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

Shah

et al. 2022

mBio

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.

Section: Methodsmentioning

confidence: 99%

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

Shah

et al. 2022

mBio

“…Western blots on samples prepared from the appropriate S. intermedius GC1825 deletion strains determined that this antibody specifically recognizes TelD but not LapD1. Generation of the α-TelC and α-EsxA antibodies have been described previously (18, 56).…”

Section: Methodsmentioning

confidence: 99%

“…Our S. intermedius gene deletion protocol was previously described in (56). In brief, deletion constructs were made using SOE PCR to fuse a spectinomycin promoter to a kanamycin resistance cassette flanked by two 1000bp fragments of DNA that are immediately adjacent to the target gene.…”

Section: Gene Deletion In S Intermedius By Allelic Replacementmentioning

confidence: 99%

Dual targeting factors are required for LXG toxin export by the bacterial type VIIb secretion system

Shah

et al. 2022

Preprint

Self Cite

Bacterial type VIIb secretion systems (T7SSb) are multi-subunit 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 co-occur with LXG effector-encoding genes. Using coimmunoprecipitation-mass spectrometry analyses, in vitro copurification experiments and T7SSb secretion assays, we find 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 modelling of DUF3130 using AlphaFold2, reveals structural similarity between these protein families and the ubiquitous WXG100 family of T7SS effectors. Interestingly, we identify 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.

“…), EssA (YueC), and EssB (YukC), are conserved across Firmicutes and may be functionally analogous to core apparatus components of the T7SSa (Figure 1). For example, EsaA may be analogous to EccB because it primarily consists of an extended extracellular domain that homodimerizes and therefore likely extends through the peptidoglycan cell wall (Klein et al, 2020b). EssA is the smallest and most inscrutable component of the T7SSb apparatus but has been demonstrated to be membrane-associated and is an essential component of the T7SSb in S. aureus (Burts et al, 2005).…”

Section: T7ssb Core Apparatus Integral Membrane Proteins: Esaa Essmentioning

confidence: 99%

“…Together with proteinprotein interaction data, there appears a possibility of the T7SSb apparatus assembling in a circular arrangement of protomers with sixfold rotational symmetry, with each subunit arranged in a similar manner as observed for the T7SSa, but compelling experimental evidence has yet to emerge. It is also interesting to note that the available structural information for T7SSb core components suggests the formation of a conduit that facilitates substrate transport across the entire cell envelope of Gram-positive bacteria to the extracellular milieu (Klein et al, 2020b), whereas the mechanism of T7SSa substrate export across the mycobacterial outer membrane may require additional protein components such as EspC (Lou et al, 2017).…”

Section: Progress Toward the Structural Resolution Of The T7ssbmentioning

confidence: 99%

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

Tran

et al. 2021

Molecular Microbiology

Self Cite

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.