Conserved Bacterial Protein Secretion System

Assignment Brief

Instructions. Answer each of the 15 questions.

Pukatzki et al. 2006 PNAS. Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system https://www.pnas.org/content/103/5/1528

1. In two or fewer sentences, what was the main objective of this study?

2. The authors use an unusual model system to measure bacterial virulence. Briefly describe this system? Name two other model systems that they could they have used and give a reason for each why the authors might have chosen not to use them.

3. The authors perform a mutagenesis screen. What is the wild type phenotype? What is the desired mutant phenotype?

4. From their screen, the authors identified several mutants in a particular gene cluster that resulted in reduced virulence. How did the authors go from a list of genes to a hypothesis about their function?

5. In Fig. 3a and 3b, the authors are measuring protein secretion. How is protein secretion being measured in 3A? In 3B? What is "bla" and why is it included in the figure?

6. In Fig. 4, what is being measured? Why do you think the authors included this experiment (i.e. what does it demonstrate that had not been demonstrated in prior figures)? Mougous et al. 2007 Nat Cell Biol. Threonine phosphorylation post-translationally regulates protein secretion in Pseudomonas aeruginosa https://www.nature.com/articles/ncb1605

7. In one sentence, what is the main "unknown" regarding the type 6 secretion system that the authors are seeking to address in this study?

8. Why do the authors use a ∆retS mutant for most of their experiments? Give two reasons why using such a mutant is problematic.

9. What is a kinase? What is a phosphatase? What are two assays that the authors use (Fig. 2) to measure the activity of the kinase/phosphatase? What is the effect of the kinase/phosphatase activity on the T6SS?

10. In Fig. 3, Fluorescence microscopy is used to assay for T6SS activity. What is being labeled? What does this labelling tell you about type 6 secretion system activity (i.e. what is the result of Fig. 3)? Basler et al. 2013 Cell. Tit-for-Tat: Type VI Secretion System Counterattack during Bacterial Cell-Cell Interactions. You are encouraged to watch the "PaperFlick" associated with paper available on the publisher`s website (it`s open-access): https://www.cell.com/cell/fulltext/S0092-8674(13)00134-7

11. In two sentences, what is the primary objective of this study?

12. In Figure 1, fluorescent spots are visible in cells. What are these spots used to indicate? Is there a difference in interpretation of what these spots mean compared to the similar fluorescent spots in Mougous et al. (2007)?

13. In Figure 3, there are three mutants being shown. What are the three mutants? What is the behavior of the T6SS in each of the three mutants?

14. Recall that Mougous et al. (2007) observed the effect of a ∆pppA deletion on T6SS activity. In this study (Basler et al.) what is the effect of a ∆pppA deletion on T6SS activity? How do the authors of this study reconcile increased T6SS-dependent secretion with reduced effect of T6SS activity (i.e. reduced cell-cell killing).

15. Why might it be important for bacteria to avoid indiscriminately killing all surrounding bacteria (i.e. why might Pseudomonas want to have this advanced regulatory system for its T6SS)? Give at least 2 reasons.

Sample Answer

Pukatzki et al. 2006 (PNAS)

• 1. Main objective (≤2 sentences):

  • The study aimed to identify and characterise a new bacterial protein secretion system in Vibrio cholerae. The authors used a non-traditional host model to link this secretion system to virulence.

• 2. Model system and alternatives:

  • They used Dictyostelium discoideum amoebae as a host because it phagocytoses bacteria in a way similar to macrophages. Alternatives could include a mouse model (more physiologically relevant but costly and ethically complex) or Caenorhabditis elegans (cheap and tractable but less representative of human innate immunity).

• 3. Wild type vs mutant phenotype:

  • Wild type V. cholerae killed Dictyostelium. The desired mutant phenotype was reduced or no killing, suggesting loss of virulence.

• 4. From genes to hypothesis:

  • After identifying mutations in a gene cluster, the authors compared gene sequences with known proteins, predicted functions bioinformatically, and hypothesised that the cluster encoded a secretion system.

• 5. Measuring protein secretion (Fig. 3A and 3B):

  • In Fig. 3A, Western blotting of culture supernatant for Hcp was used. In Fig. 3B, they fused a signal to bla (β-lactamase), which reports secretion by conferring ampicillin resistance. Bla was used as a reporter because its activity outside the cell is easy to measure.

• 6. Fig. 4 experiment:

  • They tested secretion in a heterologous E. coli system. This experiment demonstrated that the identified genes alone were sufficient to reconstitute secretion activity, strengthening the claim that the cluster encoded a novel secretion pathway.

Mougous et al. 2007 (Nature Cell Biology)

• 7. Main unknown (1 sentence):

  • The authors wanted to know how Type VI secretion system (T6SS) activity is regulated post-translationally.

• 8. Why use ∆retS mutant, and problems:

  • They used ∆retS because it upregulates T6SS, making detection easier. However, it is problematic because ∆retS alters many other pathways and does not represent wild-type physiology.

• 9. Kinase and phosphatase, assays, effects:

  • PpkA is a kinase (adds phosphate), PppA is a phosphatase (removes phosphate). They used in vitro phosphorylation assays and phospho-specific immunoblots. Phosphorylation by PpkA activates T6SS, while PppA reverses this activity.

• 10. Fig. 3 microscopy:

  • They labelled ClpV-GFP, which localises to sites of T6SS sheath disassembly. The presence and frequency of these fluorescent foci indicated T6SS firing events.

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