Transfer of T6SS_SPI-19 from Salmonella Gallinarum to Salmonella Typhimurium Lacking T6SS_SPI-6 Complements its Colonization Defect in Mice

The Type VI Secretion System (T6SS) is a multi-protein nanomachine made of 13 structural components and a variable number of accessory proteins that delivers bacterial proteins, called effectors, into cells through a contractile mechanism (Zoued et al. 2014; Coulthurst 2019). The extensive repertoire of effector activities makes the T6SS an efficient apparatus targeting prokaryotic and/or eukaryotic cells (Hernandez et al. 2020; Monjarás Feria and Valvano 2020), emerging as an environmental adaptation and pathogenesis factor for several bacteria (Records 2011; Basler 2015; Cianfanelli et al. 2016; Navarro-Garcia et al. 2019).

In the Salmonella genus, five T6SS gene clusters have been described (Blondel et al. 2009; Fookes et al. 2011; Bao et al. 2019) that belong to four distinct phylogenetic lineages (Bao et al. 2019). In addition, these gene clusters are differentially distributed among representatives of Salmonella (Blondel et al. 2009; Fookes et al. 2011).

The T6SS encoded in pathogenicity island SPI-6 (T6SS_SPI-6) in Salmonella Typhimurium (Fig. 1A) (a serotype that causes systemic infection in mice) is needed for intracellular survival within avian and murine immune cells (Parsons and Heffron 2005; Klumpp and Fuchs 2007; Mulder et al. 2012) and contributes to intestinal and systemic colonization of gavage-inoculated chickens (Pezoa et al. 2013) and mice (Mulder et al. 2012; Liu et al. 2013; Sana et al. 2016). On the other hand, current evidence indicates that the T6SS encoded in pathogenicity island SPI-19 (T6SS_SPI-19) (Fig. 1A) plays a role in gastrointestinal colonization by Salmonella enterica serotypes adapted to avian hosts, such as Salmonella Gallinarum (Blondel et al. 2010) and Salmonella Pullorum (Xian et al. 2020). Of note, the transfer of Salmonella Gallinarum T6SS_SPI-19 to a ΔT6SS_SPI-6 mutant of Salmonella Typhimurium complements its gastrointestinal colonization defect in chickens (Pezoa et al. 2013). Since these experiments were performed in chickens, whether T6SS_SPI-19 has a similar role in other hosts, such as mice, remains unknown.

Fig. 1.

In the present work, we evaluated the contribution of T6SS_SPI-19 to gastrointestinal colonization and systemic spread in mice using a mutant strain of Salmonella Typhimurium lacking the T6SS_SPI-6 and harboring the Salmonella Gallinarum T6SS_SPI-19 cloned in the R995 plasmid via the VEX-Capture technique (Wilson et al. 2004). Table I shows the bacterial strains and plasmids used in this work. The construction of Salmonella Typhimurium ΔphoN and ΔT6SS_SPI-6 mutant strains has been reported (Pezoa et al. 2013). The generation of R995 plasmid derivatives harboring gene clusters T6SS_SPI-6 (39 kb) of Salmonella Typhimurium 14028s and T6SS_SPI-19 (42 kb) of Salmonella Gallinarum 287/91 has been reported (Pezoa et al. 2013; Blondel et al. 2010).

For competition experiments, groups of five BALB/c mice (6–10-week-old females) were inoculated by oral gavage with 10⁶ CFU of a 1 : 1 mixture of the wild type and the mutant strain to be tested. The inoculum was calculated by serial ten-fold dilutions plated on LB agar plus suitable antibiotics. Mice were euthanized four days after infection, and the liver, spleen and cecum were collected and homogenized in sterile PBS. Titers of bacteria recovered from these organs were calculated as described above. A logarithmic mean ratio of mutant to wild-type CFU normalized to the input ratio was calculated with these numbers. Experiments involving mice were authorized by the Bioethics Committee of the School of Chemical and Pharmaceutical Sciences (Universidad de Chile) and were performed accordingly to the Guide to the Care and Use of Laboratory Animals, the Public Health Service Policy on the Human Care and Use of Laboratory Animals.

