Host cell killing and bacterial conjugation require overlapping sets of genes within a 22-kb region of the Legionella pneumophila genome

Abstract

A 22-kb DNA locus of Legionella pneumophila is described that contains 18 genes, 16 of which are required for macrophage killing (icm genes). In this paper two previously described icm loci were linked by the discovery of five genes located between the two loci. Four of the newly described genes are required for macrophage killing (icmMLKE) and one is dispensable. The 16 icm genes appeared to be organized as six individual genes (icmR, icmQ, icmG, icmC, icmD, and icmF), and four operons (icmTS, icmPO, icmMLKE, and icmJB). Four icm genes (icmP, icmO, icmL, and icmE) show significant sequence similarity to plasmid genes involved in conjugation, whereas the other icm genes were found not to bear any sequence similarity to database entries. We found that L. pneumophila can mediate plasmid DNA transfer at a frequency of 10 ؊3 to 10 ؊4 per donor. Strains containing null mutations in two icm genes (icmT and icmR) showed a severe reduction in conjugation frequency and macrophage killing. Strains containing an insertion in four other icm genes (icmF, icmE, icmC, and dotA) were shown to have a less severe defect in conjugation. Mutations in the other 11 icm genes had no effect on conjugation frequency. We currently do not know whether conjugation itself plays a role in macrophage killing. It is possible either that small plasmids can take advantage of an existing secretion system to be mobilized or that DNA transfer is required for human macrophage killing by L. pneumophila.Legionella pneumophila, the causative agent of Legionnaires' disease, is a facultative intracellular pathogen with a broad host range. The bacteria are able to infect, multiply within, and kill human macrophages, as well as free-living amoebae (1, 2). When inside host cells, L. pneumophila are found within a specialized phagosome that does not fuse with lysosomes (3). The bacteria multiply within the specialized phagosome, until the cell eventually lyses, releasing bacteria that can start new rounds of infection.Several years ago, a collection of 55 L. pneumophila mutants defective for macrophage killing were isolated from a large (n ϭ 4,500) pool of Tn903dIIlacZ insertions, and classified into 16 DNA hybridization groups (4). One of these groups (group 1), which contains 10 of the insertion mutants, was previously characterized as the icmA-dotA region (5, 6). More recently, five additional DNA hybridization groups were characterized as two separate regions (ref. 7; M.P. and H.A.S., unpublished results). One of the regions (6.5 kb), was shown to contain nine insertion mutations located on a single DNA hybridization group (group 3), that contains six icm genes (7). The second region (11 kb) was shown to contain 18 insertion mutations located on four contiguous DNA hybridization groups (groups 2, 6, 4, and 17), which contain an additional six icm genes (M.P. and H.A.S., unpublished results).The aim of this study was to complete the characterization of the additional DNA hybridization groups. We found that the two regions described above are conne...

Comparative assessment of virulence traits in Legionella spp.

“…The dot/icm genes have been shown to play an important role in the virulence of L. pneumophila (Segal et al, 1998;Vogel et al, 1998). We screened all the Legionella spp.…”

“…The dot/icm loci are composed of 24 genes that are involved in the assembly of a type IV secretion apparatus that is central to pathogenesis. The dot/icm loci are essential for enhancement of phagocytosis by human-derived cells (Hilbi et al, 2001), macropinocytic uptake by A/J mice-derived macrophages (Watarai et al, 2001), evasion of lysosomal fusion and intracellular replication (Segal et al, 1998;Vogel et al, 1998), induction of apoptosis (Zink et al, 2002), and pore-formationmediated lysis of the host cell and bacterial egress upon termination of intracellular replication (Alli et al, 2000;Molmeret et al, 2002b). Biphasic killing of mammalian cells by L. pneumophila (Alli et al, 2000;Gao & Abu Kwaik, 1999b) has been proposed in which apoptosis is first initiated, followed by a temporal induction of necrosis and lysis of the host upon growth transition into the postexponential phase (Alli et al, 2000;Byrne & Swanson, 1998;Kirby et al, 1998).…”

The versatile bacterial type IV secretion systems

“…4). Intriguingly, the Dot/Icm T4S system functions as a bona fide conjugation system that is closely related in ancestry to the ColIb-P9 plasmid-transfer system 5,101,102 . However, during infection L. pneumophila uses this transfer system to inject effector proteins into the phagosome, both to control biogenesis of the replicative vacuole and to modulate the activity of host factors involved in vesicle traffic.…”

Exploitation of conserved eukaryotic host cell farnesylation machinery by an F-box effector ofLegionella pneumophila

“…Although farnesyltransferase is cytosolic, it is recruited to the LCV in a Dot/Icm-dependent manner (Segal et al, 1998;Vogel et al, 1998), which may enable local farnesylation of AnkB at the LCV membrane that is ER derived (Tilney et al, 2001;Kagan and Roy, 2002). Farnesylated proteins often undergo further posttranslational modifications at the ER membrane RCE-1 and ICMT (Wright and Philips, 2006) to cleave the terminal -aaX tripeptide and methylate the terminal farnesylated cysteine residue, respectively.…”

“…Within both evolutionarily distant host cells, L. pneumophila evades endocytic fusion and intercepts ER to Golgi vesicle traffic to remodel its phagosome into an ER-derived vacuole (Kagan and Roy, 2002;Molmeret et al, 2005;Shin and Roy, 2008;Isberg et al, 2009). The Dot/Icm type IV secretion system (Segal et al, 1998;Vogel et al, 1998) injects into the host cell a cadre of 200 effectors to modulate a myriad of cellular processes to reprogram the host cell into a proliferation niche (de Felipe et al, 2008;Shin and Roy, 2008;Isberg et al, 2009). The Ankyrin B (AnkB) effector is injected into the host cell by the Dot/Icm system upon bacterial attachment to the plasma membrane and exploits an evolutionarily conserved eukaryotic machinery within mammalian and protozoan cells (Price et al, 2009).…”