Heterologous Complementation Studies With the YscX and YscY Protein Families Reveals a Specificity for Yersinia pseudotuberculosis Type III Secretion - PubMed (original) (raw)

Heterologous Complementation Studies With the YscX and YscY Protein Families Reveals a Specificity for Yersinia pseudotuberculosis Type III Secretion

Jyoti M Gurung et al. Front Cell Infect Microbiol. 2018.

Abstract

Type III secretion systems harbored by several Gram-negative bacteria are often used to deliver host-modulating effectors into infected eukaryotic cells. About 20 core proteins are needed for assembly of a secretion apparatus. Several of these proteins are genetically and functionally conserved in type III secretion systems of bacteria associated with invertebrate or vertebrate hosts. In the Ysc family of type III secretion systems are two poorly characterized protein families, the YscX family and the YscY family. In the plasmid-encoded Ysc-Yop type III secretion system of human pathogenic Yersinia species, YscX is a secreted substrate while YscY is its non-secreted cognate chaperone. Critically, neither an yscX nor yscY null mutant of Yersinia is capable of type III secretion. In this study, we show that the genetic equivalents of these proteins produced as components of other type III secretion systems of Pseudomonas aeruginosa (PscX and PscY), Aeromonas species (AscX and AscY), Vibrio species (VscX and VscY), and Photorhabdus luminescens (SctX and SctY) all possess an ability to interact with its native cognate partner and also establish cross-reciprocal binding to non-cognate partners as judged by a yeast two-hybrid assay. Moreover, a yeast three-hybrid assay also revealed that these heterodimeric complexes could maintain an interaction with YscV family members, a core membrane component of all type III secretion systems. Despite maintaining these molecular interactions, only expression of the native yscX in the near full-length yscX deletion and native yscY in the near full-length yscY deletion were able to complement for their general substrate secretion defects. Hence, YscX and YscY must have co-evolved to confer an important function specifically critical for Yersinia type III secretion.

Keywords: LcrH/SycD; T3S chaperone; YscV; protein–protein interaction; secretion hierarchy; substrate sorting.

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Figures

Figure 1

Amino acid sequence relatedness between the YscX and YscY protein families. Amino acid sequence identity was determined by BLASTP analysis (BLASTP 2.8.0+) (Altschul et al., 1997, 2005) for the YscX protein (A) and YscY protein (B) families. Numbers outside of parenthesis indicate percent amino acid identity. This is calculated from a ratio of the numbers inside parentheses; the left number indicating the number of identical residues, and the right number indication the total number of overlapping residues in a pairwise alignment. Representative sequences were retrieved from the NCBI genome database archived with the following GI reference numbers shown in parentheses: Yps, Yersinia pseudotuberculosis (YscX, 51593904 and YscY, 51593903); Ype, Yersinia pestis (YscX, 16082724 and YscY, 16082723); Yen, Yersinia enterocolitica (YscX, 4324343 and YscY, 4324342); Asa, Aeromonas salmonicida (AscX, 66947966 and AscY, 66947967); Ahy, Aeromonas hydrophilia (AscX, 46398260 and AscY, 46398261); Pae, Pseudomonas aeruginosa (PscX, 62865832 and PscY, 62865833); Plu, Photorhabdus luminescens (SctX, 36787060 and SctY, 36787059); Vha, Vibrio harveyi (VscX, 41834176 and VscY, 41834175); Vha, Vibrio parahaemolyticus (VscX, 28898438 and VscY, 28898437). Alignments of these sequences were used to generate phylogenetic trees depicting relationships between amino acid sequences of YscX-like (C) and YscY-like (D) family members. The neighbor-joining phylogenetic tree was generated using full length amino acid sequences of YscX and YscY homologs. Genetically related YscX-like and YscY-like protein sequences from Bordetella pertusis I475 were used as an outgroup. Values next to each branch node represent bootstrap percentages for 1,000 replicates. The scale bar represents the number of amino acid substitutions per site.

