Hiroshi Nikaido's research interests are in the biochemical and molecular genetic analysis of the structure and functions of bacterial membranes. Topics currently pursued include the specific and non-specific channel-forming proteins of the outer membrane, the diffusion of lipophilic compounds (inhibitors and antibiotics) across the unusually impermeable bilayer domain of the outer membrane, as well as the mechanism and regulation of multidrug efflux transport systems that pump out an incredibly wide range of compounds from bacterial cells. His research group is also studying the structure and functions of mycobacterial cell wall, which makes these bacteria intrinsically resistant to most drugs because it acts as an exceptionally efficient permeability barrier.
The most fundamental function of biological membrane is to serve as a general permeation barrier and at the same time to allow the selective permeation of certain types of molecules. They are trying to understand the molecular mechanisms underlying this function by using several systems.
All Gram-negative bacteria, including Escherichia coli, produce an extra membrane layer, called outer membrane, which is located outside the cytoplasmic membrane and the peptidoglycan (cell wall) layer. This is an ideal model for studies of this type, because sources of energy are not available at this location, and therefore the membrane allows only the passive and facilitated diffusion processes. In this system, they discovered one of the first examples of the proteins forming non-specific diffusion channels, and named it "porin." This was followed by their identification of the phage lambda receptor protein as the protein that produces channels specific for maltose and maltodextrins. They are studying the structure-function relationships in these channel-forming proteins by a variety of approaches. They believe these studies are of potential significance, not only because these channels can serve as models for other channels with more complex functions, but also because, from the practical point of view, most antibiotics have to pass through these outer membrane channels in order to be effective against Gram-negative bacteria.
Another example of an effective permeability barrier on cell surface is the cell wall of mycobacteria. They discovered that this is a bilayer of very unusual composition, with the parallel arrangement of extremely long fatty acid chains producing a structure of exceptionally low fluidity, which prevents the rapid influx of antibiotics and chemotherapeutic agents and thus contributes to the intrinsic resistance of these bacteria.
They became aware, however, during the course of these studies that permeability barriers by themselves are not sufficient to produce high levels of drug resistance. Search for additional mechanisms led to the discovery that most bacteria produce active efflux pumps that display extremely wide range of substrate specificity, a range that had not been suspected to exist earlier. For example, the AcrAB efflux pump of E. coli pumps out not only dyes and detergents but also practically all commercially important classes of antibiotics, with the sole exception of aminoglycosides, and in addition, even simple solvent molecules.
Furthermore, most Gram-negative pumps can excrete drugs directly into the external media, bypassing the outer membrane barrier: thus the slow entry of drugs through the outer membrane acts synergistically with the direct efflux process, to prevent the intracellular accumulation of noxious compounds. They are actively studying the molecular mechanisms of these fascinating transporters, as well as their physiological and genetic regulation. The most exciting news is that they have elucidated the structure of the pump with bound drug molecules in a collaborative work with the laboratory of Prof. D. E. Koshland, Jr. They found that the substrate-binding site is a vast cavity with a diameter of 35 angstroms, and that the wide substrate specificity is the result of drug binding that involves not only amino acid residues of the protein but also phospholipids within the cavity. This finding is likely to have implications in the mechanism of other multidrug efflux pumps, for example P-glycoprotein that pumps out anticancer drugs out of the cancer cells of patients, diminishing the efficacy of chemotherapy.