TheLegionella pneumophila icmGCDJBFGenes Are Required for Killing of Human Macrophages

Abstract

Previously, a collection of mutants of Legionella pneumophila that had lost the ability to multiply within and kill human macrophages was generated by Tn903dIIlacZ transposon mutagenesis and classified into DNA hybridization groups. A subset of these mutants was complemented by a plasmid, pMW100, containing a 13.5-kb genomic DNA insert. This plasmid restored the ability to multiply within and produce cytopathic effects on human macrophages to members of DNA hybridization groups II, IV, VI, and XVII. A region of the genomic insert of pMW100 was sequenced, and eight potential genes were identified and named icmE,icmG, icmC, icmD, icmJ,icmB, icmF, and tphA. None of the genes encode potential protein products with significant homology to previously characterized proteins, except for tphA, whose product has significant homology to a family of metabolite/H+ symport proteins from gram-negative bacteria. The positions of the Tn903dIIlacZ insertions within the genes were determined by nucleotide sequencing. No Tn903dIIlacZ insertions mapped toicmG, icmJ, or tphA; therefore, these loci were mutated to test whether they were required for macrophage killing. Complementation analysis was used to evaluate the roles of the potential gene products and provide information on the organization of transcriptional units within the region. The results indicate that all identified open reading frames excepttphA are required for killing of human macrophages.

The mutagenesis of a type IV secretion system locus of Piscirickettsia salmonis leads to the attenuation of the pathogen in Atlantic salmon, Salmo salar

“…Although the function of the icmB gene remains elusive, it appears to be essential for P. salmonis replication in CHSE-214 cells and to cause disease in Atlantic salmon. This hypothesis is in agreement with other bacterial models where the disruption of a similar T4SS component led to a drastic reduction in their respective virulence (Purcell & Shuman, 1998;Beare et al, 2011). We cannot discard the possibility that the phenotype exhibited by the icmB mutant may also be due to the interference with the expression of additional | 631 synteny allow to hypothesize that a duplication event could have given rise to the T4SS-2 in P. salmonis.…”

Icm/Dot‐dependent upregulation of phagocytosis by Legionella pneumophila

“…7, pathway A). In the absence of the effector, normal phagocytosis results in the formation of a phagosome that does fuse with lysosomes, eventually leading to an acidic phagolysosome, in which icm/dot mutants are degraded (Sadosky et al, 1993;Purcell and Shuman, 1998;Solomon et al, 2000). Alternatively, in a 'sequential'-type model, L. pneumophila could upregulate phagocytosis by altering aspects of cell physiology related to phagocytosis (pathway B).…”

“…The bacterial strains used for the infections were grown on CYE agar plates for 2-3 days and harboured the plasmid pMMB207 (icmD, -J, -B ), pMMB207ab (icmR, -Q, -M, -L, -K, -E, -X, dotA ), pMMB207ab-Km-14 (icmT, -S, -P, -O, -N, -G, -C, -F, tphA, dotB ) or the corresponding complementing plasmids (Table 2). Bacteria suspended in RGN medium (OD 600 of 0.3 ¼ 5 Â 10 8 JR32 icmJ::Km Purcell and Shuman (1998) bacteria ml 21 ) were centrifuged onto the HL-60 cells (700 g, 10 min) at an MOI of 100, and the infected cells were incubated for another 10 min at 378C. To kill extracellular bacteria, the infected macrophages were washed twice with PBS containing 0.1 mg ml 21 gentamicin and incubated for another 40 min in RGN containing 0.1 mg ml 21 gentamicin.…”

The Icm/Dot type-IV secretion systems ofLegionella pneumophilaandCoxiella burnetii

“…1). Region-I contains seven genes (icmV, W, and X, and dotA, B, C, and D), [4,46,47,52] and region-II contain 18 genes (icmT, S, R, Q, P, O, N, M, L, K, E, G, C, D, J, B, F and H), [3,4,[53][54][55][56] some of the genes in region-II have an icm as well as dot designation as indicated in Table 2. Eighteen of the L. pneumophila Icm/Dot proteins are homologous to the Tra/Trb proteins of IncI plasmids (such as R64) ( Table 2), and the genes that encode for these proteins are organized similarly in both systems ( Fig.…”

“…The predicted coiled-coil domain of IcmG/DotF was identified in the screen, and it was then used to identify additional substrates of the Icm/Dot system as described below. It is interesting to note that an insertion mutant in the icmG/dotF gene had a partial intracellular growth phenotype when examined in HL-60 derived human macrophages [54], and a complete null phenotype when examined in A. castellanii [49]. This result might indicate that IcmG/DotF is not the only route by which substrates are recognized by the icm/dot system.…”