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. 2008 Apr;44(4):344-61.
doi: 10.1016/j.micpath.2007.10.005. Epub 2007 Oct 24.

Molecular characterization of a functional type VI secretion system from a clinical isolate of Aeromonas hydrophila

Giovanni Suarez  1 , Johanna C Sierra, Jian Sha, Shaofei Wang, Tatiana E Erova, Amin A Fadl, Sheri M Foltz, Amy J Horneman, Ashok K Chopra
Affiliations

Molecular characterization of a functional type VI secretion system from a clinical isolate of Aeromonas hydrophila

Giovanni Suarez et al. Microb Pathog. 2008 Apr.

Abstract

Our laboratory recently molecularly characterized the type II secretion system (T2SS)-associated cytotoxic enterotoxin (Act) and the T3SS-secreted AexU effector from a diarrheal isolate SSU of Aeromonas hydrophila. The role of these toxin proteins in the pathogenesis of A. hydrophila infections was subsequently delineated in in vitro and in vivo models. In this study, we characterized the new type VI secretion system (T6SS) from isolate SSU of A. hydrophila and demonstrated its role in bacterial virulence. Study of the role of T6SS in bacterial virulence is in its infancy, and there are, accordingly, only limited, recent reports directed toward a better understanding its role in bacterial pathogenesis. We have provided evidence that the virulence-associated secretion (vas) genes vasH (Sigma 54-dependent transcriptional regulator) and vasK (encoding protein of unknown function) are essential for expression of the genes encoding the T6SS and/or they constituted important components of the T6SS. Deletion of the vasH gene prevented expression of the potential translocon hemolysin coregulated protein (Hcp) encoding gene from bacteria, while the vasK gene deletion prevented secretion but not translocation of Hcp into host cells. The secretion of Hcp was independent of the T3SS and the flagellar system. We demonstrated that secreted Hcp could bind to the murine RAW 264.7 macrophages from outside, in addition to its ability to be translocated into host cells. Further, the vasH and vasK mutants were less toxic to murine macrophages and human epithelial HeLa cells, and these mutants were more efficiently phagocytosed by macrophages. We also provided evidence that the expression of the hcp gene in the HeLa cell resulted in apoptosis of the host cells. Finally, the vasH and vasK mutants of A. hydrophila were less virulent in a septicemic mouse model of infection, and animals immunized with recombinant Hcp were protected from subsequent challenge with the wild-type (WT) bacterium. In addition, mice infected with the WT A. hydrophila had circulating antibodies to Hcp, indicating an important role of T6SS in the pathogenesis of A. hydrophila infections. Taken together, we have characterized the T6SS from Aeromonas for the first time and provided new features of this secretion system not yet known for other pathogens.

