Investigating the Secretion of HtpB, the Multifunctional Chaperonin of Legionella pneumophila

Abstract

Legionella pneumophila is a ubiquitous freshwater pathogen of unicellular eukaryotes, namely several species of amoeba. Following inhalation of Legionella-contaminated aerosols, susceptible human populations may also develop an atypical, pneumonia-like illness, as L. pneumophila opportunistically infects alveolar macrophages. One of the many virulence factors possessed by L. pneumophila is High Temperature Protein B (HtpB), a multifunctional chaperonin that has been found on the cell surface. Despite the several virulence-related roles that have been described for the surface-exposed or extracellularly released HtpB, no mechanism has yet been proposed for how this essential, typically cytoplasmic protein reaches extracytoplasmic compartments. In this work, we present evidence that Dot/Icm, a type IV secretion system of L. pneumophila, is responsible for the translocation of HtpB through a non-canonical secretion pathway. An infection model using L. pneumophila carrying a genetically tagged, recombinant HtpB demonstrated that HtpB does not reach the cytoplasm of Legionella-infected host cells without a functional Dot/Icm system. By fusing the C-terminus of HtpB to the cytoplasmic protein Icd and assaying its sub-cellular locale by western blotting, we demonstrate that the C-terminus of HtpB has affinity for the Legionella envelope membranes but is not sufficient to mediate secretion. These results, as well as prior data generated by other researchers/students in the Garduño Lab at Dalhousie University, led us to conclude that HtpB normally traffics through the periplasm of L. pneumophila and relies on the Dot/Icm system for escaping the periplasm, but crosses the inner membrane in an uncharacterized manner. This unknown mechanism by which HtpB seems to translocate across the cytoplasmic membrane may apply to other Legionella proteins and thus contribute to explain the unusual quantity and diversity of Dot/Icm effectors, therefore being of critical importance to the understanding of virulence in L. pneumophila.