PilY1 Promotes Legionella pneumophila Infection of Human Lung Tissue Explants and Contributes to Bacterial Adhesion, Host Cell Invasion, and Twitching Motility.

Julia Hoppe,Torsten Goldmann,Louisa Grimpe,Matthias Richter,Stefanie Thiem,Michael Steinert,Olga Shevchuk,Nikolaus Gaßler,Can M Ünal

doi:10.3389/fcimb.2017.00063

Abstract

Legionnaires' disease is an acute fibrinopurulent pneumonia. During infection Legionella pneumophila adheres to the alveolar lining and replicates intracellularly within recruited macrophages. Here we provide a sequence and domain composition analysis of the L. pneumophila PilY1 protein, which has a high homology to PilY1 of Pseudomonas aeruginosa. PilY1 proteins of both pathogens contain a von Willebrand factor A (vWFa) and a C-terminal PilY domain. Using cellular fractionation, we assigned the L. pneumophila PilY1 as an outer membrane protein that is only expressed during the transmissive stationary growth phase. PilY1 contributes to infection of human lung tissue explants (HLTEs). A detailed analysis using THP-1 macrophages and A549 lung epithelial cells revealed that this contribution is due to multiple effects depending on host cell type. Deletion of PilY1 resulted in a lower replication rate in THP-1 macrophages but not in A549 cells. Further on, adhesion to THP-1 macrophages and A549 epithelial cells was decreased. Additionally, the invasion into non-phagocytic A549 epithelial cells was drastically reduced when PilY1 was absent. Complementation variants of a PilY1-negative mutant revealed that the C-terminal PilY domain is essential for restoring the wild type phenotype in adhesion, while the putatively mechanosensitive vWFa domain facilitates invasion into non-phagocytic cells. Since PilY1 also promotes twitching motility of L. pneumophila, we discuss the putative contribution of this newly described virulence factor for bacterial dissemination within infected lung tissue.

Highlights

Legionella pneumophila is the causative agent of the Legionnaires’ disease, a severe form of pneumonia (Fraser et al, 1977; McDade et al, 1977; Fields et al, 2002)
At the N-terminus the PilY1 protein contains a transmembrane domain with a putative signal peptide cleavage site, similar to the Sec secretion signal peptide of P. aeruginosa PilY1 (Lewenza et al, 2005; Kuchma et al, 2010)
We propose that PilY1 functions as an adhesion factor, which influences bacterial uptake by host cells, and the successful establishment of the early Legionella-containing vacuole (LCV) by providing close cell-cell contact for effective effector translocation

Summary

Introduction

Legionella pneumophila is the causative agent of the Legionnaires’ disease, a severe form of pneumonia (Fraser et al, 1977; McDade et al, 1977; Fields et al, 2002). The development of the Legionella-containing vacuole (LCV) is pre-dominantly orchestrated by the Dot/Icm type IV secretion system, which is known to export numerous bacterial effector proteins into the host cell cytoplasm (Horwitz, 1983; Kagan and Roy, 2002; Isberg et al, 2009; Hubber and Roy, 2010). Several determinants of L. pneumophila are known to contribute to adherence and entry into different host cell types, including type IV pili, Hsp, the structural toxin RtxA, the intergrin analog LaiA and the GAG binding protein Lcl and the adenylate cyclase LadC (Garduño et al, 1998; Stone and Abu Kwaik, 1998; Cirillo et al, 2001; Chang et al, 2005; Newton et al, 2008; Duncan et al, 2011)

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