Genomic analysis of Salmonella isolated from surface water and animal sources in Chile reveals new T6SS effector protein candidates

Abstract

Type VI Secretion Systems (T6SS), widely distributed in Gram-negative bacteria, contribute to interbacterial competition and pathogenesis through the translocation of effector proteins to target cells. Salmonella harbor 5 pathogenicity islands encoding T6SS (SPI-6, SPI-19, SPI-20, SPI-21 and SPI-22), in which a limited number of effector proteins have been identified. Previous analyses by our group focused on the identification of candidate T6SS effectors and cognate immunity proteins in Salmonella genomes deposited in public databases. In this study, the analysis was centered on Salmonella isolates obtained from environmental sources in Chile. To this end, bioinformatics and comparative genomics analyses were performed using 695 genomes of Salmonella isolates representing 44 serotypes obtained from surface water and animal sources in Chile to identify new T6SS effector proteins. First, T6SS gene clusters were identified using the SecreT6 server. This analysis revealed that most isolates carry the SPI-6 T6SS gene cluster, whereas the SPI-19 and SPI-21 T6SS gene clusters were detected in isolates from a limited number of serotypes. In contrast, the SPI-20 and SPI-22 T6SS gene clusters were not detected. Subsequently, each ORF in the T6SS gene clusters identified was analyzed using bioinformatics tools for effector prediction, identification of immunity proteins and functional biochemical prediction. This analysis detected 20 of the 37 T6SS effector proteins previously reported in Salmonella. In addition, 4 new effector proteins with potential antibacterial activity were identified in SPI-6: 2 Rhs effectors with potential DNase activity (PAAR-RhsA-NucA_B and PAAR-RhsA-GH-E) and 2 effectors with potential RNase activity (PAAR-RhsA-CdiA and RhsA-CdiA). Interestingly, the repertoire of SPI-6 T6SS effectors varies among isolates of the same serotype. In SPI-19, no new effector protein was detected. Of note, some Rhs effectors of SPI-19 and SPI-6 present C-terminal ends with unknown function. The presence of cognate immunity proteins carrying domains present in bona fide immunity proteins suggests that these effectors have antibacterial activity. Finally, two new effectors were identified in SPI-21: one with potential peptidoglycan hydrolase activity and another with potential membrane pore-forming activity. Altogether, our work broadens the repertoire of Salmonella T6SS effector proteins and provides evidence that SPI-6, SPI-19 and SPI-21 T6SS gene clusters harbor a vast array of antibacterial effectors.

Keywords: Chile; Salmonella; T6SS; effector; immunity protein.

Figure 1

The SPI-6 T6SS gene cluster encodes new putative T6SS effector proteins. (A) Comparative genomic analysis of the SPI-6 T6SS cluster of S. Braenderup FA1083, S. Albany FA1443, S. Tennessee FA1455 and S. Derby FA1451. BLASTn sequence alignment was performed and visualized using EasyFig (Sullivan et al., 2011). (B) Schematic representation and distribution among Salmonella genomes of each new effector and immunity protein identified. ORFs encoding new E/I modules are highlighted in different colors according to the predicted functions. Homologs for each component were identified by BLASTn analyses as described in Materials and Methods.

Figure 2

Prevalence of ORFs encoding T6SS effectors and candidate effectors in the SPI-6 T6SS gene cluster of Chilean Salmonella isolates. A hierarchical clustering analysis was conducted using MORPHEUS, as detailed in the Materials and Methods section. The color code in the heatmap indicates the frequency of a given ORF among all isolates of a particular Salmonella serotype. The names of new T6SS candidate effectors identified in this study are highlighted in red.

Figure 3

The SPI-6 T6SS gene cluster in a number of Chilean Salmonella isolates includes two tssI-eagR-rhs gene modules in VR3. Comparative genomic analysis of the SPI-6 T6SS cluster of S. Braenderup FA1083, S. Kentucky CFSAN035145, S. Sandiego CFSAN105323 and S. Tennessee FA1455 and CFSAN070645. BLASTn sequence alignment was performed and visualized using EasyFig (Sullivan et al., 2011). ORFs encoding E/I modules are highlighted in different colors according to the confirmed or predicted functions. The tssI-eagR-rhs gene modules of the SPI-6 T6SS gene cluster are demarked by asterisks. Grayscale represents the percentage of identity between nucleotide sequences. The SPI-6 T6SS gene cluster from S. Typhimurium 14028s was used for comparative purposes.

Figure 4

The T6SS_SPI-6 effector repertoire varies among Chilean Salmonella isolates. Comparative genomic analysis of the SPI-6 T6SS cluster of selected Salmonella isolates representing different serotypes. BLASTn sequence alignment was performed and visualized using EasyFig (Sullivan et al., 2011). ORFs encoding T6SS core components are shown in blue. ORFs encoding E/I modules are highlighted in different colors according to the confirmed or predicted functions. Grayscale represents the percentage of identity between nucleotide sequences.

Figure 5

The genetic structure and repertoire of effector proteins encoded in the SPI-6 T6SS gene cluster vary among isolates of serotype S. Livingstone. Comparative genomic analysis of the SPI-6 T6SS cluster in S. Livingstone isolates. BLASTn sequence alignment was performed and visualized using EasyFig (Sullivan et al., 2011). ORFs encoding E/I modules are highlighted in different colors according to the confirmed or predicted functions. SPI-6 T6SS gene clusters from S. Typhimurium 14028s and S. Typhi CT18 were used for comparative purposes.

Figure 6

The SPI-19 T6SS gene cluster differs among Chilean Salmonella isolates and encodes putative T6SS Rhs effector proteins harboring C-terminal ends with domains of unknown function. Comparative genomic analysis of the SPI-19 T6SS gene cluster of S. Agona CFSAN116538, S. IV 43:z4,z23:- CFSAN119431 and S. Livingstone CFSAN105333. BLASTn sequence alignment was performed and visualized using EasyFig (Sullivan et al., 2011). ORFs encoding T6SS core components are shown in blue. ORFs encoding E/I modules are highlighted in different colors according to the confirmed or predicted functions. SPI-19 T6SS gene clusters from S. Dublin CT_02021853 (top) and S. Gallinarum 287/91 (bottom) were used for comparative purposes. Grayscale represents the percentage of identity between nucleotide sequences.

Figure 7

The SPI-21 T6SS gene cluster encodes new putative T6SS effector proteins. (A) Comparative genomic analysis of the SPI-21 T6SS cluster of S. enterica subsp. diarizonae 48:i:z CFSAN043227, S. enterica subsp. diarizonae 35:i:z CFSAN111176 and S. enterica subsp. diarizonae 48:i:z CFSAN119408. BLASTn sequence alignment was performed and visualized using EasyFig (Sullivan et al., 2011). SPI-21 T6SS gene cluster from S. enterica subsp. arizonae RSK2980 was used for comparative purposes. (B) Schematic representation and distribution among Salmonella genomes of each new effector and immunity protein identified. ORFs encoding new E/I modules are highlighted in different colors according to the predicted functions. Homologs for each component were identified by BLASTn analyses, as described in Materials and Methods.