| 1. |
- Lee, Danna, et al.
(author)
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Internal expression of Rhs toxins allows protection against Rhs toxin delivery by stabilizing the cognate Rhs antitoxins
- 2022
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Other publication (other academic/artistic)abstract
- Rhs elements are some of the most positively selected genes known and were recently found to function as type II TA-systems in addition to their previous role as delivered toxins. Antitoxins of type II TA systems are often inherently unstable proteins. Here we discover that the RhsI immunity proteins in Salmonella enterica serovar Typhimurium are also unstable on their own, but stabilized in the presence of their cognate toxins. This raises interesting questions regarding how such unstable immunity proteins can protect against incoming toxins delivered by neighboring attackers. In this study, we observed that one Rhs immunity protein can protect against more than one toxin. Therefore, the internal expression of RhsCT-I could be important for protection against delivered Rhs toxins in addition to regulating Salmonellagrowth in macrophages. The internal transcripts of rhsCT-I are upregulated in InSPI-2 and within macrophages by the combined action of RpoS, PhoP/Q and H-NS. These are the same regulators that control the expression of the type 6 secretion system known to deliver Rhs effectors in Salmonella. This suggests that expression of rhsCT-I is elevated under conditions that favor delivery, which further supports a role for the internal expression of Rhs proteins in protection against toxin delivery.
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| 2. |
- Stårsta, Magnus, 1991-, et al.
(author)
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Contact-dependent growth inhibition modules contain intracellularly expressed selfish genetic elements
- 2022
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Other publication (other academic/artistic)abstract
- Contact-Dependent growth Inhibition (CDI) confers a fitness advantage forbacteria with the system, by allowing delivery of toxins into cells lacking thecognate immunity protein, resulting in growth arrest. Delivery frequentlyoccurs also between siblings, which could explain how these genes aremaintained within the CDI+ population even in the absence of sensitivecompetitors. Bacteria with CDI systems often contain additional so called“orphan” CDI modules. These modules consist of a toxin not attached tooany delivery mechanism and a cognate immunity protein. How selection forsuch orphan CDI genes or for the normal CDI loci under conditionsprohibiting delivery are upheld, is not known. In this study we find thatfunctional CdiA toxin and immunity is produced from internal promoterswithin the CDI toxin gene, cdiA-CT. This expression protects cells frominhibition by delivered CDI toxins, suggesting that orphan modules could beselected to protect against competing strains with different toxin arsenal.Simultaneously, the intracellular presence of these toxins conferred a smallbut significant reduction in growth rate under nutrient limited conditions,suggesting a role for the toxin-immunity modules in growth regulation. Inaddition, loss of the genetic material was associated with a fitness cost,suggesting that selection for these genes is likely upheld by themfunctioning as selfish genetic elements in the absence of toxin delivery.
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| 3. |
- Stårsta, Magnus, 1991-, et al.
(author)
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Global regulators H-NS, RpoS and PhoP/Q regulate expression of Rhs Toxin-Antitoxin system
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Other publication (other academic/artistic)abstract
- Rhs elements are some of the most positively selected genes known. We recently discovered that the two rhs elements in Salmonella enterica serovar Typhimurium encode functional type-II TA-systems that are expressed and regulate bacterial growth during macrophage infections. However, whether these proteins have an effect on the bacterial cell when not inside the macrophage is not known. Here we investigate how expression of the rhs toxins in Salmonella enterica serovar Typhimurium is regulated and whether they can regulate growth also in laboratory media. We find that, even though expression of both rhs modules is higher in macrophages, Salmonella Typhimurium lacking both rhs toxins only grows faster compared to wildtype in LB media but not in macrophage mimicking media (InSpi-2). Thus, expression levels of the TA modules do not correlate with their effects on growth. The internal transcripts of rhsCT-I are upregulated in InSPI-2 by the combined action of RpoS, PhoP/Q and H-NS. These are the same conditions and regulators that enable expression of the type 6 secretion system, known to deliver Rhs effectors. In addition, we find that the RhsI immunity proteins are highly unstable on their own, but stabilized in the presence of their cognate toxin, suggesting that internal expression of the RhsCT-I could be important for protection against delivered Rhs toxin.
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| 4. |
- Stårsta, Magnus, et al.
(author)
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RHS-elements function as type II toxin-antitoxin modules that regulate intra-macrophage replication of Salmonella Typhimurium
- 2020
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In: PLOS Genetics. - : PUBLIC LIBRARY SCIENCE. - 1553-7390 .- 1553-7404. ; 16:2
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Journal article (peer-reviewed)abstract
- RHS elements are components of conserved toxin-delivery systems, wide-spread within the bacterial kingdom and some of the most positively selected genes known. However, very little is known about how Rhs toxins affect bacterial biology. Salmonella Typhimurium contains a full-length rhs gene and an adjacent orphan rhs gene, which lacks the conserved delivery part of the Rhs protein. Here we show that, in addition to the conventional delivery, Rhs toxin-antitoxin pairs encode for functional type-II toxin-antitoxin (TA) loci that regulate S. Typhimurium proliferation within macrophages. Mutant S. Typhimurium cells lacking both Rhs toxins proliferate 2-times better within macrophages, mainly because of an increased growth rate. Thus, in addition to providing strong positive selection for the rhs loci under conditions when there is little or no toxin delivery, internal expression of the toxin-antitoxin system regulates growth in the stressful environment found inside macrophages.
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| 5. |
- Stårsta, Magnus
(author)
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The effects of internally expressed Contact-Dependent growth Inhibition (CDI) toxins in bacteria
- 2022
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Doctoral thesis (other academic/artistic)abstract
- Bacteria, both pathogenic and non-pathogenic, have developed multiple forms of competition mechanisms to combat each other including, but not limited to, Contact-Dependent growth Inhibition (CDI) systems, Type VI Secretion Systems and the associated Rearrangement hotspot (Rhs) toxin system. These systems usually confers a great fitness advantage as they allow for precise delivery of toxic molecules into competing bacteria whilst sister cells are protected from auto-inhibition by producing a cognate immunity protein. Delivery between sister cells may serve as a form of “self-recognition” whilst maintaining selection pressure for these genes within the population. How these genes are maintained in conditions where delivery does not occur has until now not been fully understood.This thesis describes secondary functions, maintained selection pressure and regulation of Rhs and CDI systems in three parts. In Paper I, we made a novel discovery that rhs toxin and immunity genes from Salmonella enterica serovar Typhimurium are transcribed from internal transcriptional start sites independent of the full length delivery gene. This results in functional cytosolic proteins capable of regulating proliferation and growth rate of S. Typhimurium during infection of RAW264.7 cells. In Paper II, we continued our work from Paper I and studied growth effects in vitro as well as regulation of the internal expression. Our findings show that Rhs causes a small fitness cost also in rich laboratory medium and is regulated by alternate sigma factor RpoS, two-component system PhoP/Q and DNA binding protein H-NS. In Paper III, we made a discovery similar to our findings in Paper I and II by observing internally transcribed toxin and immunity genes of multiple CDI systems from E. coli regulated by RpoS. We propose that CDI toxin-immunity pairs function as selfish genetic elements that maintain gene selection whilst simultaneously retaining the ability to protect the cell from externally delivered toxins.
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