Two-tiered biological containment strategy for Lactococcus lactis-based vaccine or immunotherapy vectors

Figures & data

Table 1. Strains and plasmids generated and used in this study

Bacterial strain	Description	Growth conditions	Ref.
E. coli EC101	E. coli JM101 with repA from pWV01 integrated in chromosome	LB + 40 µg/ml Kanamycin, 37 °C, shaking	17
E. coli EC101 (pORI-PRE-POST)	Strain E. coli EC101 carrying the pORI-PRE-POST plasmid	LB + 50 µg/ml Erythromycin, 37 °C, shaking	This study
Lactococcus lactis subspecies cremoris MG1363	Plasmid free Lactococcus lactis strain	M17 + Glucose 0.5%, 30 °C, no shaking	22
Lactococcus lactis subspecies cremoris MG1363 RifR	Rifampicin resistant variant of strain L. lactis MG1363	M17 + Glucose 0.5% + 50 µg/ml Rifampicin, 30 °C, no shaking	This study
L. lactis ΔpyrG (P23:SEC-LLO)	Derivative of L. lactis subspecies cremoris MG1363 constitutively secreting LLO. Gene pyrG was replaced by hly.	M17 + Glucose 0.5% + 4 mM Cytidine, 30 °C, no shaking	2
L. lactis ΔpyrG (P23:SEC-LLO) RifR	Rifampicin variant of strain L. lactis MG1363 ΔpyrG (P23:SEC-LLO)	M17 + Glucose 0.5% + 4 mM Cytidine + 50 µg/ml Rifampicin, 30 °C, no shaking	This study
L. lactis ΔthyAΔpyrG (P23:SEC-LLO) RifR	llmg_0964 (thyA) deletion mutant of strain L. lactis ΔpyrG (P23:SEC-LLO) RifR	M17 + Glucose 0.5% + 20 µM Thymidine + 4 mM Cytidine + 50 µg/ml Rifampicin, 30 °C, no shaking	This study
L. lactis ΔthyA RifR	llmg_0964 (thyA) deletion mutant of strain L. lactis MG1363 RifR	M17 + Glucose 0.5% + 20 µM Thymidine + 50 µg/ml Rifampicin, 30 °C, no shaking	This study
Listeria monocytogenes EGDe serovar 1/2a	Wild type L. monocytogenes	BHI, 37 °C, shaking	23
Plasmid name	Description		Ref.
pORI280	A RepA- ori+ β-galactosidase-positive Erythromycin resistant (Em^r) integration vector		15
pVE6007	A temperature-sensitive RepA+ helper plasmid, Cm^r		16
pORI-PRE-POST	pORI280 derivative carrying DNA regions framing thyA gene (PRE and POST)		This study

Figure 1. Growth curves of the L. lactis variants in presence/absence of cytidine and thymidine. Strains L. lactisMG1363 (closed circles), MG1363 ΔpyrG (P23:SEC-LLO) (open circles), MG1363 ΔthyA (closed triangles), and MG1363 ΔthyAΔpyrG (P23:SEC-LLO) (open triangles) were grown in broth (A) SA, (B) SA + cytidine 4mM, (C) SA + thymidine 20µM, or (D) SA + cytidine 4mM + thymidine 20 µM. The results are expressed as the mean ± SD (n = 3).

Figure 2. Survival of the different L. lactis strains in a cytidine and thymidine deprived broth and in environmental samples. Strains L. lactis MG1363 (closed circles), MG1363 ΔpyrG (P23:SEC-LLO) (open circles), MG1363 ΔthyA (closed triangles), and MG1363 ΔthyAΔpyrG (P23:SEC-LLO)(open triangles) were used to inoculate SA broth (A), soil (B), or river water (C).The results are expressed as the mean ± SD of at least 3 independent experiments.

Figure 3. Survival of the different L. lactis strains after passing through the gastrointestinal tract. (A) Schematic representing the experimental procedure. (B) Graph expressing the survival of the lactococcal strains following administration to mice by gavage and transition through the gastrointestinal tract. Bacteria were recovered from the faeces at the indicated CFU count. Results are expressed as the mean ± SD (n = 5). a, b, c, or d indicate that the value is significantly different (P value ≤ 0.05) from that affected to strains MG1363, ΔpyrG, ΔthyA, or ΔthyAΔpyrG, respectively.

Lee P. Biocontainment strategies for live lactic acid bacteria vaccine vectors. Bioeng Bugs 2010; 1:75 - 7; http://dx.doi.org/10.4161/bbug.1.1.10594; PMID: 21327129

 PubMedGoogle Scholar

Dower WJ, Miller JF, Ragsdale CW. High efficiency transformation of E. coli by high voltage electroporation. Nucleic Acids Res 1988; 16:6127 - 45; http://dx.doi.org/10.1093/nar/16.13.6127; PMID: 3041370

 PubMed Web of Science ®Google Scholar

Bahey-El-Din M, Casey PG, Griffin BT, Gahan CG. Efficacy of a Lactococcus lactis ΔpyrG vaccine delivery platform expressing chromosomally integrated hly from Listeria monocytogenes.. Bioeng Bugs 2010; 1:66 - 74; http://dx.doi.org/10.4161/bbug.1.1.10284; PMID: 21327128

 PubMedGoogle Scholar

Geertsma ER, Poolman B. High-throughput cloning and expression in Lactococcus lactis. Protocol Exchange [Internet] 2007; Available from: http://www.nature.com/protocolexchange/protocols/287

 Google Scholar

Takiishi T, Korf H, Van Belle TL, Robert S, Grieco FA, Caluwaerts S, Galleri L, Spagnuolo I, Steidler L, Van Huynegem K, et al. Reversal of autoimmune diabetes by restoration of antigen-specific tolerance using genetically modified Lactococcus lactis in mice. J Clin Invest 2012; 122:1717 - 25; http://dx.doi.org/10.1172/JCI60530; PMID: 22484814

 PubMed Web of Science ®Google Scholar

EU Council Directive 2009/41/EC on the contained use of genetically modified micro-organisms [Internet]. Official Journal L 125, 21/05/2009 P. 0075 - 0097; Available from: http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2009:125:0075:01:EN:HTML - Last accessed May, 21st 2012.

 Google Scholar