Modified needle-tip PcrV proteins reveal distinct phenotypes relevant to the control of type III secretion and intoxication by Pseudomonas aeruginosa

Abstract

The type III secretion system (T3SS) is employed to deliver effector proteins to the cytosol of eukaryotic hosts by multiple species of Gram-negative bacteria, including Pseudomonas aeruginosa. Translocation of effectors is dependent on the proteins encoded by the pcrGVHpopBD operon. These proteins form a T3S translocator complex, composed of a needle-tip complex (PcrV), translocons (PopB and PopD), and chaperones (PcrG and PcrH). PcrV mediates the folding and insertion of PopB/PopD in host plasmic membranes, where assembled translocons form a translocation channel. Assembly of this complex and delivery of effectors through this machinery is tightly controlled by PcrV, yet the multifunctional aspects of this molecule have not been defined. In addition, PcrV is a protective antigen for P. aeruginosa infection as is the ortholog, LcrV, for Yersinia. We constructed PcrV derivatives containing in-frame linker insertions and site-specific mutations. The expression of these derivatives was regulated by a T3S-specific promoter in a pcrV-null mutant of PA103. Nine derivatives disrupted the regulation of effector secretion and constitutively released an effector protein into growth medium. Three of these regulatory mutants, in which the linker was inserted in the N-terminal globular domain, were competent for the translocation of a cytotoxin, ExoU, into eukaryotic host cells. We also isolated strains expressing a delayed-toxicity phenotype, which secrete translocators slowly despite the normal level of effector secretion. Most of the cytotoxic translocation-competent strains retained the protective epitope of PcrV derivatives, and Mab166 was able to protect erythrocytes during infection with these strains. The use of defined PcrV derivatives possessing distinct phenotypes may lead to a better understanding of the functional aspects of T3 needle-tip proteins and the development of therapeutic agents or vaccines targeting T3SS-mediated intoxication.

Figure 1. Location of the EZ-linker insertions in PcrV.

(A) Alignment of amino acid sequences and predicted secondary structures of PcrV and Y. pestis LcrV. Linker insertion sites are indicated as a triangle tag with a residue number. Site-specific point mutations are indicated with red shaded bars. Colors of predicted secondary structures (α-helix, bar; β-sheet, arrow) in PcrV correspond to the colors in the 3D models in (B). Secondary structures of LcrV are shown below the alignment (gray). N-terminal regions in the LcrV crystal structure possessing no interpretable electron density are indicated in light gray. For crystallization of LcrV, charged residues at 40 to 42 and a cysteine residue at 273 (shown in red) were replaced with alanine and serine residues, respectively. (B) Location of linker insertions in the tertiary structure models of wild-type PcrV. For modeling, LcrV (1r6f chainA) was used as a template in Swiss Model. Predicted secondary structures are colored in a succession mode. Location of linker insertions is shown as white highlights in α-helices and magenta shading in β-sheets and loops. Left versus right panels: front and back views of PcrV (rotated 180°).

Figure 2. Cytotoxicity and secretion profiles of PA103ΔpcrV host strain complemented with pcrV::EZ-linker constructs.

(A) LDH release from HeLa cells as a quantitative measurement of cytotoxicity during infection. Cell culture supernatants were assayed for LDH activity in triplicate of two independent experiments for statistical analyses. Representative strains are shown. Error bars indicate SD. (B) Secretory regulation profiles of PcrV derivatives. Bacterial cells were grown to suppress (−NTA) type III secretion and culture supernatants were subjected to Western blot analyses to quantify ExoU release. Deregulated ExoU secretion (highlighted by boxes) was quantified based on the amount of constitutively secreted ExoU by PA103ΔpcrV vector control (pUCP) as 100%. Deregulated/cytotoxic and deregulated/noncytotoxic phenotypes were shown as dotted and gray boxes, respectively. Results are representative of at least three independent experiments.

Figure 3. Characterization of translocation-competent derivative proteins and the expressing strains using a protective monoclonal antibody, Mab166.

(A) Secretion of PcrV and PcrV::EZ derivatives that contain a linker within a predicted epitope region. V-proteins released into the bacterial culture supernatant under the induced growth condition (+NTA) were detected with Mab166 as a probe. Concentrated supernatant was titrated (2-fold serial dilution) for immunoblot analysis. (B) Retention of the protective epitope analyzed by using a hemolysis-based protection assay. Sheep erythrocytes were infected with ΔpcrV mutants in the presence of 10 µg Mab166. The conformational-epitope regions of Mab166 are underlined. (C) Localization of PcrV on the bacterial cell surface detected by immunofluorescence microscopy. PcrV was probed either with rabbit polyclonal IgG alone (shown in green, top panels) or with polyclonal IgG and Mab166 (shown in red and green, respectively; lower panels). Bacterial cells were stained with DAPI (shown in blue).

