Structure of a tripartite protein complex that targets toxins to the type VII secretion system.

Type VII secretion systems are membrane-embedded nanomachines used by Gram-positive bacteria to export effector proteins from the cytoplasm to the extracellular environment. Many of these effectors are polymorphic toxins comprised of an N-terminal Leu-x-Gly (LXG) domain of unknown function and a C-terminal toxin domain that inhibits the growth of bacterial competitors. In recent work, it was shown that LXG effectors require two cognate Lap proteins for T7SS-dependent export. Here, we present the 2.6 Å structure of the LXG domain of the TelA toxin from the opportunistic pathogen Streptococcus intermedius in complex with both of its cognate Lap targeting factors. The structure reveals an elongated α-helical bundle within which each Lap protein makes extensive hydrophobic contacts with either end of the LXG domain. Remarkably, despite low overall sequence identity, we identify striking structural similarity between our LXG complex and PE-PPE heterodimers exported by the distantly related ESX type VII secretion systems of Mycobacteria implying a conserved mechanism of effector export among diverse Gram-positive bacteria. Overall, our findings demonstrate that LXG domains, in conjunction with their cognate Lap targeting factors, represent a tripartite secretion signal for a widespread family of T7SS toxins.

Bacterial type VII secretion: An important player in host-microbe and microbe-microbe interactions.

Type VII secretion systems (T7SSs) are poorly understood protein export apparatuses found in mycobacteria and many species of Gram-positive bacteria. To date, this pathway has predominantly been studied in Mycobacterium tuberculosis, where it has been shown to play an essential role in virulence; however, much less studied is an evolutionarily divergent subfamily of T7SSs referred to as the T7SSb. The T7SSb is found in the major Gram-positive phylum Firmicutes where it was recently shown to target both eukaryotic and prokaryotic cells, suggesting a dual role for this pathway in host-microbe and microbe-microbe interactions. In this review, we compare the current understanding of the molecular architectures and substrate repertoires of the well-studied mycobacterial T7SSa systems to that of recently characterized T7SSb pathways and highlight how these differences may explain the observed biological functions of this understudied protein export machine.

Dual Targeting Factors Are Required for LXG Toxin Export by the Bacterial Type VIIb Secretion System.

Bacterial type VIIb secretion systems (T7SSb) are multisubunit integral membrane protein complexes found in Firmicutes that play a role in both bacterial competition and virulence by secreting toxic effector proteins. The majority of characterized T7SSb effectors adopt a polymorphic domain architecture consisting of a conserved N-terminal Leu-X-Gly (LXG) domain and a variable C-terminal toxin domain. Recent work has started to reveal the diversity of toxic activities exhibited by LXG effectors; however, little is known about how these proteins are recruited to the T7SSb apparatus. In this work, we sought to characterize genes encoding domains of unknown function (DUFs) 3130 and 3958, which frequently cooccur with LXG effector-encoding genes. Using coimmunoprecipitation-mass spectrometry analyses, in vitro copurification experiments, and T7SSb secretion assays, we found that representative members of these protein families form heteromeric complexes with their cognate LXG domain and in doing so, function as targeting factors that promote effector export. Additionally, an X-ray crystal structure of a representative DUF3958 protein, combined with predictive modeling of DUF3130 using AlphaFold2, revealed structural similarity between these protein families and the ubiquitous WXG100 family of T7SS effectors. Interestingly, we identified a conserved FxxxD motif within DUF3130 that is reminiscent of the YxxxD/E "export arm" found in mycobacterial T7SSa substrates and mutation of this motif abrogates LXG effector secretion. Overall, our data experimentally link previously uncharacterized bacterial DUFs to type VIIb secretion and reveal a molecular signature required for LXG effector export. IMPORTANCE Type VIIb secretion systems (T7SSb) are protein secretion machines used by an array of Gram-positive bacterial genera, including Staphylococcus, Streptococcus, Bacillus, and Enterococcus. These bacteria use the T7SSb to facilitate interbacterial killing and pathogenesis through the secretion of toxins. Although the modes of toxicity for a number of these toxins have been investigated, the mechanisms by which they are recognized and secreted by T7SSb remains poorly understood. The significance of this work is the discovery of two new protein families, termed Lap1 and Lap2, that directly interact with these toxins and are required for their secretion. Overall, Lap1 and Lap2 represent two widespread families of proteins that function as targeting factors that participate in T7SSb-dependent toxin release from Gram-positive bacteria.

Structure of the Extracellular Region of the Bacterial Type VIIb Secretion System Subunit EsaA.

Gram-positive bacteria use type VII secretion systems (T7SSs) to export effector proteins that manipulate the physiology of nearby prokaryotic and eukaryotic cells. Several mycobacterial T7SSs have established roles in virulence. By contrast, the genetically distinct T7SSb pathway found in Firmicutes bacteria more often functions to mediate bacterial competition. A lack of structural information on the T7SSb has limited the understanding of effector export by this protein secretion apparatus. Here, we present the 2.4 Å crystal structure of the extracellular region of the T7SSb subunit EsaA from Streptococcus gallolyticus. Our structure reveals that homodimeric EsaA is an elongated, arrow-shaped protein with a surface-accessible "tip", which in some species of bacteria serves as a receptor for lytic bacteriophages. Because it is the only T7SSb subunit large enough to traverse the peptidoglycan layer of Firmicutes, we propose that EsaA plays a critical role in transporting effectors across the entirety of the Gram-positive cell envelope.