Shigella IpaD has a dual role: signal transduction from the type III secretion system needle tip and intracellular secretion regulation

Abstract

Type III secretion systems (T3SSs) are protein injection devices essential for the interaction of many Gram-negative bacteria with eukaryotic cells. While Shigella assembles its T3SS when the environmental conditions are appropriate for invasion, secretion is only activated after physical contact with a host cell. First, the translocators are secreted to form a pore in the host cell membrane, followed by effectors which manipulate the host cell. Secretion activation is tightly controlled by conserved T3SS components: the needle tip proteins IpaD and IpaB, the needle itself and the intracellular gatekeeper protein MxiC. To further characterize the role of IpaD during activation, we combined random mutagenesis with a genetic screen to identify ipaD mutant strains unable to respond to host cell contact. Class II mutants have an overall defect in secretion induction. They map to IpaD's C-terminal helix and likely affect activation signal generation or transmission. The Class I mutant secretes translocators prematurely and is specifically defective in IpaD secretion upon activation. A phenotypically equivalent mutant was found in mxiC. We show that IpaD and MxiC act in the same intracellular pathway. In summary, we demonstrate that IpaD has a dual role and acts at two distinct locations during secretion activation.

Figure 1

All ipaD mutants isolated cause partial defects in secretion. A. Exponential leakage of Shigella wild-type, ΔipaD mutant, complemented strain (ΔipaD/ipaD⁺) and ipaD mutants (in the ΔipaD background) isolated from a mutant library. Samples were collected as described in Experimental procedures and Silver-stained. B. Protein secretion in response to artificial inducer Congo red (CR) induction. Samples were collected as described in Experimental procedures and Silver-stained (top panel) and Western-blotted with antibodies against the translocator proteins IpaB, IpaC, IpaD and the regulator MxiC (bottom panels). C. Total protein expression levels. Samples were collected as described in Experimental procedures and Western-blotted with the indicated antibodies. Results shown are representative of at least two independent experiments.

Figure 2

The ipaD mutants show defects in HeLa invasion but have normal needle tip compositions. A. Invasion of HeLa cells was measured with a gentamicin protection assay as described in Experimental procedures. Every experiment was normalized to wild-type and the data are averages of at least three independent experiments performed at least in duplicate. Error bars indicate the standard deviation. Where the invasion is significantly different from the complemented strain ΔipaD/ipaD⁺ (P < 0.001, ANOVA with Tukey's post hoc test), ipaD mutant strains (in the ΔipaD background) are marked with ***. B. Needles from wild-type, ΔipaD mutant, complemented strain (ΔipaD/ipaD⁺) and ipaD mutants (in the ΔipaD background) overexpressing the needle protein MxiH were isolated as described in Experimental procedures and Western-blotted with antibodies against IpaB and IpaD (samples were normalized for the amount of MxiH present in preparations as detected by Silver stain, bottom panel). Results shown are representative of at least two independent experiments. For the complemented strain (ΔipaD/ipaD⁺) results from two experiments are shown to indicate experimental variability. In comparison to the complemented strain, the wild-type contains c. 50 ± 10% IpaD (average ± standard deviation), ipaDL99P contains c. 70 ± 30% IpaD, Class IIa mutants contain c. 90 ± 20% IpaD and Class IIb mutants contain c. 90 ± 40% IpaD. For IpaB, these values are 70 ± 40% for wild-type, 100 ± 70% for ipaDL99P, 150 ± 100% for Class IIa mutants and 130 ± 90% for Class IIb mutants. There are no significant differences between samples in an ANOVA (P > 0.05) for both IpaD and IpaB. C. Total protein expression levels in overnight cultures from strains overexpressing the needle protein as in B. Samples were collected as described in Experimental procedures and Western-blotted with antibodies against IpaB and IpaD.

Figure 3

Class II ipaD mutants are severely affected in induction of secretion. A. Analysis of proteins secreted by the indicated strains in response to Congo red (CR) induction. Samples from Shigella wild-type, ΔipaD mutant, complemented strain (ΔipaD/ipaD⁺) and ipaD mutants (in the ΔipaD background) were collected as described in Experimental procedures and Silver-stained. B. Total protein expression levels. Samples were collected as described in Experimental procedures and Western-blotted with an antibody against IpaD. Results shown are representative of at least two independent experiments.

