Legionella pneumophila Dot/Icm translocated substrates: a sum of parts

Abstract

Legionella pneumophila is an intracellular pathogen of freshwater amoeba and of alveolar macrophages in human hosts. After phagocytosis, L. pneumophila establishes a unique intracellular vacuolar niche that avoids entry into the lysosomal network. Critical for L. pneumophila intracellular growth is the Dot/Icm type IVB translocation system. Although over 80 substrates of the Dot/Icm apparatus have been identified, individual substrates are often genetically redundant, complicating their analysis. Deletion of critical Dot/Icm translocation system components causes a variety of defects during intracellular growth. Many of these effects on the host cell likely result from the actions of one or more Dot/Icm translocated substrates. Loss of single substrates never generates the profound effects observed in strains lacking translocation system components.

Figure 1. Measuring Dot/Icm-dependent translocation

(A) The fusion of a positive translocation signal (“+”) to the C-terminus of adenylate cyclase (Cya) results in translocation of the fusion protein into the host cytosol. Cya activity depends on the host protein calmodulin (CaM), thus the level of cAMP in infected cells is directly proportional to translocation efficiency. As is the case for (B–D) as well, translocation is not observed in Dot/Icm deficient strains or with the reporter sequence alone. (B) In the TEM1 beta-lactamase fusion assay, a reporter substrate (CCF4/AM) is loaded into host cells. Translocation of beta-lactamase fusions results in a loss of FRET due to cleavage between the FRET donor and FRET acceptor of the substrate molecule. (C) An N-terminal fragment of the known Dot/Icm substrate, SidC, is unable to translocate on its own. Translocation can be restored by the fusion of other translocated substrates (or their C-termini) to SidCΔC100. SidC remains associated with the LCV after translocation, facilitating direct immunofluorescent detection of translocation using antibodies generated to SidC. (D) The Dot/Icm translocation machinery supports interbacterial transfer of protein substrates. A donor Legionella strain contains Cre recombinase fused to a candidate translocation signal. A recipient Legionella (or E. coli) strain contains a loxP-flanked reporter site, such that Cre-mediated recombination results in the loss of a counter-selectable marker (sacB) and the generation of a functional loxP-neomycin phosphotransferase (nptII) translational fusion, thereby conferring kanamycin resistance.