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879900.8516The membrane-active polyaminoisoprenyl compound NV716 re-sensitizes Pseudomonas aeruginosa to antibiotics and reduces bacterial virulence. Pseudomonas aeruginosa is intrinsically resistant to many antibiotics due to the impermeability of its outer membrane and to the constitutive expression of efflux pumps. Here, we show that the polyaminoisoprenyl compound NV716 at sub-MIC concentrations re-sensitizes P. aeruginosa to abandoned antibiotics by binding to the lipopolysaccharides (LPS) of the outer membrane, permeabilizing this membrane and increasing antibiotic accumulation inside the bacteria. It also prevents selection of resistance to antibiotics and increases their activity against biofilms. No stable resistance could be selected to NV716-itself after serial passages with subinhibitory concentrations, but the transcriptome of the resulting daughter cells shows an upregulation of genes involved in the synthesis of lipid A and LPS, and a downregulation of quorum sensing-related genes. Accordingly, NV716 also reduces motility, virulence factors production, and biofilm formation. NV716 shows a unique and highly promising profile of activity when used alone or in combination with antibiotics against P. aeruginosa, combining in a single molecule anti-virulence and potentiator effects. Additional work is required to more thoroughly understand the various functions of NV716.202236008485
50410.8453Activation of Dithiolopyrrolone Antibiotics by Cellular Reductants. Dithiolopyrrolone (DTP) natural products are broad-spectrum antimicrobial and anticancer prodrugs. The DTP structure contains a unique bicyclic ene-disulfide that once reduced in the cell, chelates metal ions and disrupts metal homeostasis. In this work we investigate the intracellular activation of the DTPs and their resistance mechanisms in bacteria. We show that the prototypical DTP holomycin is reduced by several bacterial reductases and small-molecule thiols in vitro. To understand how bacteria develop resistance to the DTPs, we generate Staphylococcus aureus mutants that exhibit increased resistance to the hybrid DTP antibiotic thiomarinol. From these mutants we identify loss-of-function mutations in redox genes that are involved in DTP activation. This work advances the understanding of how DTPs are activated and informs development of bioreductive disulfide prodrugs.202539665630
55620.8450An ArsR/SmtB family member regulates arsenic resistance genes unusually arranged in Thermus thermophilus HB27. Arsenic resistance is commonly clustered in ars operons in bacteria; main ars operon components encode an arsenate reductase, a membrane extrusion protein, and an As-sensitive transcription factor. In the As-resistant thermophile Thermus thermophilus HB27, genes encoding homologues of these proteins are interspersed in the chromosome. In this article, we show that two adjacent genes, TtsmtB, encoding an ArsR/SmtB transcriptional repressor and, TTC0354, encoding a Zn(2+) /Cd(2+) -dependent membrane ATPase are involved in As resistance; differently from characterized ars operons, the two genes are transcribed from dedicated promoters upstream of their respective genes, whose expression is differentially regulated at transcriptional level. Mutants defective in TtsmtB or TTC0354 are more sensitive to As than the wild type, proving their role in arsenic resistance. Recombinant dimeric TtSmtB binds in vitro to both promoters, but its binding capability decreases upon interaction with arsenate and, less efficiently, with arsenite. In vivo and in vitro experiments also demonstrate that the arsenate reductase (TtArsC) is subjected to regulation by TtSmtB. We propose a model for the regulation of As resistance in T. thermophilus in which TtSmtB is the arsenate sensor responsible for the induction of TtArsC which generates arsenite exported by TTC0354 efflux protein to detoxify cells.201728696001
600930.8449Efflux pump inhibitor chlorpromazine effectively increases the susceptibility of Escherichia coli to antimicrobial peptide Brevinin-2CE. Aim: The response of E. coli ATCC8739 to Brevinin-2CE (B2CE) was evaluated as a strategy to prevent the development of antimicrobial peptide (AMP)-resistant bacteria. Methods: Gene expression levels were detected by transcriptome sequencing and RT-PCR. Target genes were knocked out using CRISPR-Cas9. MIC was measured to evaluate strain resistance. Results: Expression of acrZ and sugE were increased with B2CE stimulation. ATCC8739ΔacrZ and ATCC8739ΔsugE showed twofold and fourfold increased sensitivity, respectively. The survival rate of ATCC8739 was reduced in the presence of B2CE/chlorpromazine (CPZ). Combinations of other AMPs with CPZ also showed antibacterial effects. Conclusion: The results indicate that combinations of AMPs/efflux pump inhibitors (EPIs) may be a potential approach to combat resistant bacteria.202438683168
