Identification of Type VI Secretion Systems Effector Proteins That Contribute to Interbacterial Competition in Salmonella Dublin

Abstract

The Type VI Secretion System (T6SS) is a multiprotein device that has emerged as an important fitness and virulence factor for many Gram-negative bacteria through the injection of effector proteins into prokaryotic or eukaryotic cells via a contractile mechanism. While some effector proteins specifically target bacterial or eukaryotic cells, others can target both types of cells (trans-kingdom effectors). In Salmonella, five T6SS gene clusters have been identified within pathogenicity islands SPI-6, SPI-19, SPI-20, SPI-21, and SPI-22, which are differentially distributed among serotypes. Salmonella enterica serotype Dublin (S. Dublin) is a cattle-adapted pathogen that harbors both T6SS_SPI-6 and T6SS_SPI-19. Interestingly, while both systems have been linked to virulence and host colonization in S. Dublin, an antibacterial activity has not been detected for T6SS_SPI-6 in this serotype. In addition, there is limited information regarding the repertoire of effector proteins encoded within T6SS_SPI-6 and T6SS_SPI-19 gene clusters in S. Dublin. In the present study, we demonstrate that T6SS_SPI-6 and T6SS_SPI-19 of S. Dublin CT_02021853 contribute to interbacterial competition. Bioinformatic and comparative genomic analyses allowed us to identify genes encoding three candidate antibacterial effectors located within SPI-6 and two candidate effectors located within SPI-19. Each antibacterial effector gene is located upstream of a gene encoding a hypothetic immunity protein, thus conforming an effector/immunity (E/I) module. Of note, the genes encoding these effectors and immunity proteins are widely distributed in Salmonella genomes, suggesting a relevant role in interbacterial competition and virulence. Finally, we demonstrate that E/I modules SED_RS01930/SED_RS01935 (encoded in SPI-6), SED_RS06235/SED_RS06230, and SED_RS06335/SED_RS06340 (both encoded in SPI-19) contribute to interbacterial competition in S. Dublin CT_02021853.

Keywords: Salmonella Dublin; T6SS; effector; immunity protein; interbacterial competition.

Figure 1

Contribution of T6SS_SPI-6 and T6SS_SPI-19 to interbacterial competition by S. Dublin strain CT_02021853. Wild-type and mutant strains ΔT6SS_SPI-6, ΔT6SS_SPI-19, and ΔT6SS_SPI-6 ΔT6SS_SPI-19 of S. Dublin CT_02021853 were mixed at a ratio of 1:1 (attacker/prey) with Escherichia coli DH5α. Then, 25 μl of the mixture was incubated at 37°C for 24 h in triplicate on MacConkey agar plates. Bacterial counts recovered from each competition assay were calculated by plating serial 10-fold dilutions on LB agar plates with the appropriate antibiotics (Nal in the case of E. coli and Kan or Cam in the case of S. Dublin strains). Data show the ratio of E. coli CFU (prey) to S. Dublin CFU (attacker) normalized to the inoculum ratio and expressed as log₁₀. Error bars indicate standard error. Statistical significance was determined using a one-way ANOVA followed by Tukey’s multiple comparisons test (^*p < 0.05; ^****p < 0.0001; ns, not significant).

Figure 2

The SPI-6 T6SS gene cluster encodes novel putative Type VI Secretion Systems (T6SS) effector proteins. (A) Comparative genomic analysis of the SPI-6 T6SS cluster of S. Dublin CT_02021853 and S. Typhimurium 14028s. BLASTn sequence alignment was performed and visualized using EasyFig (Sullivan et al., 2011). (B) Schematic representation and distribution among Salmonella genomes of each novel effector and immunity protein identified. Names of genes encoding novel effectors and immunity proteins are highlighted in red and green, respectively. Homologs for each component were identified by BLASTn analyses as described in Materials and Methods.

Figure 3

The SPI-19 T6SS gene cluster encodes novel putative T6SS effector proteins. (A) Comparative genomic analysis of the SPI-19 T6SS cluster of S. Dublin CT_02021853 and S. Gallinarum SG9. BLASTn sequence alignment was performed and visualized using EasyFig (Sullivan et al., 2011). (B) Schematic representation and distribution among Salmonella genomes of each novel effector and immunity protein identified. Names of genes encoding novel effectors and immunity proteins are highlighted in red and green, respectively. Homologs for each component were identified by BLASTn analyses as described in Materials and Methods.

Figure 4

Contribution of selected effectors and effector/immunity protein pairs to interbacterial competition in S. Dublin. Bacterial suspensions of attacker and prey strains adjusted to an OD_600nm of 0.5 were mixed at a 1:1 ratio. Then, 25 μl of the mixture was incubated at 37°C for 24 h in triplicate on MacConkey agar plates. Bacterial counts recovered from each competition assay were calculated by plating serial 10-fold dilutions on LB agar plates with the appropriate antibiotics (Kan in the case of the attacker strain and Cam in the case of the prey strain). Data show the prey to attacker CFU ratio normalized to the inoculum and expressed as log₁₀. Error bars indicate standard error. Statistical significance was determined using a one-way ANOVA followed by Tukey’s multiple comparisons test (^****p < 0.0001; ns, not significant).