Polysaccharide-Targeting Lipid Nanoparticles to Kill Gram-Negative Bacteria.

The rapid increase and spread of Gram-negative bacteria resistant to many or all existing treatments threaten a return to the preantibiotic era. The presence of bacterial polysaccharides that impede the penetration of many antimicrobials and protect them from the innate immune system contributes to resistance and pathogenicity. No currently approved antibiotics target the polysaccharide regions of microbes. Here, describe monolaurin-based niosomes, the first lipid nanoparticles that can eliminate bacterial polysaccharides from hypervirulent Klebsiella pneumoniae, are described. Their combination with polymyxin B shows no cytotoxicity in vitro and is highly effective in combating K. pneumoniae infection in vivo. Comprehensive mechanistic studies have revealed that antimicrobial activity proceeds via a multimodal mechanism. Initially, lipid nanoparticles disrupt polysaccharides, then outer and inner membranes are destabilized and destroyed by polymyxin B, resulting in synergistic cell lysis. This novel lipidic nanoparticle system shows tremendous promise as a highly effective antimicrobial treatment targeting multidrug-resistant Gram-negative pathogens.

Virulence attributes of successful methicillin-resistant Staphylococcus aureus lineages.

Methicillin-resistant Staphylococcus aureus (MRSA) is a leading cause of severe and often fatal infections. MRSA epidemics have occurred in waves, whereby a previously successful lineage has been replaced by a more fit and better adapted lineage. Selection pressures in both hospital and community settings are not uniform across the globe, which has resulted in geographically distinct epidemiology. This review focuses on the mechanisms that trigger the establishment and maintenance of current, dominant MRSA lineages across the globe. While the important role of antibiotic resistance will be mentioned throughout, factors which influence the capacity of S. aureus to colonize and cause disease within a host will be the primary focus of this review. We show that while MRSA possesses a diverse arsenal of toxins including alpha-toxin, the success of a lineage involves more than just producing toxins that damage the host. Success is often attributed to the acquisition or loss of genetic elements involved in colonization and niche adaptation such as the arginine catabolic mobile element, as well as the activity of regulatory systems, and shift metabolism accordingly (e.g., the accessory genome regulator, agr). Understanding exactly how specific MRSA clones cause prolonged epidemics may reveal targets for therapies, whereby both core (e.g., the alpha toxin) and acquired virulence factors (e.g., the Panton-Valentine leukocidin) may be nullified using anti-virulence strategies.

Characterisation of key genotypic and phenotypic traits of clinical cystic fibrosis Staphylococcus aureus isolates.

Introduction. One third of people with CF in the UK are co-infected by both Staphylococcus aureus and Pseudomonas aeruginosa. Chronic bacterial infection in CF contributes to the gradual destruction of lung tissue, and eventually respiratory failure in this group.Gap Statement. The contribution of S. aureus to cystic fibrosis (CF) lung decline in the presence or absence of P. aeruginosa is unclear. Defining the molecular and phenotypic characteristics of a range of S. aureus clinical isolates will help further understand its pathogenic capabilities.Aim. Our objective was to use molecular and phenotypic tools to characterise twenty-five clinical S. aureus isolates collected from mono- and coinfection with P. aeruginosa from people with CF at the Royal Victoria Infirmary, Newcastle upon Tyne.Methodology. Genomic DNA was extracted and sequenced. Multilocus sequence typing was used to construct phylogeny from the seven housekeeping genes. A pangenome was calculated using Roary, and cluster of Orthologous groups were assigned using eggNOG-mapper which were used to determine differences within core, accessory, and unique genomes. Characterisation of sequence type, clonal complex, agr and spa types was carried out using PubMLST, eBURST, AgrVATE and spaTyper, respectively. Antibiotic resistance was determined using Kirby-Bauer disc diffusion tests. Phenotypic testing of haemolysis was carried out using ovine red blood cell agar plates and mucoid phenotypes visualised using Congo red agar.Results. Clinical strains clustered closely based on agr type, sequence type and clonal complex. COG analysis revealed statistically significant enrichment of COG families between core, accessory and unique pangenome groups. The unique genome was significantly enriched for replication, recombination and repair, and defence mechanisms. The presence of known virulence genes and toxins were high within this group, and unique genes were identified in 11 strains. Strains which were isolated from the same patient all surpassed average nucleotide identity thresholds, however, differed in phenotypic traits. Antimicrobial resistance to macrolides was significantly higher in the coinfection group.Conclusion. There is huge variation in genetic and phenotypic capabilities of S. aureus strains. Further studies on how these may differ in relation to other species in the CF lung may give insight into inter-species interactions.

