Lecture 8 - Salmonella

Question 1

Salmonella enterica is a gram ______, ______ pathogen; gamma proteobacteria.

It is __-shaped and ____ (flagella surrounding).

Can live both inside a ____ and in the ______.

It has a _____ host range. What are they?

Answer 1

negative; opportunistic

rod; motile

host (intestinal tract); environment (water, soil, plants, human/animal excretion)

broad - humans & food animals, pets and wild animals (ducks, chickens, reptiles)

Question 2

What are the diseases that salmonella enterica cause?

Answer 2

1) it is one of the leading causes of FOODBORNE GASTROENTERITIS (food-poisoning related or can contract them from pets)

2) TYPHOID FEVER (S. typhi causative agent)

Question 3

Which statement is false regarding salmonella enterica infection:

1) It has over 1.5 million cases/year in Canada and US (6 outbreaks in Canada in 2020 with 3 food-related).

2) Globally there are 90k deaths annualy.

3) Illness typically develops between 6 hours and 1 week after infection.

Answer 3

2!

270k deaths per year

Question 4

(T/F) only some parts of the world is affected by typhoid.

Answer 4

false! all parts affected.

Question 5

There are two types of Salmonella enterica serovar: ______ serovar and ______ serovar.

______ serovars (typhi, paratyphi, and sendai) are human restricted and cause _____ fever, fever, abdominal pain, transient diarrhoea, etc.

______ serovar (typhimurium, enteriditis) have a ____-range and cause ______, abdominal pain, vomitting, and inflammatory diarrhoea.

Some non-typhoidal salmonella (NTS) strains can also cause ______ infections in some parts of the world and are 20-25% lethal.

Answer 5

Typhoid; Non-typhoid

Typhoid; enteric

Non-typhoid; gastroenteritis

Bloodstream

Question 6

How does chronic carriage of Salmonella infection occur?

Where does it persist in the human body?

Answer 6

1) Chronic carriage occurs when S. enterica PERSIST in the body through 1) infection without obvious symptoms, 2) infection with symptoms

2) Gall bladder is a common reservoir

Question 7

(T/F) Bacteria can persist in body long after initial infection even if treated with antibiotics; this occurs for typhoidal serovars only.

Answer 7

False!

Though bacteria can persist in body long after initial infection even if treated with antibiotics; this occurs for BOTH typhoidal and NON-TYPHOIDAL serovars.

Question 8

What are the challenges associated with chronic carriage?

Answer 8

1) Bacteria can be SHED from host and SPREAD to others (typhoid mary - typhoid outbreaks in New York in early 1900s)

2) Antibiotic treatment CANNOT clear all bacteria from the body (ex, gallbladder). This can cause RECURRENT INFECTIONS (S. typhimurium can be invasive).

Question 9

What is reactive arthritis?

Answer 9

Inflammatory response that occurs in other parts of the body; usually AFTER Salmonella infection is cleared.

Skin lesions can be caused due to reactive arthritis.

Question 10

Which antibiotics are used as treatments for Salmonella enterica infections?

Answer 10

Quinolone, macrolide, or cephalosporin classes.

*antibiotic resistance (multi-drug) growing global problem

Question 11

Why is it important to study drug resistance Salmonella in agriculture?

Give an example.

Answer 11

Important to study because Salmonella is an environmental microbe. Antibiotic resistant Salmonella are WIDESPREAD in the environment.

Resistance profiling of Salmonella enterica derived from CHICKENS in Canada! Resistance to at least one drug class common. Multi drug resistance also observed.

Question 12

1) What are the two species of the Salmonella genus?

2) How many subspecies and serotypes are there for Salmonella enterica?

3) Which S. Enterica subspecies is primarily associated with warm-blooded animals, including humans?

Answer 12

1) Salmonella Enterica and Salmonella Bongori

2) There are 7 subspecies and over 2600 serotypes of salmonella enterica.

3) Salmonella enterica Subsp. enterica! Rest can infect warm-blooded animals as well but it is less common.

Question 13

Salmonella enterica subsp. enterica includes ________ (broad host specificity and model pathogen) and _____ (humans only).

