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

Abstract

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.

Heterogeneity of the group B streptococcal type VII secretion system and influence on colonization of the female genital tract

“…2A, Supp. Table 1), similar to those described in S. intermedius (Klein et al, 2022). GBS Lap2 proteins are not well conserved across subtypes I-III, with ≤41% identity between any two (Supp.…”

“…1C). A similar gene cluster was recently described in S. intermedius where the DUF3130 gene and the adjacent gene encode accessory proteins/chaperones required for secretion of the LXG toxins TelC and TelD (Klein et al, 2022). Consequently, these chaperones were named LXG-associated α-helical proteins (Lap).…”

“…In S. aureus, two WXG-like proteins named EsxC and EsxD can heterodimerize with EsxA and EsxB, respectively, and substrate secretion is interdependent as deletion of these WXG-like genes impacts EsxA/B export (Anderson et al, 2013). In S. intermedius, WXG-like genes adjacent to essC facilitate secretion of a downstream LXG toxin (Klein et al, 2022). We have observed that a double mutant in both these WXG-like genes in GBS subtype I did not affect EsxA secretion (data not shown); therefore, we hypothesize that GBS WXG-like proteins may function in LXG protein secretion, similar to S. intermedius.…”

“…Transmembrane protein and DUF4176 protein encoding genes, commonly observed in the vicinity of T7SS loci and toxins, appeared downstream of the LXG genes in GBS subtypes I-III. In S. aureus, cognate immunity factor esaG (DUF600) is encoded downstream of nuclease EsaD (Cao et al, 2016) and in S. intermedius, Tel immunity proteins are encoded either adjacent to (in the case of TelB, TelC, TelD) or one gene separated (in the case of TelA) from the LXG toxin (Whitney et al, 2017, Klein et al, 2022. We therefore expect that one of these downstream GBS genes encodes a subtype specific immunity factor and determining these LXG toxin-immunity factor pairs is the subject of our future work.…”