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Interaction of the Yersinia pestis type III regulatory proteins LcrG and LcrV occurs at a hydrophobic interface.

Matson JS, Nilles ML - BMC Microbiol. (2002)

Bottom Line: Our results demonstrate that the hydrophobic face of the putative helix is required for LcrV interaction.Our results demonstrate that LcrG interacts with LcrV via hydrophobic interactions located in the N-terminus of LcrG within a predicted coiled-coil motif.We also obtained preliminary evidence that the secretion blocking activity of LcrG is located between amino acids 39 and 53.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, USA. jyl_matson@und.nodak.edu

ABSTRACT

Background: Secretion of anti-host proteins by Yersinia pestis via a type III mechanism is not constitutive. The process is tightly regulated and secretion occurs only after an appropriate signal is received. The interaction of LcrG and LcrV has been demonstrated to play a pivotal role in secretion control. Previous work has shown that when LcrG is incapable of interacting with LcrV, secretion of anti-host proteins is prevented. Therefore, an understanding of how LcrG interacts with LcrV is required to evaluate how this interaction regulates the type III secretion system of Y. pestis. Additionally, information about structure-function relationships within LcrG is necessary to fully understand the role of this key regulatory protein.

Results: In this study we demonstrate that the N-terminus of LcrG is required for interaction with LcrV. The interaction likely occurs within a predicted amphipathic coiled-coil domain within LcrG. Our results demonstrate that the hydrophobic face of the putative helix is required for LcrV interaction. Additionally, we demonstrate that the LcrG homolog, PcrG, is incapable of blocking type III secretion in Y. pestis. A genetic selection was utilized to obtain a PcrG variant capable of blocking secretion. This PcrG variant allowed us to locate a region of LcrG involved in secretion blocking.

Conclusion: Our results demonstrate that LcrG interacts with LcrV via hydrophobic interactions located in the N-terminus of LcrG within a predicted coiled-coil motif. We also obtained preliminary evidence that the secretion blocking activity of LcrG is located between amino acids 39 and 53.

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Copurification of LcrG variants with His6-tagged LcrV. Cells of Y. pestis KIM8-3002.8 (ΔlcrGV2) containing plasmids pAra-HT-V and pAraG18K (lanes 1 to 3), pJM131 (LcrG S23R) (lanes 4 to 6), pJM99 (LcrG L30R) (lanes 7 to 9), or pJM89 (LcrG A16R) (lanes 10 to12) were grown in TMH with calcium and induced with 0.2% (wt/vol) arabinose prior to temperature shift to 37°C. Cultures were harvested after 4 h of growth at 37°C, and cellular extracts were disintegrated using a French press (20,000 lb/in2). Unbroken cells and large debris were removed by centrifugation (14,000 × g) for 10 min and the cleared extracts (lanes 1, 4, 7, and 10) were applied to a Talon column. Proteins that did not bind were collected as the flowthrough fraction (lanes 2, 5, 8, and 11). Proteins were eluted from the column with 50 mM imidazole and collected (lanes 3, 6, 9, and 12). All protein samples were resolved by SDS-PAGE in a 12.5% polyacrylamide gel after dilution in 2X SDS sample buffer and analyzed by immunoblotting with α-LcrG and α-LcrV. Proteins were visualized by probing with alkaline phosphatase-conjugated secondary antibodies and developing with NBT-BCIP.
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Figure 4: Copurification of LcrG variants with His6-tagged LcrV. Cells of Y. pestis KIM8-3002.8 (ΔlcrGV2) containing plasmids pAra-HT-V and pAraG18K (lanes 1 to 3), pJM131 (LcrG S23R) (lanes 4 to 6), pJM99 (LcrG L30R) (lanes 7 to 9), or pJM89 (LcrG A16R) (lanes 10 to12) were grown in TMH with calcium and induced with 0.2% (wt/vol) arabinose prior to temperature shift to 37°C. Cultures were harvested after 4 h of growth at 37°C, and cellular extracts were disintegrated using a French press (20,000 lb/in2). Unbroken cells and large debris were removed by centrifugation (14,000 × g) for 10 min and the cleared extracts (lanes 1, 4, 7, and 10) were applied to a Talon column. Proteins that did not bind were collected as the flowthrough fraction (lanes 2, 5, 8, and 11). Proteins were eluted from the column with 50 mM imidazole and collected (lanes 3, 6, 9, and 12). All protein samples were resolved by SDS-PAGE in a 12.5% polyacrylamide gel after dilution in 2X SDS sample buffer and analyzed by immunoblotting with α-LcrG and α-LcrV. Proteins were visualized by probing with alkaline phosphatase-conjugated secondary antibodies and developing with NBT-BCIP.

