A Tad-like apparatus is required for contact-dependent prey killing in predatory social bacteria
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Evaluation Summary:
In this manuscript, the authors explore mechanisms involved in predation of other bacteria by Myxococcus xanthus. They identify two gene clusters, which encode proteins with homology to proteins of the Tad pilus system and some of which are important for predation. The work represents a good starting point for understanding how Myxococcus cells may engage in contact-dependent killing of other bacteria.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Abstract
Myxococcus xanthus , a soil bacterium, predates collectively using motility to invade prey colonies. Prey lysis is mostly thought to rely on secreted factors, cocktails of antibiotics and enzymes, and direct contact with Myxococcus cells. In this study, we show that on surfaces the coupling of A-motility and contact-dependent killing is the central predatory mechanism driving effective prey colony invasion and consumption. At the molecular level, contact-dependent killing involves a newly discovered type IV filament-like machinery (Kil) that both promotes motility arrest and prey cell plasmolysis. In this process, Kil proteins assemble at the predator-prey contact site, suggesting that they allow tight contact with prey cells for their intoxication. Kil-like systems form a new class of Tad-like machineries in predatory bacteria, suggesting a conserved function in predator-prey interactions. This study further reveals a novel cell-cell interaction function for bacterial pili-like assemblages.
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Author Response
Reviewer #1 (Public Review):
In this manuscript, the authors explore mechanisms involved in the predation of other bacteria by Myxococcus xanthus. The major findings are (1) M. xanthus cells depend on gliding motility to efficiently invade an E. coli prey colony. (2) E. coli prey cells are lysed in a contact-dependent manner. (3) When M. xanthus cells make prey contact, they sometimes pause and then kill the prey cell. (4) Using a genetic screen, two gene clusters (referred to as the kil gene clusters) are identified that encode proteins, some of which have homology to those of Tad pili. Some of the Kil proteins are important for pausing of cells and killing of prey. (5) One of the suggested Kil proteins assemble to form clusters upon prey contact; however, assembly of these clusters is independent of other Kil …
Author Response
Reviewer #1 (Public Review):
In this manuscript, the authors explore mechanisms involved in the predation of other bacteria by Myxococcus xanthus. The major findings are (1) M. xanthus cells depend on gliding motility to efficiently invade an E. coli prey colony. (2) E. coli prey cells are lysed in a contact-dependent manner. (3) When M. xanthus cells make prey contact, they sometimes pause and then kill the prey cell. (4) Using a genetic screen, two gene clusters (referred to as the kil gene clusters) are identified that encode proteins, some of which have homology to those of Tad pili. Some of the Kil proteins are important for pausing of cells and killing of prey. (5) One of the suggested Kil proteins assemble to form clusters upon prey contact; however, assembly of these clusters is independent of other Kil proteins On the basis of these findings the authors suggest that the Kil proteins assemble to form a Tad pilus system and are important for pausing and prey killing. Overall, this is an interesting manuscript; however, it remains unclear what the actual function of the identified Kil proteins are.
The reviewer raised an important point because it is correct that the previous data did not formally establish that a Tad-like machinery is recruited at the prey contact site. Addressing this point was challenging because it required to either demonstrate direct interactions between KilD and Tad structural components or show that predicted Tad core components also localize dynamically upon contact with the prey. This later possibility nevertheless required to obtain functional fluorescent protein fusions, which are typically difficult to obtain for membrane proteins. Below we describe which strategies we chose to address the reviewer’s comments.
Weaknesses include
(1) The lack of genetic complementation experiments. Thus, it isunclear precisely which of the Kil proteins are important for predation.
This question is especially relevant for the cluster 2 genes, given that its functional association with the cluster 1 genes were only provided genetically in the previous version. In this cluster, we have chosen to only delete the genes annotated as Tad-like proteins, namely the two IM platform proteins CpaA and CpaG, the outer membrane accessory protein CpaB and three predicted pilin homologs. We did not attempt complementing the pili deletions given that they all show at best intermediate phenotypes when individually deleted and that a triple deletion is needed to obtain a kil-null phenotype. This strongly argues against polar effects in the pilin deletion mutants. However, we agree that it was important to show that the mutation in the Cpa homologs were not polar, demonstrating the critical function of these genes. In this version for coherence, we chose to complement core Tad components encoded in cluster 1 and 2, the secretin (cluster 1), the ATPase (cluster 1) and the two IM platform proteins (cluster 2). These complementations are now provided as proof that these genes are all essential for predation in a new Figure 5a and S4b.
(2) The Kil proteins are encoded in two gene clusters. The evidencethat these proteins make up a Tad pilus system is based on homology and that mutations in both clusters result in reduced predation. No evidence is presented that proteins encoded by these two clusters interact to form a Tad pilus machine.
