Characterization of the Yersinia enterocolitica type III secretion ATPase YscN and its regulator, YscL

Abstract

Type III secretion is a mechanism used by a broad range of gram-negative bacteria to neutralize eukaryotic defenses by enabling translocation of bacterial proteins directly into the cytoplasm of host cells. The bacterial energy source for secretion is ATP, which is consumed by an ATPase that couples ATP hydrolysis to the unfolding of secreted proteins and the dissociation of their chaperones just prior to secretion. By studying the biochemical properties of YscN and YscL of Yersinia enterocolitica, we have characterized them as the ATPase and ATPase regulator, respectively, of the type III secretion system of this organism. In vivo, YscL and YscN interact with each other, and the overexpression of glutathione S-transferase-YscL abolishes secretion and down-regulates the expression of secretion apparatus components.

FIG. 1.

Effect of GST-YscN on Y. enterocolitica type III secretion. (A) Wild-type and yscN mutant Y. enterocolitica strains bearing plasmids encoding yscN or gst-yscN were monitored for the functionality of type III secretion. Secretion was monitored by growth in medium supplemented or chelated for calcium (Ca²⁺ + or −, respectively), and supernatant (S) and pellet (P) fractions were assayed by Coomassie blue staining, SDS-PAGE, and immunoblotting with the indicated antisera (e.g., anti-YscD [αYscD]). (B) Wild-type and ΔyscN Y. enterocolitica strains bearing wild-type or mutant alleles of yscN on a plasmid were assayed for secretion by Coomassie blue staining and SDS-PAGE in the presence (+) or absence (−) of calcium. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gels.

FIG. 2.

GST-YscN binds to YscL. (A) Y. enterocolitica ΔyscN strain bearing a plasmid-encoded gst-yscN was grown at 37°C under conditions that induce the type III secretion machine and GST-YscN in the presence or absence of extracellular calcium (+Ca²⁺ or −Ca²⁺, respectively). GST-YscN was purified from this strain, and samples corresponding to the lysate (L), flowthrough (FT), wash (W), and eluate (E) fractions were collected and analyzed by Coomassie blue staining and SDS-PAGE. The white arrowhead points to GST-YscN and the filled arrowhead to GST. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gels. (B) The lysate (L) and eluate (E) fractions were analyzed by immunoblotting with the indicated antisera (e.g., anti-YscN [αYscN]). The relative intensity of immunoreactive signals in the lysate and eluate fractions (E/L) is expressed as a ratio to the right of each blot.

FIG. 3.

Complementation of ΔyscL mutant phenotypes by gst-yscL. (A) Secretion of wild-type and ΔyscL strains of Y. enterocolitica was monitored in the presence or absence (−) of a plasmid-encoded yscL or gst-yscL. Strains were grown in the presence (+) or absence (−) of calcium, and culture supernatants (S) and bacterial sediment pellets (P) were analyzed by Coomassie blue staining, SDS-PAGE, and immunoblotting with indicated antisera (e.g., anti-YscL [αYscL]). The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gel. (B) Immunoblots probing GST or YscL in strains expressing GST-YscL are the same as in panel A above, but reproduced in full. IB, immunoblotting.

FIG.4.

GST-YscL but not YscL affects type III secretion. (A) Secretion of wild-type Y. enterocolitica containing a plasmid-encoded gst-yscL was monitored as a function of an inducer for GST-YscL. Bacteria were grown in the absence (−) of extracellular calcium, and the supernatants (S) and bacterial sediment pellets (P) of each culture were scored by Coomassie blue staining, SDS-PAGE, and immunoblotting to YscL, YscD (a machine component), YopE (a secreted protein), and Npt (a cytoplasmic protein—our fractionation control). Wild-type bacteria grown without IPTG are shown in the leftmost lanes of the gel as a reference. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gel. α-YscL, anti-YscL. (B) Wild-type Y. enterocolitica bearing a plasmid-encoded gst-yscL were grown in the presence (+) or absence (−) of inducer in the absence (−) of calcium and assayed for secretion by immunoblotting. Bacteria were grown for several generations in the presence of calcium and the absence of inducer and assayed again for secretion as described above. (C) Wild-type Y. enterocolitica bearing an inducible, untagged yscL allele was grown in the absence of calcium and increasing concentrations of inducer. Secretion of proteins was monitored as described above for panel A, except anti-chloramphenicol acetyltransferase (α-CAT) antibodies were used in place of anti-Npt antibodies.

