proteins could have acquired a J domain in order to present a specific substrate(s) to an Hsp70 partner and thus capitalize upon chaperone activities when carrying out cellular functions. J-domain proteins participate in complex biological processes, such as cell-cycle control by DNA tumor viruses, regulation of protein kinases and exocytosis.

Positive control of the two-component RcsC/B signal transduction network by DjlA: a member of the DnaJ family of molecular chaperones in Escherichia coli.

The membrane-anchored DjIA protein represents the third member of the DnaJ 'J-domain' family of Escherichia coli that includes DnaJ and CbpA. DjIA possesses a J-domain at its extreme C-terminus but shares no additional homology with DnaJ. Our genetic analysis suggests that DjIA acts in concert with the RcsB/C two-component signal transduction system to augment induction of the cps (capsular polysaccharide) operon and synthesis of colanic acid mucoid capsule. The DjIA J-domain is essential for the observed stimulation of this pathway as deletion, or introduction of the mutation H233Q, within the highly conserved HPD tripeptide abolished all inducing activity. Deletion of the transmembrane anchor sequence also abolished all inducing activity. djIA is not an essential gene under all conditions tested, nor is it essential for mucoid capsule biosynthesis; however, strong overexpression leads to rapid loss of cell viability suggesting that the gene is normally tightly regulated. Northern analysis revealed that djIA message was extremely unstable but could be induced or stabilized in response to cold shock. The activation of the cps operon by DjIA is dependent upon both DnaK(Hsp70) and GrpE, and therefore we propose a role for DjIA, together with this chaperone machine, as a novel regulator of a two-component histidine kinase signal transduction pathway.

Structure-function analyses of the Ssc1p, Mdj1p, and Mge1p Saccharomyces cerevisiae mitochondrial proteins in Escherichia coli.

The DnaK, DnaJ, and GrpE proteins of Escherichia coli have been universally conserved across the biological kingdoms and work together to constitute a highly efficient molecular chaperone machine. We have examined the extent of functional conservation of Saccharomyces cerevisiae Ssc1p, Mdj1p, and Mge1p by analyzing their ability to substitute for their corresponding E. coli homologs in vivo. We found that the expression of yeast Mge1p, the GrpE homolog, allowed for the deletion of the otherwise essential grpE gene of E. coli, albeit only up to 40 degrees C. The inability of Mge1p to substitute for GrpE at very high temperatures is consistent with our previous finding that it specifically failed to stimulate DnaK's ATPase at such extreme conditions. In contrast to Mge1p, overexpression of Mdj1p, the DnaJ homolog, was lethal in E. coli. This toxicity was specifically relieved by mutations which affected the putative zinc binding region of Mdj1p. Overexpression of a truncated version of Mdj1p, containing the J- and Gly/Phe-rich domains, partially substituted for DnaJ function at high temperature. A chimeric protein, consisting of the J domain of Mdj1p coupled to the rest of DnaJ, acted as a super-DnaJ protein, functioning even more efficiently than wild-type DnaJ. In contrast to the results with Mge1p and Mdj1p, both the expression and function of Ssc1p, the DnaK homolog, were severely compromised in E. coli. We were unable to demonstrate any functional complementation by Ssc1p, even when coexpressed with its Mdj1p cochaperone in E. coli.

The T/t common exon of simian virus 40, JC, and BK polyomavirus T antigens can functionally replace the J-domain of the Escherichia coli DnaJ molecular chaperone.

The N-terminal 70 residue "J-domain" of the Escherichia coli DnaJ molecular chaperone is the defining and highly conserved feature of a large protein family. Based upon limited, yet significant, amino acid sequence homology to the J-domain, the DNA encoding the T/t common exon of the simian virus 40 (SV40), JC, or BK polyoma virus T antigen oncoproteins was used to construct J-domain replacement chimeras of the E. coli DnaJ chaperone. The virally encoded J-domains successfully substituted for the bacterial counterpart in vivo as shown by (i) complementation for viability at low and high temperature of a hypersensitive bacterial reporter strain, and (ii) the restoration of bacteriophage lambda plaque forming ability in the same strain. The amino acid change, H42Q, in the SV40 T/t and the JC virus T/t exon, which is positionally equivalent to the canonical dnaJ259 H33Q mutation within the E. coli J-domain, entirely abolished complementing activity. These results strongly suggest that the heretofore functionally undefined viral T/t common exon represents a bona fide J-domain that preserves critical features of the characteristic domain fold essential for J-domain interaction with the ATPase domain of the Hsp70 family. This finding has implications for the regulation of DNA tumor virus T antigens by molecular chaperones.

Chaperones and protein folding.

Chaperones are centrally involved in the control of protein structure, function, localization and transport. A flurry of scientific activity continues to examine the molecular nature of chaperone-substrate recognition and the role of auxiliary chaperones (cohort proteins) and small molecules that expedite these processes. Chaperones have been implicated in processes as diverse as protein secretion, nuclear transport, thermotolerance, the steroid receptor signal transduction pathway, T-cell receptor and major histocompatibility complex class I and II multimeric assembly and bacterial virulence.

Structural and functional analysis of a replication enhancer: separation of the enhancer activity from origin function by mutational dissection of the replication origin gamma of plasmid R6K.

