Multidrug-resistant plasmids repress chromosomally encoded T6SS to enable their dissemination.

Acinetobacter baumannii (Ab) is a nosocomial pathogen with one of the highest rates of multidrug resistance (MDR). This is partially due to transmissible plasmids. Many Ab strains harbor a constitutively active type VI secretion system (T6SS) that is employed to kill nonkin bacteria. T6SS and plasmid conjugation both involve cell-to-cell contact. Paradoxically, successful conjugation requires the survival of the recipient, which is the target of the T6SS. Thus, an active T6SS in either the donor or the recipient poses a challenge to plasmid conjugation. Here, we show that large conjugative MDR plasmids heavily rely on their distinctive ability to repress the T6SS of their hosts to enable their own dissemination and the conjugation of other plasmids, contributing to the propagation of MDR among Acinetobacter isolates.

A multidrug resistance plasmid contains the molecular switch for type VI secretion in Acinetobacter baumannii.

Infections with Acinetobacter baumannii, one of the most troublesome and least studied multidrug-resistant superbugs, are increasing at alarming rates. A. baumannii encodes a type VI secretion system (T6SS), an antibacterial apparatus of Gram-negative bacteria used to kill competitors. Expression of the T6SS varies among different strains of A. baumannii, for which the regulatory mechanisms are unknown. Here, we show that several multidrug-resistant strains of A. baumannii harbor a large, self-transmissible resistance plasmid that carries the negative regulators for T6SS. T6SS activity is silenced in plasmid-containing, antibiotic-resistant cells, while part of the population undergoes frequent plasmid loss and activation of the T6SS. This activation results in T6SS-mediated killing of competing bacteria but renders A. baumannii susceptible to antibiotics. Our data show that a plasmid that has evolved to harbor antibiotic resistance genes plays a role in the differentiation of cells specialized in the elimination of competing bacteria.

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

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.

Screening for Secretion of the Type VI Secretion System Protein Hcp by Enzyme-Linked Immunosorbent Assay and Colony Blot.

The bacterial type VI secretion system (T6SS) is a secretory apparatus encoded by many Gram-negative bacteria. The T6SS facilitates the secretion and injection of toxic effector proteins into host cells, providing a competitive advantage to bacteria encoding this machinery. The activity of the T6SS can be monitored by probing for the conserved tubule component Hcp, which is secreted to the supernatants by the T6SS. Detection of Hcp in culture supernatants is indicative of an active T6SS, but this secretion system is often tightly regulated or inactive under laboratory conditions and different bacterial strains display differing Hcp secretion phenotypes. Herein, we describe an enzyme-linked immunosorbent assay (ELISA) and colony blot methods to facilitate large-scale screening of isolates for Hcp secretion and, thus, T6SS activity.