Answer 13

S. Typhimurium; S. Typhi

Question 14

S. enterica serovar Typhimurium has 38 pseudogenes and one plasmid, while serovar Typhi has _____ pseudogenes and two plasmids.

What is a pseudogene? Why is this important?

Answer 14

204

Pseudogene: genetic region that resembles a protein-encoding region, but is likely non-functional. These 204 pseudogenes include effector genes. Were unneeded functions lost during the evolution to a human only host?

Question 15

In the salmonella genome, there are SPIs that encode for the T3SS. Briefly describe them.

Answer 15

SPIs (salmonella pathogenicity islands) include genes important for survival in host. They suggest frequent gene acquisition through HORIZONTAL GENE TRANSFER.

There are two T3SS encoded on SPI-1 and SPI-2, which secrete EFFECTOR proteins to mediate infection.

SPI-1 T3SS: important for early infection
SPI-2 T3SS: important for later stages of infection

Question 16

Salmonella is an ______ pathogen that resides in compartments termed _____ _____ _____.

____ are important for mediating infection; there are more than ___ produced and are secreted by the two T3SSs.

Answer 16

Intracellular; Salmonella-Containing Vacuoles (SCV)

Effectors; 30

Question 17

Salmonella infection is important in what kind of research?

Answer 17

Pathogen research!

They are commonly used MODEL organism to study: bacterial pathogenesis and host response to infection!

Question 18

Salmonella enterica is ingested through the mouth (route of entry).

It survives the low ___ of stomach. In the intestinal epithelium, it enters ___ cells and other ____ cells.

While _______ serotypes induce local inflammatory response, _____ ____ serotypes enter ______ and ___.

Answer 18

pH; M cells; epithelial

Gastroenteritis; systemic illness; macrophages; spread (to lymph nodes, liver, and spleen)

Question 19

Briefly explain the roles of SPI-1 T3SS and SPI-2 T3SS and when they are turned on.

Answer 19

SPI-1 T3SS: turned on BEFORE INFECTION; predominantly involved in INDUCING PATHOGEN UPTAKE into SCV

SPI-2 T3SS: turned on AFTER UPTAKE into SCV; predominantly involved in PROMOTING BACTERIAL SURVIVAL, REPLICATION and SPREAD

Question 20

Fill in the blanks regarding effectors:

1) Effector protein is _____ ____; proteins are only made when needed.

2) Effectors are often ______; have evolved multiple ____ to target different host processes.

3) Some effectors play important roles in ___ cell types but not ____.

4) Some effectors can function in _____ cells types, but can have ______ consequences (due to specialized pathways)

Answer 20

Intricately timed

Multifunctional; domains

some; all

multiple; different

Question 21

Match the steps of Salmonella Infection:

1) Step 1
2) Step 2
3) Step 3
4) Step 4
5) Step 5
6) Step 6
7) Step 7

A) Endocytic pathway begins to target SCVs.

B) SCVs migrate along microtubules to cell periphery (needed for cell-to-cell spread)

C) Effectors induce the formation of tubules (Salmonella induced tubules - SITs) that extend to the cell periphery.

D) SCVs migrate to the perinuclear region and multiply near golgi.

E) SPI-1 T3SS effectors induce invasion ruffle - bacterial uptake into SCV.

F) Salmonella inhibits lysosomal pathway.

G) SPI-2 T3SS effectors secreted to promote bacterial survival & replication.

Answer 21

Step 1: SPI-1 T3SS effectors induce invasion ruffle - bacterial uptake into SCV.

Step 2: Endocytic pathway begins to target SCVs.

Step 3: SPI-2 T3SS effectors secreted to promote bacterial survival & replication.

Step 4: Salmonella inhibits lysosomal pathway.

Step 5: SCVs migrate to the perinuclear region and multiply near golgi.

Step 6: Effectors induce the formation of tubules (Salmonella induced tubules - SITs) that extend to the cell periphery.

Step 7: SCVs migrate along microtubules to cell periphery (needed for cell-to-cell spread)

Question 22

How can we visualize Salmonella Infection?

Answer 22

Fluorescence microscopy: stain for both bacteria & host (SCV) and merge; often use antibodies to detect.

LAMP proteins: Markers for SCV membrane

Question 23

(T/F) Salmonella must infect different cell types for infection.

