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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.

Certain Gram‐negative bacteria use type III secretion systems to deliver effectorproteins into eukaryotic cells, serving either parasitic or mutualistic roles inside the hostcell. About 25 structural proteins are needed to assemble and deliver effector proteins.Collections of these proteins are quite well characterized, although the function ofsome continues to remain obscure. This is true for the Yersinia Ysc‐Yop systemcomponents YscX, a secreted substrate and YscY, its cognate non‐secreted chaperone.Despite recent evidence suggesting that they might coordinate Yop substrate secretion,YscX and YscY remain poorly characterized. To further investigate the function of theseproteins in the enteropathogen Y. pseudotuberculosis, we explored correlationsbetween the YscX N‐terminal segment, YscX secretion, as well as the secretion of otherYops. Analysis of a series of chimeric substrates in which the extreme YscX N‐terminushad been exchanged with equivalent functional secretion signals of other Ysc‐Yopsubstrates revealed that this segment contains non‐redundant information needed forYscX function, which includes permitting surface polymerization of the YscF needle andYops secretion. Further, in cis deletion of the YscX N‐terminus and ectopic expression ofepitope tagged YscX variants again correlated stable YscX production but not secretionto the type III secretion of Yops. Despite this, the first 5 codons were determined toconstitute a minimal signal capable of promoting secretion of the signalless ‐lactamasereporter. Hence, YscX does contain a fully equipped N‐terminal secretor domain topromote secretion of self. Nevertheless, the primary role of this N‐terminal segmentmust be to assemble an operational secretion system, and this occurs independently ofYscX secretion.

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.

Pathogenic Yersinia spp. utilizes the Ysc-Yop type III secretion system to targetYop effector proteins into the cytosol of host immune cells. Internalizedeffectors alter specific signaling pathways to neutralize immune cell-dependentphagocytosis, killing and pro-inflammatory responsiveness. This enablesextracellular bacterial multiplication and survival in immune tissue. Central tothe temporal control of Yop type III secretion is the regulator YopN. Incomplex with TyeA, YopN acts to plug the inner face of the type III secretionchannel, denying entry to other Yop substrates until after YopN has beensecreted. A +1 frameshift event in the 3-prime end of yopN results in thesynthesis of a singular secreted YopN-TyeA polypeptide chimera that retainssome regulatory function. As the C-terminal coding sequence of YopN in thishybrid product differs greatly from native sequence, we used site-directedmutagenesis to determine the functional significance of this segment. YopNtruncated at residue 287 or containing a shuffled sequence covering 288 to 293retains full function both in vitro and in vivo. Thus, the extreme C-terminus isapparently superfluous to YopN function. In contrast, a YopN varianttruncated after residue 278 was completely unstable, and these bacteria hadlost all control of T3S activity, and failed to defend against immune cell killing.Interestingly, inclusion of a shuffled sequence from residues 279 to 287recovered some T3S control over function. Hence, the YopN segmentencompassing 279 to 287 is essential for full function, although the exact aminoacid sequence is less important.