A membrane-depolarizing toxin substrate of theStaphylococcus aureusType VII secretion system mediates intra-species competition

Abstract

21 22 † for correspondence tracy.palmer@newcastle.ac.uk 23 24 25 26 42 45 proteins that are essential for virulence and immune evasion 1 . The Ess T7SS of 46Staphylococcus aureus is also required for pathogenesis in murine models of infection 2, 3, 4 , 47 and a longitudinal study of persistent S. aureus infection in the airways of a cystic fibrosis 48 patient showed that the ess T7SS genes were highly upregulated during a 13 year timespan 5 . 49It is becoming increasingly apparent, however, that in addition to having anti-eukaryotic 50 activity, the T7SS of firmicutes mediates interbacterial competition 6, 7, 8 . Some strains of S. 51aureus secrete a DNA endonuclease toxin, EsaD 6, 9 , that when overproduced leads to growth 52 inhibition of a sensitive S. aureus strain 6 . Moreover, Streptococcus intermedius exports at 53 least three LXG domain-containing toxins, TelA, TelB and TelC that mediate contact-54 dependent growth inhibition against a range of Gram positive species 7 . 55A large integral membrane ATPase of the FtsK/SpoIIIE family, termed EssC in firmicutes, is a 56 conserved component of all T7SSs and probably energizes protein secretion as well as 57 forming part of the translocation channel 10, 11, 12, 13 . Three further membrane proteins function 58 alongside the ATPase to mediate T7 protein secretion 2, 3, 13, 14, 15 . EsxA, a small secreted 59 protein of the WXG100 family, is a further conserved T7 component that is dependent on the 60 T7SS for its translocation across the membrane 2, 16 . In S. aureus the T7 structural components 61 are encoded at the ess locus. In commonly-studied S. aureus strains including Newman, 62RN6390 and USA300, the T7 substrates EsxB, EsxC, EsxD and EsaD are encoded 63 immediately downstream of essC ( Fig 1A) and are co-regulated with the genes coding for 64 machinery components 2, 3, 6, 9, 17, 18 . With the exception of EsaD the biological activities of these 65 substrates are unknown, although mutational studies have suggested that EsxB and EsxC 66 contribute to persistent infection in a murine abscess model 2, 17 . 67Despite the ess locus forming part of the core S. aureus genome, these four substrate proteins 68 are not conserved across S. aureus isolates, being found in only approximately 50% of a similar decrease in kidney abscess formation as that seen for T7 mutants in Newman and 71 USA300 2, 4, 20 , despite the fact that recognizable homologues of EsxB/EsxC/EsxD/EsaD are 72 not encoded by this strain 19 . This strongly suggests that there are further S. aureus T7 73 substrates that are yet to be identified. Here we have taken an unbiased approach to identify 74 T7 substrates using quantitative proteomic analysis of culture supernatants from S. aureus 75 RN6390 wild type and essC strains. We identify a new substrate, TspA that is encoded 76 distantly from the ess gene cluster and is found in all sequenced S. aureus strains. Further 77 analysis indicates that TspA has a toxic C-terminal domain that depolarizes membranes. 78Using a zebrafish hindbrain ventricle ...

Extreme genetic diversity in the type VII secretion system of Listeria monocytogenes suggests a role in bacterial antagonism

“…Since the identification of EsaD, a growing number of T7-secreted antibacterial toxins have been described. TspA, a second polymorphic toxin found in S. aureus, has membranedepolarizing activity that is neutralized by the TsaI immunity protein [37]. Streptococcus intermedius produces at least three antibacterial toxins, of which TelB is an NADase and TelC a lipid II phosphatase [30].…”

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

“…This distinction reflects differences in T7SS subunit composition between these two distantly related groups of Gram-positive bacteria (Klein et al, 2020). The T7SSa was originally discovered in Mycobacterium tuberculosis, where it acts as a virulence factor that facilitates immune evasion and phagosomal escape during infection, whereas the T7SSb was initially characterized in Staphylococcus aureus and has been shown to play a dual role in pathogenesis and interbacterial competition (Cao et al, 2016;Gao et al, 2004;Ohr et al, 2017;Ulhuq et al, 2020). The interkingdom-targeting capability of T7SSb has also been demonstrated in the opportunistic pathogen Streptococcus intermedius, with the antibacterial activity being attributed to the NAD + hydrolase effector TelB and the cell wall precursor degrading effector TelC (Hasegawa et al, 2017;Klein et al, 2018;Whitney et al, 2017).…”

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

“…The involvement of T7SSs in many biological processes likely reflects the diverse range of functions exhibited by T7SS substrates. Although all characterized T7SSa and T7SSb systems secrete at least one WXG100 protein, substrates from the PE/PPE and LXG families have thus far been exclusively associated with T7SSa and T7SSb systems, respectively (Figure 3) (Ates, 2020; Ulhuq et al, 2020; Whitney et al, 2017). Beyond the presence of a recognizable motif, many T7SS substrates lack known domains that suggest potential function, a problem that is further compounded by the need for many substrates to be co‐secreted with one another.…”

“…Studies in S. aureus have hinted that the T7SSb substrate landscape may contain additional toxin families beyond the LXG protein family. The S. aureus T7SSb exports both an LXG toxin, TspA, as well as EsaD, a T7SSb effector that lacks an N‐terminal LXG domain (Cao et al., 2016; Ulhuq et al, 2020). TspA acts as an interkingdom effector with membrane‐depolarizing activity and is associated with increased S. aureus virulence in zebrafish infection models, while bacterial competition experiments have implicated EsaD in interbacterial warfare, in addition to its role in mammalian infection (Cao et al., 2016; Ohr et al., 2017).…”