Role of the cell wall microenvironment in expression of a heterologous SpaP-S1 fusion protein by Streptococcus gordonii

Abstract

The charge density in the cell wall microenvironment of Gram-positive bacteria is believed to influence the expression of heterologous proteins. To test this, the expression of a SpaP-S1 fusion protein, consisting of the surface protein SpaP of Streptococcus mutans and a pertussis toxin S1 fragment, was studied in the live vaccine candidate bacterium Streptococcus gordonii. Results showed that the parent strain PM14 expressed very low levels of SpaP-S1. By comparison, the dlt mutant strain, which has a mutation in the dlt operon preventing d-alanylation of the cell wall lipoteichoic acids, and another mutant strain, OB219(pPM14), which lacks the LPXTG major surface proteins SspA and SspB, expressed more SpaP-S1 than the parent. Both the dlt mutant and the OB219(pPM14) strain had a more negatively charged cell surface than PM14, suggesting that the negative charged cell wall played a role in the increase in SpaP-S1 production. Accordingly, the addition of Ca(2+), Mg(2+), and K(+), presumably increasing the positive charge of the cell wall, led to a reduction in SpaP-S1 production, while the addition of bicarbonate resulted in an increase in SpaP-S1 production. The level of SpaP-S1 production could be correlated with the level of PrsA, a peptidyl-prolyl cis/trans isomerase, in the cells. PrsA expression appears to be regulated by the cell envelope stress two-component regulatory system LiaSR. The results collectively indicate that the charge density of the cell wall microenvironment can modulate heterologous SpaP-S1 protein expression in S. gordonii and that this modulation is mediated by the level of PrsA, whose expression is regulated by the LiaSR two-component regulatory system.

FIG. 1.

SpaP-S1 produced by S. gordonii PM14, the dlt mutant, and OB219(pPM14). (A) Western immunoblot of total cell extracts showing the full-length SpaP-S1 (arrow) and smaller-sized immunoreactive bands, presumably degradation products. (B) SDS-polyacrylamide gel (6.5%) showing equal loading of proteins in the samples. Lanes: 1, PM14; 2, dltA mutant; 3, OB219(pPM14); 4, DL1. DL1 does not carry pPM14 and thus does not produce the SpaP-S1 protein. Numbers below the blot indicate the fold increases in SpaP-S1 produced by the mutants compared to the level produced by PM14.

FIG. 2.

SpaP-S1 anchored to the cell wall of PM14, the dlt mutant, and OB219(pPM14). (A) SDS-polyacrylamide gel (7.5%) of proteins from cell walls before hot-SDS extraction. Each sample is derived from the same quantity of cell wall treated with mutanolysin, as described in Materials and Methods. (B) Western blot of SpaP-S1 in cell wall samples shown in panel A. (C) Western blot of SpaP-S1 on residual cell wall after hot-SDS extraction. Lanes: M, prestained protein markers (New England BioLabs, Mississauga, ON, Canada); 1, PM14; 2, dlt mutant; 3, OB219(pPM14). The arrow indicates the full-length SpaP-S1 protein. The lower-mass immunoreactive bands are presumably degradation products.

FIG. 3.

Effects of cations and bicarbonate on SpaP-S1 expression. (A) Immunoblot of SpaP-S1 produced by OB219(pPM14) following growth in the presence of Ca²⁺ or Mg²⁺ at the indicated concentrations. 0, no added Ca²⁺ or Mg²⁺. (B) Immunoblot of SpaP-S1 produced by OB219(pPM14) following growth in the presence of K⁺ at the indicated concentrations. 0, no addition. (C) Immunoblot of SpaP-S1 produced by S. gordonii in the presence (+) or absence (−) of 50 mM bicarbonate. Lanes: 1, PM14; 2, dlt mutant; 3, OB219(pPM14); 4, DL1. Numbers below the blots are percentages or fold changes in SpaP-S1 compared to the level for the no-addition control. Arrows indicate the full-length SpaP-S1 protein. The samples were also analyzed on 6.5% SDS-polyacrylamide gels to ensure equal loading of proteins (data not shown).

FIG. 4.

PrsA and SpaP-S1 expression by S. gordonii. (A) Immunoblot of PrsA produced by PM14 (lane 1), OB219(pPM14) (lane 2), and the dltA mutant (lane 3). (B) Immunoblots of PrsA (left) and SpaP-S1 (right) produced by RJM4 (lane 1) and the prsA mutant (lane 2). (C) Immunoblots of PrsA (left) and SpaP-S1 (right) produced by RJM4 (lane 1) and RJM4 carrying pSgPL2 (lane 2). (D) Immunoblots of PrsA (left) and SpaP-S1 (right) produced by RJM4 (lane 1) and the liaR mutant (lane 2). Arrows indicate the full-length SpaP-S1 protein, and asterisks indicate PrsA. The samples were also analyzed on 6.5% or 12.5% SDS-polyacrylamide gels to ensure equal loading of proteins (data not shown).