Abstract
Outer membrane blebs are naturally shed by Gram-negative bacteria and are candidates of interest for vaccines development. Genetic modification of bacteria to induce hyperblebbing greatly increases the yield of blebs, called Generalized Modules for Membrane Antigens (GMMA). The composition of the GMMA from hyperblebbing mutants of Shigella flexneri 2a and Shigella sonnei were quantitatively analyzed using high-sensitivity mass spectrometry with the label-free iBAQ procedure and compared to the composition of the solubilized cells of the GMMA-producing strains. There were 2306 proteins identified, 659 in GMMA and 2239 in bacteria, of which 290 (GMMA) and 1696 (bacteria) were common to both S. flexneri 2a and S. sonnei. Predicted outer membrane and periplasmic proteins constituted 95.7% and 98.7% of the protein mass of S. flexneri 2a and S. sonnei GMMA, respectively. Among the remaining proteins, small quantities of ribosomal proteins collectively accounted for more than half of the predicted cytoplasmic protein impurities in the GMMA. In GMMA, the outer membrane and periplasmic proteins were enriched 13.3-fold (S. flexneri 2a) and 8.3-fold (S. sonnei) compared to their abundance in the parent bacteria. Both periplasmic and outer membrane proteins were enriched similarly, suggesting that GMMA have a similar surface to volume ratio as the surface to periplasmic volume ratio in these mutant bacteria. Results in S. flexneri 2a and S. sonnei showed high reproducibility indicating a robust GMMA-producing process and the low contamination by cytoplasmic proteins support the use of GMMA for vaccines. Data are available via ProteomeXchange with identifier PXD002517.
Highlights
Shigellae are Gram-negative enterobacteria classified into fifty different serotypes based on the carbohydrate composition of the outer polysaccharide antigen (O antigen, OAg) of the lipopolysaccharide (LPS) (Levine et al, 2007)
In this study we used previously described hyperblebbing O antigen-deficient strains of S. sonnei 53G and S. flexneri 2a 2457T that were cured of the virulence plasmid: S. sonnei −p tolR (Berlanda Scorza et al, 2012) and S. flexneri 2a −p tolR rfbG (Rossi et al, 2014)
Analysis of solubilized whole cells and Generalized Modules for Membrane Antigens (GMMA) from the O antigen- and plasmid-negative S. flexneri 2a 2457T −p tolR rfbG (Sf2a Lysate and Shigella flexneri 2a 2457T (Sf2a) GMMA respectively), and solubilized whole cell and GMMA from the O antigen and plasmid-negative S. sonnei 53G −p tolR (Ss Lysate and Ss GMMA respectively) identified 2306 unique protein groups with an estimated abundance based on iBAQ quantification (Table S1, listed by the protein with the highest abundance in the protein group)
Summary
Shigellae are Gram-negative enterobacteria classified into fifty different serotypes based on the carbohydrate composition of the outer polysaccharide antigen (O antigen, OAg) of the lipopolysaccharide (LPS) (Levine et al, 2007). The composition of NOMV reflects the composition of the outer membrane of the donor bacteria, an important interface between the bacterial cell and its environment, and outer membrane blebs represent ideal candidates for vaccine development (Ellis and Kuehn, 2010) Blebs present these antigens in the natural membrane context and in the presence of powerful stimulators of the innate immune system (Ferrari et al, 2006; Park et al, 2011; Ellis et al, 2010) and elicit protection in animal models against bacterial infections (Alaniz et al, 2007; Schild et al, 2008; Park et al, 2011), including Shigella (Camacho et al, 2011; Mitra et al, 2013). It is possible to induce high level shedding of blebs (“gemma” in Italian), called Generalized Modules for Membrane Antigens (GMMA) (Berlanda Scorza et al, 2012), that together with high-yield, industrial processes form the bases for a practical vaccine platform (Berlanda Scorza et al, 2012; Gerke et al, 2015)
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