Molecular Basis for Immunity Protein Recognition of a Type VII Secretion System Exported Antibacterial Toxin

Abstract

Gram-positive bacteria deploy the type VII secretion system (T7SS) to facilitate interactions between eukaryotic and prokaryotic cells. In recent work, we identified the TelC protein from Streptococcus intermedius as a T7SS-exported lipid II phosphatase that mediates interbacterial competition. TelC exerts toxicity in the inner wall zone of Gram-positive bacteria; however, intercellular intoxication of sister cells does not occur because they express the TipC immunity protein. In the present study, we sought to characterize the molecular basis of self-protection by TipC. Using sub-cellular localization and protease protection assays, we show that TipC is a membrane protein with an N-terminal transmembrane segment and a C-terminal TelC-inhibitory domain that protrudes into the inner wall zone. The 1.9-Å X-ray crystal structure of a non-protective TipC paralogue reveals that the soluble domain of TipC proteins adopts a crescent-shaped fold that is composed of three α-helices and a seven-stranded β-sheet. Subsequent homology-guided mutagenesis demonstrates that a concave surface formed by the predicted β-sheet of TipC is required for both its interaction with TelC and its TelC-inhibitory activity. S. intermedius cells lacking the tipC gene are susceptible to growth inhibition by TelC delivered between cells; however, we find that the growth of this strain is unaffected by endogenous or overexpressed TelC, although the toxin accumulates in culture supernatants. Together, these data indicate that the TelC-inhibitory activity of TipC is only required for intercellularly transferred TelC and that the T7SS apparatus transports TelC across the cell envelope in a single step, bypassing the cellular compartment in which it exerts toxicity en route.

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

“…We found that at a dose of 8 × 10 7 – 2 × 10 8 of strain RN6390, the mice were asymptomatic and had almost completely cleared the bacteria from lung tissue after 48 hours, whereas a dose of 8 × 10 8 – 2 × 10 9 was lethal to all mice within 12 hours. At a dose of 3 × 10 8 , the mice developed symptoms which resolved within 12 hours, and therefore using this dosage we sought to test whether there was a difference in bacterial proliferation and dissemination dependent on the T7SS. However, after 24 hours of infection with 3 × 10 8 cfu of RN6390 or a cognate Δ essC strain, counts recovered from the lungs and livers of mice infected with the Δ essC strain were not significantly different than those from mice infected with the wild type (Fig S4).…”

“…To probe whether TspA is important for S. aureus virulence, we initially developed an immunocompetent murine model of S. aureus pneumonia. Previous reports have indicated that 2-4 × 10 8 colony forming units (cfu) of strain Newman was a suitable infectious dose, and that bacterial proliferation in lung tissue could be observed after 24 hours 38 . We found that at a dose of 8 × 10 7 – 2 × 10 8 of strain RN6390, the mice were asymptomatic and had almost completely cleared the bacteria from lung tissue after 48 hours, whereas a dose of 8 × 10 8 – 2 × 10 9 was lethal to all mice within 12 hours.…”

“…Previous reports have indicated that 2-4 × 10 8 colony forming units (cfu) of strain Newman was a suitable infectious dose, and that bacterial proliferation in lung tissue could be observed after 24 hours 38 . We found that at a dose of 8 × 10 7 – 2 × 10 8 of strain RN6390, the mice were asymptomatic and had almost completely cleared the bacteria from lung tissue after 48 hours, whereas a dose of 8 × 10 8 – 2 × 10 9 was lethal to all mice within 12 hours. At a dose of 3 × 10 8 , the mice developed symptoms which resolved within 12 hours, and therefore using this dosage we sought to test whether there was a difference in bacterial proliferation and dissemination dependent on the T7SS.…”

“…At a dose of 3 × 10 8 , the mice developed symptoms which resolved within 12 hours, and therefore using this dosage we sought to test whether there was a difference in bacterial proliferation and dissemination dependent on the T7SS. However, after 24 hours of infection with 3 × 10 8 cfu of RN6390 or a cognate Δ essC strain, counts recovered from the lungs and livers of mice infected with the Δ essC strain were not significantly different than those from mice infected with the wild type (Fig S4).…”

“…The Ess T7SS of Staphylococcus aureus is also required for pathogenesis in murine models of infection 2, 3, 4 , and a longitudinal study of persistent S. aureus infection in the airways of a cystic fibrosis patient showed that the ess T7SS genes were highly upregulated during a 13 year timespan 5 . It is becoming increasingly apparent, however, that in addition to having anti-eukaryotic activity, the T7SS of firmicutes mediates interbacterial competition 6, 7, 8 . Some strains of S. aureus secrete a DNA endonuclease toxin, EsaD 6, 9 , that when overproduced leads to growth inhibition of a sensitive S. aureus strain 6 .…”