SummaryNumerous Gram-negative bacteria use a type III, or contact dependent, secretion system to deliver proteins into the cytosol of host cells. All of these systems identified to date have been shown to have a role in pathogenesis. We have identified 13 genes on the Yersinia enterocolitica chromosome that encode a type III secretion apparatus plus two associated putative regulatory genes. In order to determine the function of this chromosomally-encoded secretion apparatus, we created an in frame deletion of a gene that has homology to the hypothesized inner membrane pore, ysaV. The ysaV mutant strain failed to secrete eight proteins, called Ysps, normally secreted by the parental strain when grown at 288C in Luria± Bertani (LB) broth supplemented with 0.4 M NaCl. Disruption of the ysaV gene had no effect on motility or phospholipase activity, suggesting this chromosomally encoded type III secretion pathway is distinct from the flagella secretion pathway of Y. enterocolitica. Deletion of the ysaV gene in a virulence plasmid positive strain had no effect on in vitro secretion of Yops by the plasmid-encoded type III secretion apparatus. Secretion of the Ysps was unaffected by the presence or absence of the virulence plasmid, suggesting the chromosomally encoded and plasmid-encoded type III secretion pathways act independently. Y. enterocolitica thus has three type III secretion pathways that appear to act independently. The ysaV mutant strain was somewhat attenuated in virulence compared with the wild type in the mouse oral model of infection (an approximately 0.9 log difference in LD 50 ). The ysaV mutant strain was nearly as virulent as the wild type when inoculated intraperitoneally in the mouse model. A ysaV probe hybridized to sequences in other Yersinia spp. and homologues were found in the incomplete Y. pestis genome sequence, indicating a possible role for this system throughout the genus.

Recombinant interferon alpha-2 (IFN-alpha2) is used clinically to treat a variety of viral diseases and cancers. IFN-alpha2 has a short circulating half-life, which necessitates frequent administration to patients. Previous studies showed that it is possible to extend the circulating half-life of IFN-alpha2 by modifying lysine residues of the protein with amine-reactive poly(ethylene glycol) (PEG) reagents. However, amine-PEGylated IFN-alpha2 comprises a heterogeneous product mixture with low specific activity due to the large number and critical locations of lysine residues in IFN-alpha2. In an effort to overcome these problems we determined the feasibility of creating site-specific, mono-PEGylated IFN-alpha2 analogues by introducing a free (unpaired) cysteine residue into the protein, followed by modification of the added cysteine residue with a maleimide-PEG reagent. IFN-alpha2 cysteine analogues were expressed in Escherichia coli and purified, and their in vitro bioactivities were measured in the human Daudi cell line growth inhibition assay. Several cysteine analogues were identified that do not significantly affect in vitro biological activity of IFN-alpha2. Certain of the cysteine analogues, but not wild-type IFN-alpha2, reacted with maleimide-PEG to produce mono-PEGylated proteins. The PEG-Q5C analogue retained high in vitro bioactivity (within 3- to 4-fold of wild-type IFN-alpha2) even when modified with 20- and 40-kDa PEGs. Pharmacokinetic experiments indicated that the 20-kDa PEG-Q5C and 40-kDa PEG-Q5C proteins have 20-fold and 40-fold longer half-lives, respectively, than IFN-alpha2 following subcutaneous administration to rats. These studies demonstrate the feasibility of using site-specific PEGylation technology to create a long-acting, mono-PEGylated IFN-alpha2 protein with high specific activity.

Recombinant interferon alpha-2 (IFN-alpha2) has proven useful for treating a variety of human cancers and viral diseases. IFN-alpha2 has a short circulating half-life in vivo, which necessitates daily or thrice weekly administration to patients. It is possible to extend the circulating half-life of IFN-alpha2 by random modification of lysine residues in the protein with polyethylene glycol (PEG); however, such preparations have heterogeneous structures and low specific activities, and may not provide optimal therapeutic benefits to patients. A long-acting, site-specific, monoPEGylated IFN-alpha2 protein has now been created by targeted attachment of a 20 kDa or a 40 kDa maleimide-PEG to a cysteine analogue of IFN-alpha2, M111C. In vitro bioactivities of the purified 20 kDa and 40 kDa PEG-M111C proteins were within 2- to 3-fold of those of wild type IFN-alpha2 and 7- to 10-fold better than that of a 40 kDa PEG IFN-alpha2 protein created using nontargeted, amine-PEGylation methodology. The 20 kDa and 40 kDa PEG-M111C proteins demonstrated 26- to 38-fold longer half-lives, respectively, than IFN-alpha2 following subcutaneous administration to rats. The 20 kDa PEG M111C protein inhibited growth of human NIH:OVCAR-3 cells transplanted into nude mice by >90%, as measured by tumor size, tumor weight, and number of animals with detectable tumors at necropsy, and was significantly more effective than a comparable dose of IFN-alpha2. These data extend our previous findings that bioactivity of IFN-alpha2 can be largely preserved by targeted attachment of PEG moieties to nonessential sites in the protein and provide evidence that site-specific PEGylated IFN-alpha2 proteins possess enhanced tumoricidal properties in vivo.