<abstract xmlns="http://www.w3.org/1999/xhtml">

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

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.

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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 (<a ref-type="bibr" href="#j_pjm-2023-017_ref_029">Zoued et al. 2014</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_009">Coulthurst 2019</a>). The extensive repertoire of effector activities makes the T6SS an efficient apparatus targeting prokaryotic and/or eukaryotic cells (<a ref-type="bibr" href="#j_pjm-2023-017_ref_011">Hernandez et al. 2020</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_017">Monjarás Feria and Valvano 2020</a>), emerging as an environmental adaptation and pathogenesis factor for several bacteria (<a ref-type="bibr" href="#j_pjm-2023-017_ref_023">Records 2011</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_003">Basler 2015</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_008">Cianfanelli et al. 2016</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_019">Navarro-Garcia et al. 2019</a>).
In the <italic>Salmonella</italic> genus, five T6SS gene clusters have been described (<a ref-type="bibr" href="#j_pjm-2023-017_ref_005">Blondel et al. 2009</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_010">Fookes et al. 2011</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_002">Bao et al. 2019</a>) that belong to four distinct phylogenetic lineages (<a ref-type="bibr" href="#j_pjm-2023-017_ref_002">Bao et al. 2019</a>). In addition, these gene clusters are differentially distributed among representatives of <italic>Salmonella</italic> (<a ref-type="bibr" href="#j_pjm-2023-017_ref_005">Blondel et al. 2009</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_010">Fookes et al. 2011</a>).
The T6SS encoded in pathogenicity island SPI-6 (T6SSSPI-6) in <italic>Salmonella</italic> Typhimurium (<a ref-type="fig" href="#j_pjm-2023-017_fig_001">Fig. 1A</a>) (a serotype that causes systemic infection in mice) is needed for intracellular survival within avian and murine immune cells (<a ref-type="bibr" href="#j_pjm-2023-017_ref_020">Parsons and Heffron 2005</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_013">Klumpp and Fuchs 2007</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_018">Mulder et al. 2012</a>) and contributes to intestinal and systemic colonization of gavage-inoculated chickens (<a ref-type="bibr" href="#j_pjm-2023-017_ref_022">Pezoa et al. 2013</a>) and mice (<a ref-type="bibr" href="#j_pjm-2023-017_ref_018">Mulder et al. 2012</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_015">Liu et al. 2013</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_024">Sana et al. 2016</a>). On the other hand, current evidence indicates that the T6SS encoded in pathogenicity island SPI-19 (T6SSSPI-19) (<a ref-type="fig" href="#j_pjm-2023-017_fig_001">Fig. 1A</a>) plays a role in gastrointestinal colonization by <italic>Salmonella enterica</italic> serotypes adapted to avian hosts, such as <italic>Salmonella</italic> Gallinarum (<a ref-type="bibr" href="#j_pjm-2023-017_ref_006">Blondel et al. 2010</a>) and <italic>Salmonella</italic> Pullorum (<a ref-type="bibr" href="#j_pjm-2023-017_ref_027">Xian et al. 2020</a>). Of note, the transfer of <italic>Salmonella</italic> Gallinarum T6SSSPI-19 to a ΔT6SSSPI-6 mutant of <italic>Salmonella</italic> Typhimurium complements its gastrointestinal colonization defect in chickens (<a ref-type="bibr" href="#j_pjm-2023-017_ref_022">Pezoa et al. 2013</a>). Since these experiments were performed in chickens, whether T6SSSPI-19 has a similar role in other hosts, such as mice, remains unknown.
<figure id="j_pjm-2023-017_fig_001" position="float" fig-type="figure">
<h2>Fig. 1.</h2>