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Figures

Figure 1
Figure 1
A. Diagram showing the genetic organization of the T6SS gene cluster of A. hydrophila SSU (II) in comparison with the similar cluster present in Vibrio cholerae N16691 (I) and A. hydrophila ATCC 7966 (III). Shown in green are the genes present only in V. cholerae and A. hydrophila SSU T6SS gene clusters. Dashed in blue are genes that were pursued during this study. Genes in cyan color are those that are present only in A. hydrophila ATCC 7966 strain (III). Genes in orange exhibit low identity (<25%) between V. cholerae and A. hydrophila SSU T6SS gene cluster. B. Two-dimensional gel electrophoresis of supernatants from A. hydrophila SSU after staining with Sypro Ruby. The spot for Hcp is highlighted, and its identity was confirmed by mass-spec analysis.
Figure 2
Figure 2
A. Production of Hcp from A. hydrophila SSU and the translocation of Hcp into host cells. HT-29 human colonic epithelial cells were infected with different A. hydrophila strains (MOI of 5) for 2 hr at 37°C in DMEM/0.5% FBS medium. The culture supernatants were TCA precipitated (supernatant fraction). The infected host cells were osmotically lysed and centrifuged to obtain soluble (cytoplasmic fraction representing translocated effectors) and insoluble fractions. The insoluble fraction was resuspended in cell lysis buffer containing 0.1% Triton X-100 and centrifuged to obtain soluble (host cell membrane fraction) and insoluble (intact bacterial pellet) fractions. The samples were run on 4–20% gradient SDS-PAGE and subjected to Western blot analyses using anti-Hcp, anti-actin, anti-DnaK and anti-calnexin antibodies. Lane 1: Untreated HT-29 cells; Lane 2: A. hydrophila WT; Lane 3: WT bacteria containing pBR322; Lane 4: ΔvasH mutant; Lane 5: ΔvasH complemented strain; Lane 6: ΔvasK mutant; Lane 7: ΔvasK complemented strain; Lane 8: ΔflhA mutant, and lane 9: ΔascV mutant B. Production and translocation of AexU are not affected by mutations in the T6SS components. HT-29 cells were infected with the WT A. hydrophila SSU (lane 1), ΔaexU mutant (lane 2), ΔvasK mutant (lane 3), ΔvasH mutant (lane 4), and ΔascV mutant (lane 5). The culture supernatants were TCA precipitated (supernatant fraction). The infected host cells were osmotically lysed and centrifuged to obtain soluble (cytoplasmic fraction) and insoluble fractions (pellet). The samples were run on 4–20% gradient SDS-PAGE and subjected to Western blot analyses using anti-AexU antibodies. C. Hcp binds to the cell membrane of RAW 264.7 murine macrophages. Supernatants from A. hydrophila Δact and Δact/ΔvasH mutants were added to RAW 264.7 cells and incubated for 2 hr at 37°C. The host cells were washed, osmotically lysed, and centrifuged to obtain soluble (cytoplasmic) and insoluble (membrane) fractions. Samples were run on a 4–20% gradient SDS-PAGE and subjected to Western blot analyses using the following antibodies: anti-calnexin, anti-actin and anti-Hcp. Control: Macrophages incubated with 1% FBS-DMEM (lanes 1 and 2); Δact: Macrophages incubated with supernatants from A. hydrophila act mutant (lanes 3 and 4); Δact/ΔvasH: Macrophages incubated with supernatants from A. hydrophila Δact/ΔvasH mutant (lanes 5 and 6). The supernatants from the Δact (lane 8) and Δact/ΔvasH (lane 9) mutants of A. hydrophila SSU were used as a control for the presence of Hcp. C=Cytoplasmic fraction from RAW 264.7 macrophages. M=Membrane fraction from RAW 264.7 macrophages.
Figure 2
Figure 2
A. Production of Hcp from A. hydrophila SSU and the translocation of Hcp into host cells. HT-29 human colonic epithelial cells were infected with different A. hydrophila strains (MOI of 5) for 2 hr at 37°C in DMEM/0.5% FBS medium. The culture supernatants were TCA precipitated (supernatant fraction). The infected host cells were osmotically lysed and centrifuged to obtain soluble (cytoplasmic fraction representing translocated effectors) and insoluble fractions. The insoluble fraction was resuspended in cell lysis buffer containing 0.1% Triton X-100 and centrifuged to obtain soluble (host cell membrane fraction) and insoluble (intact bacterial pellet) fractions. The samples were run on 4–20% gradient SDS-PAGE and subjected to Western blot analyses using anti-Hcp, anti-actin, anti-DnaK and anti-calnexin antibodies. Lane 1: Untreated HT-29 cells; Lane 2: A. hydrophila WT; Lane 3: WT bacteria containing pBR322; Lane 4: ΔvasH mutant; Lane 5: ΔvasH complemented strain; Lane 6: ΔvasK mutant; Lane 7: ΔvasK complemented strain; Lane 8: ΔflhA mutant, and lane 9: ΔascV mutant B. Production and translocation of AexU are not affected by mutations in the T6SS components. HT-29 cells were infected with the WT A. hydrophila SSU (lane 1), ΔaexU mutant (lane 2), ΔvasK mutant (lane 3), ΔvasH mutant (lane 4), and ΔascV mutant (lane 5). The culture supernatants were TCA precipitated (supernatant fraction). The infected host cells were osmotically lysed and centrifuged to obtain soluble (cytoplasmic fraction) and insoluble fractions (pellet). The samples were run on 