Mentions: To evaluate the biological significance of the changes in interaction seen in the yeast two-hybrid system between LcrV and LcrG S23R or LcrG L30R, LcrG S23R and L30R were expressed in lcrG Y. pestis where their ability to control Yops secretion was examined. Neither the S23R or the L30R mutant displayed the calcium-independent growth phenotype previously observed for LcrG A16R [20]. LcrG S23R displayed a wildtype growth and secretion phenotype while LcrG L30R was Ca2+-blind (data not shown). To further understand the phenotypes obtained, the ability of LcrG S23R and L30R to interact with LcrV was examined in Y. pestis. Wild type LcrG, LcrG S23R, LcrG L30R, and LcrG A16R were independently co-expressed with His6-tagged LcrV in a strain of Y. pestis deleted for lcrGV. The cells were French pressed and the cleared lysates were applied to Talon columns to purify His6-tagged LcrV. As previously reported [20], LcrG copurified with LcrV and LcrG A16R did not copurify with LcrV (Fig. 4, lanes 3 and 12). When LcrG S23R was coexpressed with His6-tagged LcrV, a small amount was recovered in the elution fraction from the column (Fig. 4, lane 6). This result is indicative of a weaker interaction between LcrG S23R and LcrV than the interaction between wildtype LcrG and LcrV. The weaker interaction seen in Y. pestis between LcrG S23R and LcrV corresponds with our yeast two-hybrid data for the S23R mutant, which showed an interaction with LcrV, and suggests an interaction with decreased affinity (Table 2). The interaction results for LcrG S23R provide an explanation for the failure of LcrG S23R to confer a Ca2+-independent phenotype like the previously described LcrG A16R mutant. LcrG S23R still interacts with LcrV and therefore LcrV can neutralize the LcrG-mediated secretion block. In fact, this result is consistent with the fact that very low levels of LcrG are required to provide LcrG function [20]. LcrG L30R either interacts weakly with or does not interact with His6-tagged LcrV in Y. pestis (Fig. 4, lane 9). This result also corresponds with the lack of interaction observed in the yeast two-hybrid assay for this mutant. Taken together, these results suggest that the residues found to be involved in the LcrG-LcrV interaction using two-hybrid analysis are relevant in the bacterial cell. However, the inability of L30R to function like our previously described A16R mutant was somewhat surprising. One explanation is that the protein with the L30R change is simply misfolded. The second explanation is that the secretion-blocking and the LcrV-interaction domains of LcrG overlap. Currently we do not possess the data required to differentiate between the two possibilities and we are pursuing the reasons for the failure of L30R to function like wildtype LcrG.


Interaction of the Yersinia pestis type III regulatory proteins LcrG and LcrV occurs at a hydrophobic interface.

Matson JS, Nilles ML - BMC Microbiol. (2002)

Copurification of LcrG variants with His6-tagged LcrV. Cells of Y. pestis KIM8-3002.8 (ΔlcrGV2) containing plasmids pAra-HT-V and pAraG18K (lanes 1 to 3), pJM131 (LcrG S23R) (lanes 4 to 6), pJM99 (LcrG L30R) (lanes 7 to 9), or pJM89 (LcrG A16R) (lanes 10 to12) were grown in TMH with calcium and induced with 0.2% (wt/vol) arabinose prior to temperature shift to 37°C. Cultures were harvested after 4 h of growth at 37°C, and cellular extracts were disintegrated using a French press (20,000 lb/in2). Unbroken cells and large debris were removed by centrifugation (14,000 × g) for 10 min and the cleared extracts (lanes 1, 4, 7, and 10) were applied to a Talon column. Proteins that did not bind were collected as the flowthrough fraction (lanes 2, 5, 8, and 11). Proteins were eluted from the column with 50 mM imidazole and collected (lanes 3, 6, 9, and 12). All protein samples were resolved by SDS-PAGE in a 12.5% polyacrylamide gel after dilution in 2X SDS sample buffer and analyzed by immunoblotting with α-LcrG and α-LcrV. Proteins were visualized by probing with alkaline phosphatase-conjugated secondary antibodies and developing with NBT-BCIP.
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Related In: Results  -  Collection