Figure 2

Summary of the reciprocal binding between members of the YscX and YscY protein families. Protein–protein interactions were determined using the yeast two-hybrid assay. YscX family members were fused to the GAL4 activation domain (in pGADT7), whereas YscY family members were fused to the GAL4 DNA binding domain (in pGBKT7). Pairwise transformations were performed in S. cerevisiae AH109 (Clontech Laboratories) that contained the HIS3 and ADE2 reporter genes. Strength of interactions were determined by the extent of growth on minimal medium devoid of histidine or adenine and recorded after day 4. Green shade indicates robust yeast growth (strong binary interaction), yellow shade reflects modest growth (moderate interaction) and gray shade specifies no growth (no interaction). Due to an intrinsic leakiness with the HIS3 reporter, 4 mM 3-aminotriazole was added to histidine dropout media to suppress false positives (James et al., 1996).

Figure 3

Reciprocal interactions between members of YscX, YscY, and YscV protein families. Different protein members of the YscY family were produced as recombinant GAL4 binding domain fusions from the pBridge vector. From this vector were also produced native forms of YscX protein family members to act as bridging proteins. Different protein members of the YscV family were produced as recombinant GAL4 activation domain fusions from the pGADT7 vector. As indicated, pairwise combinations of pBridge and pGADT7 derivatives were co-transformed into the yeast strain S. cerevesiae Y190. Five-fold serial dilutions of transformed yeast were grown on a synthetic dropout (SD) agar plates lacking tryptophan (Trp) and leucine (Leu) for maintenance of the plasmid pairs and methionine (Met) to induce production of YscX-like proteins. The strength of interaction was studied by growing yeast in equivalent media lacking Histidine (His). A concentration of 40 mM 3-aminotriazole was added to reduce leaky HIS3 expression. The result shown is a representative of three independent experiments.

Figure 4

Assessing complementation of YscX and/or YscY function in Yersinia type III secretion. Bacteria were grown in BHI medium under secretion-permissive conditions (absence of Ca2+). Proteins contained within intact bacteria and secreted to the culture medium (Total) or secreted free to the extracellular medium (Supernatant) were fractionated on a 12% SDS-PAGE and analyzed by immunoblotting using polyclonal rabbit anti-YopB, anti-YopD and anti-YopE antiserum. IPTG was added to a final concentration of 0.4 mM where indicated. (A) Parent Y. pseudotuberculosis and Δ_yscX_ complemented with pMMB67EHgm or pMMB67EHgm-encoded YscX family members. Strains: Parent (YPIII/pIB102); Complemented YPIII/pIB102, pMMB67EHgm (Vector);Δ_yscX_ (YPIII/pIB880); complemented YPIII/pIB880, pMMB67EHgm (Vector); complemented YPIII/pIB880, pJEB291 (YscX+); complemented YPIII/pIB880, pJEB295 (PscX+); complemented YPIII/pIB880, pMF720 (AscXAs+); complemented YPIII/pIB880, pMF722 (AscXAh+); complemented YPIII/pIB880, pMF724 (VscXVh+); complemented YPIII/pIB880, pMF725 (VscXVp+); complemented YPIII/pIB880, pMF727 (SctX+). (B) Parent Y. pseudotuberculosis and Δ_yscY_ complemented with pMMB67EHgm or pMMB67EHgm-encoded YscY family members. Strains: Parent (YPIII/pIB102); Δ_yscY_ (YPIII/pIB890); complemented YPIII/pIB890, pMMB67EHgm (Vector); complemented YPIII/pIB890, pJEB292 (YscY+); complemented YPIII/pIB890, pJEB296 (PscY+); complemented YPIII/pIB890, pMF721 (AscYAs+); complemented YPIII/pIB890, pMF723 (AscYAh+); complemented YPIII/pIB890, pMF796 (VscYVh+); complemented YPIII/pIB890, pMF726 (VscYVp+); complemented YPIII/pIB890, pMF728 (SctY+). Molecular mass values shown in parentheses were deduced from primary amino acid sequences.

