Enhanced Secretion of Biologically Active Murine Interleukin-12 by Lactococcus lactis

The novel signal peptide SLPmod was used for the secretion ofmurine interleukin-12 (mIL-12) by Lactococcus lactis. A >4-foldincrease in secretion was observed when SLPmod was used insteadof the Usp45-derived secretion signal. Oral delivery of thiscytokine using the autoinducible host L. lactis FI5876 utilizingSLPmod resulted in a significant increase in mIL-12 plasma levelsin mice.

INTRODUCTION

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INTRODUCTION

Many heterologous proteins and peptides have been successfullyexpressed in Lactococcus lactis for different biotechnologicalapplications. Despite its lack of invasiveness, L. lactis isable to deliver heterologous antigens and cytokines to the systemicand mucosal immune system (11). Cytokine mucosal delivery isdependent upon its release into the mucosa when L. lactis cellsare in contact with the mucosa-associated lymphoid tissue. Thesecretion of recombinant cytokines has generally involved fusionof the relevant protein to the signal peptide of the abundantUsp45 protein (1, 13, 14, 15); however, poor secretion efficiencyof the large murine cytokine interleukin-12 (mIL-12) was observed(1, 17). Here, we report the development of an improved secretionsystem for the delivery of large cytokines, specifically mIL-12.We have engineered the self-inducing, nisin-producing L. lactisstrain FI5876 to express mIL-12 and have compared this withan exogenously induced system using strain UKLc10.

The SLPmod signal peptide has been developed on the basis ofan alignment of lactobacillus S-layer proteins (unpublisheddata). After retrieving the respective S-layer protein sequencesfrom the UniProt database (http://www.uniprot.org/) and theremoval of incomplete sequences, the first 70 amino acids ofthe remaining proteins were aligned using ClustalW (10). Thesequences clustered into three groups (data not shown), thelargest of which contained sequences from Lactobacillus acidophilus,Lactobacillus crispatus, and Lactobacillus helveticus. Becausea clear majority of available sequences cluster into one groupand because there was a strong similarity to the sequence ofa secreted lactococcal protein (llmg_0851), the respective consensussequence was chosen as the basis for the design of SLPmod.

Construction of nisin promoter (P_nisA)-controlled mIL-12 expression vectors.

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Construction of nisin promoter...

The regions encoding the mIL-12 p40 and p35 subunits were amplifiedby PCR employing mouse spleen cDNA as a template (AMS Biotechnology,Abingdon, United Kingdom). Primer pairs 5'-ATGTGGGAGCTGGAGAAAGAC-3'and 5'-CCTGGATCCGACCCTGCAG-3' for the p40 fragment and 5'-AGGTGGAGGAGGATCTAGGGTCATTCCAGTCTC-3'and 5'-CTGGATCCTTTCAGGCGGAGC-3' for the p35 fragment were usedto create fragments of 938 bp and 578 bp, respectively. Subsequently,spliced overlap extension PCR was used to create two fragments,representing SignalPeptide_p40 under the control of the nisinA promoter or Linker_p35. These two subfragments were cut usingrestriction enzymes and cloned as PnisA_SignalPeptide_p40_Linker_p35into the shuttle vector pTG262 (12) (Fig. 1), resulting in thefragments coding for the two protein subunits being connectedby an artificial linker coding for (Gly₄-Ser)₃ (8). The signalpeptides included were either SLPmod (pFI2596) or Usp45 (pFI2602)signal peptides. Subsequently, L. lactis strain FI5876 or UKLc10was transformed with plasmid pFI2596 or pFI2602 in order togenerate the recombinant strains listed in Table 1. Bacterialgrowth conditions were as described previously (5, 7). Strainswere grown to an optical density at 600 nm of $~$ 1.0 before supernatantsand cell pellets were collected. UKLc10-derived strains wereinduced with 10 ng ml^–1 of nisin at an optical densityat 600 nm of $~$ 0.6 (optimized conditions).

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FIG. 1. Cloning strategy used for the introduction of the fragment PnisA_SignalPeptide_p40_Linker_p35 into the vector pTG262 (not to scale).

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TABLE 1. Strains used in this study

Western blot analysis was carried out on both the intracellularcontent and culture supernatant (Fig. 2), as previously described(5). Culture supernatants from FI5876 and UKLc10 derivativescarrying the plasmid pTG262, pFI2596, or pFI2602 revealed thata clear band of the expected 70 kDa was detected in the strainswith mIL-12-encoding plasmids (Fig. 2B; data shown for pFI2596).Cytokine concentration was measured by enzyme-linked immunosorbentassay. L. lactis FI10611 utilizing the SLPmod signal peptidefor secretion produced the highest level of secreted mIL-12(185 pg ml^–1). In contrast, a much lower level (40 pgml^–1) was obtained with strain FI10608 that utilized theUsp45 signal peptide. The levels of mIL-12 secretion were comparablein both FI5876 and UKLc10 mIL-12-producing derivatives (Table1).

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FIG. 2. Western blot detection of mIL-12 in the intracellular content (A) and culture supernatants (B). Lane 1, mIL-12; lane 2, L. lactis FI10614; lane 3, L. lactis FI10610; lane 4, L. lactis FI10611; lane 5, L. lactis FI10632; lanes L, MagicMark Western protein standard.

