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

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 summary Leptospirosis 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.


Introduction
In order to invade a host and establish persistent colonization, pathogens have evolved a variety of strategies to resist, circumvent, or counteract host defenses. Synthesis of detoxification enzymes or molecules to eliminate host-produced bactericidal compounds, secretion of effectors inhibiting or subverting the host innate immunity, biofilm formation enabling resistance to host defenses, are all examples of mechanisms used by pathogens depending of their lifestyle and niche.
The whole strategies used by pathogenic Leptospira for successful host colonization and virulence are not fully unraveled. These aerobic gram-negative bacteria of the spirochetal phylum are the causative agents of leptospirosis, a widespread zoonosis (1). Although recognized as a health threat among impoverished populations in developing countries and tropical areas (2), reported cases of leptospirosis are also on the rise in developed countries under temperate climates (3). Rodents are the main reservoir for leptospires as the bacteria asymptomatically colonize the proximal renal tubules of these mammals. They shed bacteria in the environment by their urine and leptospires are transmitted to other animals and humans mostly by exposure to contaminated soils and water. Once having penetrated an organism, Leptospira enter the bloodstream and rapidly disseminate to multiple tissues and organs including kidney, liver and lungs. Clinical manifestations range from a mild flu-like febrile state to more severe and fatal cases leading to hemorrhages and multiple organ failure. The lack of efficient tools and techniques for genetic manipulation of Leptospira spp. and their fastidious growth in laboratory conditions have greatly hampered and limited our understanding of their mechanisms of pathogenicity and virulence (4,5).
As part of the host innate immunity response, reactive oxygen species (ROS), i.e. superoxide anion (  O 2 -), hydrogen peroxide, (H 2 O 2 ), hydroxyl radicals (  OH), hypochlorous acid (HOCl), and nitric oxide anion (  NO) are produced upon infection by Leptospira. Indeed, the internalization of pathogenic Leptospira by macrophages and concomitant production of these oxidants have been demonstrated in vitro (6), and leptospirosis-associated oxidative stress has been observed in leptospirosis patients (7) and infected animals (8). Consistent with these findings was the demonstration that catalase, that catalyzes the degradation of H 2 O 2 , is required for Leptospira interrogans virulence (9).
Pathogenic Leptospira spp. are among the rare examples of gram-negative bacteria where defenses against peroxide stress, such as catalase, are controlled by a peroxide stress regulator (PerR) and not by OxyR (10). PerR is a peroxide-sensing transcriptional repressor that belongs to the Fur (Ferric uptake regulator) family of regulators, mostly present in grampositive bacteria (11). The B. subtilis PerR is in a DNA-binding prone conformation in the presence of a regulatory metal (Fe 2+ ) (12). Upon oxidation by H 2 O 2 , PerR releases its regulatory metal and switches to a conformation that cannot bind DNA, leading to the alleviation of gene repression (13,14).
We have conducted a structural and functional characterization of PerR in L. interrogans and showed that Leptospira PerR exhibits the typical metal-induced conformational switch controlling DNA binding and release (15). Our findings indicated that not only Leptospira PerR represses defenses against H 2 O 2 , but also a perR mutant had a decreased fitness in other host-related stress conditions including in the presence of superoxide (15). Interestingly, it was shown that perR is up-regulated when Leptospira are exposed in vitro to hydrogen peroxide (15) as well as when Leptospira are cultivated in vivo using Dialysis Membrane Chambers (DMCs) in rats (16), which strongly suggests a role of PerR in the adaptation of pathogenic Leptospira to a mammalian host.
In order to identify the mechanisms solicited by pathogenic Leptospira to adapt to oxidative stress, we have determined the global transcriptional response of L. interrogans to H 2 O 2 and assessed the role of PerR in this adaptation. This has revealed the cellular factors constituting the first-line of defense against ROS that Leptospira might encounter when infecting a mammalian host. In addition, our study has identified repair mechanisms allowing leptospires to recover from oxidative damage. Putative regulatory non-coding RNAs were also pinpointed, indicating the complexity of the regulatory network controlling the adaptive response to peroxide. We have also identified novel PerR-regulated factors involved in Leptospira survival in the presence of superoxide and assessed their role in Leptospira virulence.

