Rob Nicholas, PhD

Research Synopsis

P2Y Receptor Trafficking.

My lab is focused on defining the mechanisms by which the eight subtypes of P2Y receptors are targeted to distinct membrane domains in polarized epithelial cells. Epithelial cells, which form a tight monolayer between two compartments, have two distinct membrane surfaces: the apical membrane and the basolateral membrane. Membrane proteins can either be targeted to one of the two membrane surfaces, or they can be non-sorted. The proper targeting of proteins to either the apical or basolateral surface is critical for normal epithelial cell function. Multiple P2Y receptor subtypes are expressed in epithelial cells and are intimately involved in regulating epithelial cell physiology. We have recently begun to define the targeting sequences within each member of the P2Y receptors (Fig. 1). These experiments have utilized construction and expression of chimeric receptors in MDCK (Manin-Darby Canine Kidney) cells, a useful polarized epithelial cell line, and subsequent localization of these receptors by confocal microscopy. Our data indicate that each of the P2Y receptors achieves steady-state localization at a particular membrane surface by a different mechanism.

Antibiotic Resistance Mechanisms.

My laboratory is also interested in the mechanisms of antibiotic resistance in the pathogenic bacterium Neisseria gonorrhoeae, which is the etiologic agent for the sexually transmitted infection gonorrhoea. Although penicillin used to be the antibiotic of choice in treating a gonococcal infection, increased resistance to this antibiotic necessitated its replacement by 3rd generation cephalosporins or fluorinated quinolones for the treatment of infected individuals (resistance to fluoroquinolones has recently emerged). Resistance to penicillin and other antibiotics occurs through chromosomally-mediated alterations in endogenous genes. At least five genes are involved in mediating high level resistance to penicillin. These include penA and ponA, which encode alterations in the two essential PBPs of this organism (PBPs 1 and 2) that decrease their affinity for penicillin; mtrR, which increases expression of an efflux pump; penC, which is a spontaneously arising mutation in the PilQ secretin; and penB, which encodes mutations in the major outer membrane porin, PIB (Fig. 2). Our research utilizes biochemistry, structural biology, and genetics to define how these resistance genes synergize with one another to increase resistance to antibiotics.

Research Interests

P2Y Receptors

• P2Y Receptor trafficking in polarized epithelial cells
• Signaling mechanisms of P2Y receptors

Antibiotic Action in Neisseria gonorrhoeae

• Mechanisms of chromosomally mediated antibiotic resistance
• Structure/function of PilQ and other pilus-related proteins

Publications