Structures of Class I and Class II Transcription Complexes Reveal the Molecular Basis of RamA-Dependent Transcription Activation.

Transcription activator RamA is linked to multidrug resistance of Klebsiella pneumoniae through controlling genes that encode efflux pumps (acrA) and porin-regulating antisense RNA (micF). In bacteria, σ70 , together with activators, controls the majority of genes by recruiting RNA polymerase (RNAP) to the promoter regions. RNAP and σ70 form a holoenzyme that recognizes -35 and -10 promoter DNA consensus sites. Many activators bind upstream from the holoenzyme and can be broadly divided into two classes. RamA acts as a class I activator on acrA and class II activator on micF, respectively. The authors present biochemical and structural data on RamA in complex with RNAP-σ70 at the two promoters and the data reveal the molecular basis for how RamA assembles and interacts with core RNAP and activates transcription that contributes to antibiotic resistance. Further, comparing with CAP/TAP complexes reveals common and activator-specific features in activator binding and uncovers distinct roles of the two C-terminal domains of RNAP α subunit.

Genomic Characterization of Imipenem- and Imipenem-Relebactam-Resistant Clinical Isolates of Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an opportunistic human pathogen and a major cause of nosocomial infections. The global spread of carbapenem-resistant strains is growing rapidly and has become a major public health challenge. Imipenem-relebactam (I/R) is a novel carbapenem-beta-lactamase inhibitor combination that can overcome carbapenem resistance. In this study, we aimed to understand the mechanism underlying resistance to imipenem and imipenem-relebactam. For this purpose, we performed a genomic comparison of 40 new clinical P. aeruginosa strains with different antibiotic sensitivity patterns as well as the presence/absence of carbapenemases. Results indicated the presence of a reduced flexible genome (15% total) mostly represented by phages and defense mechanisms against them, showing an important role in evolution and pathogenicity. We found a high diversity of antibiotic resistance genes grouped in small clusters mobilized via integrative and conjugative elements and facilitated by the high homologous recombination detected. Ortholog genes were found in several pathogenic strains from distantly related taxa in different mobile elements with a global distribution. The microdiversity found in those strains without carbapenemases did not reveal a clear pattern that could be associated with carbapenem resistance, suggesting multiple mechanisms of resistance in the core genome. Our results provide new insight into the dynamics and high genomic plasticity by which clinical strains of P. aeruginosa acquire resistance. This knowledge can be applied to other multidrug-resistant microbes to create predictive frameworks for assessing common molecular mechanisms of antibiotic resistance and integrated into new strategies for their prevention. IMPORTANCE The growing emergence and spread of carbapenem-resistant pathogens worldwide exacerbate the clinical challenge of treating these infections. Given the importance of carbapenems for the treatment of infections caused by Pseudomonas aeruginosa, this study aimed to investigate the underlying genomic properties of the clinical isolates that exhibited resistance to imipenem and imipenem-relebactam. This information will enhance our ability to forecast traits of resistant strains and design reliable treatments against this important threat. Our results provide new insight into the dynamics and high genomic plasticity by which clinical strains of P. aeruginosa acquire resistance as well as offers a methodology that can be applied to many other opportunistic pathogens with broad antibiotic resistance.

Fatty acids can inhibit Staphylococcus aureus SaeS activity at the membrane independent of alterations in respiration.

In Staphylococcus aureus, the two-component system SaeRS is responsible for regulating various virulence factors essential for the success of this pathogen. SaeRS can be stimulated by neutrophil-derived products but has also recently been shown to be inactivated by the presence of free fatty acids. A mechanism for how fatty acids negatively impacts SaeRS has not been described. We found that unsaturated fatty acids, as well as fatty acids not commonly found in Staphylococcal membranes, prevent the activation of SaeRS at a lower concentration than their saturated counterparts. These fatty acids can negatively impact SaeRS without altering the respiratory capacity of the bacterium. To uncover a potential mechanism for how fatty acids impact SaeRS function/activity, we utilized a naturally occurring point mutation found in S. aureus as well as chimeric SaeS proteins. Using these tools, we identified that the native transmembrane domains of SaeS dictate the transcriptional response to fatty acids in S. aureus. Our data support a model where free fatty acids alter the activity of the two-component system SaeRS directly through the sensor kinase SaeS and is dependent on the transmembrane domains of the protein.

