Secretion of biologically active interferon-gamma inducible protein-10 (IP-10) by Lactococcus lactis

Abstract

Background: Chemokines are a large group of chemotactic cytokines that regulate and direct migration of leukocytes, activate inflammatory responses, and are involved in many other functions including regulation of tumor development. Interferon-gamma inducible-protein-10 (IP-10) is a member of the C-X-C subfamily of the chemokine family of cytokines. IP-10 specifically chemoattracts activated T lymphocytes, monocytes, and NK cells. IP-10 has been described also as a modulator of other antitumor cytokines. These properties make IP-10 a novel therapeutic molecule for the treatment of chronic and infectious diseases. Currently there are no suitable live biological systems to produce and secrete IP-10. Lactococcus lactis has been well-characterized over the years as a safe microorganism to produce heterologous proteins and to be used as a safe, live vaccine to deliver antigens and cytokines of interest. Here we report a recombinant strain of L. lactis genetically modified to produce and secrete biologically active IP-10.

Results: The IP-10 coding region was isolated from human cDNA and cloned into an L. lactis expression plasmid under the regulation of the pNis promoter. By fusion to the usp45 secretion signal, IP-10 was addressed out of the cell. Western blot analysis demonstrated that recombinant strains of L. lactis secrete IP-10 into the culture medium. Neither degradation nor incomplete forms of IP-10 were detected in the cell or supernatant fractions of L. lactis. In addition, we demonstrated that the NICE (nisin-controlled gene expression) system was able to express IP-10 "de novo" even two hours after nisin removal. This human IP-10 protein secreted by L. lactis was biological active as demonstrated by Chemotaxis assay over human CD3+T lymphocytes.

Conclusion: Expression and secretion of mature IP-10 was efficiently achieved by L. lactis forming an effective system to produce IP-10. This recombinant IP-10 is biologically active as demonstrated by its ability to chemoattract human CD3+ T lymphocytes. This strain of recombinant L. lactis represents a potentially useful tool to be used as a live vaccine in vivo.

Figure 1

Schematic design of IP-10 expression system for production and secretion by Lactococcus lactis. The scheme represents the final construction of the IP-10 inducible expression system carried by Lactococcus lactis (pSEC:huIP-10). The diagram shows the nisin-inducible promoter PnisA, the ribosome binding-site of usp45 (RBS), the usp45 signal peptide of the usp45 gene (SPusp), and the coding region for the mature moiety of IP-10. The open circle represents a rho-independent trpA transcription terminator fused just downstream to the IP-10 gene (not to scale).

Figure 2

Expression analysis of IP-10 secretion by recombinant Lactococcus lactis. Protein extracts from induced and noninduced cultures of recombinant Lactococcus lactis NZpSEC:huIP-10 were prepared from cell-free samples and analyzed by Western blotting using anti-IP-10. Mature IP-10 was detected in all the induced (+) cultures in the 10 kDa range as expected. No signal was found for noninduced cultures (-) of recombinant L. lactis NZpSEC:huIP-10. No immature or incomplete forms of IP-10 were detected. M, protein molecular marker.

Figure 3

De novo secreted IP-10 in the absence of nisin. Three separate cultures (A, B, and C) of NZpSEC:huIP-10 were induced with 10 ng/ml of nisin for 1 hour. Culture A was in the presence of nisin all the time; culture B washed and suspended in fresh medium without inducer at hour 1 and 3, and culture C was washed and suspended in fresh medium without inducer only at hour 1. All cultures were grown a total of 6 hours. Protein extracts were analyzed by Western blot at hour 1, 3, and 6. The band in culture B at hour 6 (asterisk) represents the IP-10 "de novo" specifically secreted from hour 3 to 6 in the total absence of nisin demonstrating the ability of Lactococcus lactis to keep expressing and producing IP-10 for at least three hours.

Figure 4

Secreted IP-10 by Lactococcus lactis is biologically active. Chemoattraction of L. lactis-secreted IP-10 was determined by using a Boyden chamber for chemotaxis. T lymphocytes obtained directly from human peripheral blood were stimulated with human IL-2 for 12 days. Chemotaxis assays using Boyden chambers were made with supernatants sterilized by filtration of both recombinant and wild-type Lactococcus lactis. To determine the number of chemoattracted cells, the membranes were stained with hematoxylin and the cells counted by using light microscopy at 1000 × (A). Cells in the lower chamber were incubated with specific anti-CD3+ and counted by FACS analysis (B). Supernatants of wild-type (WT) L. lactis and PBS were used as negative controls and Zymosan-activated serum (S+) as a positive control.