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.

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. 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. 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.

Acidithiobacillus ferrooxidans resists extremely high concentrations of copper. Strain ATCC 53993 is much more resistant to the metal compared with strain ATCC 23270, possibly due to the presence of a genomic island in the former one. The global response of strain ATCC 53993 to copper was analyzed using iTRAQ (isobaric tag for relative and absolute quantitation) quantitative proteomics. Sixty-seven proteins changed their levels of synthesis in the presence of the metal. On addition of CusCBA efflux system proteins, increased levels of other envelope proteins, such as a putative periplasmic glucan biosynthesis protein (MdoG) involved in the osmoregulated synthesis of glucans and a putative antigen O polymerase (Wzy), were seen in the presence of copper. The expression of A. ferrooxidansmdoG or wzy genes in a copper sensitive Escherichia coli conferred it a higher metal resistance, suggesting the possible role of these components in copper resistance of A. ferrooxidans. Transcriptional levels of genes wzy, rfaE and wzz also increased in strain ATCC 23270 grown in the presence of copper, but not in strain ATCC 53993. Additionally, in the absence of this metal, lipopolysaccharide (LPS) amounts were 3-fold higher in A. ferrooxidans ATCC 53993 compared with strain 23270. Nevertheless, both strains grown in the presence of copper contained similar LPS quantities, suggesting that strain 23270 synthesizes higher amounts of LPS to resist the metal. On the other hand, several porins diminished their levels in the presence of copper. The data presented here point to an essential role for several envelope components in the extreme copper resistance by this industrially important acidophilic bacterium.

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.

The Type VI Secretion System (T6SS) is a critical fitness and virulence factor of many Gram-negative bacteria. Five T6SS gene clusters have been described in Salmonella , each one encoded within different pathogenicity islands (i.e., SPI-6, SPI-19, SPI-20, SPI-21, and SPI-22). The events of gain and loss of these T6SS gene clusters have contributed to shape the genome evolution of different Salmonella serotypes. In addition, the differential distribution of T6SS and the ever-increasing repertoire of predicted effector proteins is likely to play an important role in the environmental fitness and pathogenic potential of different Salmonella serotypes. This review summarizes the current knowledge on the role played by T6SS in Salmonella biology, highlighting the major milestones in the field over the past two decades. We discuss the expanding repertoire of T6SS effector proteins identified to date and examine the current understanding of mechanisms controlling T6SS expression in Salmonella , focusing on host-derived cues and regulators involved. Finally, we provide a critical analysis of conflicting reports and suggest future directions for the research in the field. A better understanding of these processes could expand our knowledge of Salmonella biology, and the mechanisms behind how this versatile secretion system enables Salmonella to thrive in competitive microbial environments and contribute to host colonization.

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.