Figure 4

Impaired secretion of IpaDL99P is due to lack of regulation by MxiC. A. Secretion of IpaD after Congo red (CR) induction by a ΔipaB ΔipaD double mutant complemented with either ipaD alone or a combined ipaBΔ3 ipaD plasmid. Samples were collected as described in Experimental procedures and Western-blotted with an antibody against IpaD. B. Total protein expression levels. Samples were collected as described in Experimental procedures and Western-blotted with an antibody against IpaD. Results shown are representative of at least two independent experiments.

Figure 5

The mutations L99P and K291I affect different IpaD functions. A. Exponential leakage of the ΔipaD mutant, complemented strain (ΔipaD/ipaD⁺) and ipaD mutants (in the ΔipaD background). Samples were collected as described in Experimental procedures and Western-blotted with the indicated antibodies. B. Protein secretion in response to Congo red (CR) induction. Samples were collected as described in Experimental procedures, Silver-stained (top panel) and Western-blotted with the indicated antibodies (bottom panels). C. Total protein expression levels. Samples were collected as described in Experimental procedures and Western-blotted with the indicated antibodies. Results shown are representative of at least two independent experiments.

Figure 6

mxiC(E201K, E276K, E293K) has a phenotype similar to ipaDL99P. A. Exponential leakage. Samples were collected as described in Experimental procedures and Western-blotted with the indicated antibodies. B. Protein secretion in response to Congo red (CR) induction. Samples were collected described in Experimental procedures, Silver-stained (top panel) and Western-blotted with the indicated antibodies (bottom panels). C. Total protein expression levels. Samples were collected as described in Experimental procedures and Western-blotted with the indicated antibodies. The complemented strain ΔmxiC/mxiC⁺ was grown with 25 μM IPTG, while the ΔmxiC/mxiC(E201K, E276K, E293K) mutant was grown with 10 μM IPTG because of its higher mxiC expression level. Results shown are representative of at least two independent experiments.

Figure 7

IpaD and MxiC are both involved in the regulation of the mechanism that prevents secretion. A. Exponential leakage of the ΔipaD mutant and a ΔipaD ΔmxiC double mutant complemented with ipaD and mxiC containing plasmids. Samples were collected as described in Experimental procedures and Western-blotted with the indicated antibodies. All samples detected with the same antibody were analysed on the same gel. B. Protein secretion in response to Congo red (CR) induction. Samples were collected as described in Experimental procedures, Silver-stained (top panel) and Western-blotted with the designated antibodies (bottom panels). C. Total protein expression levels. Samples were collected as described in Experimental procedures and Western-blotted with the indicated antibodies. Bacteria were grown with 1% arabinose and 25 μM IPTG as required for expression of ipaD and mxiC respectively. Mutant mxiC(E201K, E276K, E293K) is abbreviated mxiC***. Results shown are representative of at least two independent experiments.

Figure 8

Premature secretion in ipaDL99P is MxiC-dependent. A. Exponential leakage of a ΔipaD ΔmxiC double mutant complemented with either ipaD or ipaDL99P alone or additionally with mxiC. Samples were collected as described in Experimental procedures and Western-blotted with the indicated antibodies. B. Total protein expression levels. Samples were collected as described in Experimental procedures and Western-blotted with the indicated antibodies. Bacteria were grown with 1% arabinose and 25 μM IPTG as required for expression of ipaD and mxiC respectively. Results shown are representative of at least two independent experiments.

Figure 9

Mapping of identified mutations on the IpaD structure. Mapping of identified mutations on the IpaD structure (PDB entry 2J0O chain A, Johnson et al., 28). The N-terminal domain is coloured in blue (residues 40–130), the C-terminal globular domain is coloured in red (residues 177–271) and the coiled coil domain is coloured in green (residues 131–176 and 272–321). Mutated amino acids are shown as stick models, residue L99 (mutated in the Class I mutant) is shown in white, residues mutated in the ‘strong’ Class IIa mutants (N186, N273, Q277 and K291) are shown in black and residues mutated in the ‘weak’ Class IIb (N292, T296, Q299) are shown in grey.