<figCaption><italic>In vivo</italic> competition between wild-type <italic>Salmonella</italic> Typhimurium and a ΔT6SSSPI-6 mutant complemented <italic>in trans</italic> with R995, R995 + SPI6 or R995 + SPI-19.
A) Genetic organization of <italic>Salmonella</italic> Typhimurium T6SSSPI-6 and <italic>Salmonella</italic> Gallinarum T6SSSPI-19 gene clusters. Genes encoding core components of each T6SS are shown in yellow, and the effector and immunity protein-encoding genes are shown in red and blue, respectively. B) For competition assays, groups of six- to eight-week-old female BALB/c mice were orally infected with 106 CFU of a 1 : 1 mixture of WT/R995 with ΔT6SSSPI-6/R995 (blue), ΔT6SSSPI-6/R995+SPI-6 (red) or ΔT6SSSPI-6/R995+SPI-19 (green). After four days of infection, mice were euthanized, and the cecum, liver and spleen were aseptically removed and homogenized in sterile PBS. The bacterial load recovered from each organ was determined by plating serial tenfold dilutions on LB agar plates with appropriate antibiotics. Data show the mutant to wild-type CFU ratio normalized to the inoculum and expressed as log10. Bars represent mean values ± standard error. Statistical significance was determined using a one-way ANOVA test followed by Tukey’s multiple comparisons tests (*<italic>p</italic> &lt; 0.05; ****<italic>p</italic> &lt; 0.0001; ns – not significant).</figCaption>
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In the present work, we evaluated the contribution of T6SSSPI-19 to gastrointestinal colonization and systemic spread in mice using a mutant strain of <italic>Salmonella</italic> Typhimurium lacking the T6SSSPI-6 and harboring the <italic>Salmonella</italic> Gallinarum T6SSSPI-19 cloned in the R995 plasmid via the VEX-Capture technique (<a ref-type="bibr" href="#j_pjm-2023-017_ref_026">Wilson et al. 2004</a>). <a ref-type="table" href="#j_pjm-2023-017_tab_001">Table I</a> shows the bacterial strains and plasmids used in this work. The construction of <italic>Salmonella</italic> Typhimurium Δ<italic>phoN</italic> and ΔT6SSSPI-6 mutant strains has been reported (<a ref-type="bibr" href="#j_pjm-2023-017_ref_022">Pezoa et al. 2013</a>). The generation of R995 plasmid derivatives harboring gene clusters T6SSSPI-6 (39 kb) of <italic>Salmonella</italic> Typhimurium 14028s and T6SSSPI-19 (42 kb) of <italic>Salmonella</italic> Gallinarum 287/91 has been reported (<a ref-type="bibr" href="#j_pjm-2023-017_ref_022">Pezoa et al. 2013</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_006">Blondel et al. 2010</a>).
<table-wrap id="j_pjm-2023-017_tab_001" position="float">
<label>Table I</label>
<caption>Bacterial strains and plasmids used in this study.</caption>
<table frame="hsides" rules="groups">
<thead>
<tr>
<th valign="top" align="center">Strain or plasmid</th>
<th valign="top" align="center">Features</th>
<th valign="top" align="center">Source or reference</th>
</tr>
</thead>
<tbody>
<tr>
<td valign="top" align="center" colspan="3"><italic>Salmonella</italic> Typhimurium</td>
</tr>
<tr>
<td valign="top" align="left">14028s</td>
<td valign="top" align="left">Wild-type, virulent strain</td>
<td valign="top" align="center">Laboratory collection</td>
</tr>
<tr>
<td valign="top" align="left">ΔT6SSSPI-6</td>
<td valign="top" align="left">14028s Δ(<italic>STM14_0312-STM14_0351</italic>)::Cam</td>
<td valign="top" align="center"><a ref-type="bibr" href="#j_pjm-2023-017_ref_022">Pezoa et al. 2013</a></td>
</tr>
<tr>
<td valign="top" align="left">Δ<italic>phoN</italic></td>
<td valign="top" align="left">14028s Δ(<italic>STM14_5193</italic>)::Kan</td>
<td valign="top" align="center"><a ref-type="bibr" href="#j_pjm-2023-017_ref_022">Pezoa et al. 2013</a></td>
</tr>
<tr>
<td valign="top" align="left">Δ<italic>phoN</italic>/R995</td>
<td valign="top" align="left">14028s Δ(<italic>STM14_5193</italic>)::Kan harboring plasmid R995</td>
