Optimized production of a biologically active Clostridium perfringens glycosyl hydrolase phage endolysin PlyCP41 in plants using virus-based systemic expression

Abstract

Background: Clostridium perfringens, a gram-positive, anaerobic, rod-shaped bacterium, is the third leading cause of human foodborne bacterial disease and a cause of necrotic enteritis in poultry. It is controlled using antibiotics, widespread use of which may lead to development of drug-resistant bacteria. Bacteriophage-encoded endolysins that degrade peptidoglycans in the bacterial cell wall are potential replacements for antibiotics. Phage endolysins have been identified that exhibit antibacterial activities against several Clostridium strains.

Results: An Escherichia coli codon-optimized gene encoding the glycosyl hydrolase endolysin (PlyCP41) containing a polyhistidine tag was expressed in E. coli. In addition, The E. coli optimized endolysin gene was engineered for expression in plants (PlyCP41p) and a plant codon-optimized gene (PlyCP41pc), both containing a polyhistidine tag, were expressed in Nicotiana benthamiana plants using a potato virus X (PVX)-based transient expression vector. PlyCP41p accumulated to ~ 1% total soluble protein (100μg/gm f. wt. leaf tissue) without any obvious toxic effects on plant cells, and both the purified protein and plant sap containing the protein lysed C. perfringens strain Cp39 in a plate lysis assay. Optimal systemic expression of PlyCP41p was achieved at 2 weeks-post-infection. PlyCP41pc did not accumulate to higher levels than PlyCP41p in infected tissue.

Conclusion: We demonstrated that functionally active bacteriophage PlyCP41 endolysin can be produced in systemically infected plant tissue with potential for use of crude plant sap as an effective antimicrobial agent against C. perfringens.

Keywords: Alternative antimicrobial; Bacteriophage; Clostridium perfringens; Endolysin; Nicotiana benthamiana; Plant production of recombinant proteins; Plant virus-based gene expression; Potato virus X.

Fig. 1

a Protein production in E. coli. Bacterial cultures containing pET21a: Lysin D or pET21a: PlyCP41 (PlyCP41) were induced by addition of IPTG and proteins were purified using the BugBuster reagent. Total (T), soluble (So), and inclusion body (IB) fractions were collected. Five μl aliquots were run on a protein gel. b Purification with PlyCP41 with Ni-NTA columns under native conditions. CP41, Soluble fraction from BugBuster fraction added to the Ni-NTA column; FT, flow through; W1, Wash 1; W2, Wash 2; E1, Elute 1; E2, Elute 2, E3, Elute 3; E4, Elute 4; E5, Elute 5; E6, Elute 6. Both gels were stained with SimplyBlue Safe Stain. M = Precision Plus Kaleidoscope protein standards

Fig. 2

PlyCP41p production in leaf tissue collected from virus-infected plants. Western blot using the Penta-His antibody of plant extracts from leaf discs collected 9 days’ post-infiltration CP41p (plant 1, symptomatic leaf), CP41p (plant 2, asymptomatic leaf), PVX, N.b. (healthy plant). rCP41 = 2 μg of purified recombinant PlyCP41 from bacteria. M = Precision Plus Kaleidoscope protein standards

Fig. 3

Plate lysis assay of protein samples on Clostridium perfringens Cp39 confluent plates. Spot assays were conducted as described in the Materials and Methods and a photographic image of the plate was taken 30 min after application of the samples. The contents of the wells are as follows: A1-A6, Negative control, His-purified fractions from E. coli: BL21 pET21a; B1- B4- plant virus samples in PBS buffer; B1 & B2-empty PVX virus; B3 & B4-PVX virus with PlyCP41p; B5-PBS buffer; B6-elution buffer control; C1-C4- plant virus samples in “10:90” buffer (50 mM NaH₂PO₄ pH 7.0, 30 mM NaCl, 25 mM imidazole, 3% glycerol). C1&C2-empty PVX; C3&C4-PVX with PlyCP41p; C5–10 μL PVX with PlyCP41p in PBS buffer; C6–10 μL PVX with PlyCP41p in PBS buffer; D1-D4- empty; D5–10 μL PVX with PlyCP41p in “10:90 buffer”; D6–10 μL PVX with PlyCP41p in “10:90 buffer” E1-E6- E. coli PlyCP41 fractions purified on Ni-NTA column (FT, W1, W2, E1, E2, E3); F1- F4–10 μg, 1 μg, 0.1 μg, 0.001 μg of PlyCP41; F5 10:90 buffer. Asterisks indicate the plant extracts containing the PlyCP41p protein

Fig. 4

Purification and quantitation of plant-expressed PlyCP41p. a Plant extracts were processed using Ni-NTA columns under native conditions and analyzed by Western blot of protein using the Penta-His antibody. O, leaf sample extract from virus-infected plants were added to the Ni-NTA column; FT, flow through; W1, Wash 1; W2, Wash 2; E1, Elute 1; E2, Elute 2, E3, Elute 3; E4, Elute 4. rCP41 = 2 μg of Ni-NTA purified PlyCP41 from bacteria. M = Precision Plus Kaleidoscope protein standards. b Quantitation of PlyCP41p in plant extracts. Lanes containing Ni-NTA-purified PlyCP41 from bacteria-concentrations are 0.1 μg, 0.2 μg. 0.5 μg, and 1 μg. The unpurified plant extract (Orig) and the Elution 2 fraction (E2) from Ni-NTA purifications (6/8) and (5/24). The 6/8 purification included protease inhibitor in the extraction buffer. 1 μl of a 100 μl extraction of 4 leaf discs (20 mg plant sample) M = Precision Plus Kaleidoscope protein standards

Fig. 5

PlyCP41p protein expression in N. benthamiana plants post-infiltration. Leaf disc samples were collected from infiltrated plants at 2, 3.5, 5, and 10 weeks’ post-infiltration. Western blot analysis was performed using the Penta-His conjugate. N.b., healthy N. benthamiana. M = Precision Plus Kaleidoscope protein standards. This Western blot shows that plant tissue needs to be harvested within 2 weeks’ post-inoculation (w.p.i) to achieve the highest amount of PlyCP41p protein production

Fig. 6

Comparison of the expression of PlyCp41p and PlyCp41pc in plants. Western blot of plant tissue collected 13 days post-infiltration. Neat (O.D. 600 nm = 2.7) and 1:10 (O. D at 600 nm = .27) represent dilutions of Agrobacterium cultures used to infiltrate plants with pGDPVXMCS: PlyCP41pc (CP41pc) or pGDPVXMCS:PlyCP41p mixed in a 1:10 dilution with Agrobacterium containing pGDp19. CP41p (A) designates a plant that was mechanically inoculated from a plant 22 days’ post-infiltration. This sample represents 7 days’ post-infection. rCP41 = 2 μg. M = Precision Plus Kaleidoscope protein standards