FIG. 5.

GST-YscL associates with YscN and YscQ. (A) Y. enterocolitica ΔyscL strain expressing GST-YscL was grown at 37°C under conditions that induce the type III secretion machine and GST-YscL in the presence or absence of extracellular calcium (+Ca²⁺ or −Ca²⁺, respectively). GST-YscL was purified from this strain, and samples corresponding to the lysate (L), flowthrough (FT), wash (W), and eluate (E) fractions were collected and analyzed by Coomassie staining and SDS-PAGE. The white arrowheads point to GST-YscL and the filled arrowheads to GST. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gels. (B) Lysate (L) and eluate (E) fractions were analyzed by immunoblotting with indicated antisera (e.g., anti-YscL [αYscL]). The relative intensity of immunoreactive signals in the lysate and eluate fractions (E/L) is expressed as a ratio to the right of each blot.

FIG. 6.

Purification and gel filtration analysis of _HisYscN. (A) _HisYscN purification. E. coli capable of _HisYscN expression was grown in the absence or presence of inducer (lanes 1 and 2, respectively). _HisYscN was purified on a Ni-NTA column under denaturing conditions (lane 3), renatured, and repurified (lane 4). The elution fractions were ultracentrifuged, and the supernatant was collected (lane 5). The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gel. (B) The gel filtration profile of _HisYscN was determined in the presence or absence of 5 mM AMP-PNP. Fractions (0.5 ml) were collected and probed for the relative abundance of _HisYscN by immunoblotting and analysis by densitometry as shown in the graph. The peak elution fractions of calibration standards (in kilodaltons) are indicated below the gels.

FIG. 7.

_HisYscN functions as an ATPase. (A) ATPase activity of _HisYscN was measured as a function of increasing concentrations of _HisYscN. (B) ATPase activity of _HisYscN was measured as a function of substrate concentrations. (C) Data collected in panel B presented as a Lineweaver-Burk plot. S, substrate; V, velocity of the enzymatic reaction.

FIG. 8.

_HisYscL purification and noncompetitive inhibition of _HisYscN. (A) _HisYscL was purified from E. coli, and samples corresponding to the lysate (L), flowthrough (FT), wash (W), and eluate (E) fractions were collected and analyzed by Coomassie blue staining and SDS-PAGE. The positions of molecular mass markers (in kilodaltons) are indicated to the left of the gel. (B) The effect of _HisYscL on _HisYscN activity was measured over the range of indicated concentrations of _HisYscL. Error bars indicate standard deviations derived from three independent measurements. Arrowheads identify the relative ratios of YscN (assuming it is a hexamer [YscN₆]) and YscL (assuming it is a dimer [YscL₂]) along the curve. (C) The inhibition of _HisYscN activity by _HisYscL was measured between 1 and 3 mM ATP as a function of the indicated concentrations of _HisYscL. S, substrate; V, velocity of the enzymatic reaction.

FIG. 9.

YscL can interact with itself. (A) Glutathione-Sepharose beads were charged with GST purified from an E. coli lysate. This column was incubated with an E. coli lysate containing _HisYscL, and retention of proteins by the beads was monitored in the indicated fractions by immunoblotting to YscL, GST, and the histidine tag. α-YscL, anti-YscL; Ni-HRP, horseradish peroxidase. (B) Glutathione-Sepharose beads were charged with GST-YscL and then mixed with E. coli lysate containing _HisYscL; _HisYscL binding to GST-YscL was monitored as described above for panel A.