Protein Integrated Network Analysis to Reveal Potential Drug Targets Against Extended Drug-Resistant Mycobacterium tuberculosis XDR1219.

The reconstruction and analysis of the protein-protein interaction (PPI) network is a powerful approach to understand the complex biological and molecular functions in normal and disease states of the cell. The interactome of most organisms is largely unidentified except some model organisms. The current study focused on the construction of PPI network for the human pathogen Mycobacterium tuberculosis (MTB)-resistant strain XDR1219 using computational methods. In this work, a bioinformatics approach was employed to reveal potential drug targets. The pipeline adopted the combination of an extensive integrated network analysis that led to identify 22 key proteins involved in drug resistance, resistant metabolic pathways, virulence, pathogenesis and persistency of the infection. The MTB XDR1219 interactome consists of 11,383 non-redundant PPIs among 1499 proteins covering 38% of the entire MTB XDR1219 proteome. The overall quality of the network was assessed and topological parameters of the PPI were calculated. The predicted interactions were functionally annotated and their relevance was assessed with the functional similarity. The study attempts to present the interactome of previously unidentified MTB XDR1219 and revealed potential drug targets that can be further explored by scientific community.

Latency, thermal stability, and identification of an inhibitory compound of mirolysin, a secretory protease of the human periodontopathogen Tannerella forsythia.

Mirolysin is a secretory protease of Tannerella forsythia, a member of the dysbiotic oral microbiota responsible for periodontitis. In this study, we show that mirolysin latency is achieved by a "cysteine-switch" mechanism exerted by Cys23 in the N-terminal profragment. Mutation of Cys23 shortened the time needed for activation of the zymogen from several days to 5 min. The mutation also decreased the thermal stability and autoproteolysis resistance of promirolysin. Mature mirolysin is a thermophilic enzyme and shows optimal activity at 65 °C. Through NMR-based fragment screening, we identified a small molecule (compound (cpd) 9) that blocks promirolysin maturation and functions as a competitive inhibitor (Ki = 3.2 µM), binding to the S1' subsite of the substrate-binding pocket. Cpd 9 shows superior specificity and does not interact with other T. forsythia proteases or Lys/Arg-specific proteases.

Studies of multifunctional DNA polymerase I from the extremely radiation resistant Deinococcus radiodurans: Recombinant expression, purification and characterization of the full-length protein and its large fragment.

Deinococcus radiodurans is a bacterium with extreme resistance to desiccation and radiation. Although the origins of this extreme resistance have not been fully elucidated, an efficient DNA repair machinery that includes the enzyme DNA polymerase I, is potentially crucial as part of a protection mechanism. Here we have cloned and performed small, medium, and large-scale expression of full-length D. radiodurans DNA polymerase I (DrPolI) as well as the large/Klenow fragment (DrKlenow). We then carried out functional characterization of 5' exonuclease, DNA strand displacement and polymerase activities of these proteins using gel-based and molecular beacon-based biochemical assays. With the same expression and purification strategy, we got higher yield in the production of DrKlenow than of the full-length protein, approximately 2.5 mg per liter of culture. Moreover, we detected a prominent 5' exonuclease activity of DrPolI in vitro. This activity and, DrKlenow strand displacement and DNA polymerase activities are preferentially stimulated at pH 8.0-8.5 and are reduced by addition of NaCl. Interestingly, both protein variants are more thermostable at pH 6.0-6.5. The characterization of DrPolI's multiple functions provides new insights into the enzyme's role in DNA repair pathways, and how the modulation of these functions is potentially used by D. radiodurans as a survival strategy.

A link between pH homeostasis and colistin resistance in bacteria.