Results
Leptospira transcriptional response to a sublethal concentration of hydrogen peroxide.
In order to characterize the transcriptional response of pathogenic Leptospira to hydrogen peroxide, we have exposed exponentially growing L. interrogans cells to sublethal concentrations of this oxidant. A 30 min treatment with 10 µM H 2 O 2 (in the presence of iron) was chosen during pilot experiments as having no significant effect on Leptospira viability and growth during logarithmic phase while increasing expression of H 2 O 2 -responsive genes such as perR (15). RNA sequencing was performed to assess RNA abundance and comparison with untreated cells identified a total of 21 genes with differential transcript abundance. Among those, only 12 and 1 genes were respectively up-and down-regulated by a at least two-fold with P-values ≤0.005 (See Table 1).
Under a low concentration of H 2 O 2 , LIMLP_10145, encoding a catalase, and LIMLP_02795 and LIMLP_05955, coding respectively for a cytochrome C peroxidase and for a peroxiredoxin, were up-regulated with a Log 2 FC of 1.79, 4.76 and 3.14, respectively.
The catalase encoded by LIMLP_10145 (katE) is a monofunctional heme-containing hydroperoxidase, the catalase activity of which and periplasmic localization were experimentally demonstrated in pathogenic Leptospira (9,17,18). The immediate upstream ORF (LIMLP_10150), encoding an ankyrin repeat-containing protein, was also up-regulated with a comparable fold. In bacteria such as Pseudomonas aeruginosa and Campylobacter jejuni, protein with ankyrin repeats were found to be required for the catalase activity, probably by allowing heme binding (19,20). In L. interrogans, katE and ank were organized as an operon (S1 Fig) and significant up-regulation of the ank-katE operon upon exposure to sublethal dose of H 2 O 2 was confirmed by RT-qPCR (Table 1 and   Therefore, it is very likely that LIMLP_02795 encodes a CCP with a peroxidase activity that is not involved in the methylamine metabolism pathway. In addition to these three peroxidases, whose increased expression was confirmed by RT-qPCR (Table 1 and S1 and S2 Figs), several ORFs encoding components of heme biosynthesis (LIMLP_17840-17865) were up-regulated by a 2 to 3.4-fold (Table 1). confirmed that katE, ccp, ahpC, perR, and several genes of the heme biosynthesis pathway were among the genes the expression of which was significantly up-regulated ( Fig 1A).  (Fig 1B), bearing witness to a higher number of genes with significantly and statistically changed expression than when Leptospira are exposed to sublethal dose of Fig 1A).
Differentially expressed genes were classified into COG functional categories and the obtained COG frequencies were compared to the frequency of the genes in the genome. As seen in Fig 2, the up-regulated genes were enriched in the post-translational modification, protein turnover, and chaperones categories whereas down-regulated genes mainly fell into metabolism, translation and ribosomal structure and biogenesis, coenzyme transport and metabolism, and energy production and conversion categories. and S1 Table). All these up-regulations were confirmed by RT-qPCR experiments (S1 Table).
Additional ORFs encoding cellular factors related to oxidative stress and redox maintenance were also up-regulated (Fig 3 and Table).
Major cellular pathways involved in reparation of damaged cellular components were dramatically up-regulated when Leptospira were exposed to a lethal dose of H 2 O 2 . Indeed, several genes encoding molecular chaperones had an increased expression in the presence of 1 mM H 2 O 2 (Fig 3 and S1 Table). Two ORFs encoding small heat shock proteins (sHSP), Thus, the machinery necessary for preventing protein aggregation and promoting protein refolding is solicited when Leptospira are exposed to high dose of H 2 O 2 .
Genes encoding several components of the SOS response, a regulatory network stimulated by DNA damage-inducing stress, had a higher expression in the presence of 1 mM H 2 O 2 (Fig 3 and S1 Table). Indeed, ORFs encoding the recombinase A (recA, LIMLP_08665), the DNA repair protein RecN (LIMLP_07915), the DNA polymerase IV (dinP, LIMLP_02170) as well as the repressor of the SOS response LexA1 (LIMLP_11440) were significantly up-regulated.
Other factors putatively involved in DNA repair but not under the control of LexA1 (27,28) had also an increased expression, including the DNA mismatch repair protein MutS (LIMLP_07780, Log 2 FC value of 1) and the DNA repair protein RadC (LIMLP_11400, Log 2 FC value of 3.4).
A cluster of gene encoding the ATP synthase complex (LIMLP_06050-06080) was down regulated with a Log 2 FC≤-1.2, indicating that Leptospira decrease ATP synthesis upon exposure to high dose of H 2 O 2 (S2 Table). Another metabolic pathway that was downregulated in this condition was the cobalamin (vitamine B12) biosynthesis pathway. Indeed, 15 out 17 genes of the cobI/III cluster (LIMLP_18460-18530) were significantly downregulated (with a Log 2 FC ≤-1.5, S2 Table).