Answer 23

True!

It infects phagocytic cells that take up pathogens and also non-phagocytic cells that do not uptake pathogens.

Question 24

How does Salmonella infect non-phagocytic cells?

Answer 24

Host machinery for uptake in epithelial cells (via cytoskeleton rearrangement) is present in non-phagocytic cells but ALWAYS TURNED OFF.

Uptake in non-phagocytic host cells is mediated by EFFECTOR proteins:
1) Turn on SPI-1 T3SS to secrete these effectors BEFORE infection

2) Effectors function by MIMICKING host regulatory proteins that control uptake and turn on host uptake machinery.

Effector proteins control both turning on ruffling and subsequently turning it off.

Question 25

Uptake from the extracellular environment is controlled by ___ family of small _______ (member of ___ superfamily).

___ ______ manipulated by Salmonella.

Answer 25

Rho; GTPases; Ras superfamily of GTPases

Rho GTPases

Question 26

How do Rho GTPases work?

What are GEF and GAP?

Answer 26

Ras superfamily GTPases are small G-proteins that function as MOLECULAR SWITCHES for signalling pathways.

They cycle between active (GTP-bound) and inactive (GDP-bound) forms. When active, they interact with HOST BINDING PARTNERS to mediate cellular effects.

GTPases are fine tuned using REGULATORY PROTEINS.
GEF = guanine nucleotide exchange factor –> activate
GAP = GTPase activating protein (enzyme) –> deactivate

Question 27

1) How do salmonella effectors manipulate the host for uptake?

2) What are Rho GTAases?

3) What must happen to the ruffling to complete bacterial uptake?

Answer 27

Salmonella effectors manipulate the host by:
1) Activating Rho GTPases
2) Deactivating ruffling when done

Rho GTPases act at the PLASMA MEMBRANE. When active, host binding partner proteins bind the Rho and cause ACTIN POLYMERIZATION, which causes PM to ruffle outward to mediate uptake.

Ruffling must be deactivated in order to complete bacterial uptake.

Question 28

What are the 4 salmonella effectors that control uptake?

Briefly describe what they do.

Answer 28

SopB, SopE, SopE2, and SptP

SopB (activates a host GEF), SopE (GEF mimic
= activates GTPases), SopE2 (GEF mimic) turn on localized ruffling to promote Salmonella uptake.

SptP (a GAP mimic) turns off ruffling to complete uptake by deactivating GTPases.

Question 29

What is the autophagic machinery?

Answer 29

The authophagic machinery target intracellular pathogens for degradation.

Steps of autophagy:
1) initiation
2) membrane formation
3) membrane completion
4) fusion with lysosomes
5) degradation.

Question 30

1) Which type of salmonella effectors suppress autophagy? Why?

2) Which effector suppresses autophagy at the initiation (induction) stage?

3) Why is it important to study bacterial effectors?

Answer 30

1) Both SPI-1 and SPI-2 T3SS effectors suppress autophagy. It is to the pathogen’s advantage to suppress autophagy throughout the course of infection.

2) SopF suppresses autophagy at the initiation stage. The initiation mechanism targeted is specific for antibacterial autophagy (xenophagy).

3) Researchers did not know that this mechanism existed until they studied SopF! Studying bacterial effectors can lead to the discovery of important NEW HOST PATHWAYS.

Question 31

How does SopF suppress autophagy at the initiation (induction) stage of xenophagy?

Answer 31

Background: host proteins had other known functions; not known to contribute to initiating xenophagy.

Infection induces V-ATPase on the vacuole membrane to bind Atg16L1 complex, forming a complex of complexes.

This allows Atg16L1 complex to lipidate LC3 which is critical to initiate autophagy.

SopF ADP-ribosylates the V-ATPase, disrupting its infection induced interaction with Atg16L1 complex, blocking autophagy.

Question 32

1) What is bacterial positioning important for? Which effectors control bacterial position?

2) Where is the bacteria during intermediate infection and during late infection? Why?

Answer 32

1) Bacterial positioning is important for infection. SPI-2 T3SS effectors control bacterial positioning (SseF, SseG, SifA, and PipB2).

2)
Intermediate infection: PERINUCLEAR REGION (thought to bring the pathogen in proximity to Golgi for nutrient acquisition).

