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YscX was discovered as an essential part of the Yersinia type III secretion system about 20 years ago. It is required for substrate secretion and is exported itself. Despite this central role, its precise function and mode of action remain unknown. In order to address this knowledge gap, this present study refocused attention on YscX to build on the recent advances in the understanding of YscX function. Our experiments identified an N-terminal secretion domain in YscX promoting its secretion, with the first five codons constituting a minimal signal capable of promoting secretion of the signal less β-lactamase reporter. Replacing the extreme YscX N-terminus with known secretion signals of other Ysc-Yop substrates revealed that the YscX N-terminal segment contains non-redundant information needed for YscX function. Further, both in cis deletion of the YscX N-terminus in the virulence plasmid and ectopic expression of epitope-tagged YscX variants again lead to stable YscX production but not type III secretion of Yop effector proteins. Mislocalisation of the needle components, SctI and SctF, accompanied this general defect in Yops secretion. Hence, a coupling exists between YscX secretion permissiveness and the assembly of an operational secretion system.

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.

Type III secretion systems are a prolific virulence determinant among Gram-negative bacteria. They are used to paralyze the host cell, which enables bacterial pathogens to establish often fatal infections—unless an effective therapeutic intervention is available. However, as a result of a catastrophic rise in infectious bacteria resistant to conventional antibiotics, these bacteria are again a leading cause of worldwide mortality. Hence, this report describes a pDM4-based site-directed mutagenesis strategy that is assisting in our foremost objective to better understand the fundamental workings of the T3SS, using Yersinia as a model pathogenic bacterium. Examples are given that clearly document how pDM4-mediated site-directed mutagenesis has been used to establish clean point mutations and in-frame deletion mutations that have been instrumental in identifying and understanding the molecular interactions between components of the Yersinia type III secretion system.

Yersinia bacteria target Yop effector toxins to the interior of host immune cells by the Ysc-Yop type III secretion system. A YopN-TyeA heterodimer is central to controlling Ysc-Yop targeting activity. A + 1 frameshift event in the 3-prime end of yopN can also produce a singular secreted YopN-TyeA polypeptide that retains some regulatory function even though the C-terminal coding sequence of this YopN differs greatly from wild type. Thus, this YopN C-terminal segment was analyzed for its role in type III secretion control. Bacteria producing YopN truncated after residue 278, or with altered sequence between residues 279 and 287, had lost type III secretion control and function. In contrast, YopN variants with manipulated sequence beyond residue 287 maintained full control and function. Scrutiny of the YopN-TyeA complex structure revealed that residue W₂₇₉ functioned as a likely hydrophobic contact site with TyeA. Indeed, a YopN_W279G mutant lost all ability to bind TyeA. The TyeA residue F₈ was also critical for reciprocal YopN binding. Thus, we conclude that specific hydrophobic contacts between opposing YopN and TyeA termini establishes a complex needed for regulating Ysc-Yop activity.

