A Transporter Interactome Is Essential for the Acquisition of Antimicrobial Resistance to Antibiotics

Awareness of the problem of antimicrobial resistance (AMR) has escalated and drug-resistant infections are named among the most urgent problems facing clinicians today. Our experiments here identify a transporter interactome and portray its essential function in acquisition of antimicrobial resistance. By exposing E. coli cells to consecutive increasing concentrations of the fluoroquinolone norfloxacin we generated in the laboratory highly resistant strains that carry multiple mutations, most of them identical to those identified in clinical isolates. With this experimental paradigm, we show that the MDTs function in a coordinated mode to provide an essential first-line defense mechanism, preventing the drug reaching lethal concentrations, until a number of stable efficient alterations occur that allow survival. Single-component efflux transporters remove the toxic compounds from the cytoplasm to the periplasmic space where TolC-dependent transporters expel them from the cell. We postulate a close interaction between the two types of transporters to prevent rapid leak of the hydrophobic substrates back into the cell. The findings change the prevalent concept that in Gram-negative bacteria a single multidrug transporter, AcrAB-TolC type, is responsible for the resistance. The concept of a functional interactome, the process of identification of its members, the elucidation of the nature of the interactions and its role in cell physiology will change the existing paradigms in the field. We anticipate that our work will have an impact on the present strategy searching for inhibitors of AcrAB-TolC as adjuvants of existing antibiotics and provide novel targets for this urgent undertaking.     

Uptake and fate of IAA in apple callus tissue using IAA-1-14C

Incubation of young growing and older non-growing apple callustissues in a medium containing IAA-1-¹⁴C resulted in rapid disappearanceof the IAA. In old calluses (3 months), the major portion ofIAA was lost by decarboxylation (90% after 4 hr) and very little(1.4%) was maintained by the tissue. In young calluses, after4 hr in light, decarboxylation reached 20% and absorption 35%of the labelled IAA. Some decomposition of IAA was caused byphotolysis and autoclaving (19% and 3%, respectively) but thefinal distribution of radioactivity was not affected. Factorssuch as sucrose concentration in the incubation medium, distilledwater as incubation medium, and cutting of the callus did notaffect tissue behavior. Special precautions were taken to eliminatenon-biological decomposition of IAA. Therefore, we believe thatthe rapid CO₂ evolution is of enzymatic nature. This theoryis supported by the drop in decarboxylation after killing ofthe callus, and the increase of decarboxylation with age. Noenzyme was secreted by the callus into the medium after 24 hrof incubation, and IAA decomposition in old tissues is doneprobably on the surface. Absorption of IAA increased with increasingcallus size and decarboxylation decreased. ¹ Contribution from the Agricultural Research Organization,The Volcani Center, Bet Dagan, Israel. 1973 Series, No. 274-E. (Received May 30, 1974; )     

A Method for Removing Effects of Nonspecific Binding on the Distribution of Binding Stoichiometries: Application to Mass Spectroscopy Data

There is often an interest in knowing, for a given ligand concentration, how many protein molecules have one, two, three, etc. ligands bound in a specific manner. This is a question that cannot be addressed using conventional ensemble techniques. Here, a mathematical method is presented for separating specific from nonspecific binding in nonensemble studies. The method provides a way to determine the distribution of specific binding stoichiometries at any ligand concentration when using nonensemble (e.g., single-molecule) methods. The applicability of the method is demonstrated for ADP binding to creatine kinase using mass spectroscopy data. A major advantage of our method, which can be applied to any protein-ligand system, is that no previous information regarding the mechanism of ligand interaction is required.     

A new,fast, and very sensitive bioluminescence assay for phospholipases A and C

A new, simple, and very sensitive assay for phospholipase A and C is described. The assay is based on the bioluminescence developed by the mutant of the bacterium Beneckea harveyi as a response to myristic acid released from dimyristoyl phosphatidylcholine by either phospholipase A or by a phospholipase C-lipase coupled system. It is possible to assay these enzymes at a rate corresponding to a release of as little as 1 to 2 pmol of myristic acid per minute.     

Genetic compatibility in long-term intimate relationships: partner similarity at major histocompatibility complex (MHC) genes may reduce in-pair attraction

Theoretical models from evolutionary biology predict that individual mate choice will be influenced by the extent of similarity between potential mates at the major histocompatibility complex (MHC) genes. A number of studies have sought to uncover an effect of MHC similarity on mate choice in humans, but the extent to which MHC similarity influences attraction within existing human relationships has been relatively under-explored. We investigated this question in a sample of 168 heterosexual couples that were typed and matched at 3 classical MHC markers. Findings were mixed with respect to the prediction that higher levels of MHC similarity would be linked to a reduction of in-pair attraction. In the full sample, there were no effects of MHC similarity on any of the dependent variables used to measure in-pair attraction, but there were strong and consistent effects of MHC similarity on these measures in couples with two Asian partners (N couples =44). In sum, our findings are consistent with an effect of MHC similarity on in-pair attraction within existing relationships, but they also suggest that this effect may be moderated by additional factors, particularly the ancestral background of the individual relationship partners.     

Peptide length requirement for experimental allergic encephalomyelitis in guinea pigs

Three peptides overlapping the tryptophan region of bovine CNS myelin basic protein were synthesized by the solid phase procedure of Merrifield. These were the nonapeptide H-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH, the octapeptide H-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH, and the heptapeptide H-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH. They were tested for encephalitogenic activity in guinea pigs with either Freund's complete adjuvant containing M. tuberculosis or muramyl dipeptide in incomplete Freund's adjuvant at doses of 10 microgram per animal. The results show that deletion of one or two residues from the amino-terminal end of the nonapeptide destroyed the ability of the shorter peptides to induce clinical but not histological signs of EAE.