Difference between revisions of "Part:BBa K4694008"

Latest revision as of 15:31, 12 October 2023

Sp-His-AHL-Lactonase

Usage and Biology

Quorum sensing (QS) is a form of bacterial communication allowing bacteria to detect and respond to different kinds of stimuli. QS ensures bacteria can develop into a high cell density from a low cell density [1]. Acyl homoserine lactone (AHL) are a class of quorum sensing signalling molecules. AHLs are synthesised by the Luxl enzyme and are then able to passively diffuse across the bacterial membrane in both directions. While AHLs diffuse into the cell they are recognised by the LuxR receptor, see figure below. Following binding to the receptor, this dimerised complex acts as a transcription factor on the Lux box. This activates expression of virulence-associated genes downstream, as well as the Luxl/LuxR AHL system. These virulence genes have a wide variety of functions in the cell, including regulation of biofilm formation [2, 3]. By degrading AHLs we could prevent biofilm formation by pathogenic Gram-negative bacteria. AHL-lactonase works outside the cell by degrading N-acyl homoserine lactones (AHLs) into N-acyl-homoserine.

The amino acid sequence was taken Bacillus thuringiensis serovar kurstaki (29339) [4]. The DNA sequence was codon optimised using the IDT software for L. lactis, forbidden restriction sites removed, a L. plantarum signal peptide (Lp_3050, [5]) and a 6xHis_tag flanked by GS linkers was added to the N-terminal, prefix and suffix sequences compatible with Type IIS cloning were added, and the sequence was synthesised by IDT.

For expression in L. plantarum, this CDS was inserted into plasmid pX1845 via Type IIS cloning. The plasmid has an E. coli origin of replication (pUC18) and antibiotic resistance gene (𝛽-lactamase) to allow for cloning in E. coli DH5𝛼, and an origin of replication and antibiotic resistance gene to allow for propagation in L. plantarum. Three constitutive promoters were tested: synthetic promoter P_48 [6], natural promoter from L. plantarum WCFS1 P_ldhL1 (GenBank NC_004567) and natural promoter from L. lactis P_32 [7]. The latter two promoters had integrated RBS sequences but P_48 was combined with the synthetic RBS SDOPT8 [8]. All constructs contained a terminator from L. lactis MG1363 pepN, called Lacto_term (GenBank AM406671).

For expression in E. coli, this CDS was inserted into plasmid pX1900 via Type IIS cloning. The plasmid has an E. coli origin of replication (pBR322) and antibiotic resistance gene (𝛽-lactamase) to allow for cloning and propagation within E. coli. The strong constitutive promoter BBa_J23100 combined with the strong RBS BBa_B0034 were tested as well as the IPTG inducible T7 promoter (original sequence from pET21a) were tested. All constructs contained the double terminator BBa_B0015.

Characterisation

In order to characterise this part and determine whether the enzyme would be able to inhibit biofilm formation in our modified L. plantarum we performed a series of experiments. Please refer to the Experiments page on our Wiki for the protocols.

Western Blot analysis

(Left) Western blot showing protein expression of enzymes downstream of the strong constitutive promoter J23100. Lanes 1 and 2 are ladders (PageRuler Plus and BenchMark Histagged protein ladder), 3 showing a positive control (mCherry) whilst 4 is an E. coli ‘wild type’ of DH5a (negative control). Lanes ‘7-14’ are our enzymes (7 = his-QQ7, 8 = SP-his-QQ7, 9 = his-QQ5, 10 = his-LuxS, 11 = his-AHL lactonase, 12 = SP-his-AHL lactonase, 13 = SP-his-QQ5, 14 = his-CapA

(Right) Western blot showing protein expression of our enzymes downstream with the IPTG inducible ‘T7’ promoter. Lanes 1 and 2 are ladders (PageRuler Plus and BenchMark Histagged protein ladder), 3 showing a postive control (mCherry), whilst 4 is E. coli ‘wild type’ of BL21 (DE3) (negative control). Lanes ‘7-14’ are our enzymes (7 = his-QQ7, 8 = SP-his-QQ7, 9 = his-QQ5, 10 = his-LuxS, 11 = his-AHL lactonase, 12 = SP-his-AHL lactonase, 13 = SP-his-QQ5, 14 = his-CapA). It seems like there is a lot of non-specific binding or protein overloading which has led to smearing on this western blot, making it harder to read.

