Identification and distribution of new candidate T6SS effectors encoded in Salmonella Pathogenicity Island 6.

The type VI secretion system (T6SS) is a contact-dependent contractile multiprotein apparatus widely distributed in Gram-negative bacteria. These systems can deliver different effector proteins into target bacterial and/or eukaryotic cells, contributing to the environmental fitness and virulence of many bacterial pathogens. Salmonella harbors five different T6SSs encoded in different genomic islands. The T6SS encoded in Salmonella Pathogenicity Island 6 (SPI-6) contributes to Salmonella competition with the host microbiota and its interaction with infected host cells. Despite its relevance, information regarding the total number of effector proteins encoded within SPI-6 and its distribution among different Salmonella enterica serotypes is limited. In this work, we performed bioinformatic and comparative genomics analyses of the SPI-6 T6SS gene cluster to expand our knowledge regarding the T6SS effector repertoire and the global distribution of these effectors in Salmonella. The analysis of a curated dataset of 60 Salmonella enterica genomes from the Secret6 database revealed the presence of 23 new putative T6SS effector/immunity protein (E/I) modules. These effectors were concentrated in the variable regions 1 to 3 (VR1-3) of the SPI-6 T6SS gene cluster. VR1-2 were enriched in candidate effectors with predicted peptidoglycan hydrolase activity, while VR3 was enriched in candidate effectors of the Rhs family with C-terminal extensions with predicted DNase, RNase, deaminase, or ADP-ribosyltransferase activity. A global analysis of known and candidate effector proteins in Salmonella enterica genomes from the NCBI database revealed that T6SS effector proteins are differentially distributed among Salmonella serotypes. While some effectors are present in over 200 serotypes, others are found in less than a dozen. A hierarchical clustering analysis identified Salmonella serotypes with distinct profiles of T6SS effectors and candidate effectors, highlighting the diversity of T6SS effector repertoires in Salmonella enterica. The existence of different repertoires of effector proteins suggests that different effector protein combinations may have a differential impact on the environmental fitness and pathogenic potential of these strains.

Transfer of T6SSSPI-19 from Salmonella Gallinarum to Salmonella Typhimurium Lacking T6SSSPI-6 Complements its Colonization Defect in Mice.

Salmonella genus harbors five Type VI Secretion System (T6SS) gene clusters. The T6SS encoded in SPI-6 (T6SSSPI-6) contributes to Salmonella Typhimurium colonization of chickens and mice, while the T6SS encoded in SPI-19 (T6SSSPI-19) of Salmonella Gallinarum contributes to chicken colonization. Interestingly, the T6SSSPI-19 of Salmonella Gallinarum complemented the defect in chicken colonization of a Salmonella Typhimurium strain that lacks the T6SSSPI-6, suggesting that both T6SSs are interchangeable. Here we show that the transfer of Salmonella Gallinarum T6SSSPI-19 complemented the defect in mice colonization of a Salmonella Typhimurium ΔT6SSSPI-6 strain, indicating that both T6SSs are functionally redundant during host colonization.

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

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 T6SSSPI-6 and T6SSSPI-19. Interestingly, while both systems have been linked to virulence and host colonization in S. Dublin, an antibacterial activity has not been detected for T6SSSPI-6 in this serotype. In addition, there is limited information regarding the repertoire of effector proteins encoded within T6SSSPI-6 and T6SSSPI-19 gene clusters in S. Dublin. In the present study, we demonstrate that T6SSSPI-6 and T6SSSPI-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.

Fnr and ArcA Regulate Lipid A Hydroxylation in Salmonella Enteritidis by Controlling lpxO Expression in Response to Oxygen Availability.

