Module 5: Section 2A

Question 1

Function of Two-Component Systems

Answer 1

Bacteria use two-component systems to detect environmental changes and respond through phosphorylation-based signal cascades that regulate cell activity

Question 2

Components of Two-Component Systems

Answer 2

• A sensor protein (Histidine Kinase) that detects signals
• A Response Regulator that alters gene transcription in response

Question 3

Two-Component Systems - Step 1

Answer 3

Environmental stimuli can be in the form of specific molecules, changes in pH, or temperature

Question 4

Two-Component Systems - Step 2

Answer 4

• Sensor protein (histidine kinase) in the cytoplasmic membrane has a periplasmic domain and a cytoplasmic domain that help transmit a signal across the membrane
• Periplasmic domain receives the signal and this causes the cytoplasmic domain to become phosphorylated on a histidine residue

Question 5

Two-Component Systems - Step 3

Answer 5

• Once phosphorylated, the sensor protein is activated to transfer its phosphate to a response regulator
• There is a specific response regulator for each type of histidine kinase

Question 6

Two-Component Systems - Step 4

Answer 6

• An aspartate amino acid accepts the phosphate from the sensor protein
• This activates the response regulator, altering its configuration so that it is able to bind to specific sites on the chromosome

Question 7

Response regulator

Answer 7

Is a transcriptional activator or repressor depending upon the outcome of its interaction with specific gene promoters

Question 8

Components on bacterial cell surface functions

Answer 8

• Mask the bacterium from the immune system
• Facilitate adhesion of bacterium to substrate or tissue
• Sequester iron from the external environment
• Aid in the assembly of motility structures

Question 9

Sortase System

Answer 9

The Sortase system helps Gram-positive bacteria move large molecules to their cell surface by modifying Sortase protein substrates and attaching them to the peptidoglycan

Question 10

SrtA Enzyme

Answer 10

• Gram-positive bacteria have several Sortase enzymes
• SrtA is the most similar across species and displays substrates for host cell interactions, antibody sequestration, invasion, and phage receptors

Question 11

Srt mediated secretion

Answer 11

• Mediates attachment of specific proteins to the cell surface via the LPXT region on the secreted protein
• Proteins are usually attached covalently to the PG, anchoring them to the cell surface

Question 12

First motif to be recognized by the Sortase system

Answer 12

• First motif is a specific series of amino acids at the N-terminus of the protein called the signal peptide
• This region is usually cleaved off during passage through the cytoplasmic membrane

Question 13

Second motif to be recognized by the Sortase system

Answer 13

• Second motif is a sorting signal, a series of amino acids (LPXTG) close to the C-terminus of the protein
• The region is recognized by the Sortase enzyme

Question 14

Third motif to be recognized by the Sortase system

Answer 14

• Third motif is a series of positively charged amino acids at the C-terminus of the protein
• This region helps anchor the protein in the membrane until the Sortase enzyme is finished

Question 15

Sortase-mediated secretion in gram-positive bacteria - step 1

Answer 15

• The signal peptide of the sortase substrate directs it to secretory machinery to move it into the periplasm
• The positively charged C-terminus anchors the protein to the cytoplasmic membrane surface

Question 16

Sortase-mediated secretion in gram-positive bacteria - step 2

Answer 16

The sortase enzyme recognizes the LPXTG motif

Question 17

Sortase-mediated secretion in gram-positive bacteria - step 3

Answer 17

Sortase cleaves the protein substrate

Question 18

Sortase-mediated secretion in gram-positive bacteria - step 4

Answer 18

Sortase transfers the protein substrate to a lipid intermediate, using a peptide cross bridge component of the peptidoglycan

Question 19

Sortase-mediated secretion in gram-positive bacteria - step 5

Answer 19

• The protein/lipid/pentapeptide complex is incorporated into the peptidoglycan

Question 20

Pilus Formation

Answer 20

Multiple sortases function to attach many pilin subunits together, building the pilus structure outwards from the cell surface

Question 21

Exotoxin

Answer 21

Many bacteria secrete compounds to the external environment that are toxic to their hosts

Question 22

Exotoxin example

Answer 22

• Tetanus toxin
• Botulinum toxin
• Produced by different strains of clostridium

Question 23

Endotoxin

Answer 23

Endotoxins are secreted to the outer surface of the bacterium and are only released from the bacterium if it lyses

Question 24

Endotoxin example

Answer 24

• Lipopolysaccharide component of gram-negative bacteria
• Release of LPS into the bloodstream is responsible for endotoxic shock, that can occur during septicaemia (blood poisoning)

Question 25

OMP

Answer 25

Outer membrane protein

Question 26

MFP

Answer 26

membrane fusion protein

Question 27

ABC

Answer 27

ATP-binding cassette

Question 28

Type I Secretion System

Answer 28

In Gram-negative bacteria, exotoxins cross two membranes via the Type I secretion system, where ABC, MFP, and OMP form a complex channel that secretes the toxin in one step

Question 29

Haemolysin (Type I Exotoxin Mechanism)

Answer 29

- Streptococci produce haemolysin, an exotoxin that lyses red blood cells - After secretion, toxin monomers form a donut-like complex on the RBC surface that inserts into the membrane, creating pores that release cell contents

Question 30

Type 3 Secretion System (TTSS)

Answer 30

- Some bacterial pathogens use a Type 3 secretion system to inject proteins directly into host cells, killing or altering them to help infection - This system forms a continuous conduit from the bacterial cytoplasm to the host cytoplasm and delivers “effectors” into the host cell