Subcellular Localization And Topology Of Holin Encoded By Streptococcus Suis Bacteriophage

Streptococcus suis (S. suis) type2is an important zoonotic pathogen that causes meningitis, septicemia, endocarditis, arthritis and other disease. In recent years, S. suis isolates showed serious multi-drug resistance to antibiotics, so it is difficult to control the disease by antibiotics. Developing a new method is particularly important, and the phage-based treatment strategies will be a potential one.The phage therapy techniques depends on the effects of bacterial lysis, previous studies showed that holin-lysin system encoded by phage is the biochemical basis of bacterial lysis,but the lysis mechanism by Streptococcus suis phage is still open. In this thesis, the subcellular localization, topology of holin from the phage will be revealed and the effects of different holin amino acids on the timing of bacterial lysis will be presented, which is necessary to elucidate the mechanism of bacterial lysis.In order to define the subcellular localization of holin, green fluorescent protein was chosen as a reporter gene. Firstly, the gfp gene was inserted into pET-32a (+) vector, and constructed the plasmid of p32G. Then fused the green fluorescent protein to the N-terminus of holin by linker with different length to obtain fusion gene expression vector p32GH by DNA recombinanting technology. Plasmid p32G and p32GH were transformed into E. coli BL21(DE3) pLysS cells, the recombinant bacteria were cultured by adding Dil dye and induced by IPTG, and then observed under confocal microscopy. The results showed that the green fluorescence was widely distributed in the p32G transformed cells, while the strains with fusing holin gene, whose green fluorescence was concentrated on the cell membrane. The data presented in this thesis indicate that holin was located on the cell membrane, and at the same time we found that the different length of linker has a great effect on the timing of bacterial lysis.In order to reveal the topology of holin, we use the technique of scanning cysteine accessibility mutagenesis (SCAM) was applied to analyze the distribution of key amino acids of holin in the either extracellular or intracellular of cell membrane. Based on the analytical results by three softwares for topology prediction of membrane protein, we select seven key amino acids and replaced them with cysteine by mutagenesis respectively. MPB (N-(3-Maleimidopropionyl) biocytin) was used as a probe to label cysteine side chains and Streptavidin-HRP was used for detection. The results showed that although only one amino acid was changed, enormous impact on bacterial lysis was found by analyzing these seven mutants, G35C, T80C and Y105C located in the plane of the lipid bilayer and were respectively involved in the formation of three transmembrane regions, consistent with the results predicted by biology software.To further study the bacterial lysis mechanism of holin and the interaction(s) between each transmembrane region. We mutate different sites of the nucleotide based on the sequence of holin, and thus changed a single amino acid compared with the wild type holin respectively. The growth curve of these recombinant mutants were determined after induction by IPTG, the results show that the amino acid mutation in the transmembrane regions significantly influenced the fuction of holin. When an amino acid replaced by a different amino acid, there is no regular effect on the growth curve. However, while methionine at any site of holin mutated into leucine, there are significant differences in the growth curve. Furthermore, compared with full length holin recombinant bacteria (pEXH1), the lysis time of these mutants changed differently, implying that the lysis time is sensitive to the changed sequence control of holin.The above results indicate that holin locate on the cell membrane; it is the prototype of the class I holins with three transmembrane domains, and N-terminal and C-terminal is located on both outsides of the cell membrane; It is also found that the bioactivity of holin is closely related to the encoding sequence, especially transmembrane sequence. This preliminary understanding of the topological structure of holin, will provide a theoretical basis for the development of phage-based treatment technology.