MIC 4124 8b - Salmonella II

Salmonella must infect ___ ___ ___ to cause infection

different cell types

- including non-phagocytic cells

Do non-phagocytic epithelial cells possess machinery for SCV uptake?

Yes, but it is always turned off

How do effector proteins mediate Salmonella uptake?

- Turns on SPI-1 T3SS to secrete these effectors into the host before infection

- Effectors function by mimicking host regulatory proteins that control uptake (turn on host uptake machinery)

What is another invasion process that effector proteins facilitate?

ruffling and then turning it off

Uptake of Salmonella from extracellular environment is controlled by which host protein?

Ras superfamily of small GTPases

- Rho family contribute to phagocytosis

GEF

guanine nucleotide exchange factor → activate GTPase

GAP

GTPase activating protein (enzyme) → deactivates GTPases

When active, Rho GTPases...?

interact with host binding partners to mediate cellular effects

- Only when GTPase is active (bound to GTP) will it interact with host binding proteins

Where in the host cell do Rho GTPases act?

at the plasma membrane (PM)

When Rho GTPase is active what is triggered?

host binding partner proteins bind the Rho and cause actin polymerization

this causes the plasma membrane to ruffle outward to mediate pathogen uptake

What must be deactivated in order to complete bacterial uptake?

Ruffling

Salmonella effectors manipulate the host by

- Activating Rho GTPases (to turn on ruffling)

- Deactivating ruffling when done

4 salmonella effectors that control uptake

- SopB

- SopE

- SopE2

- SptP

Which Salmonella effectors turn on localized ruffling?

SopB, SopE, SopE2

- promotes Salmonella uptake

SopB function

activates host GEF

SopE and SopE2 function

GEF mimics; activate GTPases

Which Salmonella effector turns off ruffling to complete uptake?

SptP

SptP function

GAP mimic; deactivates GTPases

SPI-2 T3SS effectors control what...?

bacterial positioning which is important for infection

SPI-2 T3SS effectors (4)

- SseF

- SseG

- SifA

- PipB2

Where is the SCV located during intermediate infection?

host perinuclear region

- Thought to bring the pathogen in proximity to Golgi for nutrient acquisition

Where is the SCV located during late infection?

Host cell periphery

- Thought to bring the pathogen in proximity to the plasma membrane for cell-to-cell spread

SseF and SseG function

help tether the SCV to Golgi by binding ACBD3

What is ACBD3?

a host protein expressed on the surface of the golgi body

Microtubule-based trafficking

mode of transportation SCVs use to move within the host cell

How do you get directionality of movement with SCVs in the host cell?

Compartments can use different motor proteins, depending on direction of transport

Motor proteins

dynein and kinesin

Dynein direction of movement

movement toward nucleus

Kinesin direction of movement

movement toward cell periphery

How do you get compartment specificity?

Different compartments have different regulatory proteins (ex, Rab GTPases)

Adapter proteins

Special host binding partners that bind to molecular motors to conjugate compartments to the microtubules

Effector proteins involved in manipulating host trafficking

- SifA (controls dynein and kinesin-dependent movement)

- PipB2 (controls Kinesin-dependent movement)

SifA function (dynein movement)

- in the intermediate stages of infection, SifA binds BLOC-2 and uses it to maintain perinuclear positioning of SCVs

BLOC-2

dynein adapter complex (3-protein complex)

- part of host dynein motor protein

- binds bacterial SifA to promote SCV trafficking

What happens to Salmonella positioning in a host cell lacking BLOC-2?

Salmonella can not maintain perinuclear localization

PipB2 function

in the late stages of infection, expression of PipB2 moves compartments to the cell periphery

How do we know PipB2's function?

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

LAMP2

host cell marker of late endosomes (usually concentrated near nucleus)

Which part of kinesin does PipB2 interact with?

light chain

SifA function (kinesin movement)

Binds to adapter protein SKIP which allows for kinesin to bind the SCV membrane surface

SITs

salmonella induced tubules

- Tubules induced by Salmonella; extend throughout the host cell along microtubules

What are the possible roles of SITs?

- Nutrient acquisition

- Cell-to-cell spread

Functions of effectors important in Salmonella-induced tubule (SIT) formation (3)

- Manipulation of host microtubule function

- Tethering to Golgi (anchor point)

- Unknown mechanism

Effectors that manipulate host microtubule function (3)

- SifA

- PipB2

- SopD2

Effectors that help tether SCV to Golgi (2)

- SseF

- SseG

Effector with unknown mechanism in SIT formation

- SteA

Which immune response do Salmonella effectors target?

NF-𝜅B Pathway

- triggered by infection

What is the NF-𝜅B Pathway?

