Agaisse, 2016 (Molecular and Cellular Mechanisms of Shigella flexneri Dissemination [Review]

Why is cell-to-cell spread critical for S. flexneri pathogenesis?

“Spreading defective but otherwise fully invasive bacterial strains are essentially avirulent, highlighting the ability of the bacteria to spread from cell to cell as a central determinant of S. flexneri pathogenesis..” (Agaisse, 2016)

What is the causative agent of bacillary dysentery and what characterizes the disease process?

"The intracellular pathogen Shigella flexneri is the causative agent of bacillary dysentery in humans. The disease is characterized by bacterial invasion of intestinal cells, dissemination within the colonic epithelium through bacterial spread from cell to cell, and massive inflammation leading to destruction of the epithelial mucosa." (Agaisse, 2016)

How does IcsA initiate actin-based motility?

IcsA is a member of the autotransporter family of proteins, which display a carboxy-terminal beta domain that forms a beta barrel channel and mediates the transfer of the (passenger) amino-terminal alpha domain across the outer membrane. The exposure of the passenger domain of IcsA to the cytosol leads to the recruitment of the nucleation-promoting factor N-WASP." (Agaisse, 2016)

How does IcsA interact with N-WASP specifically?

"IcsA interacts specifically with N-WASP, but not with other N-WASP/WAVE family members, such as WASP... Chimera experiments using WASP and N-WASP revealed that N-WASP binds IcsA through its GTPase-binding domain that normally binds Cdc42... IcsA acts as a functional mimic of Cdc42, releasing N-WASP from its auto-inhibitory conformation." (Agaisse, 2016)

What cellular factors are required for protrusion formation?

"In order to form membrane protrusions, S. flexneri utilizes the forces developed by the actin cytoskeleton to deform the plasma membrane and project into adjacent cells... Diaphanous-related formins have been identified as important determinants of protrusion formation in S. flexneri infected cells... Myosin X was recently found to localize to the plasma membrane surrounding S. flexneri protrusions and move along the bacterial sides." (Agaisse, 2016)

How does Myosin X contribute to protrusion formation?

"Myosin X depletion resulted in the formation of shorter and wider protrusions... Structure function analyses suggested that Myosin X contributes to S. flexneri protrusion formation by bridging the actin cytoskeleton and the plasma membrane, and potentially transporting unknown cargoes within protrusions." (Agaisse, 2016)

What characterizes VLP formation?

"VLPs display a continuous lining of the plasma membrane around the bacterium, but remain connected to the primary infected cells through a membranous tether. The tether results from the complete collapse of the protrusion neck, which likely reflects the collapse of the underlying actin cytoskeleton." (Agaisse, 2016)

What signaling pathways are required for VLP formation?

"The plasma membrane surrounding the protrusions formed by S. flexneri is highly enriched in phosphorylated tyrosine residues... S. flexneri dissemination through protrusion collapse and VLP formation also requires the production of phosphatidylinositol-3-phosphate (PI(3)P) in protrusions, which relies on the class II phosphatidylinositol-3 kinase PIK3C2A." (Agaisse, 2016)

What evidence shows T3SS involvement in protrusion resolution?

"The first evidence of a role for the bacterial T3SS in the resolution of S. flexneri protrusions into vacuoles came from the isolation of a transposon in the ipgC gene... The corresponding ipgC mutant grew normally in the cytosol of infected cells, where it displayed actin-based motility, but failed to spread from cell to cell." (Agaisse, 2016)

How is T3SS activity regulated during infection?

"T3 secretion is activated upon invasion, shut down in the cytosol, and re-activated in protrusions and vacuoles... When the T3SS is not active, the interaction of OspD1 with MxiE prevents transcriptional activation of the MxiE-regulated genes. As the T3SS system gets activated in protrusions or vacuoles, probably upon insertion of the translocases into the plasma membrane, the secretion of OspD1 frees up MxiE." (Agaisse, 2016)

What effector proteins are involved in vacuole escape?

