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. 1999 Nov 1;147(3):683-93.
doi: 10.1083/jcb.147.3.683.

The tripartite type III secreton of Shigella flexneri inserts IpaB and IpaC into host membranes

A Blocker  1 , P Gounon, E Larquet, K Niebuhr, V Cabiaux, C Parsot, P Sansonetti
Affiliations

The tripartite type III secreton of Shigella flexneri inserts IpaB and IpaC into host membranes

A Blocker et al. J Cell Biol. .

Abstract

Bacterial type III secretion systems serve to translocate proteins into eukaryotic cells, requiring a secreton and a translocator for proteins to pass the bacterial and host membranes. We used the contact hemolytic activity of Shigella flexneri to investigate its putative translocator. Hemolysis was caused by formation of a 25-A pore within the red blood cell (RBC) membrane. Of the five proteins secreted by Shigella upon activation of its type III secretion system, only the hydrophobic IpaB and IpaC were tightly associated with RBC membranes isolated after hemolysis. Ipa protein secretion and hemolysis were kinetically coupled processes. However, Ipa protein secretion in the immediate vicinity of RBCs was not sufficient to cause hemolysis in the absence of centrifugation. Centrifugation reduced the distance between bacterial and RBC membranes beyond a critical threshold. Electron microscopy analysis indicated that secretons were constitutively assembled at 37 degrees C before any host contact. They were composed of three parts: (a) an external needle, (b) a neck domain, and (c) a large proximal bulb. Secreton morphology did not change upon activation of secretion. In mutants of some genes encoding the secretion machinery the organelle was absent, whereas ipaB and ipaC mutants displayed normal secretons.

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Figures

Figure 1
Figure 1
Contact hemolytic activity of S. flexneri. Osmoprotective analysis of the lytic activity. (A) The hemolytic assay was performed with bacteria and sheep RBCs incubated for 1 h at 37°C; w.t. stands for strain M90T, for w.t. no spin the centrifugation step before incubation at 37°C was omitted. (B) Wild-type and ipaC or mxiD strains were incubated with sheep RBCs for 1 h at 37°C in the presence of 30 mM of the different sugars indicated. (C) Wild-type bacteria were incubated with RBCs for various times in the presence of 30 mM of the different sugars. (D) The lysis kinetics were evaluated from the data shown in B by the time necessary to obtain 50% of maximal lysis, t1/2. We used t1/2–t01/2 (t01/2 being the time without osmoprotectants) as an estimate of the time necessary for the osmoticant to diffuse inside the cell through the bacterially induced lesions. Accordingly 1/(t1/2-t01/2) is an estimate of the permeability of the sugar through the pore and was used to build a Renkin plot showing the relative permeability of the molecule versus its size. In this graph, the line was the best fitting curve determined by the computer program Cricket Graph and its intersection point with the x-axis puts the pore diameter at 2.25 nm. Consistently, PEG 3000 (estimated molecular diameter 3.2 nm) offers nearly complete osmoprotection.
Figure 2
Figure 2
Analysis of the Ipa proteins associated with RBCs membranes after contact hemolysis. RBCs membranes exposed to contact with various strains under different condition were isolated and examined for their Ipa protein content using SDS-PAGE and immunoblotting. Blots were revealed with the H16 anti-IpaB mAb, a mixture of K24, N9, H10, and J22 anti-IpaC mAbs or the 20G9 anti-IpgD and 322F7 anti-IpaA mAbs. The occurrence of contact hemolysis is indicated under each panel. (A) Ipa/Ipg protein associated with RBCs membranes after contact hemolysis with wild-type, ipaC, or mxiD strains at 37°C or 4°C. The blot probed with the anti-IpaB antibody was overexposed to visualize the small amount of IpaB associated with RBC membranes lysed by the ipaC mutant. (B) Association of IpaB and IpaC with RBC membranes isolated after contact with wild-type bacteria at 37°C and stripped, or not (mock), with 5 M NaCl (NaCl), 0.2 M carbonate, pH 11 (carbonate), and 8 M urea (urea). (C) Analysis of the association of IpaB, IpaC, IpgD, and IpaA with RBCs membranes brought into contact with various mutants.
Figure 3
Figure 3
Parallel analysis of hemolysis and Ipa protein secretion. (A) The hemolytic assay was performed with bacteria and sheep RBCs incubated for 1 h at the indicated temperatures. (B) Congo red–induced secretion in the absence of any RBCs was measured at 25, 30, and 37°C. P stands for bacterial pellet and S for supernatant. (C) Wild-type bacteria were mixed with sheep RBCs in the presence or in the absence of the indicated concentrations of sodium azide and/or 1 mg/ml of proteinase K. As a control for the activity of the protease, RBCs were also exposed to a bacterial culture supernatant containing E. coli hemolysin A (HylA) or to the same supernatant to which proteinase K had been added (HylA+protease). (D) Congo red–induced secretion was measured in the presence of increasing concentrations of azide. IpaB and IpaC content of the supernatants is shown. (E) Wild-type or mxiD bacteria expressing the afimbrial adhesin AfaE were mixed with human RBCs in the presence or in the absence of 30 μM Congo red with or without centrifugation at 1,500 g for 10 min at 10°C, and incubated for 1 h at 37°C. (F) Wild-type (w.t.) or mxiD bacteria expressing AfaE were incubated with human RBCs, saline alone, saline with 30 μM Congo red (saline + CR), or RBCs with Congo red (RBCs + CR). They were treated as in the hemolytic assay, as was an identical volume of bacteria (bacterial extract) at the same concentration. Subsequently, 20 μl of the supernatants (for the bacterial extract, the bacterial pellet was resuspended) were separated on a 10% SDS-PAGE gel, blotted and probed with anti-IpaB and anti-IpaC mAbs, respectively.
Figure 4
Figure 4
Electron microscopy analysis of the interaction between bacteria and RBCs. Derivatives of wild-type bacteria expressing the afimbrial adhesin AfaE were mixed with human RBCs and either left to sediment at (A) 1 g, or centrifuged at (B) 1,400 g for 10 min at 10°C. In C and D RBCs were mock treated or treated with sialidase, respectively, before mixing with the bacteria, and the samples were centrifuged at 160 g. The samples were fixed and prepared for transmission electron microscopy. Bar, 5 μm.
Figure 5
Figure 5
Morphological identification and analysis of the type III secretons of S. flexneri. (A) Osmotically shocked and negatively stained wild-type Shigella which had been induced to secrete with Congo red were visualized by electron microscopy. Arrows show the position of the secreton at the margin of bacteria. The neck and bulb are inside the body of the bacterium while the needle is protruding outside the outer membrane. Inset shows the periphery of partially lysed bacteria. Only the protruding needle is clearly visible. The neck is poorly distinguished and the bulb is masked inside the body of the bacterium. With the method used here one can not assume that the two lines at the margin of the bacterium are its inner and outer membranes. (B) Image of the secreton bulb resulting from alignment and averaging of the 22 best preserved secretons found in A and deduced projection density map of the averaged image at 2.8 nm resolution. (C) Typical secretons found in wild-type, ipaB, ipaC and ipaD strains. Bars: (A) 200 nm; (B) 50 nm.
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