The type VI secretion system (T6SS) is a multiprotein nanomachine composed of 13 structural components and various accessory proteins that deliver protein effectors into target cells through a contractile mechanism (Cherrak et al., 2019; Coulthurst, 2019). The T6SS needle, composed of an inner tube (made of a stack of Hcp hexamer rings) and comprising a trimer of VgrG and a PAAR protein, is wrapped into a contractile sheath formed by the polymerization of TssB/TssC subunits. These are assembled into an extended, metastable conformation (Silverman et al., 2013; Cherrak et al., 2019). Contraction of the sheath upon contact with a target cell or sensing cell envelope damage propels the needle toward the target cell (Brackmann et al., 2017). T6SS effector proteins are classified as either cargo or specialized effectors. Cargo effectors are delivered by non-covalent interaction with some core components (Coulthurst, 2019), while specialized effectors are additional domains of either VgrG, Hcp, or PAAR proteins (Durand et al., 2014; Whitney et al., 2014; Diniz and Coulthurst, 2015; Ma et al., 2017; Pissaridou et al., 2018).

The extensive repertoire of effector proteins makes the T6SS a highly versatile machine that can target prokaryotic or eukaryotic cells (Coulthurst, 2019; Monjarás Feria and Valvano, 2020). Among the antibacterial effector proteins, some target the peptidic or glycosidic bonds of the peptidoglycan (Ma and Mekalanos, 2010; Russell et al., 2012; Srikannathasan et al., 2013; Whitney et al., 2013; Berni et al., 2019; Wood et al., 2019), or the FtsZ cell division ring (Ting et al., 2018). These antibacterial effectors are encoded in bi-cistronic elements with immunity proteins (E/I pairs) that bind tightly and specifically to their cognate effector preventing self-intoxication and killing of sibling cells (Russell et al., 2012). Other T6SS effectors are eukaryote-specific, such as those targeting the actin or microtubule cytoskeleton networks (Monjarás Feria and Valvano, 2020), and others (known as trans-kingdom effectors) can target both bacterial and eukaryotic cells (Jiang et al., 2014). These effectors include those targeting conserved molecules (NAD⁺ and NADP⁺) and macromolecules (DNA, phospholipids) or forming pores in membranes (Whitney et al., 2015; Tang et al., 2018; Ahmad et al., 2019).

Many enteric pathogens (e.g., Salmonella, Shigella, and Vibrio) use the T6SS to colonize the intestinal tract of infected hosts (Sana et al., 2016; Chassaing and Cascales, 2018), while some strains of the gut commensal Bacteroides fragilis use their T6SSs only for competition against other Bacteroidales species (Coyne and Comstock, 2019). The T6SS is, therefore, a key player in bacterial warfare.

The Salmonella genus includes more than 2,600 serotypes distributed between species S. enterica and S. bongori (Issenhuth-Jeanjean et al., 2014), which differ in clinical signs and host range (Uzzau et al., 2000). Serotypes are defined based on variations in the somatic, flagellar and capsular antigens, according to the Kauffmann-White-Le Minor serotyping scheme (Grimont and Weill, 2007; Issenhuth-Jeanjean et al., 2014). Worldwide, Salmonella infections are responsible for 95.1 million cases of gastroenteritis per year (GBD 2017 Non-Typhoidal Salmonella Invasive Disease Collaborators, 2019). In addition, the World Health Organization (WHO) has also included Salmonella as a high-priority pathogen due to the emergence of strains with high levels of fluoroquinolone resistance (GBD 2017 Non-Typhoidal Salmonella Invasive Disease Collaborators, 2019). In Salmonella, 5 T6SS gene clusters have been identified within Salmonella Pathogenicity Islands (SPIs) SPI-6, SPI-19, SPI-20, SPI-21, and SPI-22 (Blondel et al., 2009; Fookes et al., 2011). These T6SSs are distributed in 4 different evolutionary lineages: T6SS_SPI-6 belongs to subtype i3, T6SS_SPI-19 to subtype i1, T6SS_SPI-22 to subtype i4a, and both T6SS_SPI-20 and T6SS_SPI-21 belong to subtype i2 (Bao et al., 2019). Besides their distinct evolutionary origin, these five T6SS gene clusters are differentially distributed among distinct serotypes, subspecies, and species of Salmonella (Blondel et al., 2009).

