A Shigella flexneri Virulence Plasmid Encoded Factor Controls Production of Outer Membrane Vesicles

Abstract

Shigella spp. use a repertoire of virulence plasmid-encoded factors to cause shigellosis. These include components of a Type III Secretion Apparatus (T3SA) that is required for invasion of epithelial cells and many genes of unknown function. We constructed an array of 99 deletion mutants comprising all genes encoded by the virulence plasmid (excluding those known to be required for plasmid maintenance) of Shigella flexneri. We screened these mutants for their ability to bind the dye Congo red: an indicator of T3SA function. This screen focused our attention on an operon encoding genes that modify the cell envelope including virK, a gene of partially characterized function. We discovered that virK is required for controlled release of proteins to the culture supernatant. Mutations in virK result in a temperature-dependent overproduction of outer membrane vesicles (OMVs). The periplasmic chaperone/protease DegP, a known regulator of OMV production in Escherichia coli (encoded by a chromosomal gene), was found to similarly control OMV production in S. flexneri. Both virK and degP show genetic interactions with mxiD, a structural component of the T3SA. Our results are consistent with a model in which VirK and DegP relieve the periplasmic stress that accompanies assembly of the T3SA.

Molecular and Cellular Mechanisms of Shigella flexneri Dissemination

“…However, enzymatic manipulation of phospholipid composition aiming at restoring normal levels of phospholipids in the outer membrane, failed to correct the plaque formation defect observed with the vpsC mutant (Hong et al, 1998 ). Interestingly, the VpsC mutant secretes elevated levels of the virulence factors IcsA, IpaB, IpaC, and IpaD (Hong et al, 1998 ) and a similar hypersecretion phenotype was reported recently for the virK mutant (Sidik et al, 2014 ). The exact function of VirK is unknown, but hypersecretion is apparently due to an increase in the release of outer membrane vesicles (OMVs).…”

“…Moreover, a similar increase in OMV release was reported for a mutant in the periplasmic chaperone DegP. As both virK and degP showed genetic interactions with mxiD , a structural component of T3SS, it was proposed that VirK and DegP may relieve the periplasmic stress associated with the assembly of the T3SS (Sidik et al, 2014 ). Also required for the proper assembly of the T3SS is the periplasmic thiol:disulfide oxidoreductase DsbA.…”

Shigella Infection Interferes with SUMOylation and Increases PML-NB Number

“…In all experiments, strains of S. flexneri bearing a plasmid encoding the gene afaE [ 55 ] were grown to mid-log phase and used to infected HeLa cells (obtained from ATCC CCL-2) at MOIs of 9.5. To ensure that Shigella entered HeLa cells we performed gentamycin protection assays in parallel to the samples that were used for analysis of proteins according to the methods described in Sidik[ 56 ]. We observed that when HeLa cells were treated with 10 μM MG132 for one hour prior to infection, Shigella entered with equal efficiency as those that were untreated.…”

A new form of actin assembly around theShigella-containing vacuole regulates its intracellular niche

“…To identify which Shigella effectors are involved in actin cocoon regulation, we screened a Shigella mutant library with single T3SS effector deletions (Sidik et al 2014). Results of nine selected mutants are presented ( Figure 5).…”

“…The following streptomycin-resistant Shigella flexneri 5a M90T-Sm (GenBank #CM001474.1) derived strains harboring the pWR100 virulence plasmid (GenBank #AL391753.1) were used: wild type M90T (WT), M90T (WT) expressing the protein afimbrial adhesin Afa-I (afaE1 gene) from E. coli (Labigne-Roussel et al, 1984), M90T (WT) expressing dsRed and AfaI (Yuen-Yan Chang), and M90T ΔicsB-2 (FXS299) (Claude Parsot). The screened library of S. flexneri single deletion T3SS effector mutants was kindly provided by John R. Rohde (Dalhousie University) as part of a pwR100 collection originating from M90T (WT) AfaI (Sidik et al, 2014). We used Salmonella enterica serovar Typhimurium (S. Typhimurium) strain SL1344 pGG2-dsRed (WT) expressing dsRed.…”