RETRACTED ARTICLE: A leptospiral AAA+ chaperone–Ntn peptidase complex, HslUV, contributes to the intracellular survival of Leptospira interrogans in hosts and the transmission of leptospirosis

Abstract

Leptospirosis caused by Leptospira is a zoonotic disease of global importance but it is considered as an emerging or re-emerging infectious disease in many areas in the world. Until now, the mechanisms about pathogenesis and transmission of Leptospira remains poorly understood. As eukaryotic and prokaryotic proteins can be denatured in adverse environments and chaperone–protease/peptidase complexes degrade these harmful proteins, we speculate that infection may also cause leptospiral protein denaturation, and the HslU and HslV proteins of L. interrogans may compose a complex to degrade denatured proteins that enhances leptospiral survival in hosts. Here we show that leptospiral HslUV is an ATP-dependent chaperone–peptidase complex containing ATPase associated with various cellular activity (AAA+) and N-terminal nucleophile (Ntn) hydrolase superfamily domains, respectively, which hydrolyzed casein and chymotrypsin-like substrates, and this hydrolysis was blocked by threonine protease inhibitors. The infection of J774A.1 macrophages caused the increase of leptospiral denatured protein aggresomes, but more aggresomes accumulated in hslUV gene-deleted mutant. The abundant denatured leptospiral proteins are involved in ribosomal structure, flagellar assembly, two-component signaling systems and transmembrane transport. Compared to the wild-type strain, infection of cells in vitro with the mutant resulted in a higher number of dead leptospires, less leptospiral colony-forming units and lower growth ability, but also displayed a lower half lethal dose, attenuated histopathological injury and decreased leptospiral loading in lungs, liver, kidneys, peripheral blood and urine in hamsters. Therefore, our findings confirmed that HslUV AAA+ chaperone–Ntn peptidase complex of L. interrogans contributes to leptospiral survival in hosts and transmission of leptospirosis.

Exoproteome from Leptospira interrogans and Host Cells during Infection: Leptospiral Virulence Factors and Cellular Proteins Involved in Stress and Inflammation

“…Infection is an interactive process that takes place between pathogens and hosts [23,35]. During this interaction, hosts raise body temperature and mount as in ammatory reaction in order to eliminate the pathogens, but conversely, prokaryotic pathogens also change their metabolism in order to respond to the adverse environment for survival in the hosts.…”

“…The trypsin-digested proteins from the hydrolyzed CSPs were identi ed by high performance liquid chromatography plus tandem mass spectrometry (LC-MS/MS) as previously described [23,53]. Brie y, 1 ug of the trypsin-digested protein preparation was dissolved in 5 μL of 0.1% formic acid (FA, Sigma) solution.…”

“…The heatmaps and clusters of the leptospiral and cellular exoproteins were generated using Complex-Heatmap package and R software [55]. GO and KEGG pathway analysis including cellular component, molecular function and biological process in gene ontology were analyzed as previously described [23,[56][57][58]].…”

“…The cells and leptospires were washed twince with PBS and then centrifuged as above. The viability of infected cells was detected by methyl thiazolyl tetrazolium (MTT) test using a Cell Proliferation Kit (Sigma) while the viability of collected leptospires was detected by confocal microscopy (Zeiss, Germany) (485/630 or 485/530 nm excitation/emission wavelengths for SYTO 9 or PI detection) using a LIVE/DEAD Bacterial Viability Kit (Invitrogen, USA) and the fold changes of green uorescence intensity (FI) were analyzed for semi-quanti cation of the leptospiral viability [23,31]. In the tests, L. interrogans strain Lai, THP-1 macrophages and HUVECs without infection were used as the controls.…”

“…Previous studies showed that the transcription and expression levels of many genes of pathogenic Leptospirainterrogans during infection of cells are signi cantly changed [14,15]. For example, the expression levels of virulence factors such as adherence factors, invasive enzymes and toxins of L. interrogans are signi cantly up-regulated during infection, but those of surface protein antigens are notably decreased [16][17][18][19][20][21][22][23][24][25]. However, it is believed that only released or surface proteins of microbes can play a direct role in the pathogenic process.…”