PMID:28637001

Citation	Won, G, Kim, B and Lee, JH (2017) A novel method to generate Salmonella Typhi Ty21a ghosts exploiting the λ phage holin-endolysin system. Oncotarget 8:48186-48195
Abstract	Human typhoid fever caused by Salmonella Typhi still poses a severe global disease burden in developing countries despite the availability of commercial vaccines. In this study, we constructed a non-living S. Typhi Ty21a vaccine candidate by employing a lambda (λ) phage-derived holin-endolysin system to efficiently construct bacterial ghosts. The lysis plasmid pJHL464 harbors an R lysis cassette that is stringently regulated by dual promoters containing cI857/λPR and ParaBAD/araC components. The plasmid was introduced into an asd gene-deleted S. Typhi Ty21a strain designated JOL1675. The in vitro expression of endolysin (~17.76 kDa) in the subsequent JOL1675 vaccine construct when grown under lysis inducible conditions was validated by immunoblotting. In scanning electron microscopy analysis, surface transmembrane tunnels and a collapsed body were visualized in the ghosts. Following 48 h of lysis, no viable JOL1675 cells remained, indicating that lysis of all cells was achieved. Subcutaneous immunizations of mice with the JOL1675 ghosts produced significantly increasing titers of serum IgG and vaginal wash secretory IgA antibodies against JOL1675 outer membrane proteins during the observational period. Further, serum collected at 6 weeks post-immunization of rabbits exhibited effective bactericidal activity against wild type S. Typhi in the presence of complement. These data showed that JOL1675 ghosts are highly immunogenic and elicit humoral and mucosal responses expected to correlate with protective immunity against S. typhi. Collectively, our findings support the conclusion that incorporating a λ phage holin-endolysin-mediated lysis construct into S. Typhi is an efficient strategy for developing a novel and safe non-living typhoid vaccine candidate.
Links	PubMed PMC5564637 Online version:10.18632/oncotarget.18383
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
LAMBD:ENLYS		GO:0044659: cytolysis by virus of host cell	ECO:0000314:		P		The R lysis cassette, including R endolysin and S holin genes was synthesized and inserted into a plasmid under regulation of arabinose and temperature sensitive promoters. The plasmid was introduced into Δasd E. coli χ6212 and the construct was named JOL1675. Under conditions inducing expression of the plasmid, presence of the R endolysin was confirmed by Western Blot and shown in Figure 2. A band at 17.7 kDa, the estimated size of the endolysin, was seen in cells with the plasmid expressed, but not in cells grown under conditions repressing expression. Lysis efficiency was confirmed by monitoring the number of viable cells, which declined to 0 after 48 hours of lysis, as shown in Figure 4. No viable cells remained, indicating that the endolysin effectively ruptured and killed the hosts.	complete CACAO 13107

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