Colistin resistance is complex and multifactorial. DbcA is an inner membrane protein belonging to the DedA superfamily required for maintaining extreme colistin resistance of Burkholderia thailandensis. The molecular mechanisms behind this remain unclear. Here, we report that ∆dbcA displays alkaline pH/bicarbonate sensitivity and propose a role of DbcA in extreme colistin resistance of B. thailandensis by maintaining cytoplasmic pH homeostasis. We found that alkaline pH or presence of sodium bicarbonate displays a synergistic effect with colistin against not only extremely colistin resistant species like B. thailandensis and Serratia marcescens, but also a majority of Gram-negative and Gram-positive bacteria tested, suggesting a link between cytoplasmic pH homeostasis and colistin resistance across species. We found that lowering the level of oxygen in the growth media or supplementation of fermentable sugars such as glucose not only alleviated alkaline pH stress, but also increased colistin resistance in most bacteria tested, likely by avoiding cytoplasmic alkalinization. Our observations suggest a previously unreported link between pH, oxygen, and colistin resistance. We propose that maintaining optimal cytoplasmic pH is required for colistin resistance in a majority of bacterial species, consistent with the emerging link between cytoplasmic pH homeostasis and antibiotic resistance.

Role of a cysteine residue in substrate entry and catalysis in MtHIBADH: Analysis by chemical modifications and site-directed mutagenesis.

Despite sharing conserved substrate-binding residues, members of 3-hydroxyisobutyrate dehydrogenase (HIBADH) superfamily show remarkable differences in substrate preference. Cysteine residues were identified within a radius of 6 Å surrounding both the active site and the substrate entry site of HIBADH enzyme from Mycobacterium tuberculosis (MtHIBADH). Chemical modification with thiol-modifying reagents, pCMB and DTNB, abrogated the dehydrogenase activity of the enzyme. The loss in activity followed pseudo-first-order kinetics as a function of the concentration of pCMB. S-HIBA (substrate) binding provided partial protection, while NAD (cofactor) binding provided ~70% protection from thiol-modifying reagent. Site-directed mutagenesis of cysteine residues present in the MtHIBADH enzyme identified the indispensable role of Cys-210 residue, located at C-terminal domain, for its dehydrogenase activity. Cys-210 mutation to serine reduced the dehydrogenase activity by ~2-fold while mutation to alanine strikingly reduced the activity by ~140-fold. C210A mutation did not perturb the state of oligomerization of the enzyme but perturbed the secondary structure content. Structural analysis revealed the involvement of Cys-210 residue in inter-chain interaction with Gln-178, which acts as hydrogen bond donor and coordinates with Cys-210 and Gly-208 of the adjacent subunit. The data demonstrate a critical role of Cys-210 residue in maintaining the conformation and rigidity of loop composed of substrate-interacting residues involved in the entry of S-HIBA substrate in MtHIBADH.

Endophytic Fungi from Dalbergia odorifera T. Chen Producing Naringenin Inhibit the Growth of Staphylococcus aureus by Interfering with Cell Membrane, DNA, and Protein.

This study focused on the antibacterial effects of the endophytic fungi producing naringenin from Dalbergia odorifera T. Chen against Staphylococcus aureus. The antibacterial activity was measured by the inhibition diameters, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC). The time-killing curve was also used to evaluate its antibacterial efficacy. The results of antibacterial activity determinations showed that endophytic fungi secondary metabolites can inhibit the growth of five pathogenic bacteria (S. aureus, Escherichia coli, Salmonella enteritidis, Pseudomonas aeruginosa, and Bacillus subtilis) and the most sensitive strain was S. aureus that had the MIC and MBC values of 0.13 and 0.50 mg/mL, respectively. The membrane permeability study was measured by a DNA leakage assay and electrical conductivity assay. Furthermore, the whole-cell protein lysates and DNA fragmentation assay was evaluated. The morphology of S. aureus treated with the endophytic fungi products was observed by scanning electron microscopy (SEM). The probable antibacterial mechanism of endophytic fungi secondary metabolites was the increased membrane permeability that leads to leaks of nucleic acids and proteins. SEM results further confirmed that the extracts can interfere with the integrity of S. aureus cell membrane and further inhibit the growth of bacteria, resulting in the death of bacteria. This study provides a new perspective for the antibacterial functions of endophytic fungi secondary metabolites for biomedical applications.