Identification of differentially non-coding RNAs in the presence of hydrogen peroxide.
In order to identify non-coding RNAs (ncRNAs) whose expression is changed in the presence of hydrogen peroxide, non-coding genome regions of RNASeq data were also analyzed. When Leptospira were exposed to 10  When Leptospira were exposed to a lethal dose of hydrogene peroxide (1 mM for 1h), a higher number of differentially expressed ncRNAs was detected. Indeed, 416 and 102 ncRNAs were up-and down-regulated, respectively. 28 ncRNAs were up-regulated with a Log 2 FC above 1.5. Rh3130 and rh3352 were the two most highly up-regulated ncRNAs with a Log 2 FC > 7 ( Table 2). A 70 bp ncRNA (rh288), which was up-regulated with a Log 2 FC of 3.81, overlapped with LIMLP_00895, an ORF located in the prophage locus 1 ( Table 2).
Several of the ncRNAS whose expression was up-or down-regulated in the presence of hydrogen peroxide were located in the vicinity or overlapped ORFs that were also up-or down-regulated in the same conditions. For instance, the rh3130 and rh859, two of the most highly up-regulated ncRNAs, were in the vicinity of Hsp20 and CCP-encoding ORFs (LIMLP_10970-10975 and LIMLP_02795, respectively), two genes whose expression was greatly increased in the presence of hydrogen peroxide (Tables 1 and 2 and Fig 3).
LIMLP_05620, LIMLP_13670, and LIMLP_13765 were three up-regulated ORFs upon exposure to hydrogen peroxide that have a up-regulated downstream ncRNA (rh1641, rh3871, and rh3894, respectively). This tendency was also observed with down-regulated ncRNAs.
Rh411, rh967, rh1101, rh1102, rh1880, rh3186, and rh4281 ncRNAs were also located downstream or upstream, or overlapped ORFs whose expression was decreased in the presence of hydrogen peroxide ( Contribution of PerR in the adaptation of pathogenic Leptospira to oxidative stress. Comparison of the transcriptome of a perR mutant with that of WT strain allowed determination of the PerR regulon in L. interrogans. In the perR mutant, 5 and 13 ORFs were up-and down-regulated, respectively, with a log 2 FC cutoff of 1 and a p-value below 0.05 (Table 3).
The ank-katE operon, encoded by LIMLP_10150-10145, ahpC, encoded by LIMLP_05955, Determining PerR regulon has allowed the identification of genes whose expression is activated, directly or indirectly, by PerR (Table 3) Interestingly, among the PerR regulon, only genes whose expression is repressed by PerR were up-regulated when Leptospira were exposed to H 2 O 2 . Indeed, the expression of the ank- In order to determine the exact contribution of PerR in the gene expression increase upon exposure to H 2 O 2 in Leptospira, the transcriptome of the perR mutant exposed to a sublethal dose of H 2 O 2 was also obtained. The ank-katE operon, whose expression is directly repressed by PerR and increased in the presence of H 2 O 2 in WT Leptospira, was not up-regulated in the presence of H 2 O 2 when perR was inactivated ( Fig 5). The amount of ank-katE operon expression in the perR mutant is in fact comparable to that in WT Leptospira exposed to a  Fig 5). Therefore, an H 2 O 2 -induced mechanism increases the expression of these two genes even in the absence of PerR, even though their expression is repressed by this regulator.
The expression of heme biosynthesis genes was not under the control of PerR and, as expected, their expression was still up-regulated in the perR mutant in the presence of H 2 O 2 ( Fig 5).
Interestingly, the ncRNA rh859, located downstream the ccp, was further up-regulated in the perR mutant upon exposure to H 2 O 2 (Log 2 FC of 1.71). This indicates that the rh859 upregulation induced by the exposure of WT Leptospira to H 2 O 2 occurs to some extent independently of the presence of PerR. When the perR mutant was exposed to 10 µM of H 2 O 2 , 10 ncRNAs were significantly down-regulated with a log 2 FC below -2 whereas only one ncRNA was significantly down-regulated when WT cells were exposed to 10 µM of For several of them (rh96, rh367, rh753, rh928, rh2114, rh2850, rh4234, and rh4918), such a down-regulation was not observed upon exposure of WT cells to 10 µM H 2 O 2 and only observed in the absence of PerR.
Altogether, these findings indicate that not all H 2 O 2 -regulated genes belong to the PerR regulon in pathogenic Leptospira and several PerR-regulated genes were not regulated by H 2 O 2 ( Fig 5). Likewise, differential expression of putative ncRNAs upon exposure to hydrogen peroxide is not necessarily exerted by PerR.