(3) The authors localize the Kil system using an NG-KilD fusion;however, there is no evidence that KilD, which is a FHA domaincontaining protein, associates with the Tad pilus machinery. In fact, KilD makes clusters independently of all other Kil proteins tested suggesting that these clusters may not report on Kil assembly and activity. An equally plausible scenario is that Myxococcus/E. coli contacts result in activation of KilD leading to the formation of foci. These foci then signal assembly of the Kil system somewhere in a cell (or maybe not). Therefore, it is not clear where and if this machinery localizes during prey contact.
These two points are related so we answer them jointly.
Showing direct interactions between Tad proteins is challenging and in fact, there is currently very little interaction data for these machineries, contrarily to Type-IV pili and Type-2 secretion systems.
For this reason, we chose a localization approach reasoning that localization of Tad core components in contact with E. coli would show that the system is assembled at the prey contact site. We now present data showing that both KilF (The ATPase encoded by cluster 1) and KilG (the CpaG homolog) both form clusters at the prey contact site similar to KilD. Since these proteins are predicted to form complementary parts of the Tad machinery and are encoded by cluster 1 and 2, we believe that these results demonstrate dynamic assembly of the machinery at the prey contact site.
(4) I did not find a description of how the mutagenesis was done.Please include a description of how the mutagenesis was done, how many mutants were screened, and in which loci the mutations (transposon insertions?) occurred. Was the screen saturated?
We did not perform an extended genetic screen to find the kil genes. We tested a number of selected mutants in predicted membrane complexes, including all A-motility genes, possible orthologs of the Caulobacter Cdcz system, a possible CDI system, T6SS genes and decarboxylase genes and the kil cluster 1 and 2 genes. Mutations in the kil cluster 1 and 2 genes were the only ones to show a killing defect so we followed up. We do not mention all the tested genes, given that they were not investigated in depth and rapidly discarded as negative candidates.
We nevertheless clarified the text to avoid any confusion.
(5) Throughout the manuscript, the authors need to tone down theirconclusions and stick to what they actually show. It is also important that the authors present their results in the context of what is already known about contact-dependent killing in M. xanthus.
We believe that this comment was mostly an objection to our inference that our data previously showed assembly of the Tad pilus at the contact site. The new data strongly reinforces this view. We nevertheless carefully rewrote the manuscript making sure that the conclusions are indeed in line with the data.
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Evaluation Summary:
In this manuscript, the authors explore mechanisms involved in predation of other bacteria by Myxococcus xanthus. They identify two gene clusters, which encode proteins with homology to proteins of the Tad pilus system and some of which are important for predation. The work represents a good starting point for understanding how Myxococcus cells may engage in contact-dependent killing of other bacteria.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their names with the authors.)
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Reviewer #1 (Public Review):
In this manuscript, the authors explore mechanisms involved in the predation of other bacteria by Myxococcus xanthus. The major findings are (1) M. xanthus cells depend on gliding motility to efficiently invade an E. coli prey colony. (2) E. coli prey cells are lysed in a contact-dependent manner. (3) When M. xanthus cells make prey contact, they sometimes pause and then kill the prey cell. (4) Using a genetic screen, two gene clusters (referred to as the kil gene clusters) are identified that encode proteins, some of which have homology to those of Tad pili. Some of the Kil proteins are important for pausing of cells and killing of prey. (5) One of the suggested Kil proteins assemble to form clusters upon prey contact; however, assembly of these clusters is independent of other Kil proteins On the basis of …
Reviewer #1 (Public Review):
In this manuscript, the authors explore mechanisms involved in the predation of other bacteria by Myxococcus xanthus. The major findings are (1) M. xanthus cells depend on gliding motility to efficiently invade an E. coli prey colony. (2) E. coli prey cells are lysed in a contact-dependent manner. (3) When M. xanthus cells make prey contact, they sometimes pause and then kill the prey cell. (4) Using a genetic screen, two gene clusters (referred to as the kil gene clusters) are identified that encode proteins, some of which have homology to those of Tad pili. Some of the Kil proteins are important for pausing of cells and killing of prey. (5) One of the suggested Kil proteins assemble to form clusters upon prey contact; however, assembly of these clusters is independent of other Kil proteins On the basis of these findings the authors suggest that the Kil proteins assemble to form a Tad pilus system and are important for pausing and prey killing. Overall, this is an interesting manuscript; however, it remains unclear what the actual function of the identified Kil proteins are.
Weaknesses include
(1) The lack of genetic complementation experiments. Thus, it is unclear precisely which of the Kil proteins are important for predation.
(2) The Kil proteins are encoded in two gene clusters. The evidence that these proteins make up a Tad pilus system is based on homology and that mutations in both clusters result in reduced predation. No evidence is presented that proteins encoded by these two clusters interact to form a Tad pilus machine.
