DsbA plays a critical and multifaceted role in the production of secreted virulence factors by the phytopathogen Erwinia carotovora subsp. atroseptica

Abstract

Erwinia carotovora subsp. atroseptica is an enterobacterial phytopathogen causing economically significant soft rot disease. Pathogenesis is mediated by multiple secreted virulence factors, many of which are secreted by the type II (Out) secretion system. DsbA catalyzes the introduction of disulfide bonds into periplasmic and secreted proteins. In this study, the extracellular proteome (secretome) of wild type E. carotovora subsp. atroseptica SCRI1043, and dsbA and out mutants, was analyzed by spectral counting mass spectrometry. This revealed that dsbA inactivation had a huge impact on the secretome and identified diverse DsbA- and Out-dependent secreted proteins, representing known, predicted, and novel candidate virulence factors. Further characterization of the dsbA mutant showed that secreted enzyme activities, motility, production of the quorum-sensing signal, and virulence were absent or substantially reduced. The impact of DsbA on secreted virulence factor production was mediated at multiple levels, including impacting on the Out secretion system and the virulence gene regulatory network. Transcriptome analyses revealed that the abundance of a broad, but defined, set of transcripts, including many virulence factors, was altered in the dsbA mutant, identifying a new virulence regulon responsive to extracytoplasmic conditions. In conclusion, DsbA plays a crucial, multifaceted role in the pathogenesis of E. carotovora subsp. atroseptica.

FIGURE 1.

Inactivation of dsbA has a large impact on the secretome of E. carotovora subsp. atroseptica SCRI1043 as shown by two-dimensional DiGE. Secreted proteins were prepared from wild type E. carotovora subsp. atroseptica (Eca) SCRI1043 (left), SCC22 (dsbA, middle), and MC4 (out, right) and labeled with different Cy dyes as indicated. The pooled sample was then separated by two-dimensional SDS-PAGE (over pH 3–10), and the fluorescent images were visualized. Proteins absent or at greatly reduced levels in both the dsbA and the out mutants are labeled in red, whereas those absent or greatly reduced in the dsbA mutant but present in the out mutant are labeled in blue. Note that equal amounts of protein were labeled in each channel, although the total amount of secreted protein recovered was much lower in the dsbA mutant than the wild type.

FIGURE 2.

The effect of DsbA inactivation on the levels of secreted pectate lyase (Pel), endoglucanase (Cel), and protease (Prt) enzyme activity. A, levels of Pel (top), Cel (middle), and Prt (bottom) activity in the culture supernatant were determined at intervals throughout growth in PMM for wild type E. carotovora subsp. atroseptica SCRI1043 (WT, dark gray bars) and SCC22 (dsbA mutant, light gray bars). B, to demonstrate complementation of the dsbA phenotypes, levels of secreted Pel (top), Cel (middle), and Prt (bottom) were measured for WT (pQE80) (vector control, white bars), WT (pSJC58) (dsbA in trans, darker gray bars), SCC22 (pQE80) (lighter gray bars), and SCC22 (pSJC58) (black bars). Secreted enzyme activities were measured as described under “Experimental Procedures.” Growth is reported as OD₆₀₀. A full key to symbols and shading is given at the base of each panel. Bars show mean ± S.E. (n ≥ 3).

FIGURE 3.

Production of OHHL is reduced in the dsbA mutant. A, levels of OHHL activity in the culture supernatant were determined at intervals throughout growth in PMM for wild type E. carotovora subsp. atroseptica SCRI1043 (WT, dark gray bars) and SCC22 (dsbA mutant, light gray bars). B, to demonstrate complementation, levels of OHHL were measured for WT (pQE80) (vector control, white bars), WT (pSJC58) (dsbA in trans, darker gray bars), SCC22 (pQE80) (lighter gray bars), and SCC22 (pSJC58) (black bars). OHHL levels were measured using the biosensor E. coli JM109 (pSB401) and are reported as light units (lu) per OD₆₀₀, relative to WT at 20 h (A) or WT (pQE80) at 25 h (B) (with these values set to 100). Growth is reported as OD₆₀₀. A full key to symbols and shading is given at the base of each panel. Bars show mean ± S.E. (n ≥ 3).

FIGURE 4.

The impact of DsbA inactivation on motility and virulence in potato tubers. A, motility plate (0.3% agar) showing the swim haloes formed by wild type E. carotovora subsp. atroseptica SCRI1043 (WT) and SCC22 (dsbA) after 18 h of incubation. B, motility of WT (pQE80) (vector control), SCC22 (pQE80), and SCC22 (pSJC58) (dsbA in trans). C, motility of wild type (dark gray bars) and SCC22 (light gray bars) in varying concentrations of Cu²⁺. B and C, motility is reported as halo area after 18 h of incubation; bars show mean ± S.E. (n ≥ 3). D, potato tuber virulence assay measuring the amount of rotted tissue generated by WT (pQE80), SCC22 (pQE80), and SCC22 (pSJC58) after 7 days of incubation (inoculum of 10⁷ cells); bars show mean ± S.E. (n = 20).

FIGURE 5.

CelV is not secreted to the medium in the dsbA mutant. A, anti-CelV Western blot of proteins in the supernatant (S) and the cell-associated (C) fractions of wild type E. carotovora subsp. atroseptica SCRI1043 (WT), SCC22 (dsbA), and MC4 (out). Protein samples were prepared from equal volumes of culture (and resuspended in equal volumes of sample buffer) for each fraction and each strain (see “Experimental Procedures”). Protein samples were separated by 12% SDS-PAGE and immunoblotted with anti-CelV antibody; four times the amount of protein sample was loaded for the supernatant than cell-associated samples. B, endoglucanase (Cel) activity was determined for supernatant and cell-associated culture samples prepared from wild type E. carotovora subsp. atroseptica SCRI1043, SCC22, MC4, and SCC29 (celV), after growth for 24 h in PMM. Bars show mean ± S.E. (n = 3).

FIGURE 6.

Expression of a dsbA-uidA fusion is modulated by quorum sensing. A, expression of dsbA-uidA in E. carotovora subsp. carotovora SCRI193 was measured during growth in LB in a wild type background (SCC20, dsbA-uidA, black bars), a carI (expI) background (SCC21, dsbA-uidA, carI, light gray bars), and in SCC21 with exogenous 5 μm OHHL (SCC21 + OHHL, dark gray bars). B, expression of dsbA-uidA in E. carotovora subsp. atroseptica SCRI1043 was measured in PMM in a wild type background (SCC33, dsbA-uidA, black bars), an expI background (SCC32, dsbA-uidA, expI, light gray bars), and in SCC32 with 5 μm OHHL added (SCC21 + OHHL, dark gray bars). Expression of dsbA-uidA was measured as β-glucuronidase activity per cell and growth reported as OD₆₀₀. A full key to symbols and shading is given at the base of the figure. Bars show mean ± S.E. (n = 3).