Role of the PerR-regulated genes in defenses against ROS and virulence in Leptospira
RNASeq experiments have allowed the identification of differentially expressed ORFs in the presence of peroxide and/or upon perR inactivation. These ORFs might encode cellular factors required for the adaptation of pathogenic Leptospira to ROS and an important question is to experimentally establish and understand the role of this factors in this adaptation.
Genetic manipulation of pathogenic Leptospira is still a challenge and functional studies in these bacteria mainly relies on random insertion transposon. Our laboratory has constructed a transposon mutant library (30) and several mutants inactivated in differentially-expressed ORFs upon exposure to H 2 O 2 or upon perR inactivation were available in our library.
Catalase, AhpC, and CCP were the peroxidases up-regulated in the presence of H 2 O 2 and repressed by PerR. Only katE and ahpC mutants were available in the transposon mutant library and we have studied the ability of these mutants to grow in the presence of H 2 O 2 and paraquat, a superoxide-generating compound. These two mutants had a comparable growth rate in EMJH medium ( Fig 6A) but when the medium was complemented with 0.5 mM H 2 O 2 , the ability of the katE mutant to divide was dramatically impaired (Fig 6B). The growth rate of the ahpC mutant in the presence of H 2 O 2 was comparable to that of the WT strain ( Fig 6B).
When the EMJH medium was complemented with 2 µM paraquat, the growth of the ahpC mutant was considerably reduced, indicating a high sensitivity to superoxide ( Fig 6C).
In other bacteria including E. coli and B. subtilis, katE is produced in higher amount during  However, when the culture medium was inoculated with Leptospira cells at the beginning of the stationary phase, Leptospira acquired a greater resistance to 2 mM H 2 O 2 as seen by their ability to grow ( Fig 7A). An even higher ability to grow in the presence of a deadly dose of H 2 O 2 was observed when the EMJH medium was inoculated with Leptospira at advanced stationary phase (Fig 7A). This indicates that Leptospira cells acquire a higher tolerance to hydrogen peroxide at stationary phase. Interestingly, this acquired tolerance to H 2 O 2 was independent of PerR since the perR mutant also acquired a higher ability to grow in the presence of 2 mM H 2 O 2 when at stationary phase ( Fig 7A). In order to determine which peroxidase was responsible for this acquired tolerance to H 2 O 2 , the survival of WT, ahpC and katE mutant strains was tested in logarithmic phase and was compared with that in stationary phase. As seen in Fig 7B,  Therefore, katE is essential for the stationary phase-acquired resistance to H 2 O 2 and this probably involves another regulation mechanism than that exerted by PerR.
Despite the fact that vicK had a reduced ability to divide in EMJH medium, this mutant strain had a slightly greater resistance to 2 µM paraquat than that of the WT (Fig 6E). In the same condition, the vicR, exbD, tonB-dpt receptor and lipl48 mutant strains had a lower ability to grow than the WT strain (Figs 6E and 6G). Altogether, these findings suggest that some of the PerR-repressed ORF are involved in Leptospira defense against superoxide.
Catalase has been shown to be essential for Leptospira virulence (9). We investigated whether other PerR-controlled genes were also required for Leptospira virulence.

