The adaptive transcriptional response of pathogenic Leptospira to peroxide reveals new defenses against infection-related oxidative stress

Abstract

Pathogenic Leptospira spp. are the causative agents of the waterborne zoonotic disease leptospirosis. During infection, Leptospira are confronted with dramatic adverse environmental changes such as deadly reactive oxygen species (ROS). Withstanding ROS produced by the host innate immunity is an important strategy evolved by pathogenic Leptospira for persisting in and colonizing hosts. In L. interrogans, genes encoding defenses against ROS are repressed by the peroxide stress regulator, PerR. In this study, RNA sequencing was performed to characterize both the L. interrogans adaptive response to low and high concentrations of hydrogen peroxide and the PerR regulon. We showed that Leptospira solicit three main peroxidase machineries (catalase, cytochrome C peroxidase and peroxiredoxin) and heme to detoxify oxidants produced during a peroxide stress. In addition, canonical molecular chaperones of the heat shock response and DNA repair proteins from the SOS response were required for Leptospira recovering from oxidative damages. Determining the PerR regulon allowed to identify the PerR-dependent mechanisms of the peroxide adaptive response and has revealed a PerR-independent regulatory network involving other transcriptional regulators, two-component systems and sigma factors as well as non-coding RNAs that putatively orchestrate, in concert with PerR, this adaptive response. In addition, we have identified other PerR-regulated genes encoding a TonB-dependent transport system, a lipoprotein (LipL48) and a two-component system (VicKR) involved in Leptospira tolerance to superoxide and that could represent the first defense mechanism against superoxide in L. interrogans, a bacterium lacking canonical superoxide dismutase. Our findings provide a comprehensive insight into the mechanisms required by pathogenic Leptospira to overcome infection-related oxidants during the arm race with a host. This will participate in framing future hypothesis-driven studies to identify and decipher novel virulence mechanisms in this life-threatening pathogen.Author summaryLeptospirosis is a zoonotic infectious disease responsible for over one million of severe cases and 60 000 fatalities annually worldwide. This neglected and emerging disease has a worldwide distribution, but it mostly affects populations from developing countries in sub-tropical areas. The causative agents of leptospirosis are pathogenic bacterial Leptospira spp. There is a considerable deficit in our knowledge of these atypical bacteria, including their virulence mechanisms. During infection, Leptospira are confronted with the deadly oxidants produced by the host tissues and immune response. Here, we have identified the cellular factors necessary for Leptospira to overcome the oxidative stress response. We found that Leptospira solicit peroxidases to detoxify oxidants as well as chaperones of the heat shock response and DNA repair proteins of the SOS response to recover from oxidative damage. Moreover, our study indicates that adaptation to oxidative stress is orchestrated by a regulatory network involving PerR and other transcriptional regulators, sigma factors, two component systems, and putative non-coding RNAs. These findings provide a comprehensive insight into the mechanisms required by pathogenic Leptospira to tolerate infection-related oxidants, helping identify novel virulence factors, developing new therapeutic targets and vaccines against leptospirosis.

The FUR-like regulators PerRA and PerRB integrate a complex regulatory network that promotes mammalian host-adaptation and virulence of Leptospira interrogans

“…Previously, Murray et al [97] and Zavala-Alvarado et al [92] independently reported that a L. Fig 4A). Based on these virulence studies, the LD 50 for the wild-type (WT) parent was  7 x 10 5 .…”

“…(Tables 1-2 and S4 Table). Surprisingly, we saw limited overlap between the DMC regulons for the double and single mutants ( Fig 6A); all of the overlapping genes were located in the TonB-related chromosomal locus dysregulated in the perRA and perRA/B mutants in vitro [91,92] Fig 7B). While LigB also was expressed at near wild-type levels in the perRB mutant cultivated in DMCs, by immunoblot, we saw a clear decrease in LigA ( Fig 7A and B).…”

“…Arrows are used to indicate significant (>3-fold, q<0.05) upregulation (↑) or repression (↓) in the WT compared to the mutant in DMCs. See Tables S2 and S3. 6 Behavior of the corresponding gene in the WT vs. perRA (A), WT vs. perRB (B) and WT vs. perRA/B (AB) RNA-Seq using L. interrogans cultivated in vitro [91,92]. Arrows are used to indicate significant (>3-fold, p<0.05) upregulation (↑) or repression (↓) in the WT compared to the corresponding mutant in vitro.…”

“…[91, 92] saw little overlap between the PerRA and PerRB regulons by RNA-Seq analysis of in vitro -cultivated organisms. Interestingly, while perRA and perRB single mutants are virulent in hamsters [89, 92], a perRA/B double mutant was avirulent [91]. Collectively, these data argue that L. interrogans requires at least one functional PerR-like regulator for infection in mammals.…”

“…89,91,92], L. interrogans perRA mutants show enhanced survival following exposure to lethal levels of H O in vitro and increased expression levels of catalase, AhpC and cytochrome c peroxidase in vitro. While inactivation of perRBhad no effect on the ability of leptospires to withstand killing by H 2 O 2 , the perRB mutant showed increased tolerance to the superoxidegenerating compound paraquat[91].…”