A mutagenesis-based approach identifies amino acids in the N-terminal part of Francisella tularensis IglE that critically control Type VI system-mediated secretion.
The Gram-negative bacterium Francisella tularensis is the etiological agent of the zoonotic disease tularemia. Its life cycle is characterized by an ability to survive within phagocytic cells through phagosomal escape and replication in the cytosol, ultimately causing inflammasome activation and host cell death. Required for these processes is the Francisella Pathogenicity Island (FPI), which encodes a Type VI secretion system (T6SS) that is active during intracellular infection. In this study, we analyzed the role of the FPI-component IglE, a lipoprotein which we previously have shown to be secreted in a T6SS-dependent manner. We demonstrate that in F. tularensis LVS, IglE is an outer membrane protein. Upon infection of J774 cells, an ΔiglE mutant failed to escape from phagosomes, and subsequently, to multiply and cause cytopathogenicity. Moreover, ΔiglE was unable to activate the inflammasome, to inhibit LPS-stimulated secretion of TNF-α, and showed marked attenuation in the mouse model. In F. novicida, IglE was required for in vitro secretion of IglC and VgrG. A mutagenesis-based approach involving frameshift mutations and alanine substitution mutations within the first ∼ 38 residues of IglE revealed that drastic changes in the sequence of the extreme N-terminus (residues 2-6) were well tolerated and, intriguingly, caused hyper-secretion of IglE during intracellular infection, while even subtle mutations further downstream lead to impaired protein function. Taken together, this study highlights the importance of IglE in F. tularensis pathogenicity, and the contribution of the N-terminus for all of the above mentioned processes.
a Department of Clinical Microbiology , Clinical Bacteriology and Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University , Umeå , Sweden.
Jeanette E
JE
Bröms
a Department of Clinical Microbiology , Clinical Bacteriology and Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University , Umeå , Sweden.
Lena
L
Meyer
a Department of Clinical Microbiology , Clinical Bacteriology and Laboratory for Molecular Infection Medicine Sweden (MIMS), Umeå University , Umeå , Sweden.
Anders
0000-0002-0768-8405
A
Sjöstedt
D000596:Amino Acids / Q000737:chemistry*; D000818:Animals; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D005604:Francisella tularensis / Q000737:chemistry / Q000235:genetics* / Q000472:pathogenicity*; D044404:Genomic Islands / Q000235:genetics; D058847:Inflammasomes; D008074:Lipoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000382:microbiology; D051379:Mice; D016296:Mutagenesis; D010588:Phagosomes / Q000382:microbiology; D014406:Tularemia / Q000382:microbiology; D000069376:Type VI Secretion Systems / Q000235:genetics; D037521:Virulence Factors / Q000378:metabolism
D000596:Amino Acids; D001425:Bacterial Outer Membrane Proteins; D058847:Inflammasomes; D008074:Lipoproteins; D000069376:Type VI Secretion Systems; D037521:Virulence Factors
Francisella pathogenicity island; Francisella tularensis; IglE; type VI secretion