Transmembrane domains of type III-secreted proteins affect bacterial-host interactions in enteropathogenic E. coli

Abstract

Many bacterial pathogens utilize a specialized secretion system, termed type III secretion system (T3SS), to translocate effector proteins into host cells and establish bacterial infection. The T3SS is anchored within the bacterial membranes and contains a long needle/filament that extends toward the host-cell and forms, at its distal end, a pore complex within the host membrane. The T3SS pore complex consists of two bacterial proteins, termed SctB and SctE, which have conflicting targeting indications; a signal sequence that targets to secretion to the extracellular environment via the T3SS, and transmembrane domains (TMDs) that target to membrane localization. In this study, we investigate whether the TMD sequences of SctB and SctE have special features that differentiate them from classical TMDs and allow them to escape bacterial membrane integration. For this purpose, we exchanged the SctB and SctE native TMDs for alternative hydrophobic sequences and found that the TMD sequences of SctB and SctE dictate membrane destination (bacterial versus host membrane). Moreover, we examined the role of the SctB TMD sequence in the activity of the full-length protein, post secretion, and found that the TMD does not serve only as a hydrophobic segment, but is also involved in the ability of the protein to translocate itself and other proteins into and across the host cell membrane.

The transmembrane domains of the type III secretion system effector Tir are involved in its secretion and cellular activities

“…We observed that the TMD2-exchanged variants, which showed reduced secretion, were enriched in the bacterial membrane compared with Tir wt -V5 and TMD1-exchanged variants ( Figure 2A ). This result is in line with our previous report showing that replacement of the translocator TMDs with an alternative hydrophobic sequence resulted in their mislocalization into the bacterial membrane ( Gershberg et al., 2021 ). Interestingly, the Tir- EscD1 -V5 variant did not display enriched membrane localization, despite containing the only TMD sequence of an actual membrane protein.…”

“…While the mechanism underlying this targeting discrimination remains largely unknown, recent studies have suggested that a major factor is the moderate hydrophobicity of the TMDs of these secreted proteins, which is sufficient for integration into eukaryotic cell membranes but is not recognized by the bacterial signal recognition particle ( Krampen et al., 2018 ). This was recently demonstrated by our group, where replacement of the TMDs of EPEC translocators (EspB and EspD) with a more hydrophobic sequence resulted in their mislocalization into the bacterial membrane and abolished their secretion ( Gershberg et al., 2021 ). In addition, the ability of Tir to escape the co-translational pathway and membrane integration mechanism was previously suggested to be mediated through interaction with its chaperone, CesT ( Elliott et al., 1999 ).…”

“…recognized by the bacterial signal recognition particle (Krampen et al, 2018). This was recently demonstrated by our group, where replacement of the TMDs of EPEC translocators (EspB and EspD) with a more hydrophobic sequence resulted in their mislocalization into the bacterial membrane and abolished their secretion (Gershberg et al, 2021). In addition, the ability of Tir to escape the cotranslational pathway and membrane integration mechanism was previously suggested to be mediated through interaction with its chaperone, CesT (Elliott et al, 1999).…”

“…We previously found that the TMDs of two TMD-containing secreted proteins are critical to promote protein secretion and prevent protein integration into the bacterial membrane ( Gershberg et al., 2021 ). To examine whether Tir TMD sequences are also critical for protein secretion, we cloned Tir labeled with a V5 tag at its C terminus (pTir wt -V5) into a plasmid and created three Tir TMD variants with an alternative hydrophobic sequence.…”

“…Tir belongs to the unique family of TMD-containing secreted proteins, which manage to escape integration into the bacterial membrane and get inserted into the host plasma membrane instead. Previous research has demonstrated that the TMDs of T3SS translocators are important for the ability of the protein to be secreted and for their postsecretion functions ( Gershberg et al., 2021 ).…”