"The VirA and IcsB mutants accumulate in double membrane vacuoles... The function of IcsB is unknown, but several activities have been attributed to VirA, including inhibition of the small-GTPase Rab1, and activation of the Calpain system, through an unknown mechanism... As the single VirA and IcsB mutants hardly displayed plaque formation defects, and the double mutant did form smaller plaques, it is likely that cooperative activities provided by various effector proteins contribute to double membrane vacuole escape." (Agaisse, 2016)

How does LPS affect S. flexneri dissemination?

"LPS-defective mutants are not able to form polar actin tails and instead polymerize actin circumferentially on the bacterial surface... IcsA localized circumferentially in O antigen defective strains, suggesting that LPS supports the maintenance of IcsA at the bacterial pole by restricting its lateral diffusion." (Agaisse, 2016)

What is the role of DnaK and FtsQ in IcsA regulation?

"Genetic screens uncovered DnaK and FtsQ as regulators of IcsA cytoplasmic chaperoning and positioning at the bacterial poles, respectively." (Agaisse, 2016)

How do periplasmic proteins affect IcsA function?

"In the periplasm, the protease/chaperone DegP functions in partnership with the chaperones Skp and SurA and mediates full exposure of the alpha domain of IcsA at the bacterial surface." (Agaisse, 2016)

What is the function of IcsP and how does it affect dissemination?

"The serine protease IcsP was proposed to contribute to the maintenance of IcsA polarity by releasing the N-terminal domain from the bacterial sides... compared to wildtype bacteria, the icsP mutant was indeed more efficient at moving in the cytosol and spreading from cell to cell, indicating that the IcsP-mediated cleavage of IcsA is in fact detrimental to S. flexneri dissemination." (Agaisse, 2016)

What is OspE2's role in cell-cell contact?

"A mutant strain defective in the expression of OspE2, a type 3 secretion effector protein injected into host cells, leads to premature rounding up of infected cells, a reflection of cell-cell contact disruption." (Agaisse, 2016)

What is VpsC and how was it identified?

"VpsC was identified as a determinant of S. flexneri dissemination in a screen for bacterial factors required for plaque formation. VpsC is part of the Vps/VacJ transporter." (Agaisse, 2016)

How does the Pst system affect dissemination?

"Mutants in the Pst system formed smaller plaque compared to wild type. The spreading defect was not related to PhoA-mediated phosphate transport, but involved the de-regulation of the PhoB regulon, which did not impact IcsA polarity or bacterial growth, at least during the first 3 h of infection." (Agaisse, 2016)

What is the role of MxiE in regulating effector proteins?

"The transcriptional activator MxiE regulates the expression of a subset of T3SS effector proteins... MxiE is regulated by the activity of the T3SS through a complex mechanism involving the chaperone IpgC, acting as MxiE co-activator, and the secreted effector OspD1, acting as MxiE inhibitor." (Agaisse, 2016)

How is IpgC involved in T3SS regulation?

"IpgC gene... encodes a molecular chaperone for IpaB and IpaC... treatment with the tyrosine kinase inhibitor Gleevec inhibited S. flexneri dissemination." (Agaisse, 2016)

What is the role of VirB in bacterial spread?

"In addition to the main transcriptional regulators of S. flexneri virulence, VirB and VirF... As opposed to N-WASP, the recruitment of TOCA-1 does not rely on IcsA, but requires the integrity of the transcriptional regulator VirB. This suggests a role for VirB-regulated virulence factors, including the T3SS, in TOCA-1 recruitment." (Agaisse, 2016)

What is MxiG's role in bacterial spread?

"Two independent studies demonstrated a role for MxiG, a structural component of the T3SS, in the resolution of protrusion into vacuoles... MxiG was shown to be required for activation of tyrosine kinase signaling in S. flexneri protrusions." (Agaisse, 2016)

How has gene loss contributed to S. flexneri virulence?

"It is noteworthy that outer membrane proteins detrimental to IcsA expression in E. coli, such as OmpT, are no longer encoded in S. flexneri spp. genomes. Deletion of 'anti-virulence' genes has been proposed to be one of the major determinants in the evolution of S. flexneri pathogenic properties, including the acquisition of IcsA-mediated actin-based motility." (Agaisse, 2016)