Figure 4. Analyses of class II mutations contributing to the noncytotoxic/constitutive-secretion phenotype.

(A) Translocation profiles of ExoU by the EZ-linker mutants during HeLa cell infection. After infection, bacterial cells (b) harvested from the cell culture medium and soluble fractions of HeLa cells (cs) were subjected to Western blot analysis. An anti-SOD antibody was used to detect SOD1 present in HeLa cell soluble fractions. (B) Western blot analysis of ExoU or ExoU-S142A release into cell culture medium during infection. Expression of PcrV::EZ derivatives or parental PcrV in bacterial fractions was detected with anti-PcrV IgG. (C) Cytotoxicity profiles of the strains that co-express the nontoxic derivatives with PcrV. Kinetics of cytotoxicity was measured by LDH release from infected HeLa cells. (D) Immunoblot analysis of secreted PcrV and PcrV derivatives (+NTA) or ExoU (±NTA). Regulation of ExoU secretion was examined when co-expressed with a chromosomal parental copy of pcrV. EZ138 contains two cysteine residues in the inserted linker, leading to the extra conformational species observed in the immunoblot (lane 7). (E) Bacterial surface localization of PcrV and class II derivatives. Surface-localized V proteins were quantified by flow cytometry.

Figure 5. Analysis of class III cytotoxic/deregulated-secretion derivatives possessing a linker insertion in the N-terminal globular domain.

(A) Hemolytic activity of EZ44, EZ52, and EZ64 mutants. The derivative proteins were expressed in the either pcrV-null (ΔV) or wild-type (PA103) strain. (B) Regulation of ExoU secretion by PcrV::EZ when the proteins were co-expressed in PA103. Under uninduced or NTA-induced conditions, the secretion of ExoU and PcrV/derivatives into growth medium (SN) and the expression of V proteins within bacterial cells were determined by Western blot analysis. (C) Bacterial surface localization of class III derivative proteins. EZ44, EZ52, and EZ64 were expressed in the pcrV-null strain (ΔV) in the presence or absence of a secretion inducer, NTA. Surface localization was quantified by flow cytometry. (D) Secretory regulation of ExoU and translocon proteins (PopB/PopD) by class III derivatives, when expressed in the pcrV-null strain (ΔV).

Figure 6. Cytotoxicity profiles of class IV site-specific mutants compared to linker-insertion strains.

(A) LDH release comparison of the point mutants (L63A and D133A) and the strains containing an EZ-linker insertion in adjacent sequence locations. (B) Cytotoxic effects on HeLa cells caused by formation of type III translocation channels at 7 h post-infection. LDH release from HeLa cells infected with PA103ΔV+pUCP, ΔV+pcrV, ΔUT+pUCP (competent type III injectisomes without the expression of any effectors) and PA103exsAΩ+pUCP (type III incompetent). The popB–deletion mutant and its complemented strain were used to represent the effect of a translocon protein on cytotoxicity. LDH release was tested in the presence (black bars) or absence (gray bars) of cytochalasin D (3 µg/ml). Cytochalasin D was added to examine the effect on the type III channel-mediated cytotoxicity and ExoU-mediated killing. *p<0.001 by t-test, compared to ΔV+pUCP. (C) The effect of cytochalasin D on the delayed cytotoxicity of class IV mutants. Cytotoxicity was measured and presented as (B).

Figure 7. Biochemical analyses of class IV derivatives containing a point mutation.

(A) Bacterial surface localization of the derivatives containing an alanine substitution quantified by flow cytometry. L63A and D133A proteins were expressed in the pcrV-null strain (ΔV) in the presence or absence of NTA. (B) Measurement of hemolytic activity of the pcrV-point mutants. Sheep erythrocytes were infected for 1 h with either PA103 or the ΔV strain expressing the L63A or D133A derivative. (C) Regulation of effector and translocator secretion by class IV derivatives expressed in the pcrV-null strain (ΔV). Under the uninduced (no NTA) or induced (2 mM NTA) condition, proteins secreted into growth medium (supernatant) were detected by Western blot analysis. Expression of the proteins within bacterial cells was shown as comparison (bacteria).

Figure 8. PcrV tertiary structure models indicating the insertion/mutation sites responsible for the phenotypic class.

(A) Class I insertions: the wild-type cytotoxic/regulated-secretion phenotype. The residue, at which an EZ-linker is inserted, is shown in black. (B) Class II insertions: the pcrV-null noncytotoxic/deregulated-secretion phenotype. (C) Class III insertions: cytotoxic/deregulated-secretion phenotype. The linker is inserted within the N-terminal globular domain. (D) Class IV mutations: delayed-cytotoxicity/regulated-secretion phenotype. Although secretion of an effector protein is well controlled, the secretion levels of translocators (PcrV derivative, PopB, and PopD) are decreased by a single amino-acid substitution.