Acinetobacter baumannii utilizes a type VI secretion system for bacterial competition

Abstract

Type VI secretion systems (T6SS) are a class of macromolecular secretion machines that are utilized by a number of bacteria for inter-bacterial competition or to elicit responses in eukaryotic cells. Acinetobacter baumannii is an opportunistic pathogen that causes severe infections in humans. These infections, including pneumonia and bacteremia, are important, as they are often associated with hospitals and medical-settings where they disproportionally affect critically ill patients like those residing in intensive care units. While it is known that A. baumannii genomes carry genes whose predicted products have homology with T6SS-associated gene products from other bacteria, and secretion of a major T6SS structural protein Hcp has been demonstrated, no additional work on an A. baumannii T6SS has been reported. Herein, we demonstrated that A. baumannii strain M2 secretes Hcp and this secretion was dependent upon TssB, an ortholog of a bacteriophage contractile sheath protein, confirming that strain M2 produces a functional T6SS. Additionally, we demonstrated that the ability of strain M2 to out-compete Escherichia coli was reliant upon the products of tssB and hcp. Collectively, our data have provided the first evidence demonstrating function in inter-bacterial competition, for a T6SS produced by A. baumannii.

Figure 1. The predicted T6SS gene cluster of A. baumannii strain M2.

A single ∼23 kb gene cluster, asaA-tssBC-hcp-tssEFG-asaB-tssM-tagFN-asaC-tssHAKL-asaDE carries 18 putative genes predicted to encode for components of a T6SS. The genome of A. baumannii strain M2 carries genes that are predicted to encode 12 of the 13 core T6SS proteins (tss, colored scarlet), 2 proteins with homology to T6SS-associated proteins in other bacteria (tag, colored black) and 5 proteins encoded by genes only identified in A. baumannii T6SS gene clusters (asa, Acinetobacter type six secretion system-associated, colored gray). Three additional genes whose products have homology to VgrG were identified in other regions of the genome (data not shown).

Figure 2. Acinetobacter baumannii strain M2 secreted Hcp.

A. Growth curves of strain M2 and the isogenic tssB and hcp mutants. Three biological replicates were performed; error bars denote the standard error of the mean of the biological replicates. B. Concentrated culture supernatants from strain M2, M2ΔtssB and M2Δhcp analyzed by SDS-PAGE and Coomassie staining. A band corresponding to the approximate molecular mass of Hcp (∼19 kDa), indicated by the arrow, was observed in the sample derived from strain M2 and was absent in the samples from M2ΔtssB and M2Δhcp. Mass spectrometry confirmed that the major protein in this band was Hcp. C. Western blot analysis of whole cell lysates and concentrated culture supernatants of A. baumannii strains. Hcp, indicated by the arrow, was detected in whole cell lysates of strain M2, M2ΔtssB and the complemented tssB mutant as well as in concentrated culture supernatants of strain M2 and the complemented tssB mutant.

Figure 3. Acinetobacter baumannii required the products of tssB and hcp to out-compete E. coli.

A. Semi-quantitative assessment of surviving E. coli after 4 h mixed with media (Control) or at a 10∶1 ratio (10 A. baumannii to 1 E. coli) with A. baumannii strain M2, M2ΔtssB or M2Δhcp. B. Quantitative assessment of the A. baumannii and E. coli DH10B populations. Data represent of 3 biological replicates each performed in duplicate. Error bars represent standard error of the mean of the biological replicates. * Indicates a significant difference (P<0.0001) between E. coli CFU observed with or without co-incubation with strain M2. The dotted line on each bar graph of panel B indicates level of detection.

Figure 4. Acinetobacter baumannii out-competes E. coli in a contact dependent manner.

Qualitative assessment of E. coli DH10B populations after 4 h of competition with the indicated A. baumannii strains or media. The backslash between strains (E.c. = E. coli, M2 = strain M2) or L (L-broth) indicate the presence of a 0.22 µm pore filter. Data represent 3 biological replicates performed in duplicate. Error bars represent standard error of the mean of the biological replicates. * Indicates a significant difference (P<0.01) in E. coli CFU between the M2+E.c./L group when compared to all other groups.