Characterization of putative chromosomal Type III secretion system LPX effector proteins of Y. pseudotuberculosis

The delivery of effector proteins into host cells via the Type III secretion system (T3SS) is a key virulence strategy shared by several Gram-negative pathogens. Once inside host cells, these effectors manipulate cellular signaling and immune responses to promote bacterial survival during infection. Members of the LPX protein family can modify and hijack the host ubiquitin system through their E3 ligase activities. So far, in Yersinia pseudotuberculosis YopM represents the only assigned member of this family. A recent phylogenetic analysis of the LPX family identified five chromosomally encoded putative LPX effector proteins in Y. pseudotuberculosis, designated as yersinia leucine rich repeat protein (Ylr) A, B, C, D, and E. All five proteins share the characteristic LPX domain structure, comprising two α-helices and a central LRR domain with the conserved consensus motif LeuX6LeuX2Ile/LeuProX3Pro. YlrA, YlrC, YlrD, and YlrE additionally possess a putative C-terminal novel E3 ligase domain (NEL) and are secreted via the virulence plasmid-encoded T3SS. In contrast, YlrB lacks this domain, and showed no detectable secretion.  The NEL domain of characterized LPX effectors is responsible for E3 ubiquitin ligase activity, enabling these effectors to modify and hijack the ubiquitin system of the host. The catalytic activity is proposed to be mediated by a conserved CEDR motif containing a catalytic cysteine, which is also present in YlrA (C396) and YlrC (C406). This study aimed to characterize the Ylr proteins by investigating their gene regulation, translocation into host cells, and functional properties.  

The present study identified promoter candidates and cis regulatory elements by in silico analysis of the upstream ylr gene regions. However, in vitro GFP reporter assays of the predicted upstream ylr gene regions revealed no detectable promoter activity under the conditions tested.  

To assess T3SS-mediated delivery into host cells, N-terminal domains of the Ylr proteins were fused to β-lactamase (29 kDa). However, no translocation was detected under the conditions tested. To overcome potential limitations of these truncated constructs, an alternative reporter assay using full-length proteins fused to the substantially smaller HiBiT tag (1.3 kDa) was successfully established for YopM, providing an additional tool for investigating delivery into host cells. Nevertheless, translocation of the Ylr proteins remained undetectable, possibly due to observed differences in protein abundance.  

In vitro ubiquitination assays demonstrated E3 ubiquitin ligase activity of YlrA and YlrC. Analysis of substitution mutants further revealed that the catalytic activity critically depends on the conserved cysteine residue within their NEL domains. In contrast, no activity was detected for YlrB, YlrD, and YlrE under the conditions tested. Furthermore, pull-down assays identified potential binding partners of YlrA and YlrC, suggesting biological functions in mitochondrial homeostasis and regulation of the host actin cytoskeleton. 

In summary, this study provides an initial characterization of these newly identified LPX effector proteins from Y. pseudotuberculosis. While in silico analysis of upstream regions provide a basis for the investigation of ylr gene regulation, the establishment of the HiBiT reporter assay offers an additional tool for investigating host cell delivery.