RESUMEN

Many bacterial pathogens employ a protein complex, termed the type III secretion system (T3SS), to inject bacterial effectors into host cells. These effectors manipulate various cellular processes to promote bacterial growth and survival. The T3SS complex adopts a nano-syringe shape that is assembled across the bacterial membranes, with an extracellular needle extending toward the host cell membrane. The assembly of the T3SS is initiated by the association of three proteins, known as SctR, SctS, and SctT, which create an entry portal to the translocation channel within the bacterial inner membrane. Using the T3SS of enteropathogenic Escherichia coli, we investigated, by mutational and functional analyses, the role of two structural construction sites formed within the SctRST complex and revealed that they are mutation-resistant components that are likely to act as seals preventing leakage of ions and metabolites rather than as substrate gates. In addition, we identified two residues in the SctS protein, Pro23, and Lys54, that are critical for the proper activity of the T3SS. We propose that Pro23 is critical for the physical orientation of the SctS transmembrane domains that create the tip of the SctRST complex and for their positioning with regard to other T3SS substructures. Surprisingly, we found that SctS Lys54, which was previously suggested to mediate the SctS self-oligomerization, is critical for T3SS activity due to its essential role in SctS-SctT hetero-interactions.

Asunto(s)

Escherichia coli Enteropatógena/metabolismo , Proteínas de Escherichia coli/metabolismo , Sistemas de Secreción Tipo III/metabolismo , Secuencias de Aminoácidos , Escherichia coli Enteropatógena/química , Escherichia coli Enteropatógena/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Unión Proteica , Dominios Proteicos , Sistemas de Secreción Tipo III/química , Sistemas de Secreción Tipo III/genética

RESUMEN

Diarrheal diseases remain a major public health concern worldwide. Many of the causative bacterial pathogens that cause these diseases have a specialized protein complex, the type III secretion system (T3SS), which delivers effector proteins directly into host cells. These effectors manipulate host cell processes for the benefit of the infecting bacteria. The T3SS structure resembles a syringe anchored within the bacterial membrane, projecting toward the host cell membrane. The entry port of the T3SS substrates, called the export apparatus, is formed by five integral membrane proteins. Among the export apparatus proteins, EscV is the largest, and as it forms a nonamer, it constitutes the largest portion of the export apparatus complex. While there are considerable data on the soluble cytoplasmic domain of EscV, our knowledge of its membrane-associated section and its transmembrane domains (TMDs) is still very limited. In this study, using an isolated genetic reporter system, we found that TMD5 and TMD6 of EscV mediate strong self-oligomerization. Substituting these TMDs within the full-length protein with a random hydrophobic sequence resulted in a complete loss of function of the T3SS, further suggesting that the EscV TMD5 and TMD6 sequences have a functional role in addition to their structural role as membrane anchors. As we observed only mild reduction in the ability of the TMD-exchanged variants to integrate into the full or intermediate T3SS complexes, we concluded that EscV TMD5 and TMD6 are not crucial for the global assembly or stability of the T3SS complex but are rather involved in promoting the necessary TMD-TMD interactions within the complex and the overall TMD orientation to allow channel opening for the entry of T3SS substrates.