<td valign="top" align="center"><a ref-type="bibr" href="#j_pjm-2023-017_ref_022">Pezoa et al. 2013</a></td>
</tr>
<tr>
<td valign="top" align="left">ΔT6SSSPI-6/R995</td>
<td valign="top" align="left">14028s Δ(<italic>STM14_0312-STM14_0351</italic>)::Cam harboring plasmid R995</td>
<td valign="top" align="center"><a ref-type="bibr" href="#j_pjm-2023-017_ref_022">Pezoa et al. 2013</a></td>
</tr>
<tr>
<td valign="top" align="left">ΔT6SSSpI-6/R995+SPI-6</td>
<td valign="top" align="left">14028s Δ(<italic>STM14_0312-STM14_0351</italic>)::Cam harboring plasmid R995+SPI-6</td>
<td valign="top" align="center"><a ref-type="bibr" href="#j_pjm-2023-017_ref_022">Pezoa et al. 2013</a></td>
</tr>
<tr>
<td valign="top" align="left">ΔT6SSSpI-6/R995+SPI-19</td>
<td valign="top" align="left">14028s Δ(<italic>STM14_0312-STM14_0351</italic>)::Cam harboring plasmid R995+SPI-19</td>
<td valign="top" align="center"><a ref-type="bibr" href="#j_pjm-2023-017_ref_022">Pezoa et al. 2013</a></td>
</tr>
<tr>
<td valign="top" align="center" colspan="3">Plasmids</td>
</tr>
<tr>
<td valign="top" align="left">R995</td>
<td valign="top" align="left">Self-transmissible, broad-host range IncP plasmid</td>
<td valign="top" align="center"><a ref-type="bibr" href="#j_pjm-2023-017_ref_026">Wilson et al. 2004</a></td>
</tr>
<tr>
<td valign="top" align="left">R995+SPI-6</td>
<td valign="top" align="left">T6SSSPI-6 gene cluster (<italic>STM14_0312-STM14_0351</italic>) from <italic>Salmonella</italic> Typhimurium 14028s cloned in plasmid R995</td>
<td valign="top" align="center"><a ref-type="bibr" href="#j_pjm-2023-017_ref_022">Pezoa et al. 2013</a></td>
</tr>
<tr>
<td valign="top" align="left">R995+SPI-19</td>
<td valign="top" align="left">T6SSSPI-19 gene cluster (<italic>SG1021-SG1056</italic>) from <italic>Salmonella</italic> Gallinarum 287/91 cloned in plasmid R995</td>
<td valign="top" align="center"><a ref-type="bibr" href="#j_pjm-2023-017_ref_006">Blondel et al. 2010</a></td>
</tr>
</tbody>
</table>
</table-wrap>
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 <italic>Salmonella</italic> Typhimurium ΔT6SSSPI-6 strain has a defect in mice colonization, as previously reported (<a ref-type="bibr" href="#j_pjm-2023-017_ref_018">Mulder et al. 2012</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_015">Liu et al. 2013</a>). The ΔT6SSSPI-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 (<a ref-type="fig" href="#j_pjm-2023-017_fig_001">Fig. 1B</a>). The colonization defect was reversed when the mutant strain was transformed with derivatives of plasmid R995 carrying either the T6SSSPI-6 or the T6SSSPI-19 gene cluster from <italic>Salmonella</italic> Typhimurium and <italic>Salmonella</italic> Gallinarum, respectively (<a ref-type="fig" href="#j_pjm-2023-017_fig_001">Fig. 1B</a>), indicating that both T6SSSPI-6 and T6SSSPI-19 are functionally interchangeable during mice colonization. Furthermore, these results, along with those reported in the chicken model (<a ref-type="bibr" href="#j_pjm-2023-017_ref_022">Pezoa et al. 2013</a>) suggest that both T6SSSPI-6 and T6SSSPI-19 are fully functional in distinct <italic>Salmonella</italic> serotypes and contribute to the colonization of different animal hosts. It is worth mentioning that the ΔT6SSSPI-6 mutant strain complemented with the T6SSSPI-19 gene cluster of <italic>Salmonella</italic> Gallinarum colonized the spleen of infected mice more efficiently than the mutant complemented with its own T6SSSPI-6 gene cluster (<a ref-type="fig" href="#j_pjm-2023-017_fig_001">Fig. 1B</a>). 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 T6SSSPI-6 and T6SSSPI-19 contribute to the systemic colonization of different <italic>Salmonella</italic> serotypes in mice and chicken (<a ref-type="bibr" href="#j_pjm-2023-017_ref_006">Blondel et al. 2010</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_022">Pezoa et al. 2013</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_021">Pezoa et al. 2014</a>). Thus, our results suggest that T6SSSPI-19 contributes more to systemic colonization than T6SSSPI-6. Further investigation is needed to elucidate this issue.