80540.8444LexR Positively Regulates the LexABC Efflux Pump Involved in Self-Resistance to the Antimicrobial Di-N-Oxide Phenazine in Lysobacter antibioticus. Myxin, a di-N-oxide phenazine isolated from the soil bacterium Lysobacter antibioticus, exhibits potent activity against various microorganisms and has the potential to be developed as an agrochemical. Antibiotic-producing microorganisms have developed self-resistance mechanisms to protect themselves from autotoxicity. Antibiotic efflux is vital for such protection. Recently, we identified a resistance-nodulation-division (RND) efflux pump, LexABC, involved in self-resistance against myxin in L. antibioticus. Expression of its genes, lexABC, was induced by myxin and was positively regulated by the LysR family transcriptional regulator LexR. The molecular mechanisms, however, have not been clear. Here, LexR was found to bind to the lexABC promoter region to directly regulate expression. Moreover, myxin enhanced this binding. Molecular docking and surface plasmon resonance analysis showed that myxin bound LexR with valine and lysine residues at positions 146 (V146) and 195 (K195), respectively. Furthermore, mutation of K195 in vivo led to downregulation of the gene lexA. These results indicated that LexR sensed and bound with myxin, thereby directly activating the expression of the LexABC efflux pump and increasing L. antibioticus resistance against myxin. IMPORTANCE Antibiotic-producing bacteria exhibit various sophisticated mechanisms for self-protection against their own secondary metabolites. RND efflux pumps that eliminate antibiotics from cells are ubiquitous in Gram-negative bacteria. Myxin is a heterocyclic N-oxide phenazine with potent antimicrobial and antitumor activities produced by the soil bacterium L. antibioticus. The RND pump LexABC contributes to the self-resistance of L. antibioticus against myxin. Herein, we report a mechanism involving the LysR family regulator LexR that binds to myxin and directly activates the LexABC pump. Further study on self-resistance mechanisms could help the investigation of strategies to deal with increasing bacterial antibiotic resistance and enable the discovery of novel natural products with resistance genes as selective markers.202337166326
54750.8437Dual role of OhrR as a repressor and an activator in response to organic hydroperoxides in Streptomyces coelicolor. Organic hydroperoxide resistance in bacteria is achieved primarily through reducing oxidized membrane lipids. The soil-inhabiting aerobic bacterium Streptomyces coelicolor contains three paralogous genes for organic hydroperoxide resistance: ohrA, ohrB, and ohrC. The ohrA gene is transcribed divergently from ohrR, which encodes a putative regulator of MarR family. Both the ohrA and ohrR genes were induced highly by various organic hydroperoxides. The ohrA gene was induced through removal of repression by OhrR, whereas the ohrR gene was induced through activation by OhrR. Reduced OhrR bound to the ohrA-ohrR intergenic region, which contains a central (primary) and two adjacent (secondary) inverted-repeat motifs that overlap with promoter elements. Organic peroxide decreased the binding affinity of OhrR for the primary site, with a concomitant decrease in cooperative binding to the adjacent secondary sites. The single cysteine C28 in OhrR was involved in sensing oxidants, as determined by substitution mutagenesis. The C28S mutant of OhrR bound to the intergenic region without any change in binding affinity in response to organic peroxides. These results lead us to propose a model for the dual action of OhrR as a repressor and an activator in S. coelicolor. Under reduced conditions, OhrR binds cooperatively to the intergenic region, repressing transcription from both genes. Upon oxidation, the binding affinity of OhrR decreases, with a concomitant loss of cooperative binding, which allows RNA polymerase to bind to both the ohrA and ohrR promoters. The loosely bound oxidized OhrR can further activate transcription from the ohrR promoter.200717586628
