Escape of TLR5 Recognition byLeptospira spp: A Rationale for Atypical Endoflagella

Abstract

26Leptospira interrogans are invasive bacteria responsible for leptospirosis, a worldwide zoonosis. They 27 possess two periplasmic endoflagella that allow their motility. L. interrogans are stealth pathogens that 28 escape the innate immune responses of the NOD-like receptors NOD1/2, and the human Toll-like 29 receptor (TLR)4, sensing peptidoglycan and lipopolysaccharide (LPS), respectively. TLR5 is another 30 receptor of bacterial cell wall components, recognizing flagellin subunits. 31To study the contribution of TLR5 in the host defense against leptospires, we infected WT and TLR5 32 deficient mice with pathogenic L. interrogans and tracked the infection by in vivo live imaging of 33 bioluminescent bacteria or by q-PCR. We did not identify any protective or inflammatory role of 34 murine TLR5 to control pathogenic Leptospira. Likewise, subsequent in vitro experiments showed 35 that infections with different live strains of L. interrogans and L. biflexa did not trigger TLR5. 36However, unexpectedly, heat-killed bacteria stimulated human and bovine TLR5, although barely 37 2 mouse TLR5. Abolition of TLR5 recognition required extensive boiling time of the bacteria or 38 proteinase K treatment, showing an unusual high stability of the leptospiral flagellins. Interestingly, 39 using antimicrobial peptides to destabilize live leptospires, we detected some TLR5 activity, 40 suggesting that TLR5 could participate in the fight against leptospires in humans or cattle. Using 41 different Leptospira strains with mutations in flagellin proteins, we further showed that neither FlaAs 42 nor Fcps participated in the recognition by TLR5, suggesting a role for the FlaBs. These have 43 structural homology to Salmonella FliC, and conserved residues important for TLR5 activation, as 44 shown by in silico analyses. Accordingly, we found that leptospires regulate the expression of FlaB 45 mRNA according to the growth phase in vitro, and that infection with L. interrogans in hamsters and 46 in mice downregulated the expression of the FlaBs but not the FlaAs subunits. 47Altogether, in contrast to different bacteria that modify their flagellin sequences to escape TLR5 48 recognition, our study suggests that the peculiar central localization and stability of the FlaB 49 monomers in the periplasmic endoflagella, associated with the downregulation of FlaB subunits in 50 hosts, constitute an efficient strategy of leptospires to escape TLR5 recognition and the immune 51 response. 53 55Among the Leptospira genus, which includes more than 60 species and 300 different serovars, 56Leptospira interrogans gathers the most pathogenic strains [2]. Rodents and other animals can carry 57 leptospires asymptomatically in the lumen of proximal renal tubules, excrete the bacteria in their urine 58 and contaminate the environment. Vertebrates get infected through skin or mucosa. In most cases, 59 humans have no symptoms or suffer from a flu-like mild disease, but may also show acute severe, 60 potentially fatal, leptospirosis. Antibiotic treatments are e...