The plasmid R6K possesses three distinct origins of replication: alpha, beta, and gamma. The replication origin gamma of plasmid R6K performs a dual function: (i) as an origin itself and (ii) as an enhancer element required in cis for the activation at a distance of the other two replication origins alpha and beta. We have dissected the gamma origin/enhancer by site-directed mutagenesis and have reached the following conclusions. The origin function can be specifically inactivated without impairing the enhancer function by insertion and/or deletion mutations near the opposite ends of the origin gamma sequence. One such mutation deleted sequences that included the left DnaA site I. The second mutation involved insertion of linker sequences that resulted in a spatial alteration between the right DnaA site II and the VIIth pi binding iteron (tandemly repeated binding sites). Other mutations that either partly or completely deleted the A+T-rich sequence adjacent to, but not including, the pi binding iterons also abrogated enhancer and origin function and suggested that pi binding sites were necessary but not sufficient for enhancer activity. Finally, the functional analysis of a set of mutants of the gamma origin/enhancer suggested that a continuous stretch of 300 base pairs is necessary for origin gamma function and that the sequences that included the binding sites for pi, DnaA, and integration host factor proteins are required in the correct stereochemical alignment to impart origin activity.

Activation in vivo of the minimal replication origin beta of plasmid R6K requires a small target sequence essential for DNA looping.

The plasmid R6K contains three distinct origins of replication: alpha, beta, and gamma. The gamma sequence is essential in cis and acts as an enhancer that activates the distant alpha and beta origins. R6K therefore represents a favorable procaryotic model system with which to unravel the biochemical mechanisms underlying selective origin activation, particularly activation involving distant sites on the same chromosome. We have discovered that plasmids containing the origins alpha and gamma required the Escherichia coli DnaA initiator protein in addition to the R6K-encoded initiator protein, Pi, and other host replisomal proteins for their maintenance in vivo. Plasmids initiating replication from origin beta required only the Pi initiator protein and other host replisomal proteins. We have exploited the differential requirement for the DnaA protein by origins gamma and beta to selectively study and localize the minimal origin beta sequences by deletion analysis as one test of a looping model of origin activation. A 64-bp region spanning the extreme -COOH terminal coding sequence of the Pi protein was found to be essential for replication in vivo in the absence of DnaA protein, consistent with the approximate physical location of the beta origin. Replication emanating from origin beta could be abolished in vivo by deletion of the 9-bp target site for Pi protein-mediated DNA looping between the gamma origin/enhancer and the distant beta origin. Electron microscopy of nascent replication intermediates generated in vivo directly confirmed our genetic localization of the beta origin. Our results strongly suggest that activation of the beta origin by a distant replication enhancer element requires a small target sequence essential for initiator protein-mediated DNA looping.

Conformational changes induced by integration host factor at origin gamma of R6K and copy number control.

We have investigated the role of integration host factor (IHF) in the replication of plasmid R6K by studying the maintainance of the plasmid in a strain of Escherichia coli that lacks both subunits of IHF and in an isogenic wild type strain and found that all three origins, alpha, beta, and gamma, were functional in the absence of IHF; however, loss of IHF reduced the copy number of those replicons initiating solely from ori gamma by 5-fold. Concomitant loss of direct repeats within the origin that bind the R6K replication initiator protein, Pi, resulted in a further reduction in copy number. Using gel mobility shift analysis, we showed that IHF bound specifically only to one site within the A/T rich region of the minimal origin adjacent to the Pi binding sites. The origin region possessed no intrinsic DNA curvature although IHF induced a strong bend upon binding. Combination footprinting with different orders of addition of Pi and IHF suggested that there was no cooperativity between the two proteins with regard to DNA binding. Hydroxyl-radical footprinting revealed hypersensitive asymmetric periodic cleavage sites within the origin region in the presence of IHF that extended over 200 base pairs and a localized perturbation of cleavage chemistry. The presence of periodic cleavages was dependent upon the presence of the wild type R6K origin sequence and was not observed when the IHF binding site was positioned adjacent to a heterologous sequence. We observed that the conformational changes induced by IHF upon binding to the R6K origin were negatively correlated with the observed decrease in copy number, and therefore, origin conformation altered by protein-DNA interaction may play an important role in the regulation of replication initiation.

Replication of plasmid R6K origin gamma in vitro. Dependence on dual initiator proteins and inhibition by transcription.

We have developed a more efficient in vitro replication system for the plasmid R6K with the objective of dissecting the mechanism of activation of replication origins at a distance. Using this in vitro system we have shown that the activation of replication origin gamma of R6K is absolutely dependent on two exogenously added initiator proteins: namely the host-encoded DnaA and the plasmid-encoded Pi proteins. Replication was inhibited by novobiocin, suggesting a requirement for DNA gyrase. Surprisingly, rifampicin stimulated in vitro replication significantly, and this stimulation was manifested in the quantitative enhancement of replication without any noticeable qualitative change in the reaction products. This result suggests that transcription at or near the gamma origin keeps it repressed. Replication intermediates that were allowed to accumulate by dideoxynucleoside triphosphate incorporation were analyzed both by restriction enzyme digestion and by electron microscopy, and both sets of analyses revealed initiation from the gamma origin resulting in theta-type replication intermediates. Further development of this system should help us to understand how DNA-protein interaction at the gamma origin/enhancer activates the distal origins alpha and beta.