Notably, most of these T6SSs have been shown to contribute to the virulence and pathogenesis of different Salmonella serotypes (Blondel et al., 2010; Mulder et al., 2012; Pezoa et al., 2013, 2014; Sana et al., 2016; Xian et al., 2020; Hespanhol et al., 2022; Sibinelli-Sousa et al., 2022). One of the most studied and widely distributed T6SS corresponds to that encoded in SPI-6. Depending on the serotype, the SPI-6 T6SS gene cluster comprises a region of ~35 to 50 kb encoding ~30 to 45 ORFs, including each of the 13 T6SS core components. The genetic architecture of the SPI-6 T6SS gene cluster is highly conserved among serotypes; nonetheless, there are structural differences restricted to three variable regions of the island (herein referred to as VR1, VR2, and VR3, Figure 1) (Blondel et al., 2009). In S. Typhimurium and S. Dublin, 9 SPI-6 T6SS effector proteins have been described to date (Russell et al., 2012; Benz et al., 2013; Whitney et al., 2013; Koskiniemi et al., 2014; Sana et al., 2016; Sibinelli-Sousa et al., 2020; Amaya et al., 2022; Jurėnas et al., 2022; Lorente-Cobo et al., 2022), most of which are encoded within these variable regions (Figure 1; Table 1).

The VR1 is located downstream of gene tssC and encodes the E/I modules Tae2/Tai2 and Tae4/Tai4. Tae2 and Tae4 are peptidoglycan hydrolases able to cleave the DD-crosslinks between D-mDAP and D-alanine or the covalent link between D-Glu and mDAP of the tetrapeptide stem, respectively, thus contributing to interbacterial competition and mice colonization (Russell et al., 2012; Sana et al., 2016). VR2 is located downstream of gene tssM and encodes many proteins of unknown function and two E/I modules with peptidoglycan hydrolase activity: Tge2/Tgi2P is predicted to have N-acetylglucosaminidase activity (Whitney et al., 2013), while Tlde1/Tldi shows L,D carboxypeptidase activity against the peptide stems of the peptidoglycan layer (Sibinelli-Sousa et al., 2020; Lorente-Cobo et al., 2022). Finally, the VR3 is located downstream of gene tssI and encodes a variable number of Rhs elements, some of them harboring endonuclease domains such as HNHc (DNase) and Ntox47 (RNase), and an ART domain (ADP-ribosyltransferase) linked to the C-terminal of these Rhs proteins (Koskiniemi et al., 2014; Amaya et al., 2022; Jurėnas et al., 2022).

Most of our knowledge regarding the presence and distribution of SPI-6 T6SS effector proteins comes from studies using reference strains of a limited number of serotypes (e.g., S. Typhimurium and S. Dublin) (Russell et al., 2012; Benz et al., 2013; Whitney et al., 2013; Koskiniemi et al., 2014; Sana et al., 2016; Sibinelli-Sousa et al., 2020; Amaya et al., 2022; Jurėnas et al., 2022; Lorente-Cobo et al., 2022). In this study, we performed a bioinformatic prediction analysis searching for putative T6SS effectors in a dataset of 60 genomes covering 37 S. enterica serotypes retrieved from the curated Secret6 database. Our analysis identified 23 new putative antibacterial effectors encoded in E/I modules within the 3 VRs of the SPI-6 T6SS gene cluster. These candidates include 5 effectors with putative peptidoglycan hydrolase activity, 16 effectors with potential nuclease activity and 2 effectors targeting the bacterial translation machinery. Finally, we expanded our analysis to include all available Salmonella genomes deposited in the NCBI database and determined the global distribution of these new putative effectors. A hierarchical clustering analysis identified that some effectors are conserved in most Salmonella serotypes. In contrast, most other effectors are differentially distributed in different serotypes. The presence of different sets of T6SS effectors suggests that distinct repertoires of these proteins may have a differential impact on the pathogenicity and environmental adaptation of Salmonella serotypes.

