The oxidative stress response and virulence of pathogenic Leptospira are controlled by the interplay of two peroxide stress regulators

Pathogenic Leptospira are the causative agents of leptospirosis, the most widespread zoonotic infectious disease. Leptospirosis is a potentially severe and life-threatening emerging disease with highest burden in sub-tropical areas and impoverish populations. Mechanisms allowing pathogenic Leptospira to survive inside a host and induce acute leptospirosis are not fully understood. The ability to resist deadly oxidants produced by the host during infection is pivotal for Leptospira virulence. We have previously shown that genes encoding defenses against oxidants in L. interrogans are repressed by PerRA (encoded by LIMLP_10155), a peroxide stress regulator of the Fur family. In this study, we describe the identification and characterization of another putative PerR-like regulator (LIMLP_05620) in L. interrogans. Protein sequence and phylogenetic analyses indicated that LIMLP_05620 displayed all the canonical PerR amino acid residues and is restricted to pathogenic Leptospira clades. We therefore named this PerR-like regulator PerRB. In L. interrogans, the PerRB regulon is distinct from that of PerRA. While a perRA mutant had a greater tolerance to peroxide, inactivating perRB led to a higher tolerance to superoxide, suggesting that these two regulators have a distinct function in the adaptation of L. interrogans to oxidative stress. The concomitant inactivation of perRA and perRB resulted in a higher tolerance to both peroxide and superoxide and, unlike the single mutants, to the loss of Leptospira virulence. Interestingly, this correlated with major changes in gene and non-coding RNA expression, only observed in the double perRAperRB mutant. Notably, several virulence-associated genes (clpB, ligA/B, and lvrAB) were repressed. By obtaining the first double mutant in a pathogenic Leptospira strain, our study has uncovered for the first time the interplay of two PerRs, not only in the adaptation of Leptospira to oxidative stress, but also in their virulence and pathogenicity, most likely through the transcriptional control of a complex regulatory network. Author summary Leptospirosis is a widespread 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. In addition to the Leptospira PerRA regulator that represses defenses against peroxide, we have identified and characterized a second PerR regulator in pathogenic Leptospira species (PerRB) that participates in Leptospira tolerance to superoxide. Phenotypic and transcriptomic analyses of single PerRA and PerRB mutants suggest that the two PerRs fulfill distinct functions in the adaptation to oxidative stress. However, concomitant inactivation of PerRA and PerRB resulted in a higher tolerance to both peroxide and superoxide, but to a loss virulence. The absence of the two PerR regulators resulted in global and major changes in the transcriptional profile, including a dramatic decrease of several virulence factor expression. Our study has demonstrated that PerRA and PerRB cooperate to orchestrate a complex regulatory network involved in Leptospira virulence.

201 Fold prediction suggests that the three Fur regulators encoded by LIMLP_05620, 202 LIMLP_04825 and LIMLP_18590 adopt the two-domain organization typical of the Fur 203 family depicted in the crystal structure of LIMLP_10155 (Fig 1C). 204 The closest relative of the PerR-encoding LIMLP_10155 is LIMLP_05620 with about 26% of 205 sequence identity, and LIMLP_04825 and LIMLP_18590 are closest relatives that share 20% 206 of sequence identity. LIMLP_05620 shares about 27% identity with the well-characterized B.

219
To get a better understanding of the evolutionary relationship of the four Fur-like 220 regulators in pathogenic Leptospira, we undertook phylogenetic analyses by searching for 221 homologous sequences of the LIMLP_10155, LIMLP_05620, LIMLP_18590 and 222 LIMLP_04825 proteins among the representative genomes present in GenBank. This revealed 223 a large phylogenetic distribution with several branches (Fig 2A). The sequences homologous 224 to the LIMLP_04825 and LIMLP_18590 proteins form two distinct groups (red and orange, 225 respectively) separated by a common ancestor. To get better definition of phylogenetic 226 relationships of PerR-like homologues, we performed analysis with only a subset of sequence 227 ( Fig 2B). This phylogenetic analysis shows two separated groups composed of the sequences 228 of LIMLP_10155 (PerRA) and LIMLP_05620 (PerRB) (see S1Fig for a more complete and 229 detailed tree).
230 The sequences of LIMLP_10155 and LIMLP_05620 ORFs from the strain L.
231 interrogans serovar Manilae were searched and compared in all available genomes from 232 the Leptospira genus (S1 Table). As seen in Fig 3,  241 S1 and S2 clades (Fig 3). LIMLP_05620 is also not found in other bacteria from the 242 Leptospirales order (Fig 2B and S1 Fig)

250
As demonstrated previously [27], when L. interrogans are exposed to a subtlethal dose 251 of H 2 O 2 (10 µM for 30 min) perRA expression is increased by a 7-fold whereas that of perRB 252 is unchanged (Fig 4). In the presence of a higher dose of H 2 O 2 (1 mM for 1h), expression of 253 both perRA and perRB was increased significantly by a 6-fold (Fig 4)  278 (108 out of 123) ranged between -1 and 1 (S2 Table). These findings indicate that the absence 279 of an active PerRB did not lead to substantial significant changes in genes expression when  On the contrary, inactivating perRB leads to a higher 366 resistance to paraquat (Fig 5 and Fig 6C). The concomitant inactivation of perRA and perRB 367 led to a greater growth in the presence of both H 2 O 2 ( Fig 6B) and paraquat (Fig 6C). 473 Interestingly, gene expression of the virulence-associated genes (lvrA, lvrB, ligA, ligB, clpB, 474 hsp15, and hsp20) was increased to the WT level (or even to a higher level) in the double 475 perRAperRB mutant strain complemented in trans only with perRB (Fig 9).

482
Intergenic regions were also analyzed to identify differentially expressed predicted 483 non-coding RNAs (ncRNAs). As observed for coding sequences, inactivation of perRB led to 484 the deregulation of only a few putative ncRNAs and most of the changes in expression were 485 below two folds (see S6  Table). Only a few of these differentially-expressed ncRNAs had an altered 494 expression in the single perRA and perRB mutant (S8 Table) [27].
495 Among the most highly differentially-expressed ncRNAs was LepncRNA38 that was located 496 downstream ccp, a highly up-regulated ORF in the perRAperRB mutant (Fig 10 and S8 497 Table). LepncRNA38 and ccp were also up-regulated in the perRA mutant [27]. The ncRNA 498 LepncRNA49, which was down-regulated in the perRAperRB mutant, overlapped with exbB 499 (LIMLP_04255), an ORF that was also down-regulated in the double perRAperRB mutant as 500 well as in the single perRA and perRB mutants (Fig 10). The down-regulated LepncRNA105 501 and LepncRNA130 ncRNAs were located downstream the hsp20-15 operon and gst, 502 respectively, three ORFs whose expression is decreased is the perRAperRB mutant (Fig 10   503 and S8 Table). It is worth noting that LepncRNA38, LepncRNA105 and LepncRNA130 are   811 The funders had no role in study design, data collection and analysis, decision to publish, or 812 preparation of the manuscript.