MIB- Lecture 8 - 7/11 - Intracellular trafficking and survival

EHEC

• pathogenic strain of e. coli → entero-haemorrhagic E. Coli

• causes gastro-intestinal disease with bloody diarrhea

  • Epithelial cells are changed upon infection

  • loss of microvilli

  • Locus of enterocyte effacement (LEE) → encodes for type III secretion system

• intimin (adhesin) → binds Tir receptor

• Translocated Intimin Receptor (Tir) → inserted via type 3 secretion → phosphorylated;

  • recruits cytoskeleton proteins

  • nucleation of actin fibers

  • pedestal is formed

• EHEC wants to adhere to epithelial cells, washed away less easily

function T3SS

• needle like-complex → inserts proteins into host cell

• It secretes effector proteins → directly from bacterial cytoplasm in the cytoplasm of the host

• Effector proteins: interfere in host-cell processes → signal transduction cascades (virulence factors)

Actin polymerization via NFPs

• NFP= Nucleation promoting factors → WASP, N-WASP, WAVEs

• Activated by the small GTPases CDC42 and Rac1

• The NPFs activate Arp2/3 which results in the formation of a branched actin network

• Tir interacts with ISRp53 → bypassing required host signalling. this results in actin being able to be made, resulting in the pedestal being formed

advantages and disadvantages of life on the inside of cells

• Advantages

  • Shielded from humoral antibodies

  • Less competing bacteria for the same resources

• disadvantages

  • Endocytosed bacteria are on route to the lysosome

  • Iron limitation

  • Antigen presentation on MHCII

  • in close contact with innate immune receptors

Salmonella and the 2 different pathogenicity islands

• T3SS used to inject molecules into host to induce uptak

• then the salmonella will enter in a vacuole

  • avoid degradation

  • avoid recognition

• salmonella has 2 different type 3 secretion systems (needs both)

• Two pathogenicity islands → SPI-1 and SPI-2

• Spi-1

  • Encodes for several bacterial proteins for secretion system and effector protein

  • Effectors translocate into host cells to mediate triggered bacterial uptake

• Spi-2

  • Encodes for several bacterial proteins for secretion system and effector protein o

  • Effectors trans-locate into host cells to mediate intracellular survival

molecular pathway how Salmonella enters the host cell

• SopE: Activates Rho GTPases directly (like Rac1 and Cdc42), which drive actin polymerization. This process forms protrusions on the host membrane, facilitating bacterial entry.

• SipA: Stabilizes actin filaments to strengthen these protrusions.

• SopB: activates GTPases indirectly via inositol phosphate metabolism, further modifying the actin structure.

• when inside SopE is inactivated by SptP

Small GTPases

• molecular switches that can be on or off

• On actin there are Ras-like proteins → Rho

• Ras-like proteins can be swithed on and of by GTP → it is on when it is bound by GTP

  • GAP= GTPase de-activating protein → switched it of by releasing the GTP (changing GTP to GDP) from the protein

  • GEF= guanine exchange factor → Switches the protein on by binding GTP

• NPF = nucleating promoting factor → is a ras-like protein and can be switch on and off → it is functional when it is switched on

  • NPF only on when GTPase is in on phase

explain the roles of SopE and SptP in entering the host cell

• Spi-1 entry into cells via actin polymerization → regulated by SopE

• SopE is a GEF → switches on → actin polymerization

• SopE is regulating the activation of the NPF → switching it on results in the actin formation for cell entry

  • you also need to turn SopE off otherwise the cell would die

• SptpP turns the NPF off

• SopE and SptP are injected simultaneously → so what regulated this?

  • SopE has higher affinity, they both compete for the same protein. Because SopE is switched off earlier (has a shorter half life) it can do its job and then it dies off and SptP can do its job

  • Spi-1 effector sopE is selectively degraded because it contains lysines → it is degraded by ubiquitination → so the actin polymerization is inhibited

  • SopE turn on is activating the actin polymerization - SptP inhibits the actin polymerization

Salmonella containing vacuole

• Salmonella secretes SopB → positive feedback loop

  • SopB activated PtdIns3P which activates Rab5

• SopB → recruitment of Rab5 and production of PI(3)P this is an early endosomal phosphoinositide

• Without Rab5 and PI(3) → no progression to late endosome

• being an early endosome is not ideal for long term →

  • type 3 secretion only activated when it is acidified

  • lower pH needed for second secretion system

salmonella avoiding degradation, Salmonella SPI-2

• Salmonella needs to fuse with lysosome → there are a lot of things that degrade in this

