The type VI secretion system in Acinetobacter baumannii clinical isolates and its roles in antimicrobial resistance acquisition.

Acinetobacter baumannii is a successful pathogen that can acquire various antibiotic resistance in a short time. However, little is known about how it can evolve from an antibiotic sensitive to a resistant phenotype. In this study, we investigated the roles of the type VI secretion system (T6SS) in the acquisition of antibiotic resistance of A. baumannii. T6SS gene cluster was found to be present in 51 of 77 A. baumannii clinical isolates, of which, it was found in 62% (8/13) of the multiple drug resistant (MDR) isolates, 90% (36/40) of the extensively drug-resistant (XDR) isolates and 26% (6/23) of the antibiotic sensitive isolates. There is a close relationship between the antimicrobial resistance and the presence of T6SS. Besides, T6SS + isolates showed lower biofilm formation activity and higher survival ability in the presence of normal human serum than T6SS- isolates. A. baumannii A152 with complete T6SS can outcompete E.coli effectively and can acquire the antibiotic resistance plasmids released by E.coli. In contrast, the T6SS core gene mutant A152Δhcp showed significantly decreased ability to acquire antimicrobial resistance plasmids from the prey bacteria. These results suggest that T6SS mediated bacterial competition plays important roles in the antimicrobial resistance of A. baumannii, which points out a new direction for us to study the antimicrobial resistance of A. baumannii.

Comparative genomics analysis of Acinetobacter baumannii multi-drug resistant and drug sensitive strains in China.

The incidence of multidrug-resistant Acinetobacter baumannii has posed a major challenge for clinical treatment. There is still a significant gap in understanding the mechanism causing multi-drug resistance (MDR). In this study, the genomes of 10 drug sensitive and 10 multi-drug resistant A.baumannii strains isolated from a hospital in China were sequenced and compared. The antibiotic resistance genes, virulence factors were determined and CRIPSR-Cas system along with prophages were detected. The results showed that MDR strains are significantly different from the drug sensitive strains in the CARD entries, patterns of sequences matching up to plasmids, VFDB entries and CRISPR-Cas system. MDR strains contain unique CARD items related to antibiotic resistance which are absent in sensitive strains. Furthermore, sequences from genomes of MDR strains can match up with plasmids from more diversified bacteria genera compared to drug sensitive strains. MDR strains also contain a lower level of CRISPR genes and larger amount of prophages, along with higher levels of spacer sequences. These findings provide new experimental evidences for the study of the antibiotic resistance mechanism of A. baumannii.

Roles of the Hcp family proteins in the pathogenicity of Salmonella typhimurium 14028s.

The type VI secretion system (T6SS) is a new secretion system that is widely distributed among Gram-negative bacteria. The core component hemolysin-coregulated protein (Hcp) can be used as both its structural protein and secretory protein or chaperone protein. Studies on Hcp are important to elucidate the overall virulence mechanism of T6SS. Salmonella typhimurium is an important foodborne pathogen. There are three copies of hcp genes identified in S. Typhimurium 14028s. This study aimed to characterize the functions of the three Hcp family proteins and to elucidate the interactions among them. The hcp gene deletion mutants were constructed by λ Red-based recombination system. Effects of hcp mutation on the pathogenicity of 14028s were studied by bacterial competition assays, Dictyostelium discoideum assays and mouse model. The three Hcp family proteins were found to play different roles. Hcp1 can affect the transcription of rpoS and type 2 flagellar gene and influence the motility of 14028s. It is also involved in the intracellular survival of 14028s in Dictyostelium discoideum; Hcp2 is involved in the early proliferative capacity of 14028s in mice and can prevent its excessive proliferation; Hcp3 did not show direct functions in these assays. Hcp1 can interact with Hcp2 and Hcp3. Deletion of one hcp gene can result in a transcription level variation in the other two hcp genes. Our findings elucidated the functions of the three Hcp family proteins in S.Typhimurium and illustrated that there are interactions between different Hcp proteins. This study will be helpful to fully understand how T6SS actions in an organism.

The type VI secretion system protein AsaA in Acinetobacter baumannii is a periplasmic protein physically interacting with TssM and required for T6SS assembly.

Type VI secretion system (T6SS) is described as a macromolecular secretion machine that is utilized for bacterial competition. The gene clusters encoding T6SS are composed of core tss genes and tag genes. However, the clusters differ greatly in different pathogens due to the great changes accumulated during the long-term evolution. In this work, we identified a novel hypothetical periplasmic protein designated as AsaA which is encoded by the first gene of the T6SS cluster in the genus Acinetobacter. By constructing asaA mutant, we delineated its relative contributions to bacterial competition and secretion of T6SS effector Hcp. Subsequently, we studied the localization of AsaA and potential proteins that may have interactions with AsaA. Our results showed that AsaA in Acinetobacter baumannii (A. baumannii) localized in the bacterial periplasmic space. Results based on bacterial two-hybrid system and protein pull-down assays indicated that it was most likely to affect the assembly or stability of T6SS by interacting with the T6SS core protein TssM. Collectively, our findings of AsaA is most likely a key step in understanding of the T6SS functions in A. baumannii.