57960.8436Control of expression of a periplasmic nickel efflux pump by periplasmic nickel concentrations. There is accumulating evidence that transenvelope efflux pumps of the resistance, nodulation, cell division protein family (RND) are excreting toxic substances from the periplasm across the outer membrane directly to the outside. This would mean that resistance of Gram-negative bacteria to organic toxins and heavy metals is in fact a two-step process: one set of resistance factors control the concentration of a toxic substance in the periplasm, another one that in the cytoplasm. Efficient periplasmic detoxification requires periplasmic toxin sensing and transduction of this signal into the cytoplasm to control expression of the periplasmic detoxification system. Such a signal transduction system was analyzed using the Cnr nickel resistance system from Cupriavidus (Wautersia, Ralstonia, Alcaligenes) metallidurans strain CH34. Resistance is based on nickel efflux mediated by the CnrCBA efflux pump encoded by the cnrYHXCBAT metal resistance determinant. The products of the three genes cnrYXH transcriptionally regulate expression of cnr. CnrY and CnrX are membrane-bound proteins probably functioning as anti sigma factors while CnrH is a cnr-specific extracytoplasmic functions (ECF) sigma factors. Experimental data provided here indicate a signal transduction chain leading from nickel in the periplasm to transcription initiation at the cnr promoters cnrYp and cnrCp, which control synthesis of the nickel efflux pump CnrCBA.200516158236
60770.8435A novel copper-sensing two-component system for inducing Dsb gene expression in bacteria. In nature, bacteria must sense copper and tightly regulate gene expression to evade copper toxicity. Here, we identify a new copper-responsive two-component system named DsbRS in the important human pathogen Pseudomonas aeruginosa; in this system, DsbS is a sensor histidine kinase, and DsbR, its cognate response regulator, directly induces the transcription of genes involved in protein disulfide bond formation (Dsb) (i.e., the dsbDEG operon and dsbB). In the absence of copper, DsbS acts as a phosphatase toward DsbR, thus blocking the transcription of Dsb genes. In the presence of copper, the metal ion directly binds to the sensor domain of DsbS, and the Cys82 residue plays a critical role in this process. The copper-binding behavior appears to inhibit the phosphatase activity of DsbS, leading to the activation of DsbR. The copper resistance of the dsbRS knock-out mutant is restored by the ectopic expression of the dsbDEG operon, which is a DsbRS major target. Strikingly, cognates of the dsbRS-dsbDEG pair are widely distributed across eubacteria. In addition, a DsbR-binding site, which contains the consensus sequence 5'-TTA-N(8)-TTAA-3', is detected in the promoter region of dsbDEG homologs in these species. These findings suggest that the regulation of Dsb genes by DsbRS represents a novel mechanism by which bacterial cells cope with copper stress.202236546013
80680.8429A two-component small multidrug resistance pump functions as a metabolic valve during nicotine catabolism by Arthrobacter nicotinovorans. The genes nepAB of a small multidrug resistance (SMR) pump were identified as part of the pAO1-encoded nicotine regulon responsible for nicotine catabolism in Arthrobacter nicotinovorans. When [(14)C]nicotine was added to the growth medium the bacteria exported the (14)C-labelled end product of nicotine catabolism, methylamine. In the presence of the proton-motive force inhibitors 2,4-dinitrophenol (DNP), carbonyl cyanide m-chlorophenylhydrazone (CCCP) or the proton ionophore nigericin, export of methylamine was inhibited and radioactivity accumulated inside the bacteria. Efflux of [(14)C]nicotine-derived radioactivity from bacteria was also inhibited in a pmfR : cmx strain with downregulated nepAB expression. Because of low amine oxidase levels in the pmfR : cmx strain, gamma-N-methylaminobutyrate, the methylamine precursor, accumulated. Complementation of this strain with the nepAB genes, carried on a plasmid, restored the efflux of nicotine breakdown products. Both NepA and NepB were required for full export activity, indicating that they form a two-component efflux pump. NepAB may function as a metabolic valve by exporting methylamine, the end product of nicotine catabolism, and, in conditions under which it accumulates, the intermediate gamma-N-methylaminobutyrate.200717464069
62190.8426Activation of ChvG-ChvI regulon by cell wall stress confers resistance to β-lactam antibiotics and initiates surface spreading in Agrobacterium tumefaciens. A core component of nearly all bacteria, the cell wall is an ideal target for broad spectrum antibiotics. Many bacteria have evolved strategies to sense and respond to antibiotics targeting cell wall synthesis, especially in the soil where antibiotic-producing bacteria compete with one another. Here we show that cell wall stress caused by both chemical and genetic inhibition of the essential, bifunctional penicillin-binding protein PBP1a prevents microcolony formation and activates the canonical host-invasion two-component system ChvG-ChvI in Agrobacterium tumefaciens. Using RNA-seq, we show that depletion of PBP1a for 6 hours results in a downregulation in transcription of flagellum-dependent motility genes and an upregulation in transcription of type VI secretion