Figure 5

Assessing functional complementation of YscX and YscY by dual expression of substrate and chaperone homologs. Bacteria were grown in BHI medium under secretion-permissive conditions (absence of Ca2+). Proteins contained within intact bacteria and secreted to the culture medium (Total) or secreted free into the extracellular medium (Supernatant) were fractionated on a 12% SDS-PAGE and analyzed by immunoblotting using polyclonal rabbit anti-YopB, anti-YopD, and anti-YopE antiserum. IPTG was added to a final concentration of 0.4 mM where indicated. Strains: Parent (YPIII/pIB102); Δ_yscXyscY_ (YPIII/pIB881); complemented YPIII/pIB881, pMMB67EHgm (Vector);complemented YPIII/pIB881, pJEB340 (YscX, YscY+); complemented YPIII/pIB881, pJEB335 (PscX, PscY+); complemented YPIII/pIB881, pMF733 (AscXAs, AscYAs+); complemented YPIII/pIB881, pMF734 (AscXAh, AscYAh+); complemented YPIII/pIB881, pMF797 (VscXVh, VscYVh+); complemented YPIII/pIB881, pMF735 (VscXVp, VscYVp+); complemented YPIII/pIB881, pMF736 (SctX, SctY+). Molecular mass values shown in parentheses were deduced from primary amino acid sequences.

Figure 6

Assessing functional complementation by codon-optimized yscX_- and yscY_-like alleles. Synthesized Yop's associated with total fractions (proteins contained within intact bacteria and secreted to the culture medium) or with supernatant (secreted free to the extracellular medium) were analyzed in Δ_yscX null-mutant complemented with YscX and codon-optimized YscX-like protein family members (A) or in Δ_yscY null-mutant complemented with YscY and codon-optimized YscY-like protein family (B). Polyclonal anti-YopB, anti-YopD and anti-YopE were used for detection of Yops. The “+” symbol indicates the addition of IPTG to a final concentration of 0.4 mM. Proteins associated with bacterial pellet from Y. pseudotuberculosis cultures harboring either pFLAG-YscX (C) or pFLAG-YscY (D) grown in secretion-permissive condition (absence of Ca2+) and supplemented with increasing concentration of IPTG were analyzed by Western immunoblot using anti-FLAG™, anti-YopB, anti-YopD, anti-YopE, and anti-DnaK. The “−” symbol indicates absence of a particular component, while the “+” symbol indicates the presence of a particular component. In the case of IPTG, the “+” symbol indicates a final concentration of 0.4 mM IPTG was added, while the filled in graduation symbol reflects an incremental increase of IPTG according to the following final concentrations of 0.01, 0.02, and 0.1 mM. Steady state accumulation in the bacterial pellet of YscX protein family (E) and YscY protein family (F) produced from codon-optimized alleles following growth of Y. pseudotuberculosis in secretion-permissive conditions (absence of Ca2+). Protein was fractionated by SDS-PAGE and detected by immunoblot using monoclonal anti-FLAG™ antiserum. The “−” symbol indicates bacterial growth in the absence of IPTG, while “+” symbol indicates a final concentration of 0.4 mM was added. Strains: Parent (YPIII/pIB102); Complemented YPIII/pIB102, pMMB208 (Vector);Δ_yscX_ (YPIII/pIB880); complemented YPIII/pIB880, pMMB208 (Vector); complemented YPIII/pIB880, pJMG242 (FLAG™-YscX+); complemented YPIII/pIB880, pJMG261(FLAG™-PscX.opt+); complemented YPIII/pIB880, pJMG262 (FLAG™-AscXAs.opt+); complemented YPIII/pIB880, pJMG263 (FLAG™-AscXAh.opt+); complemented YPIII/pIB880, pJMG264 (FLAG™-VscXVh.opt+); complemented YPIII/pIB880, pJMG265 (FLAG™-VscXVp.opt+); complemented YPIII/pIB880, pMF266 (FLAG™-SctX.opt+); Δ_yscY_ (YPIII/pIB890); complemented YPIII/pIB890, pMMB208 (Vector); complemented YPIII/pIB890, pMF800 (FLAG™-YscY+); complemented YPIII/pIB890, pJMG267 (FLAG™-PscY.opt+); complemented YPIII/pIB890, pJMG268 (FLAG™-AscYAs.opt+); complemented YPIII/pIB890, pJMG269 (FLAG™-AscYAh.opt+); complemented YPIII/pIB890, pJMG270 (FLAG™-VscYVh.opt+); complemented YPIII/pIB890, pJMG271 (FLAG™-VscYVp.opt+); complemented YPIII/pIB890, pJMG272 (FLAG™-SctY.opt+); complemented YPIII/pIB880, pJEB291 (YscX+); complemented YPIII/pIB880, pJMG293 (PscX.opt+); complemented YPIII/pIB880, pJMG294 (AscXAs.opt+); complemented YPIII/pIB880, pJMG295 (AscXAh.opt+); complemented YPIII/pIB880, pJMG296 (VscXVh.opt+); complemented YPIII/pIB880, pJMG297 (VscXVp.opt+); complemented YPIII/pIB880, pJMG298 (SctX.opt+); YPIII/pIB890, pJEB292 (YscY+); YPIII/pIB890, pJMG286 (PscY.opt+); YPIII/pIB890, pJMG287 (AscYAs.opt+); YPIII/pIB890, pJMG288 (AscYAh.opt+); YPIII/pIB890, pJMG289 (VscYVh.opt+); YPIII/pIB890, pJMG290 (VscYVp.opt+); YPIII/pIB890, pJMG291 (SctY.opt+). Molecular mass values shown in parentheses were deduced from primary amino acid sequences.