The T6SS has emerged as an important virulence and environmental fitness factor for Salmonella (Blondel et al., 2010; Mulder et al., 2012; Pezoa et al., 2013, 2014; Sana et al., 2016; Xian et al., 2020; Hespanhol et al., 2022; Sibinelli-Sousa et al., 2022). However, information regarding the complexity and diversity of effector proteins for each distinct Salmonella T6SS is still lacking. In this context, even though the T6SS encoded in SPI-6 has been shown to contribute to host colonization by S. Typhimurium and S. Dublin (Mulder et al., 2012; Pezoa et al., 2013, 2014; Sana et al., 2016) and to interbacterial competition of S. Typhimurium against the intestinal microbiota (Sibinelli-Sousa et al., 2022), only 9 effector proteins have been identified and characterized so far (Russell et al., 2012; Benz et al., 2013; Whitney et al., 2013; Koskiniemi et al., 2014; Sana et al., 2016; Sibinelli-Sousa et al., 2020; Amaya et al., 2022; Jurėnas et al., 2022; Lorente-Cobo et al., 2022).

In this study, by means of bioinformatic and comparative genomic analyses, we identified a subset of 23 new SPI-6 T6SS candidate effectors, including peptidoglycan hydrolases, DNases, RNases, deaminases, and ADP-ribosyltransferases. Despite being well conserved, the SPI-6 T6SS gene cluster encodes a variable number of ORFs of unknown function restricted to three variable regions (VR1-3), that include the T6SS effectors previously identified in this species (Blondel et al., 2009). Notably, our analysis showed that every new T6SS effector identified is encoded within one of these variable regions. An interesting observation was that all predicted peptidoglycan targeting effectors are confined to VR1 and VR2. The reason behind this observation remains unclear; however, it is possible that VR1 and VR2 are hot-spots for gene recombination during Salmonella evolution, but the lack of mobile genetic elements surrounding these regions does not support this hypothesis. Importantly, in addition to the 4 peptidoglycan targeting effectors reported so far (Tae2, Tae4, Tge2, and Tlde1) (Russell et al., 2012; Benz et al., 2013; Whitney et al., 2013; Sana et al., 2016; Sibinelli-Sousa et al., 2020; Lorente-Cobo et al., 2022), we identified 5 candidate effectors encoded in VR1 and VR2 that presumably degrade peptidoglycan, indicating that this macromolecule is a common target site for Salmonella T6SS effectors. Of note, the unannotated ORF encoded in VR1 is the first putative effector that likely cleaves the link between L-Ala and D-Glu of the peptidoglycan peptide stems reported in Salmonella and shares homology to the peptidoglycan hydrolase ChiX of Serratia marcescens (30% identity and 45.2% similarity at amino acid sequence level) (Owen et al., 2018). This finding expands the peptidoglycan target sites exploited by Salmonella T6SS effectors against competing bacteria. On the other hand, the PgP2 and TseH-like candidate effectors are predicted to have redundant peptidoglycan degrading functions with other Salmonella T6SS effectors. PgP2 is predicted to have the same L,D transpeptidase exchange activity reported for Tlde1 (Sibinelli-Sousa et al., 2020; Lorente-Cobo et al., 2022), replacing D-Ala by a non-canonical D-amino acid preventing the normal crosslink between mDAP and D-Ala. In addition, the TseH-like candidate effector is a NlpC/P60 endopeptidase family protein (Xu et al., 2010; Altindis et al., 2015; Squeglia et al., 2019) predicted to cleave the covalent link between D-Glu and mDAP, as reported for Tae4 (Benz et al., 2013). These redundant functions suggests that the peptide stems are the main peptidoglycan target sites of Salmonella T6SS effectors, as only one identified effector targets the glycoside bonds in this macromolecule corresponds to Tge2 (Whitney et al., 2013). Remarkably, most serotypes encode combinations of T6SS effectors predicted to have hydrolytic activity toward different regions of the peptidoglycan structure. We hypothesize that this assortment of seemingly redundant effectors may improve the efficiency of the bacterial killing process.