and succinoglycan biosynthesis genes, a hallmark of the ChvG-ChvI regulon. Depletion of PBP1a for 16 hours, results in differential expression of many additional genes and may promote a stress response, resembling those of sigma factors in other bacteria. Remarkably, the overproduction of succinoglycan causes cell spreading and deletion of the succinoglycan biosynthesis gene exoA restores microcolony formation. Treatment with cefsulodin phenocopies depletion of PBP1a and we correspondingly find that chvG and chvI mutants are hypersensitive to cefsulodin. This hypersensitivity only occurs in response to treatment with β-lactam antibiotics, suggesting that the ChvG-ChvI pathway may play a key role in resistance to antibiotics targeting cell wall synthesis. Finally, we provide evidence that ChvG-ChvI likely has a conserved role in conferring resistance to cell wall stress within the Alphaproteobacteria that is independent of the ChvG-ChvI repressor ExoR.202236480495
329100.8424Effect of NlpE overproduction on multidrug resistance in Escherichia coli. NlpE, an outer membrane lipoprotein, functions during envelope stress responses in Gram-negative bacteria. In this study, we report that overproduction of NlpE increases multidrug and copper resistance through activation of the genes encoding the AcrD and MdtABC multidrug efflux pumps in Escherichia coli.201020211889
11110.8424Diffusible signal factor primes plant immunity against Xanthomonas campestris pv. campestris (Xcc) via JA signaling in Arabidopsis and Brassica oleracea. BACKGROUND: Many Gram-negative bacteria use quorum sensing (QS) signal molecules to monitor their local population density and to coordinate their collective behaviors. The diffusible signal factor (DSF) family represents an intriguing type of QS signal to mediate intraspecies and interspecies communication. Recently, accumulating evidence demonstrates the role of DSF in mediating inter-kingdom communication between DSF-producing bacteria and plants. However, the regulatory mechanism of DSF during the Xanthomonas-plant interactions remain unclear. METHODS: Plants were pretreated with different concentration of DSF and subsequent inoculated with pathogen Xanthomonas campestris pv. campestris (Xcc). Pathogenicity, phynotypic analysis, transcriptome combined with metabolome analysis, genetic analysis and gene expression analysis were used to evaluate the priming effects of DSF on plant disease resistance. RESULTS: We found that the low concentration of DSF could prime plant immunity against Xcc in both Brassica oleracea and Arabidopsis thaliana. Pretreatment with DSF and subsequent pathogen invasion triggered an augmented burst of ROS by DCFH-DA and DAB staining. CAT application could attenuate the level of ROS induced by DSF. The expression of RBOHD and RBOHF were up-regulated and the activities of antioxidases POD increased after DSF treatment followed by Xcc inoculation. Transcriptome combined with metabolome analysis showed that plant hormone jasmonic acid (JA) signaling involved in DSF-primed resistance to Xcc in Arabidopsis. The expression of JA synthesis genes (AOC2, AOS, LOX2, OPR3 and JAR1), transportor gene (JAT1), regulator genes (JAZ1 and MYC2) and responsive genes (VSP2, PDF1.2 and Thi2.1) were up-regulated significantly by DSF upon Xcc challenge. The primed effects were not observed in JA relevant mutant coi1-1 and jar1-1. CONCLUSION: These results indicated that DSF-primed resistance against Xcc was dependent on the JA pathway. Our findings advanced the understanding of QS signal-mediated communication and provide a new strategy for the control of black rot in Brassica oleracea.202337404719
726120.8423Regulation of antimicrobial resistance by extracytoplasmic function (ECF) sigma factors. Extracytoplasmic function (ECF) sigma factors are a subfamily of σ(70) sigma factors that activate genes involved in stress-response functions. In many bacteria, ECF sigma factors regulate resistance to antimicrobial compounds. This review will summarize the ECF sigma factors that regulate antimicrobial resistance in model organisms and clinically relevant pathogens.201728153747
