Structure and function of the Leptospira interrogans peroxide stress regulator (PerR), an atypical PerR devoid of a structural metal-binding site

Abstract

Peroxide sensing is essential for bacterial survival during aerobic metabolism and host infection. Peroxide stress regulators (PerRs) are homodimeric transcriptional repressors with each monomer typically containing both structural and regulatory metal-binding sites. PerR binding to gene promoters is controlled by the presence of iron in the regulatory site, and iron-catalyzed oxidation of PerR by HO leads to the dissociation of PerR from DNA. In addition to a regulatory metal, most PerRs require a structural metal for proper dimeric assembly. We present here a structural and functional characterization of the PerR from the pathogenic spirochete , a rare example of PerR lacking a structural metal-binding site. studies showed that the leptospiral PerR belongs to the peroxide stimulon in pathogenic species and is involved in controlling resistance to peroxide. Moreover, a mutant had decreased fitness in other host-related stress conditions, including at 37 °C or in the presence of superoxide anion., leptospiral PerR could bind to the promoter region in a metal-dependent manner. The crystal structure of the leptospiral PerR revealed an asymmetric homodimer, with one monomer displaying complete regulatory metal coordination in the characteristic caliper-like DNA-binding conformation and the second monomer exhibiting disrupted regulatory metal coordination in an open non-DNA-binding conformation. This structure showed that leptospiral PerR assembles into a dimer in which a metal-induced conformational switch can occur independently in the two monomers. Our study demonstrates that structural metal binding is not compulsory for PerR dimeric assembly and for regulating peroxide stress.

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

“…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.…”

“…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).…”

“…Harvested cells were resuspended in 1 ml TRIzol TM (ThermoFisher Scientific) and stored at -80°C. Nucleic Acids were extracted with chloroform and precipitated with isopropanol as described earlier (15 Bioinformatics analyses were performed using the RNA-seq pipeline from Sequana (42).…”

“…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.…”

“…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.…”