Several pathogenic Gram-negative bacteria invest in sophisticated type III secretion systems (T3SS) to incapacitate their eukaryotic hosts. T3SSs can secrete protein cargo outside the bacterial cell and also target many of them into the eukaryotic cell interior. Internalized proteins promote bacterial colonization, survival and transmission, and can often cause severe disease. An example is the Ysc-Yop T3SS apparatus assembled by pathogenic Yersinia spp. A correctly assembled Ysc-Yop T3SS spans the Yersinia envelope and also protrudes from the bacterial surface. Upon host cell contact, this system is competent to secrete hydrophobic translocators that form a translocon pore in the host cell membrane to complete the delivery channel bridging both bacterial and host cells. Newly synthesized effector Yops may pass through this channel to gain entry into the host cell cytosol.As type III secretion (T3S) substrates function sequentially during infection, it is hypothesized that substrate export is temporally controlled to ensure that those required first are prioritized for secretion. On this basis three functional groups are classified as early (i.e. structural components), middle (i.e. translocators) and late (i.e. effectors). Factors considered to orchestrate the T3S of substrates are many, including the intrinsic substrate secretion signal sequences, customized chaperones, and recognition/sorting platforms at the base of the assembled T3SS. Investigating the interplay between these elements is critical for a better understanding of the molecular mechanisms governing export control during Yersinia T3S.To examine the composition of the N-terminal T3S signals of the YscX early substrate and the YopD middle substrate, these segments were altered by mutagenesis and the modified substrates analyzed for their T3S. Translational fusions between these signals and a signalless β-Lactamase were used to determine their optimal length required for efficient T3S. This revealed that YscX and YopD export is most efficiently supported by their first 15 N-terminal residues. At least for YopD, this is a peptide signal and not base upon information in the mRNA sequence. Moreover, features within and upstream of this segment contribute to their translational control. In parallel, bacteria were engineered to produce substrate chimeras where the N-terminal segments were exchanged between substrates of different classes in an effort to examine the temporal dynamics of T3S. In several cases, Yersinia producing chimeric substrates were defective in T3S activity, which could be a consequence of disturbing a pre-existing hierarchal secretion mechanism.YopN and TyeA regulatory molecules can be naturally produced as a 42 kDa YopN-TyeA hybrid, via a +1 frame shift event somewhere at the 5’-end of yopN. To study this event, Yersinia were engineered to artificially produce this hybrid, and these maintained in vitro T3S control of both middle and late substrates. However, modestly diminished directed targeting of effectors into eukaryotic cells correlated to virulence attenuation in vivo. Upon further investigation, a YopN C-terminal segment encompassing residues 278 to 287 was probably responsible, as this region is critical for YopN to control T3S, via enabling a specific interaction with TyeA.Investigated herein were molecular mechanisms to orchestrate substrate export by the T3SS of Yersinia. While N-terminal secretion signals may contribute to specific substrate order, the YopN and TyeA regulatory molecules do not appear to distinguish between the different substrate classes.

Type III secretion is a tightly controlled virulence mechanism utilized by many gram negative bacteria to colonize their eukaryotic hosts. To infect their host, human pathogenic Yersinia spp. translocate protein toxins into the host cell cytosol through a preassembled Ysc-Yop type III secretion device. Several of the Ysc-Yop components are known for their roles in controlling substrate secretion and translocation. Particularly important in this role is the YopN and TyeA heterodimer. In this study, we confirm that Y. pseudotuberculosis naturally produce a 42 kDa YopN-TyeA hybrid protein as a result of a +1 frame shift near the 3 prime of yopN mRNA, as has been previously reported for the closely related Y. pestis. To assess the biological role of this YopN-TyeA hybrid in T3SS by Y. pseudotuberculosis, we used in cis site-directed mutagenesis to engineer bacteria to either produce predominately the YopN-TyeA hybrid by introducing +1 frame shifts to yopN after codon 278 or 287, or to produce only singular YopN and TyeA polypeptides by introducing yopN sequence from Y. enterocolitica, which is known not to produce the hybrid. Significantly, the engineered 42 kDa YopN-TyeA fusions were abundantly produced, stable, and were efficiently secreted by bacteria in vitro. Moreover, these bacteria could all maintain functionally competent needle structures and controlled Yops secretion in vitro. In the presence of host cells however, bacteria producing the most genetically altered hybrids (+1 frameshift after 278 codon) had diminished control of polarized Yop translocation. This corresponded to significant attenuation in competitive survival assays in orally infected mice, although not at all to the same extent as Yersinia lacking both YopN and TyeA proteins. Based on these studies with engineered polypeptides, most likely a naturally occurring YopN-TyeA hybrid protein has the potential to influence T3S control and activity when produced during Yersinia-host cell contact.