549130.8422Extracytoplasmic function sigma factor σ(D) confers resistance to environmental stress by enhancing mycolate synthesis and modifying peptidoglycan structures in Corynebacterium glutamicum. Mycolates are α-branched, β-hydroxylated, long-chain fatty acid specifically synthesized in bacteria in the suborder Corynebacterineae of the phylum Actinobacteria. They form an outer membrane, which functions as a permeability barrier and confers pathogenic mycobacteria to resistance to antibiotics. Although the mycolate biosynthetic pathway has been intensively studied, knowledge of transcriptional regulation of genes involved in this pathway is limited. Here, we report that the extracytoplasmic function sigma factor σ(D) is a key regulator of the mycolate synthetic genes in Corynebacterium glutamicum in the suborder. Chromatin immunoprecipitation with microarray analysis detected σ(D) -binding regions in the genome, establishing a consensus promoter sequence for σ(D) recognition. The σ(D) regulon comprised acyl-CoA carboxylase subunits, acyl-AMP ligase, polyketide synthase and mycolyltransferases; they were involved in mycolate synthesis. Indeed, deletion or overexpression of sigD encoding σ(D) modified the extractable mycolate amount. Immediately downstream of sigD, rsdA encoded anti-σ(D) and was under the control of a σ(D) -dependent promoter. Another σ(D) regulon member, l,d-transpeptidase, conferred lysozyme resistance. Thus, σ(D) modifies peptidoglycan cross-linking and enhances mycolate synthesis to provide resistance to environmental stress.201829148103
760140.8419The underling mechanism of bacterial TetR/AcrR family transcriptional repressors. Bacteria transcriptional regulators are classified by their functional and sequence similarities. Member of the TetR/AcrR family is two-domain proteins including an N-terminal HTH DNA-binding motif and a C-terminal ligand recognition domain. The C-terminal ligand recognition domain can recognize the very same compounds as their target transporters transferred. TetRs act as chemical sensors to monitor both the cellular environmental dynamics and their regulated genes underlying many events, such as antibiotics production, osmotic stress, efflux pumps, multidrug resistance, metabolic modulation, and pathogenesis. Compounds targeting Mycobacterium tuberculosis ethR represent promising novel antibiotic potentiater. TetR-mediated multidrug efflux pumps regulation might be good target candidate for the discovery of better new antibiotics against drug resistance.201323602932
623150.8416The Efflux Pump MexXY/OprM Contributes to the Tolerance and Acquired Resistance of Pseudomonas aeruginosa to Colistin. The intrinsic resistance of Pseudomonas aeruginosa to polymyxins in part relies on the addition of 4-amino-4-deoxy-l-arabinose (Ara4N) molecules to the lipid A of lipopolysaccharide (LPS), through induction of operon arnBCADTEF-ugd (arn) expression. As demonstrated previously, at least three two-component regulatory systems (PmrAB, ParRS, and CprRS) are able to upregulate this operon when bacteria are exposed to colistin. In the present study, gene deletion experiments with the bioluminescent strain PAO1::lux showed that ParRS is a key element in the tolerance of P. aeruginosa to this last-resort antibiotic (i.e., resistance to early drug killing). Other loci of the ParR regulon, such as those encoding the efflux proteins MexXY (mexXY), the polyamine biosynthetic pathway PA4773-PA4774-PA4775, and Ara4N LPS modification process (arnBCADTEF-ugd), also contribute to the bacterial tolerance in an intricate way with ParRS. Furthermore, we found that both stable upregulation of the arn operon and drug-induced ParRS-dependent overexpression of the mexXY genes accounted for the elevated resistance of pmrB mutants to colistin. Deletion of the mexXY genes in a constitutively activated ParR mutant of PAO1 was associated with significantly increased expression of the genes arnA, PA4773, and pmrA in the absence of colistin exposure, thereby highlighting a functional link between the MexXY/OprM pump, the PA4773-PA4774-PA4775 pathway, and Ara4N-based modification of LPS. The role played by MexXY/OprM in the adaptation of P. aeruginosa to polymyxins opens new perspectives for restoring the susceptibility of resistant mutants through the use of efflux inhibitors.202031964794
552160.8412Aurantimycin resistance genes contribute to survival of Listeria monocytogenes during life in the environment. Bacteria can cope with toxic compounds such as antibiotics by inducing genes for their detoxification. A common detoxification strategy is compound excretion by ATP-binding cassette (ABC) transporters, which are synthesized upon compound contact. We previously identified the multidrug resistance ABC transporter LieAB in Listeria monocytogenes, a Gram-positive bacterium that occurs ubiquitously in the environment, but also causes severe infections in humans upon ingestion. Expression of the lieAB genes is strongly induced in cells lacking the PadR-type transcriptional repressor LftR, but compounds leading to relief of this repression in wild-type cells were not known. Using RNA-Seq and promoter-lacZ fusions, we demonstrate highly specific repression of the lieAB and lftRS promoters through LftR. Screening of a natural compound library yielded the depsipeptide aurantimycin A - synthesized by the soil-dwelling Streptomyces aurantiacus - as the first known naturally occurring inducer of lieAB expression. Genetic and phenotypic experiments concordantly show that aurantimycin A is a substrate of the LieAB transporter and thus, lftRS and lieAB represent the first known genetic module conferring and regulating aurantimycin A resistance. Collectively, these genes may support the survival of L. monocytogenes when it comes into contact with antibiotic-producing bacteria in the soil.201930648305
