The Serratia marcescens hemolysin is secreted but not activated by stable protoplast-type L-forms of Proteus mirabilis

Abstract

The outer-membrane protein ShlB of Serratia marcescens activates and secretes hemolytic ShlA into the culture medium. Without ShlB, inactive ShlA (termed ShlA*) remains in the periplasm. Since Proteus mirabilis L-form cells lack an outer membrane and a periplasm, it was of interest to determine in which compartment recombinant ShlA* and ShlB are localized and whether ShlB activates ShlA*. The cloned shlB and shlA genes were transcribed in P. mirabilis stable L-form cells by the temperature-inducible phage T7 RNA polymerase. Radiolabeling, Western blotting, and complementation with C-terminally truncated ShlA (ShlA255) identified inactive ShlA* in the culture supernatant. ShlB remained cell-bound and did not activate ShlA without integration in an outer membrane. Although hemolytic ShlA added to L-form cells had access to the cytoplasmic membrane, it did not affect L-form cells. Synthesis of the large ShlA protein (165 kDa) in P. mirabilis L-form cells under phage T7 promoter control demonstrates that L-form cells are suitable for the synthesis and secretion of large recombinant proteins. This property and the easy isolation of released proteins make L-form cells suitable for the biotechnological production of proteins.

The Family of Serratia Type Pore Forming Toxins

“…Inactive ShlA* is secreted by the L-form cells, and ShlB is associated with the cytoplasmic membrane. Addition of hemolytic ShlA to the L-form cells has no effect, which suggests that the prokaryotic cytoplasmic membrane is resistant to ShlA [42]. Apparently, ShlA need an eukaryotic undefined factor for pore formation which is lacking in prokaryotic membranes.…”

“…These data implicate an explanation for the resistance of prokaryotic membranes against ShlA. Serratia and Proteus strains exhibit up to 90 % PE in the outer, and about 75 % PE in the inner membrane [42] but no negative charge in the form of PS rendering these organisms resistant to ShlA treatment. The mechanism of pore formation is not well understood, but one can speculate that the negatively charged PS-heads on the inside of eukaryotic membranes my play an essential role in the orientation and stabilization of the ShlA pore.…”

“…Activation during secretion across the outer membrane by ShlB could be a means to protect the bacterial producer cells. To test this hypothesis, stable protoplast-type L-forms of Proteus mirabilis that do not contain an outer membrane were transformed with a plasmid carrying the shlA shlB genes [42]. Inactive ShlA* is secreted by the L-form cells, and ShlB is associated with the cytoplasmic membrane.…”

Cytotoxic Action of Serratia marcescens Hemolysin on Human Epithelial Cells

“…Since ShlA lyses all mammalian erythrocytes tested, there is no evidence for a high-affinity binding site. However, the cytoplasmic membranes of gram-negative prokaryotes are not affected by ShlA (40).…”

Activation of Serratia marcescens Hemolysin through a Conformational Change

“…It also acts as a cytotoxin on epithelial cells and fibroblasts, where it causes ATP depletion and potassium efflux (15). The ShlA toxin is secreted across the outer membrane by the ShlB protein, encoded by the shlB gene (7,19,24,27,30), thereby distinguishing this type of secretion from all other known secretion systems (3,4). Homologous hemolysins are formed by Proteus mirabilis (33), Yersinia pestis (23), Haemophilus ducreyi (22,32), and Edwardsiella tarda (10,17,31).…”