Characterization of leptospiral Extracellular vesicles in stress conditions

Abstract

Pathogenic Leptospira spp. is a causative agent of leptospirosis, a worldwide zoonosis with public health concern especially in the urban slum of metropolis and rural areas in tropical and subtropical countries. The pathogenesis of leptospirosis remains elusive. Extracellular vesicles (ECVs), which pinch off from the bacterial membranes, simultaneously harbor multiple active molecules that may serve as a secretion system, communication tool, and vaccine candidates. Recently, chemically induced leptospiral ECVs were studied and used as vaccine candidates. However, the naturally produced leptospiral ECVs has not been characterized. This study aimed to identify proteins in leptospiral ECVs produced under stress conditions including temperature shift to 37°C and physiologic osmolarity, which mimicked the host environment, in comparison to in vitro growth at 30°C. The leptospiral ECVs produced under each condition were isolated and purified from intact cells using combined methods of centrifugation, filtration, ultracentrifugation, and sucrose density gradient centrifugation resulting in nanosized spherical vesicles as shown by transmission electron microscopy. To identify and relatively quantify proteins in these leptospiral ECVs, dimethylation labeling coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed. A total of 690 proteins were identified and predicted their subcellular localization with bioinformatics tools including PSORTb v3.0.2, CELLO, Gneg-mPLoc, SOSUI, and SignalP. Of these, the majority (399 proteins, 57.9%) were predicted as cytoplasmic proteins followed by 103 (14.8%) outer membrane proteins, 101 (14.5%) inner membrane proteins, 36 (5.1%) unknown, 27 (4%) periplasmic proteins, and 24 (3.6%) extracellular proteins. Based on KEGG pathway analysis the identified proteins were biologically categorized into unidentified group (49.6%) followed by transcription (10.6%), and carbohydrate metabolism (9.1%). Relative quantification of protein abundance showed differential expression of proteins cargoes. In response to temperature shift, 83 proteins significantly up- and downregulated (55 and 28, respectively) (p< 0.05) of which diaminopimelate decarboxylase was the most up-regulated (3.9-fold). Under physiologic osmolarity, 106 proteins were differentially expressed with 17 up-regulated and 89 down-regulated proteins (p< 0.05) of which transketolase alpha subunit protein was the most up-regulated (1.52-fold). In addition, sulfate ABC transporter periplasmic sulphate-binding protein was the most up-regulated (2.9 fold) under temperature shift of all 89 proteins differentially expressed between the stress conditions. Moreover, known virulence factors, such as Lig proteins, LipL21, LipL32, LipL41, and hemolysin, as well as hypothetical proteins were found in leptospiral ECVs. In conclusion, leptospiral ECVs produced in response to stress conditions harboring differentially expressed proteins that may play a role in the pathogenesis of leptospirosis and survival of leptospires in the host.