Lipid A is the bioactive component of lipopolysaccharide, and presents a dynamic structure that undergoes modifications in response to environmental signals. Many of these structural modifications influence Salmonella virulence. This is the case of lipid A hydroxylation, a modification catalyzed by the dioxygenase LpxO. Although it has been established that oxygen is required for lipid A hydroxylation acting as substrate of LpxO in Salmonella, an additional regulatory role for oxygen in lpxO expression has not been described. The existence of this regulation could be relevant considering that Salmonella faces low oxygen tension during infection. This condition leads to an adaptive response by changing the expression of numerous genes, and transcription factors Fnr and ArcA are major regulators of this process. In this work, we describe for the first time that lipid A hydroxylation and lpxO expression are modulated by oxygen availability in Salmonella enterica serovar Enteritidis (S. Enteritidis). Biochemical and genetic analyses indicate that this process is regulated by Fnr and ArcA controlling the expression of lpxO. In addition, according to our results, this regulation occurs by direct binding of both transcription factors to specific elements present in the lpxO promoter region. Altogether, our observations revealed a novel role for oxygen acting as an environment signal controlling lipid A hydroxylation in S. Enteritidis.

Differential roles for pathogenicity islands SPI-13 and SPI-8 in the interaction of Salmonella Enteritidis and Salmonella Typhi with murine and human macrophages.

BACKGROUND: Salmonella pathogenicity island (SPI)-13 is conserved in many serovars of S. enterica, including S. Enteritidis, S. Typhimurium and S. Gallinarum. However, it is absent in typhoid serovars such as S. Typhi and Paratyphi A, which carry SPI-8 at the same genomic location. Because the interaction with macrophages is a critical step in Salmonella pathogenicity, in this study we investigated the role played by SPI-13 and SPI-8 in the interaction of S. Enteritidis and S. Typhi with cultured murine (RAW264.7) and human (THP-1) macrophages. RESULTS: Our results showed that SPI-13 was required for internalization of S. Enteritidis in murine but not human macrophages. On the other hand, SPI-8 was not required for the interaction of S. Typhi with human or murine macrophages. Of note, the presence of an intact copy of SPI-13 in a S. Typhi mutant carrying a deletion of SPI-8 did not improve its ability to be internalized by, or survive in human or murine macrophages. CONCLUSIONS: Altogether, our results point out to different roles for SPI-13 and SPI-8 during Salmonella infection. While SPI-13 contributes to the interaction of S. Enteritidis with murine macrophages, SPI-8 is not required in the interaction of S. Typhi with murine or human macrophages. We hypothesized that typhoid serovars have lost SPI-13 and maintained SPI-8 to improve their fitness during another phase of human infection.

Differential roles for pathogenicity islands SPI-13 and SPI-8 in the interaction of Salmonella Enteritidis and Salmonella Typhi with murine and human macrophages

BACKGROUND: Salmonella pathogenicity island (SPI)-13 is conserved in many serovars of S. enterica, including S. Enteritidis, S. Typhimurium and S. Gallinarum. However, it is absent in typhoid serovars such as S. Typhi and Paratyphi A, which carry SPI-8 at the same genomic location. Because the interaction with macrophages is a critical step in Salmonella pathogenicity, in this study we investigated the role played by SPI-13 and SPI-8 in the interaction of S. Enteritidis and S. Typhi with cultured murine (RAW264.7) and human (THP-1) macrophages. RESULTS: Our results showed that SPI-13 was required for internalization of S. Enteritidis in murine but not human macrophages. On the other hand, SPI-8 was not required for the interaction of S. Typhi with human or murine macrophages. Of note, the presence of an intact copy of SPI-13 in a S. Typhi mutant carrying a deletion of SPI-8 did not improve its ability to be internalized by, or survive in human or murine macrophages. CONCLUSIONS: Altogether, our results point out to different roles for SPI-13 and SPI-8 during Salmonella infection. While SPI-13 contributes to the interaction of S. Enteritidis with murine macrophages, SPI-8 is not required in the interaction of S. Typhi with murine or human macrophages. We hypothesized that typhoid serovars have lost SPI-13 and maintained SPI-8 to improve their fitness during another phase of human infection.