A Clinically Selected Staphylococcus aureus clpP Mutant Survives Daptomycin Treatment by Reducing Binding of the Antibiotic and Adapting a Rod-Shaped Morphology.

Daptomycin is a last-resort antibiotic used for the treatment of infections caused by Gram-positive antibiotic-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA). Treatment failure is commonly linked to accumulation of point mutations; however, the contribution of single mutations to resistance and the mechanisms underlying resistance remain incompletely understood. Here, we show that a single nucleotide polymorphism (SNP) selected during daptomycin therapy inactivates the highly conserved ClpP protease and is causing reduced susceptibility of MRSA to daptomycin, vancomycin, and ß-lactam antibiotics as well as decreased expression of virulence factors. Super-resolution microscopy demonstrated that inactivation of ClpP reduced binding of daptomycin to the septal site and diminished membrane damage. In both the parental strain and the clpP strain, daptomycin inhibited the inward progression of septum synthesis, eventually leading to lysis and death of the parental strain while surviving clpP cells were able to continue synthesis of the peripheral cell wall in the presence of 10× MIC daptomycin, resulting in a rod-shaped morphology. To our knowledge, this is the first demonstration that synthesis of the outer cell wall continues in the presence of daptomycin. Collectively, our data provide novel insight into the mechanisms behind bacterial killing and resistance to this important antibiotic. Also, the study emphasizes that treatment with last-line antibiotics is selective for mutations that, like the SNP in clpP, favor antibiotic resistance over virulence gene expression.

A polytherapy based approach to combat antimicrobial resistance using cubosomes.

A depleted antimicrobial drug pipeline combined with an increasing prevalence of Gram-negative 'superbugs' has increased interest in nano therapies to treat antibiotic resistance. As cubosomes and polymyxins disrupt the outer membrane of Gram-negative bacteria via different mechanisms, we herein examine the antimicrobial activity of polymyxin-loaded cubosomes and explore an alternative strategy via the polytherapy treatment of pathogens with cubosomes in combination with polymyxin. The polytherapy treatment substantially increases antimicrobial activity compared to polymyxin B-loaded cubosomes or polymyxin and cubosomes alone. Confocal microscopy and neutron reflectometry suggest the superior polytherapy activity is achieved via a two-step process. Firstly, electrostatic interactions between polymyxin and lipid A initially destabilize the outer membrane. Subsequently, an influx of cubosomes results in further membrane disruption via a lipid exchange process. These findings demonstrate that nanoparticle-based polytherapy treatments may potentially serve as improved alternatives to the conventional use of drug-loaded lipid nanoparticles for the treatment of "superbugs".

The Membrane Composition Defines the Spatial Organization and Function of a Major Acinetobacter baumannii Drug Efflux System.

