The Gram-negative permeability barrier: tipping the balance of the in and the out.

Gram-negative bacteria are intrinsically resistant to many antibiotics, due in large part to the permeability barrier formed by their cell envelope. The complex and synergistic interplay of the two Gram-negative membranes and active efflux prevents the accumulation of a diverse range of compounds that are effective against Gram-positive bacteria. A lack of detailed information on how components of the cell envelope contribute to this has been identified as a key barrier to the rational development of new antibiotics with efficacy against Gram-negative species. This review describes the current understanding of the role of the different components of the Gram-negative cell envelope in preventing compound accumulation and the state of efforts to describe properties that allow compounds to overcome this barrier and apply them to the development of new broad-spectrum antibiotics.

AadT, a new weapon in Acinetobacter's fight against antibiotics.

Genes encoding a novel multidrug efflux pump, AadT, from the Drug:H+ antiporter 2 family, were discovered in Acinetobacter multidrug resistance plasmids. Here, we profiled the antimicrobial resistance potential, and examined the distribution of these genes. aadT homologs were found in many Acinetobacter and other Gram-negative species and were typically adjacent to novel variants of adeAB(C), which encodes a major tripartite efflux pump in Acinetobacter. The AadT pump decreased bacterial susceptibility to at least eight diverse antimicrobials, including antibiotics (erythromycin and tetracycline), biocides (chlorhexidine), and dyes (ethidium bromide and DAPI) and was able to mediate ethidium transport. These results show that AadT is a multidrug efflux pump in the Acinetobacter resistance arsenal and may cooperate with variants of AdeAB(C).

Breaching the Barrier: Genome-Wide Investigation into the Role of a Primary Amine in Promoting E. coli Outer-Membrane Passage and Growth Inhibition by Ampicillin.

Gram-negative bacteria are problematic for antibiotic development due to the low permeability of their cell envelopes. To rationally design new antibiotics capable of breaching this barrier, more information is required about the specific components of the cell envelope that prevent the passage of compounds with different physiochemical properties. Ampicillin and benzylpenicillin are ß-lactam antibiotics with identical chemical structures except for a clever synthetic addition of a primary amine group in ampicillin, which promotes its accumulation in Gram-negatives. Previous work showed that ampicillin is better able to pass through the outer membrane porin OmpF in Escherichia coli compared to benzylpenicillin. It is not known, however, how the primary amine may affect interaction with other cell envelope components. This study applied TraDIS to identify genes that affect E. coli fitness in the presence of equivalent subinhibitory concentrations of ampicillin and benzylpenicillin, with a focus on the cell envelope. Insertions that compromised the outer membrane, particularly the lipopolysaccharide layer, were found to decrease fitness under benzylpenicillin exposure, but had less effect on fitness under ampicillin treatment. These results align with expectations if benzylpenicillin is poorly able to pass through porins. Disruption of genes encoding the AcrAB-TolC efflux system were detrimental to survival under both antibiotics, but particularly ampicillin. Indeed, insertions in these genes and regulators of acrAB-tolC expression were differentially selected under ampicillin treatment to a greater extent than insertions in ompF. These results suggest that maintaining ampicillin efflux may be more significant to E. coli survival than full inhibition of OmpF-mediated uptake. IMPORTANCE Due to the growing antibiotic resistance crisis, there is a critical need to develop new antibiotics, particularly compounds capable of targeting high-priority antibiotic-resistant Gram-negative pathogens. In order to develop new compounds capable of overcoming resistance a greater understanding of how Gram-negative bacteria are able to prevent the uptake and accumulation of many antibiotics is required. This study used a novel genome wide approach to investigate the significance of a primary amine group as a chemical feature that promotes the uptake and accumulation of compounds in the Gram-negative model organism Escherichia coli. The results support previous biochemical observations that the primary amine promotes passage through the outer membrane porin OmpF, but also highlight active efflux as a major resistance factor.

Increasing the PACE of characterising novel transporters by functional genomics.

Since the late 1990's the genome sequences for thousands of species of bacteria have been released into public databases. The release of each new genome sequence typically revealed the presence of tens to hundreds of uncharacterised genes encoding putative membrane proteins and more recently, microbial metagenomics has revealed countless more of these uncharacterised genes. Given the importance of small molecule efflux in bacteria, it is likely that a significant proportion of these genes encode for novel efflux proteins, but the elucidation of these functions is challenging. We used transcriptomics to predict that the function of a gene encoding a hypothetical membrane protein is in efflux-mediated antimicrobial resistance. We subsequently confirmed this function and the likely native substrates of the pump by using detailed biochemical and biophysical analyses. Functional studies of homologs of the protein from other bacterial species determined that the protein is a prototype for a family of multidrug efflux pumps - the Proteobacterial Antimicrobial Compound Efflux (PACE) family. The general functional genomics approach used here, and its expansion to functional metagenomics, will very likely reveal the identities of more efflux pumps and other transport proteins of scientific, clinical and commercial interest in the future.