Late infection: CELL PERIPHERY (thought to bring the pathogen in proximity to the plasma membrane for cell-to-cell spread)

Question 33

_____ and ____ help tether SCV to Golgi.

Answer 33

SseF; SseG

*possibly though an interaction with ACBD3.

Question 34

1) What is trafficking?

2) Which proteins are involved? What are their functions?

Answer 34

1) Movement of intracellular compartments along MICROTUBULES.

2) Compartments move using MOTOR proteins, such as dynein and kinesin.

Dynein: movement toward nucleus
Kinesin: movement toward cell periphery

Question 35

Fill in the blanks regarding compartment specificity:

1) Compartments use ____ ____ (ex. Rab GTPases) to control trafficking.

2) Different compartments have ______ regulatory proteins.

3) Special ____ ______ proteins called ____ proteins bind the molecular motors to conjugate compartments to ________.

4) ____ and ____ manipulate host trafficking (microtubules) to control the positioning of the SCV and host organelles.

Answer 35

Regulatory proteins

Different

Host binding; Adapter; microtubules

SifA & PipB2

Question 36

During the ______ stages of infection, SifA binds ____ adapter complex _____. It is used to maintain ______ positioning of SCVs.

What happens when you deplete member of BLOC-2 complex?

Answer 36

intermediate; dynein; BLOC-2; perinuclear

Bacteria are father from nucleus!

Question 37

During the _____ stages of infection, SCVs migrate to the cell periphery. Expression of _____ moves compartments to the cell periphery.

This effector protein interacts with the ___ chain of _____.

How do we know this?

Answer 37

later; PipB2

light; kinesin (PipB2 controls kinesin-dependent movement)

Normally, LAMP2+ (SCV marker) compartments are concentrated near the nucleus; expression of the effector PipB2 causes them to move to the periphery.

Question 38

How does SifA help PipB2 control Kinesin dependent movement to the cell periphery?

Answer 38

SifA, a multifunctional effector, also contributes by binding to the adapter protein SKIP (which helps conjugate compartment to the kinesin complex to help it move).

Question 39

What are SITs? What are their possible roles?

Answer 39

Salmonella-Induced Tubules (SITs); tubules induced by Salmonella that extend throughout the host cell along microtubules and are positive for different marker proteins.

Possible roles:
1) Nutrient acquisition
2) Cell-to-cell spread

Question 40

Which effectors are important for SIT formation?

Answer 40

SifA, PipB2, SopD2 –> manipulation of host microtubule formation

SseF, SseG -> tethering to Golgi (anchor point)

SteA -> unknown

Question 41

1) What is the ULK1 complex? What is it regulated by?

2) What are the roles of SPI-2 T3SS effectors, SseF and SseG, later during infection?

Answer 41

ULK1 complex can initiate general autophagy (which includes xenophagy. The GTPase Rab1 regulates ULK1 complex formation; its role is to RECRUIT ULK1 to the initiation complex. Rab1 is activated by a GEF called TRAAPPIII.

SseF and SseG, mulifunctional SPI-2 T3SS effectors, target autophagy later during infection at the initiation stage. They bind Rab1 and block TRAAPPIIIfrom binding; autophagy cannot be activated.

*SopF blocks autophagy early in the infection
*SseF and SseG block autophagy later in the infection

Question 42

Infections can trigger the _____ pathway; transcription factors (TFs) control genes associated with the ____ and _____ immune responses.

What do salmonella effectors do?

Answer 42

NF-kb; adaptive; immune

Expression of Salmonella effectors cause a decrease in TF activity.

Effectors PipA, GogA and GtgA are proteases that cleave TFs p65 and ReIB, inhibiting gene expression!

Question 43

How does Salmonella infection impair antigen presentation? Which effectors are invovled?

Answer 43

SPI-2 T3SS effectors suppress antigen presentation in Dendritic Cells (DCs).

Dendritic cells present antigens to T cells to activate. Antigen presentation involves loading of antigen peptides onto MHC surface protein.

Before loading, antigens in compartments must traffic along microtubules to get to dendritic cell periphery.