Acinetobacter baumannii is one of the world's most problematic nosocomial pathogens. The combination of its intrinsic resistance and ability to acquire resistance markers allow this organism to adjust to antibiotic treatment. Despite being the primary barrier against antibiotic stress, our understanding of the A. baumannii membrane composition and its impact on resistance remains limited. In this study, we explored how the incorporation of host-derived polyunsaturated fatty acids (PUFAs) is associated with increased antibiotic susceptibility. Functional analyses of primary A. baumannii efflux systems indicated that AdeB-mediated antibiotic resistance was impacted by PUFA treatment. Molecular dynamics simulations of AdeB identified a specific morphological disruption of AdeB when positioned in the PUFA-enriched membrane. Collectively, we have shown that PUFAs can impact antibiotic efficacy via a vital relationship with antibiotic efflux pumps. Furthermore, this work has revealed that A. baumannii's unconditional desire for fatty acids may present a possible weakness in its multidrug resistance capacity. IMPORTANCE Antimicrobial resistance is an emerging global health crisis. Consequently, we have a critical need to prolong our current arsenal of antibiotics, in addition to the development of novel treatment options. Due to their relatively high abundance at the host-pathogen interface, PUFAs and other fatty acid species not commonly synthesized by A. baumannii may be actively acquired by A. baumannii during infection and change the biophysical properties of the membrane beyond that studied in standard laboratory culturing media. Our work illustrates how the membrane phospholipid composition impacts membrane protein function, which includes an important multidrug efflux system in extensively-drug-resistant A. baumannii. This work emphasizes the need to consider including host-derived fatty acids in in vitro analyses of A. baumannii. On a broader scope, this study presents new findings on the potential health benefits of PUFA in individuals at risk of contracting A. baumannii infections or those undergoing antibiotic treatment.

The Resistance to Host Antimicrobial Peptides in Infections Caused by Daptomycin-Resistant Staphylococcus aureus.

Daptomycin is an important antibiotic for the treatment of infections caused by Staphylococcus aureus. The emergence of daptomycin resistance in S. aureus is associated with treatment failure and persistent infections with poor clinical outcomes. Here, we investigated host innate immune responses against clinically derived, daptomycin-resistant (DAP-R) and -susceptible S. aureus paired isolates using a zebrafish infection model. We showed that the control of DAP-R S. aureus infections was attenuated in vivo due to cross-resistance to host cationic antimicrobial peptides. These data provide mechanistic understanding into persistent infections caused by DAP-R S. aureus and provide crucial insights into the adaptive evolution of this troublesome pathogen.

Phytantriol-Based Cubosome Formulation as an Antimicrobial against Lipopolysaccharide-Deficient Gram-Negative Bacteria.

Treatment of multidrug-resistant (MDR) bacterial infections increasingly relies on last-line antibiotics, such as polymyxins, with the urgent need for discovery of new antimicrobials. Nanotechnology-based antimicrobials have gained significant importance to prevent the catastrophic emergence of MDR over the past decade. In this study, phytantriol-based nanoparticles, named cubosomes, were prepared and examined in vitro by minimum inhibitory concentration (MIC) and time-kill assays against Gram-negative bacteria: Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa. Phytantriol-based cubosomes were highly bactericidal against polymyxin-resistant, lipopolysaccharide (LPS)-deficient A. baumannii strains. Small-angle neutron scattering (SANS) was employed to understand the structural changes in biomimetic membranes that replicate the composition of these LPS-deficient strains upon treatment with cubosomes. Additionally, to further understand the membrane-cubosome interface, neutron reflectivity (NR) was used to investigate the interaction of cubosomes with model bacterial membranes on a solid support. These results reveal that cubosomes might be a new strategy for combating LPS-deficient Gram-negative pathogens.

A mouse model of Staphylococcus aureus small intestinal infection.

Introduction. Staphylococcus aureus is a recognised cause of foodborne intoxication and antibiotic-associated diarrhoea (AAD), which are both mediated by staphylococcal enterotoxins. However, unlike foodborne intoxication, AAD appears to require infection of the host. While S. aureus intoxication is widely studied, little is known about S. aureus pathogenesis in the context of gastrointestinal infection.Aim. To develop a mouse model of S. aureus gastrointestinal infection.Methodology. An established AAD mouse model was adapted for S. aureus infection, and damage observed via histopathological analysis and immunostaining of intestinal tissues.Results. Various strains colonised the mouse model, and analysis showed that although clinical signs of disease were not seen, S. aureus infection induced damage in the small intestine, disrupting host structures essential for epithelial integrity. Studies using a staphylococcal enterotoxin B mutant showed that this toxin may contribute to damage during gastrointestinal infection.Conclusion. This work presents a new mouse model of S. aureus gastrointestinal infection, while also providing insight into the pathogenesis of S. aureus in the gut.

