Essential genes from Arctic bacteria used to construct stable, temperature-sensitive bacterial vaccines

Abstract

All bacteria share a set of evolutionarily conserved essential genes that encode products that are required for viability. The great diversity of environments that bacteria inhabit, including environments at extreme temperatures, place adaptive pressure on essential genes. We sought to use this evolutionary diversity of essential genes to engineer bacterial pathogens to be stably temperature-sensitive, and thus useful as live vaccines. We isolated essential genes from bacteria found in the Arctic and substituted them for their counterparts into pathogens of mammals. We found that substitution of nine different essential genes from psychrophilic (cold-loving) bacteria into mammalian pathogenic bacteria resulted in strains that died below their normal-temperature growth limits. Substitution of three different psychrophilic gene orthologs of ligA , which encode NAD-dependent DNA ligase, resulted in bacterial strains that died at 33, 35, and 37 °C. One ligA gene was shown to render Francisella tularensis , Salmonella enterica , and Mycobacterium smegmati s temperature-sensitive, demonstrating that this gene functions in both Gram-negative and Gram-positive lineage bacteria. Three temperature-sensitive F. tularensis strains were shown to induce protective immunity after vaccination at a cool body site. About half of the genes that could be tested were unable to mutate to temperature-resistant forms at detectable levels. These results show that psychrophilic essential genes can be used to create a unique class of bacterial temperature-sensitive vaccines for important human pathogens, such as S. enterica and Mycobacterium tuberculosis .

Temperature Sensitivity Conferred by ligA Alleles from Psychrophilic Bacteria upon Substitution in Mesophilic Bacteria and a Yeast Species

“…Temperature-resistant variants of S3 ligA had mutations that introduced changes at the N terminus of the LigA product (Table 2), and we were surprised to find that the mutations were identical to those previously found in temperature-resistant mutants of S1 ligA (26). We noted that both of the S1 and S3 alleles had the lowest predicted RBS strengths (86 arbitrary units [AU]) of all of the alleles and wondered if the mutations leading to temperature resistance affected the strengths of the RBSs.…”

“…Predictably, these cold adaptation and heat lability properties extend to essential proteins. In our previous work, we have found that psychrophilic forms of DNA ligase are inactivated in a temperature range appropriate for engineering mesophilic bacterial to temperature-sensitive (TS) forms that can be used for live vaccines (26). In this work, we have discovered new alleles of ligA conferring psychrophilic properties and show that they impart a range of TS phenotypes in bacteria.…”

“…The ligA alleles C2, P6, and S1 and the corresponding F. novicida strains carrying these alleles have been described previously (26) and were isolated from the ocean psychrophilic strains Colwellia psychrerythraea 34H (C2), Pseudoalteromonas haloplanktis TAC125 (P6), and our isolate of Shewanella frigidimarina (S1) (26). The other ligA alleles were isolated from bacteria found in ocean waters collected at the following global positioning system (GPS) coordinates: C1, 82°32.04N, 62°45.86W; P3, 82°32N, 62°46.4W; S2, 48°20.26N, 123°36.24W; S3, 45°55.24=N, 129°59.48W; P4, 35°52.16N, 114°39.73W; P5, 82°32N, 62°46.43W; P7, 64°47.9N, 168°36W; S4, 48°20.26N, 123°36.24W.…”

“…The exchange of the psychrophilic ligA for the F. novicida homologue was accomplished via a Campbell-like integration followed by excision as previously described (26). In essence, the transformation step was used to select for the integration of the plasmid; after cointegrants were identified, the merodiploids were cultured in the presence of 10% sucrose and plated on agar medium with sucrose (see schematic in Fig.…”

Temperature-Sensitive Salmonella enterica Serovar Enteritidis PT13a Expressing Essential Proteins of Psychrophilic Bacteria

“…Intracellular growth assays were performed essentially as described previously (26), using the mouse macrophage-like cell line J774. Growth of bacterial strains and temperature shifts were done as previously described (27). PT13a strains were grown in microtiter plates, and the A 595 was measured every 15 min with a BioTek EL808 plate reader.…”