Ginkgo biloba exocarp extracts inhibit S. aureus and MRSA by disrupting biofilms and affecting gene expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Ginkgo biloba L. fruit, also known as Bai Guo, Ya Jiao Zi (in pinyin Chinese), and ginkgo nut (in English), has been used for many years as an important material in Chinese traditional medicine to treat coughs and asthma and as a disinfectant, as described in the Compendium of Materia Medica (Ben Cao Gang Mu, pinyin in Chinese), an old herbal book. Ginkgo nuts are used to treat phlegm-associated asthma, astringent gasp, frequent urination, gonorrhoea and turgidity; consumed raw to reduce phlegm and treat hangovers; and used as a disinfectant and insecticide. A similar record was also found in Sheng Nong's herbal classic (Shen Nong Ben Cao Jing, pinyin in Chinese). Recent research has shown that Ginkgo biloba L. exocarp extract (GBEE) can unblock blood vessels and improve brain function and exhibits antitumour and antibacterial activities. AIM OF STUDY: To investigate the inhibitory effect of Ginkgo biloba L. exocarp extract (GBEE) on methicillin-resistant S. aureus (MRSA) biofilms and assess its associated molecular mechanism. MATERIALS AND METHODS: The antibacterial effects of GBEE on S. aureus and MRSA were determined using the broth microdilution method. The growth curves of bacteria treated with or without GBEE were generated by measuring the CFU (colony forming unit) of cultures at different time points. The effects of GBEE on bacterial biofilm formation and mature biofilm disruption were determined by crystal violet staining. Quantitative polymerase chain reaction (qPCR) was used to measure the effects of GBEE on the gene expression profiles of MRSA biofilm-related factors at 6, 8, 12, 16 and 24 h. RESULTS: The minimum inhibitory concentration (MIC) of GBEE on S. aureus and MRSA was 4 µg/mL, and the minimum bactericidal concentration (MBC) was 8 µg/ml. Moreover, GBEE (4-12 µg/mL) inhibited S. aureus and MRSA biofilm formation in a dose-dependent manner. Interestingly, GBEE also destroyed mature biofilms of S. aureus and MRSA at 12 µg/ml. The expression of the MRSA biofilm-associated factor icaA and sarA were downregulated after 6 h of treatment with GBEE, while sigB was downregulated after 12 h. MeanwhileMeanwhile, icaR was upregulated at 12 h. In addition, GBEE also downregulated the virulence gene hld and inhibited the synthesis of staphyloxanthin. CONCLUSIONS: GBEE has excellent antibacterial effects against S. aureus and MRSA and inhibits their biofilm-forming ability by altering related gene expression.

Cefoperazone/sulbactam: New composites against multiresistant gram negative bacteria?

Sulbactam, a class A ß-lactamase inhibitor, added to cefoperazone either at a fixed 8 mg/L level of sulbactam or at a level of fixed cefoperazone: sulbactam ratio (2:1) would constitute a combination form of cefoperazone/sulbactam, which has better activities against Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii than cefoperazone alone. Cefoperazone/sulbactam (1:1 or 1:2) has greater in-vitro activity against most multidrug-resistant organisms (ESBL- and AmpC-producing Enterobacteriaceae and carbapenem-resistant A. baumannii except for carbapenem-resistant P. aeruginosa) than a 2:1 ratio. However, increased sulbactam concentration may induce AmpC production. Besides, sulbactam concentration might not be readily achievable in serum if the susceptibility rates were defined by the breakpoints of higher sulbactam composites, such as ≤16/16 (1:1) or 16/32 (1:2) mg/L. Carbapenemases (KPC-, OXA-type enzymes and metallo-ß-lactamases) can't be inhibited by sulbactam. Some in-vitro studies showed that increasing sulbactam composites of cefoperazone/sulbactam had no effect on carbapenem-resistant P. aeruginosa, suggesting the presence of carbapenemases or AmpC overproduction that could not be overcome by increasing sulbactam levels to recover cefoperazone activity. Sulbactam alone has good intrinsic activity against carbapenem-resistant Acinetobacter strains sometimes even in the presence of carbapenemase genes, suggesting unsteady levels of carbapenemases. In conclusion, appropriate composites of cefoperazone and ß-lactamase inhibitor sulbactam may expand the clinical use if the pharmacokinetic optimization could be achieved in the human serum.

Phenoxyalkylimidazoles with an oxadiazole moiety are subject to efflux in Mycobacterium tuberculosis.