The Reprolysin_4 domain found in some candidate effectors is present in zinc-binding metallo-peptidases harboring the binding motif HExxGHxxGxxH of family M12B peptidases. Of note, this motif is also present in the T6SS antibacterial effector SED_RS06335 with putative peptidoglycan hydrolase activity encoded in SPI-19 of Salmonella Dublin CT_02021853 (Amaya et al., 2022). The last candidate effector targeting the peptidoglycan identified in our study harbors the Peptidase_M64 protein domain that is also present in the IgA proteinase of Clostridium ramosum (Kosowska et al., 2002), recently reclassified as Thomasclavelia ramosa (Lawson et al., 2023). The putative immunity proteins of Reprolysin_4 and Peptidase_M64 have a signal peptide and a transmembrane domain, respectively. This suggests that both candidate effectors target the bacterial periplasm.

On the other hand, the VR3 of the SPI-6 T6SS gene cluster encodes a wide variety of effector proteins including domains found in DNases, RNases, deaminases and ADP-ribosyltransferases. Interestingly, most of these domains are fused to the C-terminal of Rhs proteins contributing to diversify the molecular targets of T6SSs in Salmonella. This was not unexpected since we have previously shown that the VR3 of SPI-6 encodes a variable number of Rhs elements (Blondel et al., 2009; Amaya et al., 2022) and many Rhs proteins have C-terminal polymorphic endonuclease domains associated with T6SS effectors in Salmonella and other bacteria (Zhang et al., 2012; Koskiniemi et al., 2014; Amaya et al., 2022). It is known that Rhs proteins have YD-peptide repeats, which fold into a large β-cage structure that surrounds and protects the C-terminal toxin domain increasing T6SS secretion efficiency (Donato et al., 2020; Jurėnas et al., 2021; Günther et al., 2022). This could explain why many T6SS effectors are associated to these elements.

Altogether, our work expands the repertoire of Salmonella T6SS effectors and provides evidence that the SPI-6 T6SS gene cluster harbors a great diversity of antibacterial effectors encoded in three variable regions. One interesting finding of our study is that peptidoglycan hydrolyzing effectors restricted to VR1 and VR2 are highly conserved in Salmonella genomes, while effectors targeting nucleic acids and the translation machinery encoded in VR3 are broadly distributed in Salmonella serotypes. This suggests that different repertoires of effectors could have an impact on the pathogenic potential and environmental fitness of these bacteria. Importantly, although this study increases the number of putative Salmonella antibacterial effectors against competing bacteria, we could not rule out that those targeting nucleic acids encoded in VR3 may also affect eukaryotic cells. This is an important knowledge gap, since no T6SS effector protein identified to date in Salmonella has been confirmed to target eukaryotic organisms, despite the clear contribution of Salmonella T6SSs to intracellular replication, survival and cytotoxicity inside the host immune cells (Mulder et al., 2012; Blondel et al., 2013; Schroll et al., 2019). Further research is required to address this issue. Finally, we are currently performing experimental work to confirm that each of the 23 candidates identified in our study correspond to bona fide T6SS effector proteins.

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding author.

CB, FA, PB, CS, and DP: conceptualization, formal analysis, validation, writing-original draft preparation, writing review and editing, resources, project administration, and funding acquisition. CB and DP: methodology, investigation, and visualization. CS and DP: supervision. All authors contributed to the article and approved the submitted version.

DP was supported by Fondo Concursable Proyectos de Investigación Regulares UDLA 2023 DI-13/23. CS was supported by FONDECYT grant 1212075. CB was supported by FONDECYT grant 1201805, ECOS-ANID ECOS200037 and HHMI-Gulbenkian International Research Scholar Grant #55008749. FA was supported by CONICYT/ANID fellowship 21191925.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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