Pathogenic Leptospires Modulate Protein Expression and Post-translational Modifications in Response to Mammalian Host Signals

Abstract

Pathogenic species of Leptospira cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Reservoir hosts of leptospirosis, including rodents, dogs, and cattle, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. Whilst little is known about how Leptospira adapt to and persist within a reservoir host, in vitro studies suggest that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. We applied the dialysis membrane chamber (DMC) peritoneal implant model to compare the whole cell proteome of in vivo derived leptospires with that of leptospires cultivated in vitro at 30°C and 37°C by 2-dimensional difference in-gel electrophoresis (2-D DIGE). Of 1,735 protein spots aligned across 9 2-D DIGE gels, 202 protein spots were differentially expressed (p < 0.05, fold change >1.25 or < −1.25) across all three conditions. Differentially expressed proteins were excised for identification by mass spectrometry. Data are available via ProteomeXchange with identifier PXD006995. The greatest differences were detected when DMC-cultivated leptospires were compared with IV30- or IV37-cultivated leptospires, including the increased expression of multiple isoforms of Loa22, a known virulence factor. Unexpectedly, 20 protein isoforms of LipL32 and 7 isoforms of LipL41 were uniformly identified by DIGE as differentially expressed, suggesting that unique post-translational modifications (PTMs) are operative in response to mammalian host conditions. To test this hypothesis, a rat model of persistent renal colonization was used to isolate leptospires directly from the urine of experimentally infected rats. Comparison of urinary derived leptospires to IV30 leptospires by 2-D immunoblotting confirmed that modification of proteins with trimethyllysine and acetyllysine occurs to a different degree in response to mammalian host signals encountered during persistent renal colonization. These results provide novel insights into differential protein and PTMs present in response to mammalian host signals which can be used to further define the unique equilibrium that exists between pathogenic leptospires and their reservoir host of infection.

Inbred Rats as a Model to Study Persistent Renal Leptospirosis and Associated Cellular Immune Responsiveness

“…At this time, a lymphocyte-rich inflammatory infiltrate in association with leptospires in infected kidney sections was apparent; however, it was also apparent that leptospiral organisms were also detected within tubules devoid of any immune response. It remains unclear if such responses can ultimately be successful enough to remove organisms and eliminate renal excretion, or whether leptospires can continue to evade detection and reactivity as hypothesized by differential expression of antigens (Nally et al, 2007 , 2011 , 2017 ; Monahan et al, 2008 ; Witchell et al, 2014 ).…”

“…Rat kidneys were harvested at approximately 8 weeks post-infection and immediately fixed by immersion in neutral buffered 10% formalin, processed routinely, embedded in paraffin, cut into 4 μm sections, and stained with hematoxylin and eosin (HE). Immunohistochemistry was performed on paraffin-embedded tissue sections using antiserum generated against outer membrane vesicles (OMV) of Leptospira species or with anti-LipL32 (Nally et al, 2004 , 2017 ). After dewaxing, tissue sections were blocked with 10% normal goat serum in PBS for 30 mins at room temperature.…”

“…Experimentally infected rats persistently excrete large numbers of leptospires in urine which has allowed for the characterization of urinary derived leptospires compared to its in vitro cultivated counterpart (Bonilla-Santiago and Nally, 2011 ). Leptospires excreted from renal tubules modify their protein and antigen expression, and regulate expression of protein post-translational modifications, a function hypothesized to help evade host immune responses (Nally et al, 2005 , 2011 , 2017 ; Monahan et al, 2008 ; Witchell et al, 2014 ). Antibodies from experimentally infected rats react with a larger number of antigens expressed by in vitro cultivated leptospires compared to urinary derived leptospires (Monahan et al, 2008 ).…”

Recent findings related to immune responses against leptospirosis and novel strategies to prevent infection

“…Then following mass spectrometry analysis of proteins separated by 2-D gel electrophoresis, they showed that LoA22, an outer lipoprotein important for virulence, LipL32, LipL41 and chaperone proteins, are more abundant in vivo. In addition, these proteins were identified to have post-translational modifications, such as phosphorylation or tri-methylations, not observed in vitro when leptospires were grown in EMJH culture medium at 30 C or 37 C [30]. Therefore, PTM of leptospiral proteins may constitute a hurdle to generate effective vaccines from in vitro cultured bacteria, since candidate proteins will be devoid of these modifications and the antibodies elicited may not recognize the corresponding modified proteins expressed in the host.…”

Leptospiral 3-hydroxyacyl-CoA dehydrogenase as an early urinary biomarker of leptospirosis

“…Recently, Nally et al. reported that pathogenic leptospires modulate protein post-translational modifications in response to mammalian host signals [18] .…”