Abstract
Pathogens deliver complex arsenals of translocated effector proteins to host cells during infection, but the extent to which these proteins are regulated once inside the eukaryotic cell remains poorly defined. Among all bacterial pathogens, Legionella pneumophila maintains the largest known set of translocated substrates, delivering over 300 proteins to the host cell via its Type IVB, Icm/Dot translocation system. Backed by a few notable examples of effector–effector regulation in L. pneumophila, we sought to define the extent of this phenomenon through a systematic analysis of effector–effector functional interaction. We used Saccharomyces cerevisiae, an established proxy for the eukaryotic host, to query > 108,000 pairwise genetic interactions between two compatible expression libraries of ~330 L. pneumophila‐translocated substrates. While capturing all known examples of effector–effector suppression, we identify fourteen novel translocated substrates that suppress the activity of other bacterial effectors and one pair with synergistic activities. In at least nine instances, this regulation is direct—a hallmark of an emerging class of proteins called metaeffectors, or “effectors of effectors”. Through detailed structural and functional analysis, we show that metaeffector activity derives from a diverse range of mechanisms, shapes evolution, and can be used to reveal important aspects of each cognate effector's function. Metaeffectors, along with other, indirect, forms of effector–effector modulation, may be a common feature of many intracellular pathogens—with unrealized potential to inform our understanding of how pathogens regulate their interactions with the host cell.
Highlights
The concept of effector-based modulation of host pathways is central to the current molecular understanding of microbial pathogenesis
Over 200 Icm/Dot-translocated substrate (IDTS) remained uncharacterized in yeast, so we generated a library of 330 yeast strains in which each strain expresses one IDTS on a high-copy HIS3 plasmid under control of a galactose-inducible promoter
It was recently shown that the cognate effector of LupA, LegC3, interferes with vesicle trafficking by inhibiting the formation of endogenous trans-SNARE complexes during vacuolar fusion (Bennett et al, 2013) and forms a stable and functional SNARE acceptor complexes with two other IDTS YlfA/LegC7 and YlfB/LegC2 and the mammalian R-SNARE VAMP4 (Shi et al, 2016)
Summary
The concept of effector-based modulation of host pathways is central to the current molecular understanding of microbial pathogenesis. In this view, effector activity is directed against host factors and is regulated by changes to effector expression or through modulation of translocation efficiency. There are, several ways that translocated proteins might functionally interact once inside the host (Shames & Finlay, 2012). The regulatory complexity provided by effector–effector interactions may add another layer to the program of pathogenesis, helping balance host perturbation with the maintenance of a replicative niche. Pathogens that strongly rely on their hosts for replication and translocate large numbers of proteins are candidates for possessing such effector complexity (Ensminger, 2016).
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