Type III secretion enables bacteria to intoxicate eukaryotic cells with anti-host effectors. A class of secreted cargo are the two hydrophobic translocators that form a translocon pore in the host cell plasma membrane through which the translocated effectors may gain cellular entry. In pathogenic Yersinia, YopB and YopD shape this translocon pore. Here, four in cis yopD mutations were constructed to disrupt a predicted α-helix motif at the C-terminus. Mutants YopD_(I262P) and YopD_(K267P) poorly localized Yop effectors into target eukaryotic cells and failed to resist uptake and killing by immune cells. These defects were due to deficiencies in host-membrane insertion of the YopD-YopB translocon. Mutants YopD_(A263P) and YopD_(A270P) had no measurable in vitro translocation defect, even though they formed smaller translocon pores in erythrocyte membranes. Despite this, all four mutants were attenuated in a mouse infection model. Hence, YopD variants have been generated that can spawn translocons capable of targeting effectors in vitro, yet were bereft of any lethal effect in vivo. Therefore, Yop translocators may possess other in vivo functions that extend beyond being a portal for effector delivery into host cells.

Pathogenic Yersinia all harbor a virulence plasmid-encoded Ysc–Yop T3SS. In this system, translocator function is performed by the hydrophobic proteins YopB and YopD. With the goal to better understand how YopD orchestrates yop-regulatory control, translocon pore formation and Yop effector translocation, we performed an in silico prediction of coiled-coil motifs in YopD and YopD-like sequences from other bacteria. Of interest was a predicted N-terminal coiled-coil that occurred solely in Yersinia YopD sequences. To investigate if this unique feature was biologically relevant, two in cis point mutations were generated with a view to disrupting this putative structure. Both mutants maintained full T3SS function in vitro in terms of environmental control of Yops synthesis and secretion, effector toxin translocation and evasion of phagocytosis and killing by cultured immune cells. However, these same mutants were attenuated for virulence in a murine oral-infection model. The cause of this tardy disease progression is unclear. However, these data indicate that any structural flaw in this element unique to the N-terminus will subtly compromise an aspect of YopD biology. Sub-optimal T3SSs are then formed that are unable to fortify Yersinia against attack by the host innate and adaptive immune response.

Type III secretion systems (T3SSs) secrete needle components, pore-forming translocators, and the translocated effectors. In part, effector recognition by a T3SS involves their N-terminal amino acids and their 5′ mRNA. To investigate whether similar molecular constraints influence translocator secretion, we scrutinized this region within YopD from Yersinia pseudotuberculosis. Mutations in the 5′ end of yopD that resulted in specific disruption of the mRNA sequence did not affect YopD secretion. On the other hand, a few mutations affecting the protein sequence reduced secretion. Translational reporter fusions identified the first five codons as a minimal N-terminal secretion signal and also indicated that the YopD N terminus might be important for yopD translation control. Hybrid proteins in which the N terminus of YopD was exchanged with the equivalent region of the YopE effector or the YopB translocator were also constructed. While the in vitro secretion profile was unaltered, these modified bacteria were all compromised with respect to T3SS activity in the presence of immune cells. Thus, the YopD N terminus does harbor a secretion signal that may also incorporate mechanisms of yopD translation control. This signal tolerates a high degree of variation while still maintaining secretion competence suggestive of inherent structural peculiarities that make it distinct from secretion signals of other T3SS substrates.

Type III secretion enables bacteria to intoxicate eukaryotic cells with anti-host effectors. A class of secreted cargo are the two hydrophobic translocators that form a translocon pore in the host cell plasma membrane through which the translocated effectors may gain cellular entry. In pathogenic Yersinia, YopB and YopD shape this translocon pore. Here, four in cis yopD mutations were constructed to disrupt a predicted a-helix motif at the C-terminus. Mutants YopD_I262P and YopD_K267P poorly localised Yop effectors into target eukaryotic cells and failed to resist uptake and killing by immune cells. These defects were due to deficiencies in host-membrane insertion of the YopD-YopB translocon. Mutants YopD_A263P and YopD_A270P had no measurable in vitro translocation defect, even though they formed smaller translocon pores in erythrocyte membranes. Despite this, all four mutants were attenuated in a mouse infection model. Hence, YopD variants have been generated that can spawn translocons capable of targeting effectors in vitro, yet were bereft of any lethal effect in vivo. It is therefore probable that an active translocon makes a range of contributions during bacteria-host cell contact that extends beyond effector delivery per se.