Radioiodinated Capsids Facilitate In Vivo Non-Invasive Tracking of Adeno-Associated Gene Transfer Vectors.

Viral vector mediated gene therapy has become commonplace in clinical trials for a wide range of inherited disorders. Successful gene transfer depends on a number of factors, of which tissue tropism is among the most important. To date, definitive mapping of the spatial and temporal distribution of viral vectors in vivo has generally required postmortem examination of tissue. Here we present two methods for radiolabeling adeno-associated virus (AAV), one of the most commonly used viral vectors for gene therapy trials, and demonstrate their potential usefulness in the development of surrogate markers for vector delivery during the first week after administration. Specifically, we labeled adeno-associated virus serotype 10 expressing the coding sequences for the CLN2 gene implicated in late infantile neuronal ceroid lipofuscinosis with iodine-124. Using direct (Iodogen) and indirect (modified Bolton-Hunter) methods, we observed the vector in the murine brain for up to one week using positron emission tomography. Capsid radioiodination of viral vectors enables non-invasive, whole body, in vivo evaluation of spatial and temporal vector distribution that should inform methods for efficacious gene therapy over a broad range of applications.

Growth-phase-dependent expression of the cyclopeptide antibiotic microcin J25.

Microcin J25 is a 2,107-Da, plasmid-encoded, cyclopeptide antibiotic produced by Escherichia coli. We have isolated lacZ fusions to mcjA (encoding the 58-amino-acid microcin precursor) and mcjB and mcjC (which are required for microcin maturation), and the regulation of these fusions was used to identify factors that control the expression of these genes. The mcjA gene was found to be dramatically induced as cells entered the stationary phase. Expression of mcjA could be induced by resuspending uninduced exponential-phase cells in spent supernatant obtained from an early-stationary-phase culture. Induction of mcjA expression was not dependent on high cell density, pH changes, anaerobiosis, or the buildup of some inducer. A starvation for carbon and inorganic phosphate induced mcjA expression, while under nitrogen limitation there was no induction at all. These results taken together suggest that stationary-phase induction of mcjA is triggered by nutrient depletion. The mcjB and mcjC genes were also regulated by the growth phase of the culture, but in contrast to mcjA, they showed substantial expression already during exponential growth. Induction of the microcin genes was demonstrated to be independent of RpoS, the cyclic AMP-Crp complex, OmpR, and H-NS. Instead, we found that the growth-phase-dependent expression of mcjA, mcjB, and mcjC may be explained by the concerted action of the positively acting transition state regulators ppGpp, Lrp, and integration host factor. Measurements of microcin J25 production by strains defective in these global regulators showed a good correlation with the reduced expression of the fusions in such mutant backgrounds.

Escherichia coli outer membrane protein TolC is involved in production of the peptide antibiotic microcin J25.

A Tn5 insertion in tolC eliminated microcin J25 production. The mutation had little effect on the expression of the microcin structural gene and presumably acted by blocking microcin secretion. The tolC mutants carrying multiple copies of the microcin genes were less immune to the microcin. TolC is thus likely a component of a microcin export complex containing the McjD immunity protein, an ABC exporter.

The cyclic structure of microcin J25, a 21-residue peptide antibiotic from Escherichia coli.

Microcin J25 (MccJ25) is the single representative of the immunity group J of the microcin group of peptide antibiotics produced by Enterobacteriaceae. It induces bacterial filamentation in susceptible cells in a non-SOS-dependent pathway [R. A. Salomon and R. Farias (1992) J. Bacteriol. 174, 7428-7435]. MccJ25 was purified to homogeneity from the growth medium of a microcin-overproducing Escherichia coli strain by reverse-phase HPLC. Based on amino acid composition and absolute configuration determination, liquid secondary ion and electrospray mass spectrometry, extensive two-dimensional NMR, enzymatic and chemical degradations studies, the structure of MccJ25 was elucidated as a 21-residue peptide, cyclo(-Val1-Gly-Ile-Gly-Thr- Pro-Ile-Ser-Phe-Tyr-Gly-Gly-Gly-Ala-Gly-His-Val-Pro-Glu-Tyr-Phe21- ). Although MccJ25 showed high resistance to most of endoproteases, linearization by thermolysin occurred from cleavage at the Phe21-Val1 bond and led to a single peptide, MccJ25-L. While MccJ25 exhibited remarkable antibiotic activity towards Salmonella newport and several E. coli strains (minimal inhibitory concentrations ranging between 0.01 and 0.2 microgram.mL-1), the thermolysin-linearized microcin showed a dramatic decrease of the activity, indicating that the cyclic structure is essential for the MccJ25 biological properties. As MccJ25 is ribosomally synthesized as a larger peptide precursor endowed with an N-terminal extremity, the present study shows that removal of this extension and head-tail cyclization of the resulting propeptide are the only post-translational modifications involved in the maturation of MccJ25, that appears as the first cyclic microcin.