Physiological Functions of Bacterial "Multidrug" Efflux Pumps.

Bacterial multidrug efflux pumps have come to prominence in human and veterinary pathogenesis because they help bacteria protect themselves against the antimicrobials used to overcome their infections. However, it is increasingly realized that many, probably most, such pumps have physiological roles that are distinct from protection of bacteria against antimicrobials administered by humans. Here we undertake a broad survey of the proteins involved, allied to detailed examples of their evolution, energetics, structures, chemical recognition, and molecular mechanisms, together with the experimental strategies that enable rapid and economical progress in understanding their true physiological roles. Once these roles are established, the knowledge can be harnessed to design more effective drugs, improve existing microbial production of drugs for clinical practice and of feedstocks for commercial exploitation, and even develop more sustainable biological processes that avoid, for example, utilization of petroleum.

NPC1-mTORC1 Signaling Couples Cholesterol Sensing to Organelle Homeostasis and Is a Targetable Pathway in Niemann-Pick Type C.

Lysosomes promote cellular homeostasis through macromolecular hydrolysis within their lumen and metabolic signaling by the mTORC1 kinase on their limiting membranes. Both hydrolytic and signaling functions require precise regulation of lysosomal cholesterol content. In Niemann-Pick type C (NPC), loss of the cholesterol exporter, NPC1, causes cholesterol accumulation within lysosomes, leading to mTORC1 hyperactivation, disrupted mitochondrial function, and neurodegeneration. The compositional and functional alterations in NPC lysosomes and nature of aberrant cholesterol-mTORC1 signaling contribution to organelle pathogenesis are not understood. Through proteomic profiling of NPC lysosomes, we find pronounced proteolytic impairment compounded with hydrolase depletion, enhanced membrane damage, and defective mitophagy. Genetic and pharmacologic mTORC1 inhibition restores lysosomal proteolysis without correcting cholesterol storage, implicating aberrant mTORC1 as a pathogenic driver downstream of cholesterol accumulation. Consistently, mTORC1 inhibition ameliorates mitochondrial dysfunction in a neuronal model of NPC. Thus, cholesterol-mTORC1 signaling controls organelle homeostasis and is a targetable pathway in NPC.

Tunability of DNA Polymerase Stability during Eukaryotic DNA Replication.

Structural and biochemical studies have revealed the basic principles of how the replisome duplicates genomic DNA, but little is known about its dynamics during DNA replication. We reconstitute the 34 proteins needed to form the S. cerevisiae replisome and show how changing local concentrations of the key DNA polymerases tunes the ability of the complex to efficiently recycle these proteins or to dynamically exchange them. Particularly, we demonstrate redundancy of the Pol α-primase DNA polymerase activity in replication and show that Pol α-primase and the lagging-strand Pol δ can be re-used within the replisome to support the synthesis of large numbers of Okazaki fragments. This unexpected malleability of the replisome might allow it to deal with barriers and resource challenges during replication of large genomes.

A randomized control trial of group counseling in a naltrexone treatment program.

This study evaluated the additional effectiveness of a 12-week manualized group counseling program over a structured naltrexone treatment program. The randomized controlled trial, the first of its kind in Australia, was conducted at Turning Point Alcohol and Drug Centre, Melbourne, Australia. Ninety-seven participants received a 50 mg dose of naltrexone daily and were randomized to either the experimental (n = 52) or control (n = 45) conditions. The experimental group received a structured group counseling program, which used a cognitive-behavioral relapse prevention approach. Using intention-to-treat analyses, there was only one statistically significant difference between the groups, with the control group reporting a significantly higher level of physical functioning at Week 6. All participants improved significantly in their level of heroin use and in psychosocial functioning between Baseline and Weeks 6, 12, and 24. It is not possible to conclude from these results whether or not group counseling provides additional benefit to naltrexone treatment.

Naltrexone maintenance for heroin dependence: uptake, attrition and retention.

With naltrexone registered only recently in Australia in 1999, it is important to examine the rate of uptake of naltrexone treatment, early attrition and retention rates during treatment, in order to inform the way naltrexone is used in Australian practice. Of 317 people screened for the study, 97 participants were recruited post-withdrawal from opiates and were inducted to naltrexone after a period of at least 5 days of abstinence. While in treatment, participants received a 50-mg dose of naltrexone daily, with daily dispensing for the first 7 days, and weekly dispensing for the following 11 weeks. For the naltrexone-treated sample as a whole, the rate of uptake of naltrexone treatment was 30%, with 30% retained in treatment for the entire 12-week program. Attrition from treatment was found to be steady throughout the 12 weeks. The authors conclude that further research is required to improve withdrawal and naltrexone induction techniques and to improve medication compliance and treatment retention.