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

Legionella pneumophila Secretes a Mitochondrial Carrier Protein during Infection

The Mitochondrial Carrier Family (MCF) is a signature group of integral membrane proteins that transport metabolites across the mitochondrial inner membrane in eukaryotes. MCF proteins are characterized by six transmembrane segments that assemble to form a highly-selective channel for metabolite transport. We discovered a novel MCF member, termed Legionella nucleotide carrier Protein (LncP), encoded in the genome of Legionella pneumophila, the causative agent of Legionnaire's disease. LncP was secreted via the bacterial Dot/Icm type IV secretion system into macrophages and assembled in the mitochondrial inner membrane. In a yeast cellular system, LncP induced a dominant-negative phenotype that was rescued by deleting an endogenous ATP carrier. Substrate transport studies on purified LncP reconstituted in liposomes revealed that it catalyzes unidirectional transport and exchange of ATP transport across membranes, thereby supporting a role for LncP as an ATP transporter. A hidden Markov model revealed further MCF proteins in the intracellular pathogens, Legionella longbeachae and Neorickettsia sennetsu, thereby challenging the notion that MCF proteins exist exclusively in eukaryotic organisms

Neisserial pathogenesis: Key factors in disease

© 2012 Dr. Jhih-Hang JiangThe Gram-negative bacteria, Neisseria meningitidis and Neisseria gonorrhoeae, are human pathogens that cause meningococcal meningitis and sexually transmitted gonorrhea respectively. During infection, the abundant outer membrane protein PorB is translocated from bacteria to host cells as a virulence factor for the advantage of pathogenic Neisserial species. Mitochondria are reported to be a primary target of PorB where it has a role in interfering with host cell apoptosis regulation although a role in induction and inhibition of apoptosis has also been suggested. Determining the exact localization of PorB in mitochondria and the molecular mechanisms behind the targeting of PorB into mitochondria stands as a key to deciphering how pathogenic Neisserial species manipulate mitochondrial function during infection. The understanding of the PorB targeting is hindered by the lack of a suitable assay to accurately measure the assembly of the β-barrel protein, PorB, in mitochondria. In Chapter 2 of this thesis, a novel assay combining in vitro mitochondrial import and semi-native gel analysis was developed to measure the folding and assembly of PorB in mitochondria. Studies using this novel assay indicated that PorB was folded and assembled in the mitochondrial outer membrane. The assembly of PorB in mitochondria was facilitated by the intermembrane space chaperones, the small TIMs, and the core subunit of sorting and assembly machinery (SAM), Sam50. Sam50 is the homolog of BamA, which is also the core subunit of β-barrel assembly machinery (BAM) in Gram-negative bacteria, which indicates that PorB targets to the mitochondrial outer membrane via an evolutionary conserved pathway. Although the translocation of PorB from pathogenic Neisserial species to host cells was first described in 1984, it is still not clear how the pathogens deliver PorB to host cells. Outer membrane vesicles (OMVs) containing outer membrane proteins are continuously released from the Neisserial cell surface. Furthermore, OMVs from other Gram-negative bacteria can deliver bacterial proteins to host cells. In Chapter 3, OMVs from N. gonorrhoeae were isolated and the protein contents were identified by mass spectrometry coupled with bioinformatic analysis. Several proteins were specifically present in OMVs when compared to the protein profile of outer membranes, and these proteins may have a role in Neisserial pathogenesis. The treatment of host cells with gonococcal OMVs followed by immunofluorescence microscopy for the detection of PorB indicated that PorB was delivered to the proximity of mitochondria via OMVs, describing a potential mechanism for pathogenic Neisserial species to release PorB to host cells. The release of OMVs from pathogenic Neisseria was first reported in 1982, but the role of Neisserial OMVs during pathogenesis is unclear. In Chapter 4, the effects of OMVs on host cells were investigated. The results suggested that the treatment of host cells with OMVs resulted in compromised metabolic activity, loss of mitochondrial membrane potential, morphological changes and cell death. Autophagy was also triggered in host cells by OMVs, possibly as a defense mechanism or for the recycling of damaged mitochondria. Therefore, the Neisseria derived vesicles may have a role in gonococcal infection to affect host cell fate, possibly by delivering virulence factors

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

Draft Genome Sequences of Clinical Daptomycin-Nonsusceptible Methicillin-Resistant Staphylococcus aureus Strain APS211 and Its Daptomycin-Susceptible Progenitor APS210

To assess the genetic factors contributing to daptomycin resistance in Staphylococcus aureus, the draft genome of a clinically derived daptomycin-nonsusceptible isolate APS211 was generated and compared to the draft sequence of its susceptible progenitor strain APS210. Four genetic differences were identified including a previously described mutation within the mprF gene

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

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

Virulence attributes of successful methicillin-resistant Staphylococcus aureus lineages.

SUMMARYMethicillin-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