(3) The authors localize the Kil system using an NG-KilD fusion; however, there is no evidence that KilD, which is a FHA domain-containing protein, associates with the Tad pilus machinery. In fact, KilD makes clusters independently of all other Kil proteins tested suggesting that these clusters may not report on Kil assembly and activity. An equally plausible scenario is that Myxococcus/E. coli contacts result in activation of KilD leading to the formation of foci. These foci then signal assembly of the Kil system somewhere in a cell (or maybe not). Therefore, it is not clear where and if this machinery localizes during prey contact.
(4) I did not find a description of how the mutagenesis was done. Please include a description of how the mutagenesis was done, how many mutants were screened, and in which loci the mutations (transposon insertions?) occurred. Was the screen saturated?
(5) Throughout the manuscript, the authors need to tone down their conclusions and stick to what they actually show. It is also important that the authors present their results in the context of what is already known about contact-dependent killing in M. xanthus.
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Reviewer #2 (Public Review):
This works has clear novelty and describes aspects on how Myxococcus xanthus can kill other bacterial cells.
It starts by demonstrating that A-motility (Agl-Glt system) is needed to invade adjacent colony. These A-motile cells are able to kill a prey (E. coli) likely by making holes in the peptidoglycan layer, but the killing does not directly involve the A-motility system.Based on this, the authors did use an elegant approach to identify mutants that can invade but cannot kill. Reported hits lie within two gene clusters encoding Tad proteins, which in other bacteria such as Pseudomonas aeruginosa are involved in the assembly of a Tad pilus which promotes bacterial attachment. The cluster 1 encodes the prepilin peptidase, the secretin and the ATPase, while cluster 2 encodes the inner membrane platform, …
Reviewer #2 (Public Review):
This works has clear novelty and describes aspects on how Myxococcus xanthus can kill other bacterial cells.
It starts by demonstrating that A-motility (Agl-Glt system) is needed to invade adjacent colony. These A-motile cells are able to kill a prey (E. coli) likely by making holes in the peptidoglycan layer, but the killing does not directly involve the A-motility system.Based on this, the authors did use an elegant approach to identify mutants that can invade but cannot kill. Reported hits lie within two gene clusters encoding Tad proteins, which in other bacteria such as Pseudomonas aeruginosa are involved in the assembly of a Tad pilus which promotes bacterial attachment. The cluster 1 encodes the prepilin peptidase, the secretin and the ATPase, while cluster 2 encodes the inner membrane platform, major and minor pilins. Both clusters encode additional genes of unknown function. It is then shown that this Tad-like system, called Kil system, can trigger target cell lysis probably via the recruitment of other systems allowing delivery of toxic elements into prey cells. Despite not having data supporting what could contribute to the toxicity, it is shown that the Kil system is actually assembling at the site of contact with the prey cell. Finally, the authors also showed that the Myxococcus Kil system allows killing of a wide range of bacteria which are not necessarily phylogenetically-related.
In conclusion, this work brought novel and original concepts, some of which would definitively deserve further investigation in subsequent studies.
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Reviewer #3 (Public Review):
The authors set out to identify factors involved in bacterial predation and have identified a set of genes that are homologous to secretion systems. The study focuses on M. xanthus as the predator with E. coli as the prey source. The authors demonstrate that M. xanthus A-Motility (focal adhesion mediated motility) is required for efficient penetration of E. coli under the conditions of their assays. The authors identified two genes that encode proteins thought to assemble into a type IV filament-like machine, designated "Kil". This system inhibits motility of prey cells and stimulates lysis of those cells. They show that the Kil apparatus assembles near contact sites between predator and prey cells using protein-fusion constructs. However, there are limitations based on experimental design that diminish …
Reviewer #3 (Public Review):
The authors set out to identify factors involved in bacterial predation and have identified a set of genes that are homologous to secretion systems. The study focuses on M. xanthus as the predator with E. coli as the prey source. The authors demonstrate that M. xanthus A-Motility (focal adhesion mediated motility) is required for efficient penetration of E. coli under the conditions of their assays. The authors identified two genes that encode proteins thought to assemble into a type IV filament-like machine, designated "Kil". This system inhibits motility of prey cells and stimulates lysis of those cells. They show that the Kil apparatus assembles near contact sites between predator and prey cells using protein-fusion constructs. However, there are limitations based on experimental design that diminish support for the stated conclusions. The assay used to identify genes of interest was conducted in aqueous media where the motility system of interest is not required. Futhermore, the microscopic techniques used here do not allow for the visualization of pores or precise localization of machinery thought to be involved in the mechanism for delivery of toxins from predator to prey cells.
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