Daptomycin-resistant Staphylococcus aureus clinical isolates are poorly sensed by dendritic cells.

Methicillin-resistant Staphylococcus aureus (MRSA) presents an increasing threat to public health, with antimicrobial resistance on the rise and infections endemic in the hospital setting. Despite a global research effort to understand and combat antimicrobial resistance, less work has focused on understanding the nuances in the immunopathogenesis of clinical strains. In particular, there is a surprising gap of knowledge in the literature pertaining to how clinical strains are recognized by dendritic cells (DCs). Here, we show that the activation of DCs is compromised in response to MRSA strains resistant to the last-line antibiotic daptomycin. We found a significant reduction in the secretion of proinflammatory cytokines including tumor necrosis factor-α, interleukin-6, regulated upon activation, normal T cell expressed, and secreted and macrophage inflammatory protein-1ß, as well as decreased expression of CD80 by DCs responding to daptomycin-resistant MRSA. We further demonstrate that this phenotype is coincident with the acquisition of specific point mutations in the cardiolipin synthase gene cls2, and, partly, in the bifunctional lysylphosphatidylglycerol flippase/synthetase gene mprF, which are genes that are often mutated in clinical daptomycin-resistant strains. Therefore, throughout infection and antibiotic therapy, MRSA has the capacity to not only develop further antibiotic resistance, but also develop resistance to immunological recognition by DCs, because of single amino acid point mutations occurring under the selective pressures of both host immunity and antibiotic therapy. Understanding the diversity of clinical MRSA isolates and the nuances in their immune recognition will have important implications for future therapeutics and the treatment of these infections.

Detection of Antimicrobial Resistance-Related Changes in Biochemical Composition of Staphylococcus aureus by Means of Atomic Force Microscopy-Infrared Spectroscopy.

The development of antimicrobial resistance (AMR) resulting from widespread antibiotic usage is occurring at an alarming pace, much faster than our understanding of the mechanisms behind resistance. Knowledge about resistance-related phenotypic and genotypic changes is critical for the development of new drugs. Here, we identify changes in the chemical composition of Staphylococcus aureus associated with the development of resistance to last resort drugs, vancomycin and daptomycin, using a novel, single cell, nanoscale technique, atomic force microscopy-infrared spectroscopy (AFM-IR), combined with chemometric analysis. We utilized paired clinical isolates, with the parent (susceptible) strain isolated prior to treatment and the daughter (resistant) strain obtained from the same patient after drug admission and clinical failure. We observed an increase in the amount of nonintracellular carbohydrates, indicating thickening or changes in the packing of the cell wall, as well as changes in the phospholipid content in relation to vancomycin resistance and daptomycin nonsusceptibility, respectively.

A plasmid-encoded peptide from Staphylococcus aureus induces anti-myeloperoxidase nephritogenic autoimmunity.

Autoreactivity to myeloperoxidase (MPO) causes anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV), with rapidly progressive glomerulonephritis. Here, we show that a Staphylococcus aureus peptide, homologous to an immunodominant MPO T-cell epitope (MPO409-428), can induce anti-MPO autoimmunity. The peptide (6PGD391-410) is part of a plasmid-encoded 6-phosphogluconate dehydrogenase found in some S. aureus strains. It induces anti-MPO T-cell autoimmunity and MPO-ANCA in mice, whereas related sequences do not. Mice immunized with 6PGD391-410, or with S. aureus containing a plasmid expressing 6PGD391-410, develop glomerulonephritis when MPO is deposited in glomeruli. The peptide induces anti-MPO autoreactivity in the context of three MHC class II allomorphs. Furthermore, we show that 6PGD391-410 is immunogenic in humans, as healthy human and AAV patient sera contain anti-6PGD and anti-6PGD391-410 antibodies. Therefore, our results support the idea that bacterial plasmids might have a function in autoimmune disease.

