Posted: November 23rd, 2022

Sectretion systems

Sectretion systems

Bacterial secretion systems (Type I, Type II, Type III, Type IV, Type V and Type VI) are used by Gram-negative bacteria to secrete proteins outside the cell, or in some cases into another bacterial or eukaryotic cell.

Vibrio parahaemolyticus is a Gram-negative bacterium that inhabits warm, brackish coastal waters. It is found as a free-swimming organism in these waters, or attached to sediments, zooplankton, fish, or shellfish. V. parahaemolyticus is a human pathogen and can cause acute gastroenteritis due to the consumption of contaminated raw or undercooked seafood.

In common with many Vibrio species, V. parahaemolyticus strains contain a number of secretion systems that are important for their free-living and pathogenic lifestyles.

You are researching the V. parahaemolyticus strain ATCC 17802. The genome of this strain has been sequenced and is available in IMG (Genome ID = 2721755783). As part of your research you decide to analyse the secretome of this strain: that is, the set of proteins that are expressed by ATCC 17802 and secreted into the extracellular space.

ATCC 17802 was cultured in Luria-Bertani broth (pH 8.5, 3% NaCl) at 37°C to mid-logarithmic phase. The bacterial cells were then removed by centrifuging and the extracellular proteins in the supernatant media were precipitated, separated by 2D PAGE and identified by LC-MS/MS Analysis. Table 1 (below) contains a subset of the identified extracellular proteins.

Table 1. Subset of proteins identified in the secretome of Vibrio parahaemolyticus strain ATCC 17802.

IMG Gene Locus Tag

Protein name

Ga0174928_112082

Asparaginase

Ga0174928_112444

Dihydrolipoyl dehydrogenase

Ga0174928_112183

Flagellar protein FliS

Ga0174928_121489

Flagellar protein FliS

Ga0174928_111901

Haemolysin-type calcium-binding repeat-containing protein

Ga0174928_12327

Putative exonuclease SbcC. Putative effector protein.

Ga0174928_111601

Type III secretion protein C (YscC outer membrane pore)

Ga0174928_111590

Type III secretion effector protein VopS (Vibrio outer protein S)

Ga0174928_111343

Type VI secretion system secreted protein Hcp

Ga0174928_12322

Type VI secretion system secreted protein Hcp

Ga0174928_111344

Type VI secretion system secreted protein VgrG

Ga0174928_12323

Type VI secretion system secreted protein VgrG

Ga0174928_12324

Zn-binding Pro-Ala-Ala-Arg (PAAR) domain-containing protein, incolved in TypeVI secretion

Question 3.1. The haemolysin-type calcium-binding repeat-containing protein encoded by Ga0174928_111901 is a serralysin-like metalloprotease that contains several haemolysin-type calcium-binding repeat motifs (repeats-in-toxin (RTX) motifs) in the carboxy-terminal portion of the protein. Which type of bacterial secretion system is most likely used to secrete this protein? Briefly explain your choice. (2 marks)

Question 3.2.

The Hcp and VgrG proteins identified in the ATCC 17802 secretome are proteins that are exported via the Type 6 secretion system (T6SS). The proteins that make up the T6SS machinery have been characterised in the Cluster of Orthologous Groups (COG) categories.

Table 2 is a list of the required Type VI Secretion System (T6SS) proteins and COG descriptors for each protein. Use the COG descriptors in Table 2 and the Function Cart tool in IMG to fill in Table 3 to identify genes encoding these T6SS components within the genome of ATCC 17802. (5 marks)

Table 2. List of the required Type VI Secretion System (T6SS) proteins, alternative names and Cluster of Orthologous Groups (COG) descriptors.

