Delivery of Yop effector proteins by pathogenic Yersinia across the eukaryotic cell membrane requires LcrV, YopB and YopD. These proteins were also required for channel formation in infected erythrocytes and, using different osmolytes, the contact‐dependent haemolysis assay was used to study channel size. Channels associated with LcrV were around 3 nm, whereas the homologous PcrV protein of Pseudomonas aeruginosa induced channels of around 2 nm in diameter. In lipid bilayer membranes, purified LcrV and PcrV induced a stepwise conductance increase of 3 nS and 1 nS, respectively, in 1 M KCl. The regions important for channel size were localized to amino acids 127–195 of LcrV and to amino acids 106–173 of PcrV. The size of the channel correlated with the ability to translocate Yop effectors into host cells. We suggest that LcrV is a size‐determining structural component of the Yop translocon.
Bacteria that infect humans and animals face a hard combat with the host´s immune system and in order to establish infection, pathogenic bacteria has evolved mechanisms to avoid being cleared from the host tissue. Many Gram-negatives carry a Type 3 secretion (T3S) system that is used to deliver effector proteins (toxins) into host cells. The toxins exhibit a broad range of intra cellular effects that has in common that they increase the chances of the bacteria to establish infection, multiply in infected tissue or spread to other tissues or hosts. The object of this study was to analyse the mechanisms behind the T3S effectors delivery into target cells. Two bacterial proteins, LcrV and YopD, which are involved in the translocation of effectors were analyzed by mutagenesis. LcrV was found to affect the efficiency of the translocation, probably by determining the size of the pore in the target cell membrane through which the effectors pass. Truncated variants of the multi-functional YopD revealed that defined regions of the protein were important for pore-formation and translocation. Effectors and translocators were demonstrated to form complexes with free acyl chains (lipids) at the bacterial surface. These complexes –termed Yop-lipid complexes, (YLC)– are released from the surface and can act as discrete units that are able to promote translocation of effectors even when separated from the bacterium from which they originate. These findings shed new light on the process of effector translocation by Yersinia and possibly by other gram-negative bacterial pathogens with a similar T3S setup.