Autonomous translocation and intracellular trafficking of the cell-penetrating and immune-suppressive effector protein YopM

Abstract

Extracellular Gram-negative pathogenic bacteria target essential cytoplasmic processes of eukaryotic cells by using effector protein delivery systems such as the type III secretion system (T3SS). These secretion systems directly inject effector proteins into the host cell cytoplasm. Among the T3SS-dependent Yop proteins of pathogenic Yersinia, the function of the effector protein YopM remains enigmatic. In a recent study, we demonstrated that recombinant YopM from Yersinia enterocolitica enters host cells autonomously without the presence of bacteria and thus identified YopM as a novel bacterial cell-penetrating protein. Following entry YopM down-regulates expression of pro-inflammatory cytokines such as tumor necrosis factor α. These properties earmark YopM for further development as a novel anti-inflammatory therapeutic. To elucidate the uptake and intracellular targeting mechanisms of this bacterial cell-penetrating protein, we analyzed possible routes of internalization employing ultra-cryo electron microscopy. Our results reveal that under physiological conditions, YopM enters cells predominantly by exploiting endocytic pathways. Interestingly, YopM was detected free in the cytosol and inside the nucleus. We could not observe any colocalization of YopM with secretory membranes, which excludes retrograde transport as the mechanism for cytosolic release. However, our findings indicate that direct membrane penetration and/or an endosomal escape of YopM contribute to the cytosolic and nuclear localization of the protein. Surprisingly, even when endocytosis is blocked, YopM was found to be associated with endosomes. This suggests an intracellular endosome-associated transport of YopM.

Bacterium-Derived Cell-Penetrating Peptides Deliver Gentamicin To Kill Intracellular Pathogens

“…These data suggest that ␣1H and ␣2H employ direct translocation as an alternative or even parallel uptake mechanism to enter HeLa cells. This is in accordance with our previous findings for full-length YopM which primarily enters cells by endocytosis but also employs direct membrane penetration as an entry mechanism (28,29). In contrast, Tat uptake was completely inhibited at 4°C in HeLa cells, suggesting that direct translocation of Tat can be excluded in this cell line.…”

“…In particular, two CPPs derived from the Y. enterocolitica effector protein YopM were employed and their functional properties were characterized. We had previously shown that the Y. enterocolitica-derived YopM protein can autonomously translocate into eukaryotic cells independently of the T3SS (28)(29)(30). Autonomously cell-penetrating recombinant YopM is functional and downregulates the expression of proinflammatory cytokines (4,34), suggesting a dual use of YopM as a delivery vehicle for cargo molecules and as a biological therapeutic for immunomodulation (31,32).…”

Distribution and Evolution of Yersinia Leucine-Rich Repeat Proteins

“…Therefore, the lack of coexpression of the genes with yopM does not exclude possible translocation of the LRR proteins through the Ysa T3SS. Alternatively, the LRR proteins may also enter host cells and act as effectors by other secretion mechanisms, such as autotranslocation (10). The LRR genes were not detected in the chromosomes of all Yersinia species, possibly due to acquisition of the ancestral genes after species diversification had occurred.…”

“…Boland et al sequenced a homologous yopM gene from Y. enterocolitica strain W22703 and demonstrated that the N-terminal 100 residues gave YopM the ability to translocate into the host cell cytoplasm through the Ysc type III secretion system (T3SS) (8). YopM also showed the ability to enter the host cell cytoplasm by autonomous translocation independently of T3SSs (9,10). In another study, YopM-specific antibodies did not slow the progression of experimental plague, indicating that YopM may have a major intracellular function (11).…”

“…The C terminus and an internal portion of YopM were essential for the interactions with RSK1 and RSK2, respectively, and for the expression of proinflammatory cytokines (17)(18)(19). The interaction of YopM with RSKs and the resultant consequences may explain the observation of interference of YopM with host innate immunity (10,(20)(21)(22)(23). LaRock and Cookson showed that YopM inhibits caspase-1 activity and alters inflammasome assembly and processing (24).…”