The Vps/VacJ ABC Transporter Is Required for Intercellular Spread of Shigella flexneri

Abstract

The Vps/VacJ ABC transporter system is proposed to function in maintaining the lipid asymmetry of the outer membrane. Mutations in vps or vacJ in Shigella flexneri resulted in increased sensitivity to lysis by the detergent sodium dodecyl sulfate (SDS), and the vpsC mutant showed minor differences in its phospholipid profile compared to the wild type. vpsC mutants were unable to form plaques in cultured epithelial cells, but this was not due to a failure to invade, to replicate intracellularly, or to polymerize actin via IcsA for movement within epithelial cells. The addition of the outer membrane phospholipase gene pldA on a multicopy plasmid in a vpsC or vacJ mutant restored its resistance to SDS, suggesting a restoration of lipid asymmetry to the outer membrane. However, the pldA plasmid did not restore the mutant's ability to form plaques in tissue culture cells. Increased PldA levels also failed to restore the mutant's phospholipid profile to that of the wild type. We propose a dual function of the Vps/VacJ ABC transporter system in S. flexneri in both the maintenance of lipid asymmetry in the outer membrane and the intercellular spread of the bacteria between adjacent epithelial cells. Shigella flexneri is the causative agent of bacillary dysentery in humans. The bacteria invade the colonic epithelium, replicate intracellularly, and spread cell to cell, provoking the acute inflammatory response that is characteristic of the disease (1). While much is known about the initial invasion of epithelial cells by Shigella, less is understood about the requirements for intracellular replication and intercellular spread.Many of the genes required for pathogenesis of S. flexneri are carried on a large plasmid (2, 3). Plasmid-encoded virulence factors include the invasion plasmid antigen (Ipa) effector proteins required for S. flexneri to enter the host cell and escape from the vacuole (4-6). These effector proteins are secreted by a type III secretion system (TTSS) that is also encoded on the virulence plasmid. When the effectors contact epithelial cells, they induce cytoskeletal changes leading to internalization of the bacteria (7,8). Once engulfed by the epithelial cell, the bacteria lyse the vacuole and replicate inside the cytosol of the host cell (6).Within the host cell cytoplasm, S. flexneri uses another virulence plasmid-encoded protein, IcsA, to polymerize actin at one pole of the cell, resulting in the propulsion of the bacteria throughout the host cell (9, 10). This actin-based motility also promotes movement of the bacteria into adjacent epithelial cells. Intercellular movement results in S. flexneri cells being surrounded by a double membrane, one from the cell which the bacteria are exiting and another from the cell which they are entering (11). The secreted effector proteins are required for the bacteria to escape this double-membrane-bound vacuole, and once free in the cytoplasm, the bacteria repeat the cycle of replication and intercellular spread (12, 13).An understanding of the mechanisms of invas...

How Do the Virulence Factors of Shigella Work Together to Cause Disease?

“…VacJ is also encoded on the chromosome, and a vacJ − knockout is incapable of escaping into the recipient cell cytoplasm, suggesting that VacJ also plays a role in intercellular spread (Suzuki et al, 1994). Carpenter et al (2014) describe a Vps/VacJ ABC transporter, which maintains asymmetry of lipids in the outer membrane and in the context of Shigella infection is required for lysis of the double membrane vacuole. Transformation of vps / vacJ knockouts with a plasmid expressing pldA , a phospholipase in other Gram negative bacteria, was able to restore the maintenance of outer membrane lipid asymmetry but was unable to lyse the double membrane vacuole, indicating that these two functions of the proposed Vps/VacJ ABC transporter are separate (Carpenter et al, 2014).…”

“…Carpenter et al (2014) describe a Vps/VacJ ABC transporter, which maintains asymmetry of lipids in the outer membrane and in the context of Shigella infection is required for lysis of the double membrane vacuole. Transformation of vps / vacJ knockouts with a plasmid expressing pldA , a phospholipase in other Gram negative bacteria, was able to restore the maintenance of outer membrane lipid asymmetry but was unable to lyse the double membrane vacuole, indicating that these two functions of the proposed Vps/VacJ ABC transporter are separate (Carpenter et al, 2014). Another substrate may therefore be transported across the membrane to induce vacuolar lysis, however this is yet to be discovered.…”

Genome‐wide identification of tolerance mechanisms toward p‐coumaric acid in Pseudomonas putida

“…VacJ has been characterized as an outer membrane protein associated to an ABC transporter system in other organisms, such as E. coli and Shigella flexneri , in which it has been proposed to be involved in the maintenance of outer membrane stability in the presence of membrane disruptors such as SDS (Carpenter et al, 2014; Malinverni & Silhavy, 2009). Its deletion has been shown to increase permeability of the outer membrane (Malinverni & Silhavy, 2009) and increase the formation of vesicles in E. coli and other gram‐negative bacteria (Roier et al, 2016).…”

The Mla pathway is critical for Pseudomonas aeruginosa resistance to outer membrane permeabilization and host innate immune clearance

“…The Mla system is involved in maintaining OM lipid asymmetry and is comprised of seven proteins that are distributed across the cell envelope [6, 7]. VacJ (also known as MlaA) and other components of the Mla pathway have been shown to play a role in intracellular spread of Shigella flexneri [8, 9], for survival of Haemophilus influenzae during lung infections and in serum [10, 11], resistance to surfactants in Escherichia coli [6, 12, 13] and Pseudomonas putida [14], antibiotic resistance in P. aeruginosa [15, 16] and Salmonella enterica serovar Typhimurium [17], and has a possible role in the creation of outer membrane vesicles [18]. VacJ has also been explored as a possible vaccine target in Pasteurella multocida [19].…”