Discussion
Reactive oxidative species are powerful and efficient weapons used by the host innate immunity response to eliminate pathogens. The ability of pathogenic Leptospira to detoxify hydrogen peroxide, one of the ROS produced upon Leptospira infection and pathogenicity, is essential for these pathogenic bacteria virulence (9). Because Leptospira are also environmental aerobic bacteria, they will also face low concentrations of ROS endogenously produced through the respiratory chain or present in the outside environment. The present study has used RNASeq technology to determine the adaptive response of pathogenic  (Fig 9B),   (Fig. 9). Also, the ncRNA rh3130 might be involved in the up-regulation of the sHsp-encoding operon upon exposure to H 2 O 2 .
Therefore, our study has unveiled the complexity of the regulatory network involved in the leptospiral adaptive response to oxidative stress.
Comparison of the transcriptome of the perR mutant determined in this study with that  . This is quite intriguing as it is generally believed that all aerobic bacteria do have a SOD. One fundamental question is to understand the mechanism these pathogenic bacteria use to detoxify superoxide produced endogenously during the respiratory chain or exogenously by phagocytic cells during infection. Our study is the first to identify cellular factor in pathogenic Leptospira involved in survival in the presence of superoxide-generating compound. AhpC could detoxify H 2 O 2 produced upon the reduction of superoxide, but the exact function of ExbD, the TBDT, and LipL48 in superoxide detoxification is still unclear.
In bacteria, ExbD is part of inner membrane complex TonB/ExbD/ExbB that uses proton motive force to provide the energy necessary by TBDT for uptake of an iron source. The presence of LipL48-encoding ORF in the same operon as the TBDT strongly suggests that these two proteins are functionally linked. This TBDT machinery could be solicited for iron uptake when iron concentration inside Leptospira is low as a result of the presence of ROS.
Indeed, one can imagine that Leptospira lower the level of free intracellular iron to prevent worsening the production of ROS inside the cells by the Fenton reaction. Consistent with this is the down-regulation of some of the gene encoding this TBDT machinery upon exposure to H 2 O 2 (Fig. 1). Alternatively, the TBDT machinery could also be involved in the uptake of another metal (such as zinc or manganese) that could be used by a ROS detoxification enzyme or even act by itself as ROS scavenger. Indeed, manganese has been shown to scavenge superoxide in Lactobacillus plantarum and Neisseria gonorrhoeae, independently to any SOD activity (39,40).
None of the mutants inactivated in these ORF exhibited a dramatic reduction in virulence, suggesting that these mechanisms do not have a pivotal role in Leptospira during infection or that redundant activities compensate for their absence. Experiments using other infection routes (ocular or subcutaneous routes) might result in different outcomes.
In conclusion, the present study has revealed, for the first time, the genome-wide general adaptive response to peroxide in pathogenic Leptospira, unfolding putative biological pathways Leptospira have evolved to overcome the deadly effect of ROS. Peroxide adaptive response involves detoxifying enzymes, molecular chaperones, DNA repair machinery, and transporters. This adaptive response also engages a large number of non-annotated and sometimes Leptospira specific ORFs reflecting the submerged part of the iceberg in these bacteria physiology. We have also uncovered a regulatory network of transcriptional regulators, sigma factors, two component systems and non-coding RNA that orchestrate together with PerR the peroxide adaptive response.

Materials and Methods
Bacterial strains and growth condition L. interrogans serovar Manilae strain L495 and transposon mutant strains (see S3 Bioinformatics analyses were performed using the RNA-seq pipeline from Sequana (42).
Reads were cleaned of adapter sequences and low-quality sequences using cutadapt version DESeq2 using the default parameters and statistical tests for differential expression were performed applying the independent filtering algorithm. A generalized linear model including the replicate effect as blocking factor was set in order to test for the differential expression between Leptospira samples. Raw p-values were adjusted for multiple testing according to the Benjamini and Hochberg (BH) procedure (48) and genes with an adjusted p-value lower than 0.005 and a Log 2 FC higher than 1 or lower than -1 were considered differentially expressed.
The Fisher statistical test was used for the COG (Clusters of Orthologous Groups) classification.

Quantitative RT-qPCR experiments
cDNA synthesis was performed with the cDNA synthesis kit (Biorad) according to the manufacturer's recommendation. Quantitative PCR was conducted with the SsoFast EvaGreen Supermix (Biorad) as previously described (9,15). Gene expression was measured using flaB (LIMLP_09410) as a reference gene.

Non-coding RNA identification
Sequencing data from the Leptospira WT and perR mutant strains incubated in the absence or presence of H 2 O 2 were processed with Trimmomatic (49) to remove low-quality bases and adapter contaminations. BWA mem (version 0.7.12) was used to discard the reads matching Leptospira rRNA, tRNA or polyA sequences and to assign the resulting reads to Leptospira replicons. Then Rockhopper (50) was used to re-align reads corresponding to separate replicons and to assemble transcripts models. The output was filtered to retain all transcripts longer than 50 nucleotides not overlapping within 10 nucleotides with NCBI annotated genes on the same orientation, and showing a minimum Rockhopper raw count value of 50 in at least two isolates. This high-quality set of 778 new sRNA was subjected to differential expression analysis with Rockhopper, adopting a Benjamini-Hochberg adjusted P-value threshold of 0.01. For each non-coding RNAs, putative function was identified by BLAST using the Rfam database (51).                Fig 1A).