The Tip of the VgrG Spike Is Essential to Functional Type VI Secretion System Assembly in Acinetobacter baumannii

Abstract

The type VI secretion system (T6SS) is a critical weapon in bacterial warfare between Gram-negative bacteria. Although invaluable for niche establishment, this machine represents an energetic burden to its host bacterium. Acinetobacter baumannii is an opportunistic pathogen that poses a serious threat to public health due to its high rates of multidrug resistance. In some A. baumannii strains, the T6SS is transcriptionally downregulated by large multidrug resistance plasmids. Other strains, such as the clinical isolate AbCAN2, express T6SS-related genes but lack T6SS activity under laboratory conditions, despite not harboring these plasmids. This suggests that alternative mechanisms exist to repress the T6SS. Here, we used a transposon mutagenesis approach in AbCAN2 to identify novel T6SS repressors. Our screen revealed that the T6SS of this strain is inhibited by a homolog of VgrG, an essential structural component of all T6SSs reported to date. We named this protein inhibitory VgrG (VgrGi). Biochemical and in silico analyses demonstrated that the unprecedented inhibitory capability of VgrGi is due to a single amino acid mutation in a widely conserved C-terminal domain of unknown function, DUF2345. We also show that unlike in other bacteria, the C terminus of VgrG is essential for functional T6SS assembly in A. baumannii. Our study provides insight into the architectural requirements underlying functional assembly of the T6SS of A. baumannii. We propose that T6SS-inactivating point mutations are beneficial to the host bacterium, since they eliminate the energy cost associated with maintaining a functional T6SS, which appears to be unnecessary for A. baumannii virulence. IMPORTANCE Despite the clinical relevance of A. baumannii, little is known about its fundamental biology. Here, we show that a single amino acid mutation in VgrG, a critical T6SS structural protein, abrogates T6SS function. Given that this mutation was found in a clinical isolate, we propose that the T6SS of A. baumannii is probably not involved in virulence; this idea is supported by multiple genomic analyses showing that the majority of clinical A. baumannii strains lack proteins essential to the T6SS. We also show that, unlike in other species, the C terminus of VgrG is a unique architectural requirement for functional T6SS assembly in A. baumannii, suggesting that over evolutionary time, bacteria have developed changes to their T6SS architecture, leading to specialized systems.

Peptidoglycan editing provides immunity to Acinetobacter baumannii during bacterial warfare

“…Crude PG samples were treated with α-amylase (1 mg/ml) for 1 hour at 37°C and then with Pronase (2 mg/ml) for overnight at 60°C to remove trapped high–molecular weight glycogen and PG-associated proteins, respectively. Further PG preparation steps were performed as previously described ( 41 ). Briefly, muropeptides were released from PG by the muramidase cellosyl (Hoechst, Frankfurt am Main, Germany), reduced by sodium borohydride, and separated on a 250 mm by 4.6 mm 3-μm ProntoSIL 120-3-6C18 AQ reversed-phase column (Bischoff, Leonberg, Germany).…”

“…PG from exponential and stationary cells from A. baumannii strains was isolated and analyzed by HPLC as previously described (41,42). Briefly, A. baumannii strains were grown at 37°C in 500 ml of LB media to a final OD 600 (optical density at 600 nm) of 0.6 for exponential and grown for 24 hours for stationary phase samples.…”

“…Strains from the most common lineages of clinical A. baumannii strains, such as international clone 1 (IC1), IC2, and IC3, do not carry the racK gene. This is reminiscent of the loss of the T6SS via genetic disruptions to the T6SS gene locus or the accumulation of inactivating point mutations in ~40% of A. baumannii clinical isolates (40,41). Both PG editing and a functional T6SS are crucial in the environment but appear to impose a fitness cost during infection.…”

Recent Advances in Genetic Tools for Acinetobacter baumannii

“…Summary of selection markers used in clinical isolates of A. baumannii Jacobs et al, 2014;Gallagher et al, 2015;Xu Q. et al, 2019;Lopez et al, 2020;Pérez-Varela et al, 2020. …”

Table_1.docx

“…This vector also contains the Acinetobacter specific rpoD promoter to control transposase expression. A different marinerbased system, pSAM::OmpAp+Tn903, has been applied to ATCC R 17978 TM (Di Venanzio et al, 2019) and the Canadian clinical isolate, AbCAN2 (Lopez et al, 2020). An AbCAN2 transposon mutant library provided the basis for determining the essentiality of the Vgr spike protein in the type VI secretion system (T6SS) and its role in interbacterial competition (Lopez et al, 2020).…”