4–20% gradient SDS-PAGE and subjected to Western blot analyses using anti-AexU antibodies. C. Hcp binds to the cell membrane of RAW 264.7 murine macrophages. Supernatants from A. hydrophila Δact and Δact/ΔvasH mutants were added to RAW 264.7 cells and incubated for 2 hr at 37°C. The host cells were washed, osmotically lysed, and centrifuged to obtain soluble (cytoplasmic) and insoluble (membrane) fractions. Samples were run on a 4–20% gradient SDS-PAGE and subjected to Western blot analyses using the following antibodies: anti-calnexin, anti-actin and anti-Hcp. Control: Macrophages incubated with 1% FBS-DMEM (lanes 1 and 2); Δact: Macrophages incubated with supernatants from A. hydrophila act mutant (lanes 3 and 4); Δact/ΔvasH: Macrophages incubated with supernatants from A. hydrophila Δact/ΔvasH mutant (lanes 5 and 6). The supernatants from the Δact (lane 8) and Δact/ΔvasH (lane 9) mutants of A. hydrophila SSU were used as a control for the presence of Hcp. C=Cytoplasmic fraction from RAW 264.7 macrophages. M=Membrane fraction from RAW 264.7 macrophages.
Figure 3
Figure 3
A. Phagocytosis is enhanced in A. hydrophila ΔvasH and ΔvasK mutants. RAW 264.7 murine macrophages were infected at an MOI of 5 with A. hydrophila strains, namely, WT, ΔvasH and ΔvasK mutants. Thirty minutes after infection, the cells were washed and treated with 100 μg/ml of gentamicin for 1 hr. Then, RAW 264.7 cells were washed and lysed with water. The bacteria were plated at different dilutions and the colony forming units were determined.* denotes statistically significant values (p<0.01) compared to the parent strain (WT). B. Cytotoxicity associated with the T6SS. RAW 264.7 cells (Panel I) and HeLa cells (Panel II) were infected with Δact/ΔvasH mutant (solid triangle) and Δact/ΔvasK mutant (open triangle) double-knockout mutants and their parental strain Δact mutant (solid square) at an MOI 0.5. At different time points, cytotoxicity was measured by the lactate dehydrogenase (LDH) enzyme release assay. *** or ♦♦♦ denotes statistically significant values (p<0.001) compared to the parent strain Δact. * denotes statistically significant values (p<0.05) compared to the parent strain Δact. Three independent experiments in duplicate wells were performed.
Figure 4
Figure 4
A. Expression and production of Hcp in transfected HeLa Tet-Off cells. Panel I. Western blot analysis showing production of Hcp in whole cell lysates of HeLa Tet-Off cells after 24 hr of transfection with pBI-EGFP-hcp (lane 2) or pBI-EGFP (empty vector) (lane 1) plasmid. Recombinant Hcp was used as a positive control (lane 3). Panel II. Expression of the hcp gene in HeLa Tet-Off cells after permeabilization and intracellular staining using anti-Hcp antibodies. Mouse pre-immune serum was used as an isotype control. The cells were acquired using a FACScan flow cytometer and analyzed using WinMDI software, gated on EGFP-positive cells. B. Induction of apoptosis in HeLa Tet-Off cells transfected with the hcp gene. Panel I. Detection by ELISA of cytoplasmic nucleosomes in HeLa Tet-Off cells transfected with hcp after 24 hr. *** denotes statistically significant values (p<0.001) compared to those in cells transfected with the pBI-EGFP (empty vector) plasmid. Standard deviations were calculated from duplicate samples from one representative experiment. A minimum of three experiments were performed with similar results. Panel II. Colorimetric caspase 3 detection in total lysates of HeLa Tet-Off cells transfected with the hcp gene after 24 hr. Figures are representative of three independent experiments. *** denotes statistically significant values (p<0.001) compared to those of cells transfected with the pBI-EGFP (empty vector) plasmid. Standard deviations were calculated from duplicate assays from one experiment. A minimum of three experiments were performed with similar results.
Figure 5
Figure 5
Role of Hcp during A. hydrophila infection in a mouse model. A. Swiss Webster mice were infected i.p., with WT A. hydrophila at a dose of 1 LD50 (1 × 105 cfu/100μl). After 2 weeks of infection, sera from the surviving mice were collected and pooled. The sera was diluted 1:100 and used as a source of primary antibodies in Western blot analysis against rHcp. Pre-immune serum was used as a negative control. B. Groups of 10 Swiss Webster mice were infected i.p. (8 × 106 cfu) with A. hydrophila: WT (solid circles), ΔvasH mutant (open circle) and ΔvasK mutant (solid triangle). Same doses of the complemented strains were also used, ΔvasH/C (open triangle) and ΔvasK/C (solid square). Deaths were recorded for 16 days post-infection. The bacterial doses represented approximately 2 LD50 of WT A. hydrophila. * denotes statistically significant values (p<0.001) of the mutants (vasH and ΔvasK) compared to the WT bacterium and of ΔvasH mutant compared to the ΔvasH/C strain (p<0.05) using the Fisher exact test. The death curve for WT A. hydrophila with pBR322 vector alone was similar to that of the WT bacterium (without the vector) (not shown).
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