Figure 1.
Maximum Likelihood Phylogenetic tree of Salmonella based on concatenated MLST loci.
The relationships of the isolates shown in the enlarged region were produced using a Maximum Likelihood phylogenetic tree of S. bongori based on the whole genome alignments produced by mapping sequence reads to the reference genome S. bongori 12419 (see [82]). The location for the root for the tree for the enlarged region was determined by using S. arizonae as an outgroup. The S. bongori isolates shown represent 21 different serotypes (SV), inferred by the coloured circles, the tree branches are coloured by BAPS cluster (SNP counts for each branch and strain labels for each node are in Figure S1).
Figure 2.
Events in the evolutionary history of Salmonella bongori and S. Typhi, a phenotypically and evolutionarily distant member of S. enterica.
Traits shared by the common ancestor are depicted in blue; those unique to S. bongori are shown in red and those unique to S. Typhi in green. Arrows, Salmonella Pathogenicity Islands (SPIs); extended ovals, fimbriae; circles, effectors; small ovals and needle complexes, secretion systems. Metabolic pathways: lines, enzymatic reactions; open squares, carbohydrates; ovals, pyrimidines; open circles, other substrates; filled shapes, phosphorylated. Novel effectors acquired by S. bongori are secreted by the type III secretion system encoded on SPI-1. SPI-3a and 3b carry the same genes in both organisms but are fused into one island in S. Typhi. SPI-5a also carries the same genes in both organisms, but a further 3 kb (termed SPI-5b) has fused to SPI-5a in S. Typhi. *indicates a pseudogene.
Table 1.
Distribution of known SPIs in the salmonellae.
Table 2.
Distribution of type three secretion systems encoded by SPI-1 (T3SS-1) and SPI-2 (T3SS-2) and their secreted effectors in the salmonellae.
Figure 3.
Salmonella bongori harbours a novel and phylogenetically distinct T6SS.
A. Schematic representation of the SPI-22 T6SS locus. Coding sequences are represented as blocked arrows showing the direction of their transcription. Conserved core T6SS components are represented with a different color. B. DNA-based comparison of the T6SS encoded in SPI-22 and the CTS2 locus of Citrobacter rodentium strain ICC168. The analysis was performed by TBLASTX with WebACT and visualized with ACT software. C. Evolutionary relationships of Salmonella T6SS loci. A distance tree (neighbour-joining) was calculated from concatenated VipA and VipB protein sequences of previously identified T6SS gene clusters, including the novel SPI-22 T6SS locus. Each of the four major phylogenetic groups is shown in the nodes labeled A to D. Bootstrap support values (% from 3,000 replicates) were: A, 99%; B, 80%; C, 99% and D, 99%. For brevity species or serovar names are used only.
Table 3.
Novel candidate T3SS effector proteins.
Figure 4.
S. bongori translocates five novel effector proteins into the host cell.
A. The translocation of TEM1-fusions of the putative new effectors, the positive controls SopB and SopD, and the negative control FabI into HeLa cells by S. bongori wild type (black bars) or the T3SS-deficient mutant S. bongori ΔinvA (white bars) was measured using a Fluostar Optima plate reader. The translocation rate is expressed as fold increase of the emission ratio 450/520 nm of each sample in relation to the emission ratio of uninfected cells. SboA-, SboC-, SboD-, SboH- and SboI-TEM1, along with SopB- and SopD-TEM1, but not FabI-TEM1 were translocated into the host cell. Error bars represent mean standard deviation (SD). Similar results were obtained in three independent experiments. B. The translocated effector SboI shows cytoplasmic distribution throughout strongly infected cells and a ring-like staining around bacteria reminiscent of a vacuolar membrane. HeLa cells were infected with S. bongori wild type or ΔinvA expressing SboI or FabI fused to four HA-tags (HAx4) for 2 h and processed for immunofluorescence microscopy (DNA - blue, HA-tag - green, LPS - red, actin - white). Bar = 5 µm.
Figure 5.
The chimeric effector SboH combines features of NleH1 and SopA and reduces host cell detachment during infection.
A–C. To compare the phenotypes of SboH and NleH1 HeLa cells were transfected with pRK5-nleH1, pRK5-sboH or the control plasmid pEGFP-N1 and processed. A. SboH inhibits tunicamycin (TUN) and brefeldin A (BFA) induced caspase-3 activation. Immunofluorescence analysis of transfected HeLa cells treated with 5 µg/mL TUN or 10 µg/mL BFA for 18 h and stained for DNA (blue), Myc-tagged effectors (green) and activated caspase-3 (red). GFP, but not SboH and NleH1 expressing cells frequently stain positive for activated caspase-3. (Images are representative of both TUN and BFA treated cells). Bar = 10 µm. B. The level of inhibition of caspase-3 activation by SboH or NleH1 was quantified by immunofluorescence counting of transfected cells. C. SboH and NleH1 are targeted to different subcellular locations. Transfected HeLa cells were stained for DNA (blue), Myc-tagged effector (green) and mitochondria (red) and analysed by immunofluorescence microscopy. SboH nearly exclusively co-localises with the mitochondrial marker MitoTracker, whereas NleH1 shows plasma membrane and perinuclear localisation. Scale bar = 10 µm. D. Cells infected with S. bongori ΔsboH show increased cell detachment. Quantification of HeLa cells lost following 5 h infection with S. bongori wild type, ΔinvA mutant, ΔsboH mutant and ΔsboH pSboH complemented strain. Staurosporine (STS) was used as a positive control to induce cell detachment. The one-way ANOVA Test using Bonferroni correction was used on data from five independent experiments and showed that the differences between S. bongori wild type and ΔsboH, as well as between S. bongori ΔsboH and the complemented strain are significant (* p-value<0.001). There was no significant difference between S. bongori wild type and the complemented strain (# p-value >0.05).