Antibiotic resistance and host immune evasion in Staphylococcus aureus mediated by a metabolic adaptation.

Staphylococcus aureus is a notorious human bacterial pathogen with considerable capacity to develop antibiotic resistance. We have observed that human infections caused by highly drug-resistant S. aureus are more prolonged, complicated, and difficult to eradicate. Here we describe a metabolic adaptation strategy used by clinical S. aureus strains that leads to resistance to the last-line antibiotic, daptomycin, and simultaneously affects host innate immunity. This response was characterized by a change in anionic membrane phospholipid composition induced by point mutations in the phospholipid biosynthesis gene, cls2, encoding cardiolipin synthase. Single cls2 point mutations were sufficient for daptomycin resistance, antibiotic treatment failure, and persistent infection. These phenotypes were mediated by enhanced cardiolipin biosynthesis, leading to increased bacterial membrane cardiolipin and reduced phosphatidylglycerol. The changes in membrane phospholipid profile led to modifications in membrane structure that impaired daptomycin penetration and membrane disruption. The cls2 point mutations also allowed S. aureus to evade neutrophil chemotaxis, mediated by the reduction in bacterial membrane phosphatidylglycerol, a previously undescribed bacterial-driven chemoattractant. Together, these data illustrate a metabolic strategy used by S. aureus to circumvent antibiotic and immune attack and provide crucial insights into membrane-based therapeutic targeting of this troublesome pathogen.

Identification of Novel Acinetobacter baumannii Host Fatty Acid Stress Adaptation Strategies.

Free fatty acids hold important immune-modulatory roles during infection. However, the host's long-chain polyunsaturated fatty acids, not commonly found in the membranes of bacterial pathogens, also have significant broad-spectrum antibacterial potential. Of these, the omega-6 fatty acid arachidonic acid (AA) and the omega-3 fatty acid decosahexaenoic acid (DHA) are highly abundant; hence, we investigated their effects on the multidrug-resistant human pathogen Acinetobacter baumannii Our analyses reveal that AA and DHA incorporate into the A. baumannii bacterial membrane and impact bacterial fitness and membrane integrity, with DHA having a more pronounced effect. Through transcriptional profiling and mutant analyses, we show that the A. baumannii ß-oxidation pathway plays a protective role against AA and DHA, by limiting their incorporation into the phospholipids of the bacterial membrane. Furthermore, our study identified a second bacterial membrane protection system mediated by the AdeIJK efflux system, which modulates the lipid content of the membrane via direct efflux of lipids other than AA and DHA, thereby providing a novel function for this major efflux system in A. baumannii This is the first study to examine the antimicrobial effects of host fatty acids on A. baumannii and highlights the potential of AA and DHA to protect against A. baumannii infections.IMPORTANCE A shift in the Western diet since the industrial revolution has resulted in a dramatic increase in the consumption of omega-6 fatty acids, with a concurrent decrease in the consumption of omega-3 fatty acids. This decrease in omega-3 fatty acid consumption has been associated with significant disease burden, including increased susceptibility to infectious diseases. Here we provide evidence that DHA, an omega-3 fatty acid, has superior antimicrobial effects upon the highly drug-resistant pathogen Acinetobacter baumannii, thereby providing insights into one of the potential health benefits of omega-3 fatty acids. The identification and characterization of two novel bacterial membrane protective mechanisms against host fatty acids provide important insights into A. baumannii adaptation during disease. Furthermore, we describe a novel role for the major multidrug efflux system AdeIJK in A. baumannii membrane maintenance and lipid transport. This core function, beyond drug efflux, increases the appeal of AdeIJK as a therapeutic target.

Evolution of Daptomycin Resistance in Coagulase-Negative Staphylococci Involves Mutations of the Essential Two-Component Regulator WalKR.

