Evolution of RND efflux pumps in the development of a successful pathogen.

AIMS: This study examined the origins and evolution of the AdeABC, AdeFGH and AdeIJK efflux pumps in the Acinetobacter genus, including human and animal pathogens and species from non-clinical environments. METHODS: Comparative genome analyses were performed using the reference sequences for 70 Acinetobacter species to identify putative orthologs of AdeABC, AdeFGH and AdeIJK and their regulators. Sequence similarities and the genomic locations of coding sequences were correlated with phylogeny to infer modes of evolution. Intraspecies variation was assessed in species of interest using up to 236 complete genome sequences. Mutants overproducing adeIJK in A. baylyi were examined to identify regulators of this system in a non A. baumannii species. RESULTS: The results indicate that adeIJK has been a stable part of Acinetobacter genomes since the genesis of this genus, whereas adeABC and adeFGH were carried by less than half of the species, but showed some lineage specificity. The organisation and local genetic contexts of adeABC loci were particularly variable to the sub-species level, suggesting frequent recombination. Cognate regulatory systems were almost always found in the genomes of species encoding pumps. Mutations in adeN, which encodes a repressor of adeIJK, were selected by antibiotic exposure in A. baylyi, similar to previous findings in pathogenic lineages. CONCLUSIONS: The multidrug efflux capacity of clinical Acinetobacter strains stems from accessory and core genetic features. AdeIJK is likely to have ancient core function(s) that have promoted its maintenance, whereas recent antibiotic use may be driving the evolution of the AdeABC pump.

Perivascular macrophages mediate neutrophil recruitment during bacterial skin infection.

Transendothelial migration of neutrophils in postcapillary venules is a key event in the inflammatory response against pathogens and tissue damage. The precise regulation of this process is incompletely understood. We report that perivascular macrophages are critical for neutrophil migration into skin infected with the pathogen Staphylococcus aureus. Using multiphoton intravital microscopy we showed that neutrophils extravasate from inflamed dermal venules in close proximity to perivascular macrophages, which are a major source of neutrophil chemoattractants. The virulence factor α-hemolysin produced by S. aureus lyses perivascular macrophages, which leads to decreased neutrophil transmigration. Our data illustrate a previously unrecognized role for perivascular macrophages in neutrophil recruitment to inflamed skin and indicate that S. aureus uses hemolysin-dependent killing of these cells as an immune evasion strategy.

Roles of DHA2 family transporters in drug resistance and iron homeostasis in Acinetobacter spp.

BACKGROUND: Acinetobacter baumannii is a major cause of nosocomial infections worldwide due to its fitness within clinical settings and recalcitrance to conventional therapies. The drug: H(+) antiporter 2 (DHA2) family export systems encoded by A. baumannii were investigated for their roles in promoting the success ofthis organism as a human pathogen. METHODS: Bioinformatic tools were used to identify the DHA2 family transporters encoded by Acinetobacter spp. and establish their phylogenetic relationships. The drug resistance phenotypes conferred by the transporters were tested using both heterologously expressed proteins in Escherichia coli and Acinetobacter deletion mutants. The transcriptional responses of DHA2 family transporter genes to their substrates were established by qRT-PCR. RESULTS: Six highly conserved DHA2 family proteins were identified in A. baumannii. Drug resistance phenotypes were established for two DHA2 family transporters. The expression of a third DHA2 family protein is highly responsive to the availability of iron. The gene encoding this protein is located within a putative siderophore biosynthesis locus, suggesting a physiological role in iron uptake, possibly via the export of a siderophore. CONCLUSIONS: These results highlight functions for DHA2 family proteins in both drug resistance and the maintenance of stable cellular physiology, emphasizing their importance in A. baumannii infections.

Segrosome structure revealed by a complex of ParR with centromere DNA.

The stable inheritance of genetic material depends on accurate DNA partition. Plasmids serve as tractable model systems to study DNA segregation because they require only a DNA centromere, a centromere-binding protein and a force-generating ATPase. The centromeres of partition (par) systems typically consist of a tandem arrangement of direct repeats. The best-characterized par system contains a centromere-binding protein called ParR and an ATPase called ParM. In the first step of segregation, multiple ParR proteins interact with the centromere repeats to form a large nucleoprotein complex of unknown structure called the segrosome, which binds ParM filaments. pSK41 ParR binds a centromere consisting of multiple 20-base-pair (bp) tandem repeats to mediate both transcription autoregulation and segregation. Here we report the structure of the pSK41 segrosome revealed in the crystal structure of a ParR-DNA complex. In the crystals, the 20-mer tandem repeats stack pseudo-continuously to generate the full-length centromere with the ribbon-helix-helix (RHH) fold of ParR binding successive DNA repeats as dimer-of-dimers. Remarkably, the dimer-of-dimers assemble in a continuous protein super-helical array, wrapping the DNA about its positive convex surface to form a large segrosome with an open, solenoid-shaped structure, suggesting a mechanism for ParM capture and subsequent plasmid segregation.