Table 1.
Salmonella strains analysed for the presence of st313-td.
Table 2.
Strains and plasmids used for mutagenesis and derived isolates.
Figure 1.
Genetic structure of ST313-GI.
The genomic organization is shown with a scale in kb. Arrow above the scale represents predicted ORFs and their transcription directions. ORF 21 corresponds to the virulence gene st313-td and is highlighted with a rectangle. Twenty-one ORFs encode phage-related functions: integration and excision: int and xis, respectively, Ea region: eaa and eae, recombination: abc2 (Anti-RecBCD), erf and kil, regulatory functions: reg, structural: gp54, anti-restriction: ral, inmunity: c1 (repressor) and cro (antirepressor), transcription: ta (transcriptional activator), replication: repl, Nin region: nin, antitermination: Q (Q protein). The sequence data have been extracted from the sequenced genome of the strain S. Typhimurium D23580, accession no. FN424405.1. The location of ST313-GI within this genome is 368767_386754. The location of st313-td is; 377825_378748 (locus_tag = ”STMMW_03531” and protein id: “YP_005231421.1”). All the genes within ST313-GI have been annotated in the genome of D23580. The function of the encoded proteins was determined by using the Uniprot database (http://www.uniprot.org/). See additional Table S2 for more details.
Table 3.
Competitive index analysis of S. Typhimurium mutants.
Figure 2.
Infection of J774 mouse macrophages.
Intracellular survival/replication, A) at t = 1 h post-infection with opsonisation, B) at t = 1 h post-infection without opsonisation, and C) at t = 20 h post-infection with opsonisation, and with regards to t = 1 h post-uptake (the intracellular survival at t = 1 h for all the tested isolates is regarded as 1.0). Strains tested were: S. Typhimurium WT 02–03/002, 4/74 and 14028 and the mutant isolates: Δst313-td, 02-03/002-C (pAHF4, complemented strain), 4/74-C (pAHF4) and ΔssaV. The experiments were repeated at least 4 times with similar results and shown is an average of these. Errorbars indicate standard deviation. Statistical significance (**** = P<0,00001; ** = P<0,001) was determined by one-way ANOVA analysis with pair-wise comparison of means.
Figure 3.
Cytotoxicity towards J774 mouse macrophages.
Measurement of cytotoxicity levels, A) at t = 1 h post-infection and B) t = 20 h post-infection without opsonization, respectively. Strains tested for cytotoxicity were: S. Typhimurium WT 02–03/002, 4/74 and 14028 and the mutant isolates: Δst313-td, 02-03/002-C (pAHF4, complemented strain), 4/74-C (pAHF4) and ΔssaV. The citotoxicity level is based on the release of LDH (lactate dehydrogenase). The experiments were repeated at least four times with similar results and shown is an average of these. Errorbars indicate standard deviation. Statistical significance was determined by one-way ANOVA analysis with pair-wise comparison of means.
Figure 4.
Infection of the human epithelial Int407 cells.
Invasion of monolayes of Int407 cells was performed and invaded bacteria were enumerated at t = 2 h post-infection. Strains tested were: S. Typhimurium WT 02–03/002 and 4/74, and the mutant isolates: Δst313-td, 02-03/002-C (pAHF4, complemented strain), 4/74-C (pAHF4) and invH201::TnphoA. The experiments were repeated at least 4 times with similar results and shown is an average of these. Error bars indicate standard deviation. Statistical significance was determined by one-way ANOVA analysis with pair-wise comparison of means.