TABLE 2.

Abilities of IpaD deletion mutations to restore invasiveness and contact-mediated hemolysis activity to S. flexneri SF622

StrainaRelative invasionb (%)Contact-mediated hemolysis (%)cIpaD secretiond
SF622 (ipaD)0.1 ± 0.2e0.0 ± 0e
M90T123 ± 12100 ± 5+
BS547 (mxiM)0.0 ± 0e0.0 ± 0e
IpaD100 ± 16100 ± 0.1+
IpaDΔ1-200.0 ± 0e0.0 ± 0e
IpaDΔ41-8095.0 ± 1547 ± 5f+
IpaDΔ81-120101.0 ± 1132 ± 2f+
IpaDΔ121-1601.0 ± 1e0.0 ± 0e+
IpaDΔ161-1803.0 ± 0.2e0.0 ± 0e+
IpaDΔ201-2401.0 ± 0.4e0.0 ± 0e+
IpaDΔ241-2803.0 ± 0.2e0.0 ± 0e+
IpaDΔ281-3200.1 ± 0.2e0.0 ± 0e+
IpaDΔ321-3323.0 ± 0.1e0.0 ± 0e++
IpaDΔ328-3320.1 ± 0.1e0.0 ± 0e++
IpaDSC321AS147 ± 10100 ± 0.1++
SipD0.0 ± 0e0.0 ± 0eNot done
  • a Strains that have IpaD or an IpaD deletion mutation listed are actually S. flexneri SF622 (a nonpolar ipaD mutant) expressing the gene for that particular IpaD derivative.

  • b Restoration of invasion is presented relative to invasion by SF622 transformed with pWPsf4D±the standard deviation (n = 3). The data shown are from one of three representative experiments.

  • c Contact-mediated hemolysis is presented as a percentage of complete hemolysis, such as that which occurs upon the addition of water (n = 3 from one of five representative experiments).

  • d IpaD secretion was measured by a semiquantitative ELISA system (n = 5) with overnight culture supernatants (also see Fig. 1). The antibodies used here 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. Whole bacterial lysates were also used to determine whether the IpaD derivatives described here were similarly synthesized (Fig. 1). −, no secretion; +, normal secretion; ++, increased secretion.

  • e P < 0.001 by a paired Student t test.

  • f P < 0.05 by a paired Student t test.