T6SS-associated genes have been identified previously in other Acinetobacter
and in A. baumannii
, but a function for these T6SS was not elucidated. In this study, we provided the first evidence assigning a function to the A. baumannii
T6SS in inter-bacterial competition.
Analysis of concentrated culture supernatants showed that the T6SS structural and effector protein, Hcp, accumulated in supernatants of cultures of A. baumannii
strain M2 grown under standard laboratory conditions, and was the predominant protein species observed by Coomassie-stained SDS-PAGE of culture supernatants (). In addition, Hcp secretion was dependent upon tssB,
a gene that is predicted to encode a T6SS sheath protein that is required for T6SS function in other bacteria 
. Western blot analysis confirmed secretion of Hcp into the culture supernatant of strain M2 and the complemented hcp
mutant, which indicated that the tssB
mutation did not affect expression of the downstream hcp
gene. In addition, the tssB
mutant produced Hcp but was unable to secrete it (.C). Attempts to complement the Hcp secretion phenotype in the hcp
mutant were unsuccessful. This is likely due to polar effects on down stream genes in the T6SS gene cluster. As secretion of Hcp is the hallmark of a functional T6SS 
, these results indicated that A. baumannii
strain M2, indeed, does produce a functional T6SS. We assume, as has been postulated by others, that detection of Hcp in culture supernatants was due to Hcp being sheared off from the surface of cells that express T6SS 
In order to identify a function for the A. baumannii
T6SS we assessed the potential of strain M2 and the isogenic tssB
mutants to outcompete E. coli.
In this assay, strain M2 elicited a 5.3-log decrease in E. coli
CFU during co-incubation, which was tssB
-dependent (). The CFU of A. baumannii
strains was unaffected by co-incubation with E. coli
(). These data are similar to those reported for T6SS-dependent competition-phenotypes of Serratia marcescens, Vibrio cholerae
and P. aeruginosa
, strongly supporting the role for the A. baumannii
T6SS in bacterial competition.
The T6SS-dependent competition between strain M2 and E. coli
was observed, by a 6-log reduction in E. coli
CFU when mixed and incubated together above a filter with 0.22 µm pores (). Competition was abrogated when strain M2 and E. coli
were separated by a filter. These results indicated that competition between A. baumannii
and E. coli
was dependent on cell-to-cell contact. In other systems, the antibacterial action mediated by T6SS has also been shown to be contact dependent 
. These data provide further evidence that A. baumannii
competition with E. coli
was, indeed, mediated by a T6SS.
A small number of T6SS effectors, exhibiting toxicity against bacteria have been identified. In Pseudomonas aeruginosa,
for example, the toxic effectors, Tse1, Tse2 and Tse3 are injected into target cells via a T6SS 
. Tse1 and Tse3 are active in the periplasm and have been shown to cleave peptidoglycan. Cells expressing the cognate immunity proteins to Tse1 and Tse3, Tsi1 and Tsi2, are protected from these effects. Tse2 appears to cause quiescence when expressed in the cytoplasm in the absence the Tse2 immunity protein, Tsi2 
. In S. marcescens,
the T6SS effectors Ssp1 and Ssp2 are toxic to target bacteria when localized to the periplasm in the absence of the immunity proteins Rap1a and Rap2a respectively. The mechanism behind the effect Ssp1 and Ssp2 has on target bacteria has yet to be elucidated 
. We and others have attempted to identify potential T6SS effectors in A. baumannii
strains using bioinformatics approaches. Russell et al.,
using a heuristic method, identified a potential effector 
. This predicted T6SS effector was found in one A. baumannii
strain, strain SDF, encoded by a gene carried on a plasmid that is not conserved amongst Acinetobacter
spp. Thus, the specific mechanism behind T6SS-dependent inter-bacterial competition in A. baumannii
The ability to compete with other bacteria, via T6SS, could be important both in and ex vivo for the success of A. baumannii as a pathogen. One can imagine a ventilation tube in an intubated patient becoming colonized with a variety of bacteria including A. baumannii. In order to colonize this surface, thrive and eventually cause disease in the patient, it is logical to assume these bacteria would compete for this niche. Similarly, in patients with infected wounds or that are afflicted with pneumonia; a mixed population of bacteria at these sites would compete to maintain the species. We postulate that A. baumannii utilizes its T6SS to compete with bacteria in these environments. With respect to the role of T6SS in disease, specifically T6SS-mediated interactions with eukaryotic cells, we welcome the idea that the A. baumannii T6SS plays a direct role in virulence but, as of yet, do not have evidence for such a role.
In conclusion, we have provided evidence that the A. baumannii T6SS is functional and that it plays a role in competition with other bacterial species. The specific mechanism behind this competition has yet to be elucidated. Additional studies are needed to determine if the T6SS produced by A. baumannii confers a fitness advantage, when faced with bacterial competitors during the course of colonization and disease. The study of biogenesis, function and regulation of A. baumannii T6SS, although in its infancy, has the potential to make a significant impact on our understanding of how A. baumannii may persist amongst mixed populations during survival in the medical environment or during the course of disease.
During revision of this manuscript, Weber et al.
described the type VI secretion system in A. baumannii ATCC 17978 
. Although the gene clusters characterized by both laboratories are very similar, Weber et al.
did not observe Type VI secretion system-dependent killing of E. coli
. Additional work will be required to identify the basis for the differences observed in the two studies.