Enzyme activity assays

(Left) Scatter plot of C4-HSL concentration over 24 hours for AHL lactonase and mCherry.

(Right) Scatter plot of C4-HSL concentration over 24 hours for AHL lactonase and LuxS.

The left graph shows that the degradation was increased by approximately 2.5 times what was observed when cell lysate from the E. coli transformed with mCherry was used. The right graph shows that the degrading action of AHL lactonase compared to LuxS was over 4 times greater. A one-tailed Wilcoxon signed rank test was carried out to validate this and we obtained a p=0.027 for the left graph and p=0.002 for the right graph. As the p value is less than 0.05, we can reject the null hypothesis (that the samples follow the same distribution) and conclude that there is a significant difference in the distributions of the two samples.

Conclusion

From the left and right graph we have shown that there was a significantly increased rate of C4-HSL degradation by AHL lactonase, which is part of the quorum sensing (QS) system in P. aeruginosa (9) - a bacterium which is responsible for catheter-associated biofilm formation (10). Meaning we have successfully shown that we can produce an enzyme which can target the QS system of a biofilm-forming species.

A band at 36KDa for mCherry, with no bands in our negative control validated our western blot. The his-LuxS SP-his-AHL lactonase proteins expressed under control of the PJ21300 and T7 promoters were visualised at 18.8 KDa and 33.8 KDa respectively. A further band on lane 10 was seen at ~38 KDa suggesting dimerisation.

References

[1] Waters CM, Bassler BL. 2005. Quorum Sensing: Cell-to-Cell Communication in Bacteria. Annual Review of Cell and Developmental Biology. 21, 319-46.

[2] Coquant G, Grill J-P, Seksik P. 2020. Impact of N-Acyl-Homoserine Lactones, Quorum Sensing Molecules, on Gut Immunity. Frontiers in Immunology. 11.

[3] Zhou L, Zhang Y, Ge Y, Zhu X, Pan J. 2020. Regulatory Mechanisms and Promising Applications of Quorum Sensing-Inhibiting Agents in Control of Bacterial Biofilm Formation. Frontiers in Microbiology. 11.

[4] Liu, D., Momb, J., Thomas, P. W., Moulin, A., Petsko, G. A., Fast, W. & Ringe, D. 2008. Mechanism of the quorum-quenching lactonase (AiiA) from Bacillus thuringiensis 1. Product-bound structures. Biochemistry, 47, 7706-14.

[5] Ben-David, Y., Morais, S., Stern, J., Mizrahi, I. & Bayer, E. A. 2019. Cell-surface display of designer cellulosomes by Lactobacillus plantarum. Methods Enzymol., 617, 241-263.

[6] Rud, I., Jensen, P. R., Naterstad, K. & Axelsson, L. 2006. A synthetic promoter library for constitutive gene expression in Lactobacillus plantarum. Microbiol., 152, 1011-1019.

[7]Liu, W. B., Lin, Z. W., Zhou, Y. & Ye, B. C. 2021. Overexpression of Capsular Polysaccharide Biosynthesis Protein in Lactobacillus plantarum P1 to Enhance Capsular Polysaccharide Production for Di-n-butyl Phthalate Adsorption. J. Microbiol. Biotechnol., 31, 1545-1551.

[8] Tauer, C., Heinl, S., Egger, E., Heiss, S. & Grabherr, R. 2014. Tuning constitutive recombinant gene expression in Lactobacillus plantarum. Microbiol. Cell. Fact., 13, 150.

[9] Boşgelmez-Tinaz G, Ulusoy S. Characterization of N-butanoyl-L-homoserine lactone (C4-HSL) deficient clinical isolates of Pseudomonas aeruginosa. Microb Pathog. 2008;44(1):13-9.

[10] Cole SJ, Records AR, Orr MW, Linden SB, Lee VT. Catheter-associated urinary tract infection by Pseudomonas aeruginosa is mediated by exopolysaccharide-independent biofilms. Infect Immun. 2014;82(5):2048-58.

Sequence and Features