Immune response that includes transcription factors (TFs) that control genes associated with both the innate and adaptive immune responses

Effectors that target the NF-𝜅B Pathway (3)

- PipA

- GogA

- GtgA

PipA, GogA & GtgA function

proteases that cleave TFs p65 & RelB (part of NF-𝜅B pathway) → inhibit gene expression

PipA, GogA & GtgA triple knockout

Triple knockout in all genes (PipA, GogA, GtgA) was susceptible to NF-KB. Complementation restored resistance

How does a Salmonella infection impair antigen presentation?

SPI-2 T3SS effectors suppress antigen presentation in dendritic cells (DCs) by inhibiting loading of MHC-II

Dendritic cells

immune cells that detect pathogens; capture, degrade, and present antigens to T cells to activate them

- load antigens to MHC-II surface proteins

- Part of adaptive immune response

Before loading antigens in compartments to MHC-II surface proteins, compartments...?

Compartments must traffic along microtubules to get to cell periphery. Therefore, disruption of normal trafficking is likely to impair loading of peptides on MHC-II

How many effector proteins are involved in inhbiting MHC-II loading? Which are known?

7 effectors contribute

5 are known

- SifA, PipB2, SopD2, SseF and SseG

Salmonella enterica spp. tyhpi host

S. typhi only infects humans. It is human adapted

Evolutionary history of Salmonella lineages

SPI-1 T3SS and SPI-1 effectors are present in all salmonella strains (appeared early in Salmonella evolution)

S. enterica vs. S. bongori

S. Bongori have different pathogenicity islands and effectors than S. enterica

- S. bongori: (SPI-22 - T6SS, effectors)

- S. enterica: (SPI-2 - T3SS, SPI-2 effectors)

S. typhi pseudogenes

204 pseudogenes = functional loss of almost half of effectors

Could introducing a single functional effector could allow S. Typhi replicate in mouse macrophages?

Yes, adding GtgE (protease that degrades some Rab GTPases) allowed S. typhi to survive

Which Rab GTPases does GtgE degrade?

Rab32, Rab38, and Rab29

GtgE role in broad host range Salmonella

Other Salmonella can infect other mammals; use GtgE to degrade Rab32 to suppress BLOC-3 Complex function

Rab32 and BLOC-3 role in host cell

host protein complexes that control trafficking of antimicrobial cargo pathway

Which two effectors are important for broad host specificity of Salmonella

- GtgE

- SopD2

SopD2 and GtgE work together to target Rab32 and both are needed to survive in non-human hosts

Experimental Test with S. Typhimurium (broad range host)

S. Typhimurium can replicate in mice. What genes are important?

- Researchers took a knockout strain of gtgE and made a series of double knockouts of effectors that are pseudogenes in S. Typhi

- wild-type eliminated all Rab32 function, but not double knockout with the effector sopD2

How does SopD2 target Rab32?

SopD2 is a GAP mimic that hydrolyzes GTP to inactivate Rab32

- SopD2 caused more GTP hydrolysis when exposed to Rab32-GTP

- Active site mutant (predicted) resulted in less GTP hydrolysis

Rab32 in other hosts vs. humans

Rab32 is important for host defense against bacterial pathogens; trafficking of antimicrobial cargo

- pathway important in many hosts, except humans

- So, S. enterica typhi lacking functional GtgE can survive in humans only

GtgE and SopD2 in S. typhi

Both are pseudogenes in S. typhi and are non-functional (cannot inactivate Rab32)

Specialized virulence factors that S. typhi evolved to target humans (2)

1. Vi antigen

2. Typhoid toxin (exotoxin)

How was Vi antigen gained in S. typhi?

horizontal gene transfer; it's not in S. Typhimurium or S. Paratyphi genomes

Vi antigen

polysaccharide capsule (viaB gene locus) that aids in immune evasion

- protection against neutrophil & phagocytic respiratory burst

- capsular polysaccharide blocks host antibodies

- upregulated during systemic infection

S. paratyphi similar phenotype to Vi antigen

S. paratyphi lacks Vi antigen, but can also evade host immune system

- Mechanism: very long O-antigen chains on LPS to block host antibodies

- Example of convergent evolution of two pathogens

Typhoid Toxin is found in which Salmonella subspecies?

unique to S. Typhi and S. Paratyphi

Typhoid toxin

exotoxin that targets human cells specifically

- recognizes surface glycoprotein sialoglycans with acetyl neuraminic acid termini (preferentially expressed human cells)

- can act on many different cell types

When is typhoid toxin secreted?

secreted after uptake, from SCV within compartments

Typhpoid toxin belongs to which toxin family?

AB toxin family

Which genes encode typhoid toxin?

pltB, pltA, cdtB

- all 3 genes essential for function

Where must typhoid toxin be transported to function?

outside of the host cell

it is secreted into SCV lumen by Salmonella than exported in compartments

Typhoid Toxin Structure

A2B5

- PltB: five subunits

- PltA: two subunits

- CdtB: cytolethal distending toxin (exotoxin)

CdtB

deoxyribonuclease, causes DNA damage; arrests in G2/M phase of cell cycle