Tss/Tag

Alternative Name

COG

TssH

Clp, VasG

0542

TssD

Hcp

3157

TssL

IcmH, DotU, ImpK, VasF

3455

TssI

VgrG

3501

TssA

ImpA, VasJ/VasL

3515

TssB

ImpB, VipA

3516

TssC

ImpC, ImpD, VipB

3517

TssE

HsiF, ImpF

3518

TssF

ImpG, VasA

3519

TssG

ImpH ,VasB

3520

TssJ

Lip, SciN, VasD

3521

TssK

ImpJ

3522

TssM

IcmF, ImpL, VasK

3523

TagD

PAAR domain protein

4104

Table 3: List of genes that encode T6SS components in the genome of Vibrio parahaemolyticus strain ATCC 17802.

Tss/Tag descriptor

Alternative name

COG descriptor

IMG Locus Tag

TssH

Clp, VasG

0542

TssD

Hcp

3157

TssL

IcmH, DotU, ImpK, VasF

3455

TssI

VgrG

3501

TssA

ImpA, VasJ/VasL

3515

TssB

ImpB, VipA

3516

TssC

ImpC, ImpD, VipB

3517

TssE

HsiF, ImpF

3518

TssF

ImpG, VasA

3519

TssG

ImpH ,VasB

3520

TssJ

Lip, SciN, VasD

3521

TssK

ImpJ

3522

TssM

IcmF, ImpL, VasK

3523

TagD

PAAR domain protein

4104

Question 3.3. V. parahaemolyticus strain ATCC 17802 contains components for more than one T6SS. What information from Table 1 (Subset of proteins identified in the secretome of Vibrio parahaemolyticus strain ATCC 17802) indicates that both these T6SSs are functional and genes encoding both these T6SSs are expressed under laboratory conditions? (2 marks)

Question 3.4. Figure 1 (below) is a diagram of the T6SS. Identify the components that are indicated at the following places: (5 marks)

A. B.

C. D.

E.F.

G.H.

I. J.

Figure 1. Diagram of the Type Six Secretion System

Question 3.5. Is the target cell illustrated in Figure 1 likely to be a Gram-negative bacterial cell or a eukaryotic cell? Explain your answer. (2 marks)

Question 3.6. The V. parahaemolyticus strain ATCC 17802 gene with locus tag Ga0174928_12327 encodes a putative exonuclease SbcC. This locus is downstream of the vgrG1 gene (locus tag Ga0174928_12323) in an operon. This putative exonuclease SbcC is present in the secretome of ATCC 17802 (Table 1) and has been identified as a putative effector protein. It is not present in the secretome of a ΔT6SS1/ ΔT6SS2 mutant of ATCC 17802.

You have created a knockout mutation in the gene encoding the exonuclease SbcC. Describe how you would set up an experiment to determine whether this protein is a bactericidal effector protein.

Tss/Tag descriptor

Alternative name

COG descriptor

IMG Locus Tag

TssH

Clp, VasG

0542

Ga0174928_11529

Ga0174928_11991
Ga0174928_111342

Ga0174928_1232131

TssD

Hcp

3157

Ga0174928_111343

Ga0174928_12322

TssL

IcmH, DotU, ImpK, VasF

3455

Ga0174928_111363

Ga0174928_12309

TssI

VgrG

3501

Ga0174928_111344

Ga0174928_12323

TssA

ImpA, VasJ/VasL

3515

Ga0174928_12313

TssB

ImpB, VipA

3516

Ga0174928_111351

Ga0174928_12314

TssC

ImpC, ImpD, VipB

3517

Ga0174928_111352

Ga0174928_12315

Ga0174928_12316

TssE

HsiF, ImpF

3518

Ga0174928_111353

Ga0174928_12318

TssF

ImpG, VasA

3519

Ga0174928_111354

Ga0174928_12319

TssG

ImpH ,VasB

3520

Ga0174928_111355

Ga0174928_12320

TssJ

Lip, SciN, VasD

3521

Ga0174928_111361

Ga0174928_12307

TssK

ImpJ

3522

Ga0174928_111362

Ga0174928_12308

TssM

IcmF, ImpL, VasK

3523

Ga0174928_111357

Ga0174928_12310

TagD

PAAR domain protein

4104

Ga174928_12324

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