Salmonella SPI-2 effectors suppress antigen presentation in dendritic cells by inhibiting loading of MHC-11 by disrupting normal trafficking. Seven effectors contribute; five of these manipulate MICROTUBULE-based trafficking of intracellular compartments (SifA, PipB2, SopD2, SseF and SseG).

Question 44

(T/F) While most salmonella, even most of S. enterica subsp. enterica have broad host range, S. Typhi subgroup is human adapted.

Answer 44

True!

Question 45

(T/F) SPI-1 T3SS and its effectors are present in all salmonella strains (enterica and bongori), but SPI-2 and its effectors are acquired in S. enterica after divergence.

Answer 45

True!

Question 46

How did S. Typhi become human specific? How did it lose the ability to survive in other hosts?

Answer 46

S. Typhi has functional loss of almost half of effectors (pseudogenes)!

Could one or more be responsible for the inability to survive in other hosts?

Question 47

What is GtgE? How did researchers find that GtgE is important for broad host specificity of Salmonella?

Answer 47

GtgE is a protease that degrades some Rab GTPases.

The introduced a single effector (GtgE) in S. Typhi to see if it replicates in MOUSE macrophages.

It did replicate!! GtgE degrades Rab32 to suppress BLOC-3 Complex formation.

Question 48

Two effectors are important for broad host specificity of Salmonella, one of them being GtgE (protease). What is the second one?

How was this found?

Answer 48

SopD2! Both SopD2 and GtgE work together to target and suppress Rab32 and are needed to survive in non-human hosts.

S. Typhimurium can replicate in mice, unlike S. Typhi. Researchers wanted to know which genes were important for that. They took knockout strain of gtgE and made a series of double knockouts of effectors that are pseudogenes in S. Typhi. The found that double knockout with the effector sopD2 eliminated all Rab32 function!!

Question 49

How does SopD2 target Rab32 in S. Typhi?

Answer 49

SopD2 is a GAP that hydrolyzes GTP to inactivate Rab32.

Question 50

1) What is Rab32 important for and in which hosts?

2) How does this allow S. Typhi to only infect humans?

Answer 50

1) Rab32 is important for HOST DEFENSE against bacterial pathogens in many hosts EXCEPT HUMANS.

2) Most serovars of Salmonella Enterica have functional effectors GtgE and SopD2 to inactivate Rab32. S. Typhi has pseudogenes of GtgE and SopD2 and thus cannot inactivate Rab32. Thus, it can not survive in non-human hosts. It can only survive in humans, where Rab32 is not essential.

Question 51

S. Typhi has evolved specialized virulence factors to target humans. What are the two?

Answer 51

1) Vi Antigen
2) Typhoid toxin

Question 52

1) How was Vi antigen acquired in S. Typhi?

2) What is it?

Answer 52

Horizontal gene transfer (not in S. Typhimurium or S. Paratyphi genomes).

Polysaccharide capsule (viaB gene locus) that blocks recognition of bacteria; up-regulated during systemic infection.

Immune evasion: neutrophil and phagocytic respiratory burst.

Question 53

S. ____ can also cause a similar phenotype but it has no Vi antigen found in S. Typhi.

Mechanism: very long O-antigen chains on LPS that block recognition of the bacteria.

Example of _______ evolution of two pathogens.

Answer 53

S. paratyphi

Convergent

Question 54

1) Which species is the Typhoid toxin unique to?

2) When is it secreted?

3) What family is it part of? What genes are essential for its function?

4) What is its mechanism of action?

Answer 54

1. unique to S. Typhi and S. paratyphi
2. Secreted into SCV lumen after uptake; exported in compartments; toxin needs to be exported outside the host cell in order to function.
3. AB toxin family - A2B5 (pltB, pltA and cdtB all 3 genes essential for function)
4. It targets HUMAN CELLS specifically. It recognizes surface glycoprotein sialoglycans with acetyl neuraminic acid termini (preferentially expressed human cells). It can act on many different cell types.

Question 55

What is the CdtB of the Typhoid Toxin?

How many subunits of PltB and PItA does the Typhoid toxin have?

Answer 55

1) CdtB (cytolethal distending toxin; exotoxin) - deoxyribonuclease, causes DNA damage, arrests in G2/M phase of cell cycle.

2) Five pItB subunits and two pItA subunits.