Bacterial secretion systems (Type I, Type II, Type III, Type IV, Type V and Type

Burdened by Homework? Let us write your essays and assignments Order This Now

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.

Burdened by Homework? Let us write your essays and assignments Order This Now

Published
Categorized as Biology

Leave a comment