Outer membrane vesicles isolated from two clinical Acinetobacter baumannii strains exhibit different toxicity and proteome characteristics
Introduction
Acinetobacter baumannii is a strictly aerobic, non-motile, Gram-negative, non-fermentative, oxidase-negative, catalase-positive bacterium that has emerged as one of the most troublesome pathogens worldwide [1]. This pathogen has primarily been implicated in a diverse range of nosocomial infections, such as pneumonia, skin and urinary tract infections, and bacteremia, particularly in immunocompromised individuals [2]. A. baumannii has acquired resistance to a wide spectrum of antibiotics used in clinical practice, which often makes treatment extremely difficult [3]. Although many studies have shown that multidrug-resistant A. baumannii (MDRAb) leads to poorer outcomes compared with sensitive bacteria [4], [5], [6], [7], [8], [9], [10], the clinical impact of A. baumannii infections and the contribution of multidrug resistance remain controversial. The enhanced virulence of MDRAb has become a great concern in recent years [11], [12].
The secretion of virulence factors through general secretory pathways in Gram-negative pathogens is a ubiquitous phenomenon [13]. Outer membrane vesicles (OMVs) have been proposed as vehicles for protein secretion distinct from type I to VI secretion [14]. These spherical bilayered OMVs are released from the outer membrane and range from 20 to 200 nm in diameter [15], [16], [17], [18]. Unlike other secretory systems, OMVs carry insoluble membrane proteins, proteolytically unstable enzymes, and other nonprotein molecules. In some sense, OMVs comprise complexes of pathogen-associated molecular patterns (PAMPs), involving LPS, flagellin, CpG DNA and other outer membrane proteins and envelope lipids [17], [19]. OMVs could induce potent inflammatory responses in host cells through the cumulative effects of vesicle-associated proteins and LPS [20], [21], [22].
In the present study, we isolated OMVs from two A. baumannii strains, MDRAb A38 and non-MDRAb 5806. Cytotoxicity and innate immune response induction and proteome characteristics were compared between the OMVs from these two strains to obtain insight into the toxic characteristics of OMVs from different A. baumannii strains. To our knowledge, this is the first study concerning the toxic discrepancies of A. baumannii OMVs.
Section snippets
Bacterial strains
Two A. baumannii strains were isolated from the sputum of patients hospitalized in the intensive care unit (ICU) at the Third Affiliated Hospital of Sun Yat-sen University. Multilocus sequence typing (MLST) was performed to establish species type, and the disc diffusion method was used to determine the resistance phenotype in accordance with the Clinical and Laboratory Standards Institute (CLSI; M100-S22, 2012).
Preparation of OMVs from culture supernatants
For each strain, OMVs were isolated according to the methods of Wai et al., with
Characteristics of the two A. baumannii strains and OMVs
Using the disc diffusion method, we characterized A38 as a polymyxin-sensitive MDRAb and 5806 as a non-MDRAb. MLST confirmed that A38 belongs to ST457 and 5806 belongs to ST893, which is a new ST type identified in the present study and was added to the database (http://pubmlst.org/abaumannii/). The two A. baumannii strains were grown to the early stationary phase in liquid media (OD600 ≈ 1.0), and the OMVs were harvested. Examination of the transmission electron microscopy images of negatively
Discussion
A. baumannii is a tenacious pathogen that induces nosocomial infections associated with high morbidity and mortality. However, the debate concerning the clinical impact of A. baumannii infections and the contribution of multidrug resistance remains unresolved. The increased morbidity and mortality associated with MDRAb has been associated with inappropriate empirical antimicrobial therapy and more severe illness [4], [26]. The enhanced virulence of MDRAb has recently become of great concern [11]
Conclusion
In the present study, we showed that the clinical MDRAb strain A38 produced more OMVs with enhanced cytotoxicity and the increased ability to induce innate immune responses compared with the non-MDRAb strain 5806. LC-MS/MS analysis revealed more virulence factors enriched in A38 OMVs. Based on these results, we postulate that OMVs might contribute to the worse outcome of MDRAbs compared with non-MDRAbs, reflecting the enhanced toxicity and increased abundance of these strains. Further studies
Acknowledgments
The authors would like to thank Peng-yu Ji, Feng Wu, and Zhen Liu for assistance with cell culture and real-time PCR, and Professor Guo-bao Tian for kindly gifting A549 cells and critically reading the manuscript. This work was supported through a grant from the Specialized Research Fund for the Doctoral Program of Higher Education of China (No. 20130171110077).
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