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Figure 4: Copurification of LcrG variants with His6-tagged LcrV. Cells of Y. pestis KIM8-3002.8 (ΔlcrGV2) containing plasmids pAra-HT-V and pAraG18K (lanes 1 to 3), pJM131 (LcrG S23R) (lanes 4 to 6), pJM99 (LcrG L30R) (lanes 7 to 9), or pJM89 (LcrG A16R) (lanes 10 to12) were grown in TMH with calcium and induced with 0.2% (wt/vol) arabinose prior to temperature shift to 37°C. Cultures were harvested after 4 h of growth at 37°C, and cellular extracts were disintegrated using a French press (20,000 lb/in2). Unbroken cells and large debris were removed by centrifugation (14,000 × g) for 10 min and the cleared extracts (lanes 1, 4, 7, and 10) were applied to a Talon column. Proteins that did not bind were collected as the flowthrough fraction (lanes 2, 5, 8, and 11). Proteins were eluted from the column with 50 mM imidazole and collected (lanes 3, 6, 9, and 12). All protein samples were resolved by SDS-PAGE in a 12.5% polyacrylamide gel after dilution in 2X SDS sample buffer and analyzed by immunoblotting with α-LcrG and α-LcrV. Proteins were visualized by probing with alkaline phosphatase-conjugated secondary antibodies and developing with NBT-BCIP.
Mentions: To evaluate the biological significance of the changes in interaction seen in the yeast two-hybrid system between LcrV and LcrG S23R or LcrG L30R, LcrG S23R and L30R were expressed in lcrG Y. pestis where their ability to control Yops secretion was examined. Neither the S23R or the L30R mutant displayed the calcium-independent growth phenotype previously observed for LcrG A16R [20]. LcrG S23R displayed a wildtype growth and secretion phenotype while LcrG L30R was Ca2+-blind (data not shown). To further understand the phenotypes obtained, the ability of LcrG S23R and L30R to interact with LcrV was examined in Y. pestis. Wild type LcrG, LcrG S23R, LcrG L30R, and LcrG A16R were independently co-expressed with His6-tagged LcrV in a strain of Y. pestis deleted for lcrGV. The cells were French pressed and the cleared lysates were applied to Talon columns to purify His6-tagged LcrV. As previously reported [20], LcrG copurified with LcrV and LcrG A16R did not copurify with LcrV (Fig. 4, lanes 3 and 12). When LcrG S23R was coexpressed with His6-tagged LcrV, a small amount was recovered in the elution fraction from the column (Fig. 4, lane 6). This result is indicative of a weaker interaction between LcrG S23R and LcrV than the interaction between wildtype LcrG and LcrV. The weaker interaction seen in Y. pestis between LcrG S23R and LcrV corresponds with our yeast two-hybrid data for the S23R mutant, which showed an interaction with LcrV, and suggests an interaction with decreased affinity (Table 2). The interaction results for LcrG S23R provide an explanation for the failure of LcrG S23R to confer a Ca2+-independent phenotype like the previously described LcrG A16R mutant. LcrG S23R still interacts with LcrV and therefore LcrV can neutralize the LcrG-mediated secretion block. In fact, this result is consistent with the fact that very low levels of LcrG are required to provide LcrG function [20]. LcrG L30R either interacts weakly with or does not interact with His6-tagged LcrV in Y. pestis (Fig. 4, lane 9). This result also corresponds with the lack of interaction observed in the yeast two-hybrid assay for this mutant. Taken together, these results suggest that the residues found to be involved in the LcrG-LcrV interaction using two-hybrid analysis are relevant in the bacterial cell. However, the inability of L30R to function like our previously described A16R mutant was somewhat surprising. One explanation is that the protein with the L30R change is simply misfolded. The second explanation is that the secretion-blocking and the LcrV-interaction domains of LcrG overlap. Currently we do not possess the data required to differentiate between the two possibilities and we are pursuing the reasons for the failure of L30R to function like wildtype LcrG.

Bottom Line: Our results demonstrate that the hydrophobic face of the putative helix is required for LcrV interaction.Our results demonstrate that LcrG interacts with LcrV via hydrophobic interactions located in the N-terminus of LcrG within a predicted coiled-coil motif.We also obtained preliminary evidence that the secretion blocking activity of LcrG is located between amino acids 39 and 53.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, ND 58202, USA. jyl_matson@und.nodak.edu

ABSTRACT

Background: Secretion of anti-host proteins by Yersinia pestis via a type III mechanism is not constitutive. The process is tightly regulated and secretion occurs only after an appropriate signal is received. The interaction of LcrG and LcrV has been demonstrated to play a pivotal role in secretion control. Previous work has shown that when LcrG is incapable of interacting with LcrV, secretion of anti-host proteins is prevented. Therefore, an understanding of how LcrG interacts with LcrV is required to evaluate how this interaction regulates the type III secretion system of Y. pestis. Additionally, information about structure-function relationships within LcrG is necessary to fully understand the role of this key regulatory protein.

Results: In this study we demonstrate that the N-terminus of LcrG is required for interaction with LcrV. The interaction likely occurs within a predicted amphipathic coiled-coil domain within LcrG. Our results demonstrate that the hydrophobic face of the putative helix is required for LcrV interaction. Additionally, we demonstrate that the LcrG homolog, PcrG, is incapable of blocking type III secretion in Y. pestis. A genetic selection was utilized to obtain a PcrG variant capable of blocking secretion. This PcrG variant allowed us to locate a region of LcrG involved in secretion blocking.

Conclusion: Our results demonstrate that LcrG interacts with LcrV via hydrophobic interactions located in the N-terminus of LcrG within a predicted coiled-coil motif. We also obtained preliminary evidence that the secretion blocking activity of LcrG is located between amino acids 39 and 53.

Show MeSH
Related in: MedlinePlus