Diverse mechanisms of metaeffector activity in an intracellular bacterial pathogen, Legionella pneumophila

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 effectoreffector 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.

The Legionella pneumophila effector Ceg4 is a phosphotyrosine phosphatase that attenuates activation of eukaryotic MAPK pathways

“…In these assays, Ceg4 demonstrated the highest activity toward phosphotyrosine ( Figure 2C, see also Supplemental Figure S1E). To determine if Ceg4 is active against protein substrates, we screened for its ability to remove the phosphate group from a selection of 53 phosphopeptides, chosen for their importance to signaling in Saccharomyces cerevisiae, which has been successfully used as a model eukaryotic system in characterization of bacterial effector functions including those from Legionella (30,59). This set included pSer-, pThr-and pTyrcontaining sequences.…”

Legionella pneumophila regulates the activity of UBE 2N by deamidase‐mediated deubiquitination

“…Although the exact source remains unknown, the evolutionary pressure that led to the acquisition of the deubiquitinase activity of MvcA likely came from host cells in which the inhibitory effects of MavC need to be relieved for balanced infection. The regulation of UBE2N activity by MavC and MvcA with opposite biochemical function extends the list of L. pneumophila effectors that function to regulate the activity of another effector (Urbanus et al, 2016;Qiu & Luo, 2017b). Such effectors, also called meta-effectors (Kubori et al, 2010), often function to impose spatiotemporal regulation of the activity of other effectors by potentially diverse mechanisms (Urbanus et al, 2016).…”

Combinatorial actions of bacterial effectors revealed by exploiting genetic tools in yeast

“…Effector–effector balance could also change over time through differential effector half‐life, much as actin rearrangement is modulated during Salmonella infection (Kubori & Galán, ). The Urbanus et al () study also elegantly illustrates the multidisciplinary power of integrating high‐throughput genetic and cell biology tools in molecular microbiology.…”

“…Urbanus et al () extend this work to all pairwise combinations of 330 L. pneumophila type 4 secreted effectors, resulting in the comprehensive analysis of 108,000 potential interactions. Using techniques developed for SGA screens, they mated yeast expressing arrayed effector libraries to co‐express all possible pairs of effectors (Fig ).…”

“… So far, Legionella pneumophila effectors have been analysed at the individual protein level. In their recent work, Ensminger and colleagues (Urbanus et al , ) map all Legionella effector–effector interactions and describe mechanisms used by “meta‐effectors” to modulate target effector activity.

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