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.     

Tuning RNA Flexibility with Helix Length and Junction Sequence

The increasing awareness of RNA’s central role in biology calls for a new understanding of how RNAs, like proteins, recognize biological partners. Because RNA is inherently flexible, it assumes a variety of conformations. This conformational flexibility can be a critical aspect of how RNA attracts and binds molecular partners. Structurally, RNA consists of rigid basepaired duplexes, separated by flexible non-basepaired regions. Here, using an RNA system consisting of two short helices, connected by a single-stranded (non-basepaired) junction, we explore the role of helix length and junction sequence in determining the range of conformations available to a model RNA. Single-molecule Förster resonance energy transfer reports on the RNA conformation as a function of either mono- or divalent ion concentration. Electrostatic repulsion between helices dominates at low salt concentration, whereas junction sequence effects determine the conformations at high salt concentration. Near physiological salt concentrations, RNA conformation is sensitive to both helix length and junction sequence, suggesting a means for sensitively tuning RNA conformations.     

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.     

Twinning in a colony of vervet monkeys (Cercopithecus aethiops sabaeus)

From 1975 through 1991, three sets of twins were born from a total of 693 live and stillborn births (0.43%) at the UCLA/Sepulveda Veterans Administration Medical Center Nonhuman Primate Research Laboratory, CA. None of the twin's mothers were related. Positive patrilineal relationships have not been established; however, a brother (not a twin) of the first set of twins may have fathered the third set. All twins were born to multiparous females and, in each case, only one infant survived beyond 4 days. One set of twins was dizygotic; the genetic status of the others is unknown. © 1994 Wiley-Liss, Inc.     

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.     

Uptake and intra-inclusion accumulation of exogenous immunoglobulin by <Emphasis Type="Italic">Chlamydia</Emphasis>-infected cells

Background  

Obligate intracellular pathogens belonging to the Chlamydiaceae family possess a number of mechanisms by which to manipulate the host cell and surrounding environment. Such capabilities include the inhibition of apoptosis, down-regulation of major histocompatability complex (MHC) and CD1/d gene expression, and the acquisition of host-synthesized nutrients. It is also documented that a limited number of host-derived macromolecules such as β-catenin and sphingomyelin accumulate within the inclusion.     

Synaptic inputs to the omega neuron of the cricket Teleogryllus oceanicus: differences in EPSP waveforms evoke by low and high sound frequencies

EPSP waveforms were recorded from the omega neuron of Teleogryllus oceanicus for 5 kHz and ultrasonic sound stimuli. EPSPs in response to 5 kHz stimuli were smooth in shape and increased in amplitude with increasing stimulus intensity, while responses to ultrasound consisted of series' of large, discrete, unitary EPSPs, which increased in frequency with stimulus intensity.The hypothesis that a few, synaptically potent receptors might account for ultrasound sensitivity was tested by examining temporal coupling between ultrasound responses of the omega neuron and of another ultrasound-sensitive neuron, INT-1. INT-1 spikes were temporally correlated both to omega neuron spikes and to the large EPSPs recorded in the omega neuron. Coupling was not apparent for 5 kHz stimuli.The omega neuron encodes the intensity of 5 kHz and ultrasonic stimuli with similar resolution. Response latencies are markedly shorter for ultrasonic stimuli.These findings suggest that 5 kHz information is carried by a relatively large number of receptors, each of which has only a small effect on central neurons, while ultrasound information is carried by a few, synaptically potent, receptors.