Comparative genomic analysis uncovers 3 novel loci encoding type six secretion systems differentially distributed in Salmonella serotypes

Abstract

Background: The recently described Type VI Secretion System (T6SS) represents a new paradigm of protein secretion in bacteria. A number of bioinformatic studies have been conducted to identify T6SS gene clusters in the available bacterial genome sequences. According to these studies, Salmonella harbors a unique T6SS encoded in the Salmonella Pathogenicity Island 6 (SPI-6). Since these studies only considered few Salmonella genomes, the present work aimed to identify novel T6SS loci by in silico analysis of every genome sequence of Salmonella available.

Results: The analysis of sequencing data from 44 completed or in progress Salmonella genome projects allowed the identification of 3 novel T6SS loci. These clusters are located in differentially-distributed genomic islands we designated SPI-19, SPI-20 and SPI-21, respectively. SPI-19 was identified in a subset of S. enterica serotypes including Dublin, Weltevreden, Agona, Gallinarum and Enteritidis. In the later, an internal deletion eliminated most of the island. On the other hand, SPI-20 and SPI-21 were restricted to S. enterica subspecies arizonae (IIIa) serotype 62:z4,z23:-. Remarkably, SPI-21 encodes a VgrG protein containing a C-terminal extension similar to S-type pyocins of Pseudomonas aeruginosa. This is not only the first evolved VgrG described in Salmonella, but also the first evolved VgrG including a pyocin domain described so far in the literature. In addition, the data indicate that SPI-6 T6SS is widely distributed in S. enterica and absent in serotypes Enteritidis, Gallinarum, Agona, Javiana, Paratyphi B, Virchow, IIIa 62:z4,z23:- and IIIb 61:1,v:1,5,(7). Interestingly, while some serotypes harbor multiple T6SS (Dublin, Weltvreden and IIIa 62:z4,z23:-) others do not encode for any (Enteritidis, Paratyphi B, Javiana, Virchow and IIIb 61:1,v:1,5,(7)). Comparative and phylogenetic analyses indicate that the 4 T6SS loci in Salmonella have a distinct evolutionary history. Finally, we identified an orphan Hcp-like protein containing the Hcp/COG3157 domain linked to a C-terminal extension. We propose to designate this and related proteins as "evolved Hcps".

Conclusion: Altogether, our data suggest that (i) the Salmonella T6SS loci were acquired by independent lateral transfer events and (ii) evolved to contribute in the adaptation of the serotypes to different lifestyles and environments, including animal hosts. Notably, the presence of an evolved VgrG protein related to pyocins suggests a novel role for T6SS in bacterial killing. Future studies on the roles of the identified T6SS loci will expand our knowledge on Salmonella pathogenesis and host specificity.

Figure 1

Gene organization of T6SS gene clusters in Salmonella. Schematic representation of the T6SS loci identified in Salmonella, including the previously described T6SS encoded in SPI-6. One representative of each T6SS-encoding island is shown. ORFs are represented as blocked arrows showing the direction of their transcription. Conserved core, accessory and additional components are represented with a different color.

Figure 2

Comparative analysis of SPI-19 in Salmonella. DNA-based comparison of SPI-19 in serotypes Agona strain SL483, Dublin strain CT_02021853, Weltevreden strain SL484, Gallinarum strain 287/91 and Enteritidis strain P125109. BLASTN analysis was performed using WebACT and displayed with the ACT software.

Figure 3

Genomic context of SPI-20 and SPI-21 in S. IIIa 62:z4,z23:-. DNA-based comparison of the genomic surrounding of SPI-20 and SPI-21 in S. IIIa 62:z4,z23:- strain RKS2980 and the corresponding region in the genome of S. Typhimurium strain LT2. BLASTN analysis was performed using WebACT and displayed with the ACT software. The location of key genetic elements is indicated. tRNA-encoding genes are indicated by blue arrows. saf and stb correspond to Salmonella fimbrial operons. SPI: Salmonella Pathogenicity Island.

Figure 4

VgrG homologs encoded in SPI-21. (A) Schematic representation of three VgrG proteins encoded in SPI-21. Conserved protein domains are represented in color. (B) Homology between the C-terminal extension of SARI_02603 and Usp in UPEC strains. SARI_02603 [residues 841–1201 (1201 total)] and Usp [residues 241–593 (593 total)] were aligned using the multiple sequence alignment tool ClustalW2. Identical and conserved residues are indicated by shaded boxes. Consensus residues are indicated below alignment with an asterix. S-type pyocin and HNH domains are indicated in green and red color boxes, respectively.

Figure 5

Genetic architecture of SPI-6 T6SS in Salmonella enterica serotypes. DNA-based comparison of SPI-6 T6SS in 13 different serotypes of S. enterica was performed by BLASTN analysis with WebACT and visualized with ACT software. The three major regions presenting structural differences between serotypes are indicated.

Figure 6

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 3 novel Salmonella T6SS loci. 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%.

Figure 7

Comparative analysis of Salmonella T6SS clusters. DNA-based comparison of the T6SS encoded in SPI-6, SPI-19, SPI-20 and SPI-21 and phylogenetically-related T6SS loci. The analysis was performed by BLASTN with WebACT and visualized with ACT software. (A) Comparison of SPI-6 T6SS in serotype Typhimurium strain LT2 with gene cluster tss-2 in Burkholderia pseudomallei strain 1106a and h16_A0645-h16_A0657 in Ralstonia eutropha strain H16. (B) Comparison of SPI-19 in serotype Gallinarum strain 287/91 with the OI#7 island in Escherichia coli O157:H7 strain Sakai and island AGI-1 in APEC strain O1. (C) Comparison of SPI-20 and SPI-21 in S. IIIa 62:z4,z23:- strain RKS2980 with the T6SS gene cluster within PAI-metV in UPEC strain CFT073.

Figure 8

Evolutionary relationships of Hcp-like protein. A distance tree (neighbour-joining) was calculated from the alignment of every Hcp-like protein identified in Salmonella. The analysis also included Hcp proteins of Pseudomonas aeruginosa strain PA01 and Vibrio cholerae strain V52. Each of the major groups of Hcp phylogeny is shown in the nodes labeled A to D. Bootstrap support values (% from 3,000 replicates) were: A, 98%; B, 94%; C, 98% and D, 80%.