The phenoxyalkylimidazoles (PAI) are an attractive chemical series with potent anti-tubercular activity targeting Mycobacterium tuberculosis respiration. Our aim was to determine if the PAI compounds are subject to efflux. Two analogs containing an oxadiazole had improved potency in the presence of the efflux inhibitors reserpine and carbonyl cyanide m-chlorophenylhydrazine, whereas the potency of analogs with a diazole was not affected.

Importance of Reviewing Antibiotic Courses by 48 Hours: Risk Factors for Third-Generation Cephalosporin Resistance Among AmpC Harboring Organisms in Urine and Respiratory Cultures.

BACKGROUND: Citrobacter, Enterobacter, Morganella, and Serratia (AmpC organisms) species can exhibit third-generation cephalosporin (TGC) resistance after TGC exposure. We aimed to assess if institutional TGC utilization correlated with institutional AmpC organism susceptibility and if prior TGC exposure ≤48 hours were associated with TGC resistance in the first culture of a future infection episode caused by an AmpC organism. METHODS: A 5-year retrospective cohort study was performed, including AmpC organisms isolated from pediatric urinary and respiratory tract cultures at an institution with TGC courses reviewed by the antimicrobial stewardship program at 48 hours. Correlations were assessed by Pearson's correlation. Multivariable logistic regression identified factors independently associated with TGC resistance in a subcohort of infection episodes. RESULTS: Among 654 cultures, AmpC organism TGC susceptibility increased from 74% in 2013 to 89.3% in 2017, and this correlated with a 26.1% decrease in TGC utilization (R = -0.906; P = 0.034). Among 275 AmpC organism infections, 21.1% were resistant. Resistance occurred in 13.6%, 17.4%, and 56.5% of infections with no exposure, ≤48 hours, and >48 hours of TGC exposure in the past 30 days, respectively. TGC exposure ≤48 hours was not associated with resistance (odds ratio [OR], 1.26; 95% confidence interval [CI], 0.32-4.94; P = 0.74), whereas, TGC exposure >48 hours was (OR, 8.7; 95% CI, 3.67-20.6; P < 0.001). Infections in 2017 were less likely to be resistant (OR, 0.25; 95% CI, 0.08-0.8; P = 0.019). CONCLUSIONS: Decreased TGC utilization, likely related to antimicrobial stewardship, correlated with increased AmpC organism susceptibility. Limiting TGC exposure to ≤48 hours when possible may reduce AmpC organism resistance in future infections.

Culture Conversion in Patients Treated with Bedaquiline and/or Delamanid. A Prospective Multicountry Study.

Rationale: Bedaquiline and delamanid offer the possibility of more effective and less toxic treatment for multidrug-resistant (MDR) tuberculosis (TB). With this treatment, however, some patients remain at high risk for an unfavorable treatment outcome. The endTB Observational Study is the largest multicountry cohort of patients with rifampin-resistant TB or MDR-TB treated in routine care with delamanid- and/or bedaquiline-containing regimens according to World Health Organization guidance.Objectives: We report the frequency of sputum culture conversion within 6 months of treatment initiation and the risk factors for nonconversion.Methods: We included patients with a positive baseline culture who initiated a first endTB regimen before April 2018. Two consecutive negative cultures collected 15 days or more apart constituted culture conversion. We used generalized mixed models to derive marginal predictions for the probability of culture conversion in key subgroups.Measurements and Main Results: A total of 1,109 patients initiated a multidrug treatment containing bedaquiline (63%), delamanid (27%), or both (10%). Of these, 939 (85%) experienced culture conversion within 6 months. In adjusted analyses, patients with HIV had a lower probability of conversion (0.73; 95% confidence interval [CI], 0.62-0.84) than patients without HIV (0.84; 95% CI, 0.79-0.90; P = 0.03). Patients with both cavitary disease and highly positive sputum smear had a lower probability of conversion (0.68; 95% CI, 0.57-0.79) relative to patients without either (0.89; 95% CI, 0.84-0.95; P = 0.0004). Hepatitis C infection, diabetes mellitus or glucose intolerance, and baseline resistance were not associated with conversion.Conclusions: Frequent sputum conversion in patients with rifampin-resistant TB or MDR-TB who were treated with bedaquiline and/or delamanid underscores the need for urgent expanded access to these drugs. There is a need to optimize treatment for patients with HIV and extensive disease.