Coagulase-negative staphylococci (CoNS) represent one of the major causes of health care- and medical device-associated infections. Emerging antimicrobial resistance has complicated the treatment of systemic infections caused by CoNS. Here, we describe the prevalence of antimicrobial resistance in clinical CoNS strains from a tertiary care hospital over a 4-year period, and we observed a significant increase in resistance to daptomycin. Notably, Staphylococcus capitis accounted for the majority of these daptomycin-resistant (DAP-R) CoNS. To further investigate the mechanisms of daptomycin resistance in CoNS, daptomycin-susceptible clinical strains of S. capitis and Staphylococcus epidermidis underwent in vitro daptomycin exposure to generate DAP-R CoNS mutants. Unlike that seen with Staphylococcus aureus, alteration of cell surface charge was not observed in the DAP-R CoNS strains, but biofilm formation was compromised. Whole-genome sequencing analysis of the DAP-R CoNS strains identified single nucleotide polymorphisms (SNPs) in walKR, the essential two-component regulatory system controlling cell wall biogenesis. PCR and sequencing of walK and walR from 17 DAP-R CoNS clinical isolates identified seven nonsynonymous mutations. The results were confirmed by the recreation of the walK SNP in S. epidermidis, which resulted in reduced susceptibility to daptomycin and vancomycin. This study highlights the significance of CoNS in evolving daptomycin resistance and showed that walKR is shared among the staphylococcal species and is involved in antibiotic resistance development. Notably, we did not observe mutations in genes responsible for phospholipid biosynthesis or an altered cell surface charge, suggesting that reduced daptomycin susceptibility in CoNS may emerge in a fashion distinct from that in S. aureus.

In vivo atomic force microscopy-infrared spectroscopy of bacteria.

A new experimental platform for probing nanoscale molecular changes in living bacteria using atomic force microscopy-infrared (AFM-IR) spectroscopy is demonstrated. This near-field technique is eminently suited to the study of single bacterial cells. Here, we report its application to monitor dynamical changes occurring in the cell wall during cell division in Staphylococcus aureus using AFM to demonstrate the division of the cell and AFM-IR to record spectra showing the thickening of the septum. This work was followed by an investigation into single cells, with particular emphasis on cell-wall signatures, in several bacterial species. Specifically, mainly cell wall components from S. aureus and Escherichia coli containing complex carbohydrate and phosphodiester groups, including peptidoglycans and teichoic acid, could be identified and mapped at nanometre spatial resolution. Principal component analysis of AFM-IR spectra of six living bacterial species enabled the discrimination of Gram-positive from Gram-negative bacteria based on spectral bands originating mainly from the cell wall components. The ability to monitor in vivo molecular changes during cellular processes in bacteria at the nanoscale opens a new platform to study environmental influences and other factors that affect bacterial chemistry.

Outer membrane vesicles from Neisseria gonorrhoeae target PorB to mitochondria and induce apoptosis.

Neisseria gonorrhoeae causes the sexually transmitted disease gonorrhoea by evading innate immunity. Colonizing the mucosa of the reproductive tract depends on the bacterial outer membrane porin, PorB, which is essential for ion and nutrient uptake. PorB is also targeted to host mitochondria and regulates apoptosis pathways to promote infections. How PorB traffics from the outer membrane of N. gonorrhoeae to mitochondria and whether it modulates innate immune cells, such as macrophages, remains unclear. Here, we show that N. gonorrhoeae secretes PorB via outer membrane vesicles (OMVs). Purified OMVs contained primarily outer membrane proteins including oligomeric PorB. The porin was targeted to mitochondria of macrophages after exposure to purified OMVs and wild type N. gonorrhoeae. This was associated with loss of mitochondrial membrane potential, release of cytochrome c, activation of apoptotic caspases and cell death in a time-dependent manner. Consistent with this, OMV-induced macrophage death was prevented with the pan-caspase inhibitor, Q-VD-PH. This shows that N. gonorrhoeae utilizes OMVs to target PorB to mitochondria and to induce apoptosis in macrophages, thus affecting innate immunity.

Vancomycin susceptibility in methicillin-resistant Staphylococcus aureus is mediated by YycHI activation of the WalRK essential two-component regulatory system.