Notably, the different sets of effector proteins encoded in T6SSSPI-6 and T6SSSPI-19 gene clusters may play similar roles during host colonization by <italic>Salmonella</italic>, 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) (<a ref-type="bibr" href="#j_pjm-2023-017_ref_004">Benz et al. 2013</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_028">Zhang et al. 2013</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_024">Sana et al. 2016</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_025">Sibinelli-Sousa et al. 2020</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_016">Lorente Cobo et al. 2022</a>) and an Rhs-main toxin (<a ref-type="bibr" href="#j_pjm-2023-017_ref_014">Koskiniemi et al. 2014</a>; <a ref-type="bibr" href="#j_pjm-2023-017_ref_012">Jurėnas et al. 2022</a>) encoded within the T6SSSPI-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 <italic>Salmonella</italic> Gallinarum T6SSSPI-19 gene cluster encodes two copies SG1045 and SG1048 of an effector gene homologous to SED_RS06335 of <italic>Salmonella</italic> Dublin (<a ref-type="bibr" href="#j_pjm-2023-017_ref_001">Amaya et al. 2022</a>). SED_RS06335 is the only predicted <italic>Salmonella</italic> T6SSSPI-19 effector protein with putative peptidoglycan hydrolase activity whose contribution to interbacterial competition has been experimentally demonstrated (<a ref-type="bibr" href="#j_pjm-2023-017_ref_001">Amaya et al. 2022</a>). Thus, the effect of the T6SSSPI-19 during <italic>Salmonella</italic> 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 T6SSSPI-6 and T6SSSPI-19 can be functionally interchangeable during host colonization. Besides, since a role for T6SSSPI-19 in <italic>Salmonella</italic> Gallinarum interaction with immune cells has been described (<a ref-type="bibr" href="#j_pjm-2023-017_ref_007">Blondel et al. 2013</a>), we cannot rule out the contribution of unidentified T6SSSPI-19 effectors associated with this interaction during host colonization.
Altogether, we have shown that both T6SSSPI-6 and T6SSSPI-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 (<a ref-type="bibr" href="#j_pjm-2023-017_ref_026">Wilson et al. 2004</a>) 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.
</div>

Transfer of T6SSSPI-19 from <italic>Salmonella</italic> Gallinarum to <italic>Salmonella</italic> Typhimurium Lacking T6SSSPI-6 Complements its Colonization Defect in Mice

Polish Journal of Microbiology

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

Transfer of T6SS from  Gallinarum to  Typhimurium Lacking T6SS 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...

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

Article Category: SHORT COMMUNICATION

Published Online: Jun 14, 2023

Page range: 215 - 219

Received: Jan 23, 2023

Accepted: Mar 27, 2023

DOI: https://doi.org/10.33073/pjm-2023-017

Keywords
S Typhimurium, T6SS, SPI-19, mice, colonization

© 2023 David Pezoa et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

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

Article Category: SHORT COMMUNICATION

Published Online: Jun 14, 2023

Page range: 215 - 219

Received: Jan 23, 2023

Accepted: Mar 27, 2023

DOI: https://doi.org/10.33073/pjm-2023-017

KeywordsS Typhimurium, T6SS, SPI-19, mice, colonization

© 2023 David Pezoa et al., published by Sciendo

This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.

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

Keywords
S Typhimurium, T6SS, SPI-19, mice, colonization