Interestingly, the buffering of media with 2% NaHCO₂ led toa further threefold increase in mIL-12 production. Furthermore,an additional plasmid (pFI2595) encoding mature mIL-12 (no signalpeptide) was also included. The resulting Western blot of theintracellular contents of FI10610 and FI10614 revealed a strongsingle band in each (Fig. 2A), indicating the presence of anintact cytokine, unlike the result published by Bermúdez-Humaránet al. (1), which showed a smeared range of bands, suggestingthat degradation had occurred.

Using the bioactivity assay described previously (1), the additionof culture supernatants of an L. lactis strain secreting mIL-12to mouse splenocytes resulted in the induction of IFN- ${gamma}$ production.These induction levels were comparable to those obtained usingstandard mIL-12, indicating that bacterial produced cytokinewas biologically active.

Animal study and plasma levels of mIL-12.

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Animal study and plasma...

Five groups of C3H/HeJ mice were intragastrically treated bygavage with 50 µl of 2% NaHCO₂ containing 10⁹ CFU, aspreviously described (6), of freshly prepared L. lactis cellsof either the parental strains UKLc10 and FI5876 carrying thecontrol vector (FI10724 and FI10632), their mIL-12 engineeredderivatives (FI10615 and FI10611), or a buffer solution (2%NaHCO₂). Plasma samples were collected 6 h after gavage treatment,and mIL-12 levels were measured. Wilcoxon rank tests were usedfor statistical analysis of the data. A significant (P <0.05) increase in mIL-12 levels was obtained only when the mIL-12-secretingFI5876 strain was used (FI10611, an autoinduced strain). Independentstudies were carried out, and a mean value of 40 ± 11pg ml^–1 was obtained when animals had been treated bygavage with FI10611. In contrast, a mean value of 30 ±10 pg ml^–1 was found when mice had been treated by gavageeither with the mIL-12-secreting UKLc10 strain (FI10615, requiringexogenous induction) or the controls.

In this study, the use of a more effective signal peptide hasenhanced the secretion of biologically active mIL-12 by L. lactis.In comparison to the secretion achieved when the signal peptideof Usp45 was used, the presence of the alternative SLPmod signalpeptide lead to a >4-fold increase in mIL-12 secretion, whichin turn resulted in an eightfold increase in IFN- ${gamma}$ productionin the biological activity assays using mouse splenocytes (datanot shown). The new SLPmod signal peptide will be useful forthe heterologous production of proteins not only in L. lactisbut potentially also in other gram-positive bacteria, wherean enhancement of protein secretion is required.

Here, two L. lactis strains, FI5876 and UKLc10, were engineeredto secrete mIL-12 by using a nisin-controlled gene expressionsystem (3, 4, 9). The strong inducible nisin promoter requiresthe presence of nisin in the media in order to be activated.We compared the abilities of the mIL-12-secreting derivativestrains of FI5876 (autoinducible) and UKLc10 (inducible) toinfluence the plasma levels of mIL-12 after their oral administrationin mice. Similar mIL-12 levels of secretion were detected inthe culture supernatants of the respective FI5876 and UKLc10derivative strains. However, as predicted, elevated mIL-12 plasmalevels in mice were obtained only when the nisin-producing strainFI10611 was employed because nisin is likely to be present whenthe metabolically active strain is in contact with the intestinalmucosa. This local supply of nisin (2) is able to autoinduceP_nisA, leading to the subsequent secretion of mIL-12 and itsdelivery to the mucosal tissue. In contrast, L. lactis UKLc10derivatives are non-nisin-producing strains that depend uponthe exogenous addition of nisin in order to produce mIL-12.We conclude that a nisin-producing strain is the ideal hostfor oral delivery.

Intragastric administration of a recombinant noncolonizing L.lactis strain that secretes biologically active mIL-12 couldbe of great benefit in reducing toxic side effects associatedwith systemic delivery. In addition, mucosal delivery of mIL-12has been shown to act as an adjuvant in vaccine delivery, whichmight be enhanced by the application of the FI10611 strain developedhere.

ACKNOWLEDGMENTS

This work was partially supported by an Intra-European MarieCurie Fellowship.

We are grateful to Jeffrey Temblay (IFR) and Simon Deakin (DMU,University East of Anglia, United Kingdom) for advice and technicalassistance. We also thank Carmen Pin (IFR) for the statisticalanalysis performed. We thank Bernhard Henrich (University ofKaiserslautern, Germany) for providing plasmid pUK200 and thelactococcal strain UKLc10.

FOOTNOTES

* Corresponding author. Mailing address: Commensals and Microflora, BBSRC Institute of Food Research, Norwich Research Park, Colney, Norwich NR4 7UA, United Kingdom. Phone: 441603-255000. Fax: 441603-255288. E-mail for A. Fernandez: antonio.fernandezdumont{at}efsa.europa.eu. E-mail for N. Horn: nikki.horn{at}bbsrc.ac.uk

Published ahead of print on 5 December 2008.

REFERENCES

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