Characterization of an Antibacterial Agent Targeting Ferrous Iron Transport Protein FeoB against Staphylococcus aureus and Gram-Positive Bacteria.

The emergence of multidrug-resistant Staphylococcus aureus strains has become a serious clinical problem. Iron is absolutely required for the bacterial growth, virulence associated with colonization, and survival from the host immune system. The FeoB protein is a major iron permease in bacterial ferrous iron transport systems (Feo) that has been shown to play a crucial role in virulence of some pathogenic bacteria. However, FeoB is still uncharacterized in Gram-positive pathogens, and its effects on S. aureus pathogenesis are unknown. In this study, we identified a novel inhibitor, GW3965·HCl, that targets FeoB in S. aureus. The molecule effectively inhibited FeoB in vitro enzyme activity, bacterial growth, and virulence factor expression. Genome-editing and metabolomic analyses revealed that GW3965·HCl inhibited FeoB function and affected the associated mechanisms with reduced iron availability in S. aureus. Gentamicin resistance and Caenorhabditis elegans infection assays further demonstrated the power of GW3965·HCl as a safe and efficient antibacterial agent. In addition to S. aureus, GW3965·HCl also presented its effectiveness on inhibition of the FeoB activity and growth of Gram-positive bacteria. This novel inhibitor will provide new insight for developing a next-generation antibacterial therapy.

Structural and Functional Study of the Klebsiella pneumoniae VapBC Toxin-Antitoxin System, Including the Development of an Inhibitor That Activates VapC.

Klebsiella pneumoniae is one of the most critical opportunistic pathogens. TA systems are promising drug targets because they are related to the survival of bacterial pathogens. However, structural information on TA systems in K. pneumoniae remains lacking; therefore, it is necessary to explore this information for the development of antibacterial agents. Here, we present the first crystal structure of the VapBC complex from K. pneumoniae at a resolution of 2.00 Å. We determined the toxin inhibitory mechanism of the VapB antitoxin through an Mg2+ switch, in which Mg2+ is displaced by R79 of VapB. This inhibitory mechanism of the active site is a novel finding and the first to be identified in a bacterial TA system. Furthermore, inhibitors, including peptides and small molecules, that activate the VapC toxin were discovered and investigated. These inhibitors can act as antimicrobial agents by disrupting the VapBC complex and activating VapC. Our comprehensive investigation of the K. pneumoniae VapBC system will help elucidate an unsolved conundrum in VapBC systems and develop potential antimicrobial agents.

7X multiplexed, optofluidic detection of nucleic acids for antibiotic-resistance bacterial screening.

Rapid and accurate diagnosis of bacterial infections resistant to multiple antibiotics requires development of new bio-sensors for differentiated detection of multiple targets. This work demonstrates 7x multiplexed detection for antibiotic-resistance bacterial screening on an optofluidic platform. We utilize spectrally multiplexed multi-spot excitation for simultaneous detection of nucleic acid strands corresponding to bacterial targets and resistance genes. This is enabled by multi-mode interference (MMI) waveguides integrated in an optofluidic device. We employ a combinatorial three-color labeling scheme for the nucleic acid assays to scale up their multiplexing capability to seven different nucleic acids, representing three species and four resistance genes.

The 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. Leptospira are challenged by numerous adverse conditions, including deadly reactive oxygen species (ROS), when infecting their hosts. 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 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 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 damage. Identification of the PerR regulon upon exposure to H2O2 allowed to define the contribution of this regulator in the oxidative stress response. This study 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, the oxidative stress response. We have shown that PerR-regulated genes encoding a TonB-dependent transporter and a two-component system (VicKR) are involved in Leptospira tolerance to superoxide. This could represent the first defense mechanism against superoxide in L. interrogans, a bacterium lacking canonical superoxide dismutase. Our findings provide an insight into the mechanisms required by pathogenic Leptospira to overcome oxidative damage during infection-related conditions. This will participate in framing future hypothesis-driven studies to identify and decipher novel virulence mechanisms in this life-threatening pathogen.