Our competition experiments showed that the Salmonella Typhimurium ΔT6SS_SPI-6 strain has a defect in mice colonization, as previously reported (Mulder et al. 2012; Liu et al. 2013). The ΔT6SS_SPI-6 mutant strain was recovered 1.3-, 2.5-, and 2.2-fold less often than the wild-type strain in the cecum, liver and spleen, respectively, at four days post-infection (Fig. 1B). The colonization defect was reversed when the mutant strain was transformed with derivatives of plasmid R995 carrying either the T6SS_SPI-6 or the T6SS_SPI-19 gene cluster from Salmonella Typhimurium and Salmonella Gallinarum, respectively (Fig. 1B), indicating that both T6SS_SPI-6 and T6SS_SPI-19 are functionally interchangeable during mice colonization. Furthermore, these results, along with those reported in the chicken model (Pezoa et al. 2013) suggest that both T6SS_SPI-6 and T6SS_SPI-19 are fully functional in distinct Salmonella serotypes and contribute to the colonization of different animal hosts. It is worth mentioning that the ΔT6SS_SPI-6 mutant strain complemented with the T6SS_SPI-19 gene cluster of Salmonella Gallinarum colonized the spleen of infected mice more efficiently than the mutant complemented with its own T6SS_SPI-6 gene cluster (Fig. 1B). A similar trend was observed when liver colonization was evaluated. Although there is no easy explanation for this observation, it has been reported that both T6SS_SPI-6 and T6SS_SPI-19 contribute to the systemic colonization of different Salmonella serotypes in mice and chicken (Blondel et al. 2010; Pezoa et al. 2013; Pezoa et al. 2014). Thus, our results suggest that T6SS_SPI-19 contributes more to systemic colonization than T6SS_SPI-6. Further investigation is needed to elucidate this issue.

Notably, the different sets of effector proteins encoded in T6SS_SPI-6 and T6SS_SPI-19 gene clusters may play similar roles during host colonization by Salmonella, despite differences in resident microbiota and the immune system of mammals and birds. In this context, previous studies have characterized effector proteins with peptidoglycan hydrolase activity (Tae4, Tlde1) (Benz et al. 2013; Zhang et al. 2013; Sana et al. 2016; Sibinelli-Sousa et al. 2020; Lorente Cobo et al. 2022) and an Rhs-main toxin (Koskiniemi et al. 2014; Jurėnas et al. 2022) encoded within the T6SS_SPI-6 gene cluster that can explain the contribution of this system to animal colonization. In addition, a recent comparative genomics analysis also revealed that the Salmonella Gallinarum T6SS_SPI-19 gene cluster encodes two copies SG1045 and SG1048 of an effector gene homologous to SED_RS06335 of Salmonella Dublin (Amaya et al. 2022). SED_RS06335 is the only predicted Salmonella T6SS_SPI-19 effector protein with putative peptidoglycan hydrolase activity whose contribution to interbacterial competition has been experimentally demonstrated (Amaya et al. 2022). Thus, the effect of the T6SS_SPI-19 during Salmonella Typhimurium host colonization could be partly mediated by the antibacterial activity of these putative effectors SG1045 and SG1048 against members of the intestinal microbiota. Altogether, the presence of effector proteins in both T6SS gene clusters with peptidoglycan as a common bacterial target site strongly suggests that T6SS_SPI-6 and T6SS_SPI-19 can be functionally interchangeable during host colonization. Besides, since a role for T6SS_SPI-19 in Salmonella Gallinarum interaction with immune cells has been described (Blondel et al. 2013), we cannot rule out the contribution of unidentified T6SS_SPI-19 effectors associated with this interaction during host colonization.

Altogether, we have shown that both T6SS_SPI-6 and T6SS_SPI-19 are functionally interchangeable during mice colonization, suggesting that both T6SS contribute similarly to host colonization despite having different repertoires of effector proteins. Importantly, our study highlights the utility of the VEX-Capture technique (Wilson et al. 2004) in studying the contribution of specialized protein secretion systems encoded within discrete gene clusters to bacterial pathogenesis. The main limitation of the current study is the need for more information on the contribution of specific effectors to the observed phenotypes and the molecular mechanisms involved. Further research is required to address this limitation.