IpaD of Shigella flexneri is independently required for regulation of Ipa protein secretion and efficient insertion of IpaB and IpaC into host membranes

Abstract

Shigella flexneri causes human dysentery after invading the cells of the colonic epithelium. The best-studied effectors of Shigella entry into colonocytes are the invasion plasmid antigens IpaC and IpaB. These proteins are exported via a type III secretion system (TTSS) to form a pore in the host membrane that may allow the translocation of other effectors into the host cytoplasm. TTSS-mediated secretion of IpaD is also required for translocation pore formation, bacterial invasion, and virulence, but the mechanistic role of this protein is unclear. IpaD is also known to be involved in controlling Ipa protein secretion, but here it is shown that this activity can be separated from its requirement for cellular invasion. Amino acids 40 to 120 of IpaD are not essential for IpaD-dependent invasion; however, deletions in this region still lead to constitutive IpaB/IpaC secretion. Meanwhile, a central deletion causes only a partial loss of control of Ipa secretion but completely eliminates IpaD's invasion function, indicating that IpaD's role in invasion is not a direct outcome of its ability to control Ipa secretion. As shigellae expressing ipaD N-terminal deletion mutations have reduced contact-mediated hemolysis activity and are less efficient at introducing IpaB and IpaC into erythrocyte membranes, it is possible that IpaD is responsible for insertion of IpaB/IpaC pores into target cell membranes. While efficient insertion of IpaB/IpaC pores is needed for optimal invasion efficiency, it may be especially important for Ipa-dependent membrane disruption and thus for efficient vacuolar escape and intercellular spread.

FIG. 1.

The IpaD deletion mutations are synthesized in S. flexneri SF622, and all but IpaD^Δ1-20 are secreted into the bacterial culture supernatant (CS). Overnight cultures were used to prepare culture supernatants and whole-cell extracts (WCE). The presence or absence of IpaD was then determined by immunoblot analysis. The antibodies used were a mixture of rabbit antisera (numbers 18, 20, and 23) generated against three different IpaD-derived peptides composed of amino acids 55 to 70, 102 to 115, and 280 to 295 (38) that were derived from different regions of the protein so that they could recognize all of the deletion mutations described here. The negative control for production of IpaD was S. flexneri SF622 harboring pWPsf4 (no ipaD insert), and the negative control for secretion was S. flexneri BS547 (lacking a functional TTSS). Positive controls for synthesis and secretion were wild-type S. flexneri M90T and SF622 harboring pWPsf4D (for expressing a wild-type copy of ipaD). The relative amount of IpaD either synthesized or secreted was roughly estimated on the basis of the intensity and size of the resulting IpaD bands. A nonspecific band present in the whole-cell extracts (indicated by the asterisk) provides a crude control for protein loading, while equal volumes of culture supernatant were loaded onto the culture supernatant lanes. The bands corresponding to IpaD^Δ321-332 and IpaD^Δ328-332 migrate slower than the other IpaD deletion mutant proteins because they have only 12 and 5 amino acids deleted, respectively (arrows). The complete experiment was performed in toto twice (with most parts being performed at least three times) with identical results.

FIG. 2.

Secretion of IpaC is increased in S. flexneri expressing mutant forms of ipaD. An ELISA was used to allow initial detection of IpaC quantities in the culture supernatants of S. flexneri strain SF622 expressing ipaD harboring different deletions. The primary antibody used here was a mouse monoclonal antibody that recognizes an epitope precisely mapped to the central hydrophilic region of IpaC (37). All analyses were performed in triplicate and are presented as an average ± standard deviation (n = 5) from one of two nearly equal experiments in which the upper limit with respect to linearity for the IpaC protein is approached but not exceeded. On the basis of rough standard curves with purified recombinant IpaC, all of the values observed for the detection of IpaC are within the linear range of detection by this ELISA system, although the higher IpaC values are near the upper range of linear detection. The asterisks indicate P values (Student t test) of ≤0.10.

FIG. 3.

SDS-PAGE analysis of IpaB and IpaC secreted into culture supernatants by SF622 expressing different ipaD deletion mutations. The strains of S. flexneri shown were grown overnight in TSB, and the proteins present in culture supernatants were trichloroacetic acid precipitated, separated on SDS-10% polyacrylamide gels, and stained with Coomassie blue. The level of secretion of IpaB and IpaC is compared to that for SF622 harboring pWPsf4 without an ipaC insert (designated SF622) and SF622 harboring pWPsf4D for expressing wild-type ipaD (IpaD). Wild-type S. flexneri M90T and S. flexneri BS547 (mxiM mutant lacking a functional TTSS) were also included as controls. The latter of these was performed in an experiment separate from the one with all of the deletion mutations but with SF622, IpaD, and M90T. The overall experiment was performed three times with identical results.

FIG. 4.

Kinetics of contact-mediated hemolysis by S. flexneri. A typical contact-mediated hemolysis reaction was performed, and the extent of hemoglobin release was monitored as a function of time (up to 60 min). The percentage of hemolysis was determined for wild-type S. flexneri M90T (filled triangles) or S. flexneri SF622 expressing ipaD (open circles), ipaD^Δ41-80 (closed squares), or ipaD^Δ81-120 (open squares). Negative controls included S. flexneri SF622 harboring pWPsf4 (no ipaD insert; filled circles) and S. flexneri BS547 (defective TTSS; open triangles). Complete hemolysis was determined by the addition of water. The values shown are the average of triplicates (± the standard deviation) and are from a representative experiment performed three times.

FIG. 5.

Influence of different-sized osmoprotectants on the ability for S. flexneri expressing different forms of ipaD to lyse RBC. A typical contact-mediated hemolysis reaction was performed in the presence of different osmoprotectants with wild-type S. flexneri M90T (open circles) or S. flexneri SF622 expressing ipaD (filled circles), ipaD^Δ41-80 (filled triangles), or ipaD^Δ81-120 (open triangles). Hemolysis in the absence of osmoprotectant is indicated by PBS, and all of the values shown are relative to complete hemolysis, such as that which occurs with the addition of water. The values shown are an average of triplicates ± the standard deviation (n = 3) from a single experiment performed three times.

FIG. 6.

IpaD controls the efficiency of IpaB and IpaC insertion into erythrocyte membranes. The amount of IpaB and IpaC present in prepared RBC membranes was determined by immunoblot analysis with anti-IpaB and anti-IpaC antibodies. M90T (lane 1) and SF622 expressing ipaD (lane 2) inserted similar amounts of these proteins into the membranes. In contrast, SF622 expressing ipaD^Δ41-80 or ipaD^Δ81-120 inserted much lower levels of IpaB and IpaC into the membranes (lanes 3 and 4, respectively). SF622 not producing any IpaD failed to insert detectable amounts of IpaB or IpaC into these membranes (lane 6), as did the mxiD TTSS apparatus mutant (lane 5) (1, 33). This experiment was performed three times with similar results.