The treatment of infections caused by methicillin-resistant Staphylococcus aureus is complicated by the emergence of strains with intermediate-level resistance to vancomycin (termed VISA). We have characterised a molecular pathway involved in the in vivo evolution of VISA mediated by the regulatory proteins YycH and YycI. In contrast to their function in other bacterial species, we report a positive role for these auxiliary proteins in regulation of the two-component regulator WalRK. Transcriptional profiling of yycH and yycI deletion mutants revealed downregulation of the 'WalRK regulon' including cell wall hydrolase genes atlA and sle1, with functional autolysis assays supporting these data by showing an impaired autolytic phenotype for each deletion strain. Using bacterial-two hybrid assays, we showed that YycH and YycI interact, and that YycHI also interacts with the sensor kinase WalK, forming a ternary protein complex. Mutation to YycH or YycI associated with clinical VISA strains had a deleterious impact on the YycHI/WalK complex, suggesting that the interaction is important for the regulation of WalRK. Taken together, we have described a novel antibiotic resistance strategy for the human pathogen S. aureus, whereby YycHI mutations are selected for in vivo leading to reduced WalRK activation, impaired cell wall turnover and ultimately reduced vancomycin efficacy.

Cell death caused by the synergistic effects of zinc and dopamine is mediated by a stress sensor gene Gadd45b - implication in the pathogenesis of Parkinson's disease.

The pathogenesis of Parkinson's disease (PD) is not completely understood, Zinc (Zn(2+) ) and dopamine (DA) have been shown to involve in the degeneration of dopaminergic cells. By microarray analysis, we identified Gadd45b as a candidate molecule that mediates Zn(2+) and DA-induced cell death; the mRNA and protein levels of Gadd45b are increased by Zn(2+) treatment and raised to an even higher level by Zn(2+) plus DA treatment. Zn(2+) plus DA treatment-induced PC12 cell death was enhanced when there was over-expression of Gadd45b and was decreased by knock down of Gadd45b. MAPK p38 and JNK signaling was able to cross-talk with Gadd45b during Zn(2+) and DA treatment. The synergistic effects of Zn(2+) and DA on PC12 cell death can be accounted for by an activation of the Gadd45b-induced cell death pathway and an inhibition of p38/JNK survival pathway. Furthermore, the in vivo results show that the levels of Gadd45b protein expression and phosphorylation of p38 were increased in the substantia nigra by the infusion of Zn(2+) /DA in the mouse brain and the level of Gadd45b mRNA is significantly higher in the substantia nigra of male PD patients than normal controls. The novel role of Gadd45b and its interactions with JNK and p38 will help our understanding of the pathogenesis of PD and help the development of future treatments for PD. Zinc and dopamine are implicated in the degeneration of dopaminergic neurons. We previously demonstrated that zinc and dopamine induced synergistic effects on PC12 cell death. Results from this study show that these synergistic effects can be accounted for by activation of the Gadd45b-induced cell death pathway and inhibition of the p38/JNK survival pathway. We provide in vitro and in vivo evidence to support a novel role for Gadd45b in the pathogenesis of Parkinson's disease.

Daptomycin-Nonsusceptible Staphylococcus aureus: The Role of Combination Therapy with Daptomycin and Gentamicin.

Reduced susceptibility to daptomycin in Staphylococcus aureus has now been described, leading to clinical failures. Here we determined the impact of daptomycin and gentamicin combination therapy on bactericidal activity and resistance emergence using daptomycin-susceptible and -resistant isolates with mutations linked to previous daptomycin or vancomycin exposure. Enhanced killing of S. aureus was observed when gentamicin was combined with daptomycin, most commonly with daptomycin concentrations below the peak serum free-drug concentrations achieved with standard dosing. Synergy was seen with daptomycin-susceptible isolates and with isolates resistant to vancomycin and daptomycin. Combination therapy also prevented the emergence of resistance. Daptomycin and gentamicin combination therapy may provide the synergy required to prevent emergence of resistance when daptomycin levels are below peak serum concentrations as would be found in deep-seated, complicated infections.