  • SPI-2 is activated at an acidic pH

  • SPI-2 secreted effectors regulate membrane recruitment containing vacuole for growth nutrients and avoidance of degradation -

  • SPI-2 secreted effector down regulate MHCII to prevent detection

  • SPI-2 encodes a Type III secretion system → required for the survival in epithelial cells and macrophages

How does salmonella avoid degradation, Rab 5, 7 and 9

• Salmonella interferes with transport route to the golgi of the lysosomes

  • Rab9 → the function is transport proteins from the trans golgi network to late endosomes

• Rab9 is essential for delivery of cathepsins to lysosomes from the Golgi

• Cathepsins are proteases responsible for protein degradation in lysosomes

• Salmonella SifA binds and thus inactivates the proteins SKIP and Rab9(GTP) → reducing lysosomal degradation

• intracellular trafficking

  • Early to late endosome → Rab5

  • Late endosome to lysosome → Rab7

• FYCO1 and RILP are Rab7 effectors regulating lysosome formation

• Salmonella SopD2 binds with Rab7 (then FYCO1 and RILP can not bind anymore) effector recruitment → no lysosomes can be formed

how is autophagy prevented - salmonella

• salmonella is flagged with ubiquitin

• autophagosome is formed, this autophagosome is tagged with LC3 → surrounds salmonella and fuses with lysosome to degrade it

• Salmonella escapes this by secreting SseL → this enzyme removes ubiquitin → preventing the autophagy

Avoiding recognition (salmonella)

• MHC-II antigen presentation → causes the recognition of Salmonella

• Avoiding recognition: Salmonella SPI-effector SteD (protein) reduces surface MHC-II

  • It activates MARCH8 and this adds ubiquitinate to MHC-II and causes the degradation

Listeria monocytogenes

• gram positive bacteria causes listeriosis. symptoms; fever, muscle pain, headache, stomach - gastrointestinal problems

• It can grow and reproduce itself inside the host cells → virulent foodborne pathogens

• Efficient in placental invasion and fetal infection

• It uses Zipper to enter the host → via host surface proteins

Internalization and spread of listeria monocytogenes

• It enters via zipper mechanism; uses receptors on host cells to enter → InIA and InIB trigger endocytosis

• InlA important for the internalization of listera via E. cadherin

  • First it ubiquitinates E-cadherin

  • It recruits Clathrin mediated machinery → so it can start endocytosis

  • Then it remodels actin via Arp2/3 and endocytosis starts

• InlB is important for the internalization of Listeria via Met receptor

  • expressed in brain and placenta

  • It ubiquitinates the Met receptors

  • It recruits Clathrin mediated machinery → so it can start endocytosis

  • Then it remodels actin (Arp2/3) and endocytosis starts

• LLO (produced by listeria) → allows listeria to escape vacuole into host cell cytosol. listeria makes ActA → recruits Arp2/3 → produces actin polymerization → tail behind bacteria → bacteria propel through cytoplasm → listeria is pushed into neighbouring cells

Escape into cytosol advantages and disadvantages

advantages;

• shielded from humoral antibodies

• less competing bacteria for resources

• nutritional heaven

disadvantages

• autophagy

• innate immune system

How does Listeria avoid lysosomal degradation (escape from phagosome)

• secretion of LLO, a pore forming protein

• secretion of bacterial phospholipase A and B (PlcA, PlcB)

• LLO makes pore in membrane needs cholesterol, bacteria also have a membrane, bacteria are not a target because they do not have cholesterol

how do salmonella, EHEC, listeria all manipulate ARP2/3 complex

• TIR → important for initiating actin polymerization at the site of bacterial entry

• SopE (salmonella) → activates Rac/CDC42 signaling pathway → leads to actin polymerization via N-WASP → drives internalization of salmonella

• ActA (listeria) → this protein produced by listeria mimics WAVE complex → activating ARP2/3 → leads to actin polymerization

• m marinum also uses actin tails to spread around cells

Escape autophagy - Listeria

• Listeria prevents the binding of autophagy adapter molecules

• InIK (produced by listeria) binds MVP (major vault protein) this prevents the recruitment of autophagy adaptor proteins (P62). no recruitment. no formation of phagophore

• p62 is necessary to link ubiquitinated pathogens to autophagic machinery (LC3 protein) for degradation

overview slide