Characterization of the amp genes involved in the regulation of beta-lactamase expression in Pseudomonas aeruginosa

Antibiotic resistance, production of alginate and virulence factors, and altered host immune responses are the hallmarks of chronic Pseudomonas aeruginosa infection. Failure of antibiotic therapy has been attributed to the emergence of P. aeruginosa strains that produce β-lactamase constitutively. In Enterobacteriaceae, β-lactamase induction involves four genes with known functions: ampC, ampR, ampD, and ampG, encoding the enzyme, transcriptional regulator, amidase and permease, respectively. In addition to all these amp genes, P. aeruginosa possesses two ampG paralogs, designated ampG and ampP. In this study, P. aeruginosa ampC, ampR, ampG and ampP were analyzed. Inactivation of ampC in the prototypic PAO1 failed to abolish the β-lactamase activity leading to the discovery of P. aeruginosa oxacillinase PoxB. Cloning and expression of poxB in Escherichia coli confers β-lactam resistance. Both AmpC and PoxB contribute to P. aeruginosa resistance against a wide spectrum of β-lactam antibiotics. The expression of PoxB and AmpC is regulated by a LysR-type transcriptional regulator AmpR that up-regulates AmpC but down-regulates PoxB activities. Analyses of P. aeruginosa ampR mutant demonstrate that AmpR is a global regulator that modulates the expressions of Las and Rhl quorum sensing (QS) systems, and the production of pyocyanin, LasA protease and LasB elastase. Introduction of the ampR mutation into an alginate-producing strain reveals the presence of a complex co-regulatory network between antibiotic resistance, QS alginate and other virulence factor production. Using phoA and lacZ protein fusion analyses, AmpR, AmpG and AmpP were localized to the inner membrane with one, 16 and 10 transmembrane helices, respectively. AmpR has a cytoplasmic DNA-binding and a periplasmic substrate binding domains. AmpG and AmpP are essential for the maximal expression of β-lactamase. Analysis of the murein breakdown products suggests that AmpG exports UDP-N-acetylmuramyl-L-alanine-γ-D-glutamate-meso-diaminopimelic acid-D-alanine-D-alanine (UDP-MurNAc-pentapeptide), the corepressor of AmpR, whereas AmpP imports N-acetylglucosaminyl-beta-1,4-anhydro-N-acetylmuramic acid-Ala-γ-D-Glu-meso-diaminopimelic acid (GlcNAc-anhMurNAc-tripeptide) and GlcNAc-anhMurNAc-pentapeptide, the co-inducers of AmpR. This study reveals a complex interaction between the Amp proteins and murein breakdown products involved in P. aeruginosa β-lactamase induction. In summary, this dissertation takes us a little closer to understanding the P. aeruginosa complex co-regulatory mechanism in the development of β-lactam resistance and establishment of chronic infection. ^

Melanocytes in the developing and adult atrioventricular valves of the murine heart

The Neural Crest (NC) is a multipotential group of cells that arises from the dorsal aspect of the neural tube early in development. It is well established that a group of NC cells named Cardiac Neural Crest (CNC) migrates to the heart and plays a critical role in the remodeling of the aortic arch arteries and septation of the outflow tract. In this study, using the mouse mutant Pax3sp/sp that has CNC deficits I have identified a putative novel role for the CNC in regulating apoptosis in the atrioventricular (AV) endocardial cushion. The AV endocardial cushion undergoes remodeling to give rise to the cardiac AV valves. Using a transgenic mouse that carries the LacZ reporter gene under the control of the Dopachrome tautomerase promoter (Dct-LacZ), I found that another NC derived population, melanocyte precursors, also contribute to the AV endocardial cushion and developing AV valves. The analysis of Dct-LacZ embryos at different stages showed that NC cells already committed to the melanocytic fate migrate to the heart along the same initial pathway taken by those that will populate the skin. Hypopigmented mice carrying mutations in the Kit and Endothelin receptor b genes, that are critical for the proper development of skin melanocytes, do not have cardiac melanocytes indicating that cardiac and skin melanocyte precursors share the same initial signaling requirements. The analysis of murine adult hearts showed that melanocytes are mostly found in the atrial sides of the tricuspid and mitral valve leaflets. The distribution of melanocytes in the AV valves corresponds exactly to areas of high Versican B expression, a proteoglycan essential for the process of AV valve remodeling. To evaluate a potential role for melanocytes in the AV valves, a nanoindentation analysis of the tricuspid valves of wild type, hypopigmented and hyperpigmented mice was performed. The storage modulus, a measure of stiffness, for the leaflets obtained from hyperpigmented mice was considerably higher (10.5GPa) than that for the leaflets from wild type (7.5GPa) and hypopigmented animals (between 3.5 and 5.5 GPa) suggesting that melanocytes may contribute to the mechanical properties of the AV valves.

Screening for AmpR-Specific Inhibitors to Combat P. aeruginosa Infections

Pseudomonas aeruginosa, a Gram-negative bacterium, an opportunistic pathogen that infects individuals suffering from reduced immunity or damaged tissue. The treatment of these infections has become a major problem due to its increasing antibiotic resistance. Many multi-drug resistant isolates of P. aeruginosa can thwart most antibiotic classes including ?- lactams, fluoroquinolones, and aminoglycosides. Its ability to combat ?-lactams is in part due to expression of AmpC, a major chromosomally encoded ?-lactamase. The expression of ampC is positively regulated by AmpR. Besides antibiotic resistance, AmpR is an important regulator of various factors that are required for establishing acute and chronic infections. Loss of ampR makes P. aeruginosa susceptible to ?-lactams and less virulent than the wild type. We hypothesize that AmpR is a potential therapeutic target. In the absence of new drugs in the pipeline, the aim of this study is to find an AmpR-specific inhibitor to assist and improve the use of currently available ?- lactam treatment. A small-molecule library from Torrey Pines Institute will be used in this study. Two reporter systems, lux and lacZ, fused to a PampC promotor will be used to assess AmpR activity. Positive hits will be those that inhibit 50% PampC activity in the presence of sub inhibitory concentration of imipenem, a ?- lactam. The top positive hits will be screened for their ability to cause human cell-cytotoxicity. The non-cytotoxic hits will be assessed for their ability to affect P. aeruginosa virulence and antibiotic resistance using various in vitro assays. Determination of potential AmpR inhibitors will prove to be useful in fighting off infections and may save countless patients suffering from these infections.