A Conserved α-Helix Essential for a Type VI Secretion-Like System of Francisella tularensis

Abstract

Francisella tularensis harbors genes with similarity to genes encoding components of a type VI secretion system (T6SS) recently identified in several gram-negative bacteria. These genes include iglA and iglB encoding IglA and IglB, homologues of which are conserved in most T6SSs. We used a yeast two-hybrid system to study the interaction of the Igl proteins of F. tularensis LVS. We identified a region of IglA, encompassing residues 33 to 132, necessary for efficient binding to IglB, as well as for IglAB protein stability and intramacrophage growth. In particular, residues 103 to 122, overlapping a highly conserved ␣-helix, played an absolutely essential role. Point mutations within this domain caused modest defects in IglA-IglB binding in the yeast Saccharomyces cerevisiae but markedly impaired intramacrophage replication and phagosomal escape, resulting in severe attenuation of LVS in mice. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity. This interaction may be universal to type VI secretion, since IglAB homologues of Yersinia pseudotuberculosis, Pseudomonas aeruginosa, Vibrio cholerae, Salmonella enterica serovar Typhimurium, and Escherichia coli were also shown to interact in yeast, and the interaction was dependent on preservation of the same ␣-helix. Heterologous interactions between nonnative IglAB proteins further supported the notion of a conserved binding site. Thus, IglA-IglB complex formation is clearly crucial for Francisella pathogenicity, and the same interaction is conserved in other human pathogens.Francisella tularensis is a gram-negative facultative intracellular bacterial pathogen capable of causing a severe disease, tularemia, in many mammalian species (22). Human infections are caused mainly by two subspecies, the more virulent organism F. tularensis subsp. tularensis (type A) found predominantly in North America and the less virulent organism F. tularensis subsp. holarctica (type B) found in North America, Europe, and Asia (34, 43). While little is known about the molecular mechanisms of Francisella pathogenesis, a key strategy appears to be its ability to survive and replicate within macrophages (42,46). Francisella-containing vacuoles have been reported to evade phagosome-lysosome fusion, followed by bacterial escape into the cytoplasm (8, 16). Several genes necessary for intramacrophage survival, as well as growth within the amoeba Acanthamoeba castellanii, a putative natural reservoir of F. tularensis, have been identified. Many of these genes, including the members of the iglABCD operon, are located in a 34-kb Francisella pathogenicity island (FPI) (reviewed in reference 31), and they are regulated by the global regulator MglA (4, 23). Almost all of the proteins of the FPI are essentially conserved across subspecies. Studies have shown that IglC and IglD are required for F. tularensis to replicate within the cytosol of macrophages (24, 37). While IglC was shown to be essential for bacterial escape from the phagosome into the cytoplasm (24, 38), the r...

BcsKC Is an Essential Protein for the Type VI Secretion System Activity in Burkholderia cenocepacia That Forms an Outer Membrane Complex with BcsLB

“…show that BcsK C homologs interact with a small T6SS protein homologous to BcsL B from B. cenocepacia (32,45). To investigate whether BcsK C also interacts with BcsL B or with other proteins, we performed pulldown assays using cell lysates from B. cenocepacia K56-2 ⌬atsR ⌬bcsK C (pEL-1), K56-2 ⌬atsR ⌬bcsK C (pBcsK C-FLAG ), and K56-2 ⌬atsR ⌬bcsK C (pBcsK C⌬40 -189-FLAG ).…”

Identification of a Novel Francisella tularensis Factor Required for Intramacrophage Survival and Subversion of Innate Immune Response

“…F. tularensis lacks type III and IV secretion systems; however, it does contain a type IV pilus biogenesis system that secretes soluble proteins via type II-like secretion machinery (9 -12). Additionally, a type VI secretion system encoded by Francisella pathogenicity island has recently been identified in F. tularensis, which is required for intracellular survival and modulation of host cell signaling (13,14). F. tularensis also encodes a functional type I secretion system that is required for pathogenesis (11).…”

Identification of two substrates of FTS_1067 protein – An essential virulence factor of Francisella tularensis

“…The results shown in Figure 3 are from experiment where all tested proteins were fused to the C-terminus of either T18 or T25 adenylate cyclase domains. Only the test of the interaction between IglA and IglB proteins, which has been previously described [19], provided a positive signal ( Figure 3 -I and J).…”

“…As another positive control pUT18C and pKT25 plasmids carrying F. tularensis iglA, and iglB genes, respectively, were used. IglA-IglB interaction has been previously demonstrated in F. tularensis [19].…”