Figure 7

Assessing YscX-like and YscY-like homolog function in the presence of a fully intact type III secretion system. Bacteria were grown in BHI medium under secretion-permissive condition (absence of Ca2+). Proteins contained within intact bacteria and secreted to the culture medium (Total) or secreted free into the extracellular medium (Supernatant) were fractionated on a 12% SDS-PAGE and analyzed by immunoblotting using polyclonal rabbit anti-YopB, anti-YopD, and anti-YopE antiserum. IPTG was added to a final concentration of 0.4 mM where indicated. Strains: Parent (YPIII/pIB102); Δ_yscX_ (YPIII/pIB880); Complemented YPIII/pIB102, pMMB67EHgm (Vector); Complemented YPIII/pIB102, pJEB291 (YscX+); complemented YPIII/pIB102, pJMG293 (PscX.opt+); complemented YPIII/pIB102, pJMG294 (AscXAs.opt+); complemented YPIII/pIB102, pJMG295 (AscXAh.opt+); complemented YPIII/pIB102, pJMG296 (VscXVh.opt+); complemented YPIII/pIB102, pJMG297 (VscXVp.opt+); complemented YPIII/pIB102, pJMG298 (SctX.opt+); Δ_yscY_ (YPIII/pIB890); YPIII/pIB102, pJEB292 (YscY+); YPIII/pIB102, pJMG286 (PscY.opt+); YPIII/pIB102, pJMG287 (AscYAs.opt+); YPIII/pIB102, pJMG288 (AscYAh.opt+); YPIII/pIB102, pJMG289 (VscYVh.opt+); YPIII/pIB102, pJMG290 (VscYVp.opt+); YPIII/pIB102, pJMG291 (SctY.opt+). Molecular mass values shown in parentheses were deduced from primary amino acid sequences.

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Heterologous Complementation Studies With the YscX and YscY Protein Families Reveals a Specificity for Yersinia pseudotuberculosis Type III Secretion - PubMed (original) (raw)