730170.8412How intracellular bacteria survive: surface modifications that promote resistance to host innate immune responses. Bacterial pathogens regulate the expression of virulence factors in response to environmental signals. In the case of salmonellae, many virulence factors are regulated via PhoP/PhoQ, a two-component signal transduction system that is repressed by magnesium and calcium in vitro. PhoP/PhoQ-activated genes promote intracellular survival within macrophages, whereas PhoP-repressed genes promote entrance into epithelial cells and macrophages by macropinocytosis and stimulate epithelial cell cytokine production. PhoP-activated genes include those that alter the cell envelope through structural alterations of lipopolysaccharide and lipid A, the bioactive component of lipopolysaccharide. PhoP-activated changes in the bacterial envelope likely promote intracellular survival by increasing resistance to host cationic antimicrobial peptides and decreasing host cell cytokine production.199910081503
8797180.8408Presence of quorum-sensing systems associated with multidrug resistance and biofilm formation in Bacteroides fragilis. Bacteroides fragilis constitutes 1-2% of the natural microbiota of the human digestive tract and is the predominant anaerobic opportunistic pathogen in gastrointestinal infections. Most bacteria use quorum sensing (QS) to monitor cell density in relation to other cells and their environment. In Gram-negative bacteria, the LuxRI system is common. The luxR gene encodes a transcriptional activator inducible by type I acyl-homoserine lactone autoinducers (e.g., N-[3-oxohexanoyl] homoserine lactone and hexanoyl homoserine lactone [C6-HSL]). This study investigated the presence of QS system(s) in B. fragilis. The genome of American-type culture collection strain no. ATCC25285 was searched for QS genes. The strain was grown to late exponential phase in the presence or absence of synthetic C6-HSL and C8-HSL or natural homoserine lactones from cell-free supernatants from spent growth cultures of other bacteria. Growth, susceptibility to antimicrobial agents, efflux pump gene (bmeB) expression, and biofilm formation were measured. Nine luxR and no luxI orthologues were found. C6-HSL and supernatants from Yersinia enterocolitica, Vibrio cholerae, and Pseudomonas aeruginosa caused a significant (1) reduction in cellular density and (2) increases in expression of four putative luxR genes, bmeB3, bmeB6, bmeB7, and bmeB10, resistance to various antibiotics, which was reduced by carbonyl cyanide-m-chlorophenyl hydrazone (CCCP, an uncoupler that dissipates the transmembrane proton gradient, which is also the driving force of resistance nodulation division efflux pumps) and (3) increase in biofilm formation. Susceptibility of ATCC25285 to C6-HSL was also reduced by CCCP. These data suggest that (1) B. fragilis contains putative luxR orthologues, which could respond to exogenous homoserine lactones and modulate biofilm formation, bmeB efflux pump expression, and susceptibility to antibiotics, and (2) BmeB efflux pumps could transport homoserine lactones.200818188535
6006190.8408Missense Mutations in the CrrB Protein Mediate Odilorhabdin Derivative Resistance in Klebsiella pneumoniae. NOSO-502 is a preclinical antibiotic candidate of the Odilorhabdin class. This compound exhibits activity against Enterobacteriaceae pathogens, including carbapenemase-producing bacteria and most of the Colistin (CST)-resistant strains. Among a collection of CST-resistant Klebsiella pneumoniae strains harboring mutations on genes pmrAB, mgrB, phoPQ, and crrB, only those bearing mutations in gene crrB were found to be resistant to NOSO-502.CrrB is a histidine kinase which acts with the response regulator CrrA to modulate the PmrAB system, which finally induces the restructuring of the lipopolysaccharide present on the outer membrane and thus leading to CST resistance. Moreover, crrB mutations also enhance the transcription of neighboring genes such as H239_3063, an ABC transporter transmembrane region; H239_3064, a putative efflux pump also known as KexD; and H239_3065, a N-acetyltransferase.To elucidate the mechanism of resistance to NOSO-502 induced by CrrB missense mutations in K. pneumoniae, mutants of NCTC 13442 and ATCC BAA-2146 strains resistant to NOSO-502 and CST with single amino acid substitutions in CrrB (S8N, F33Y, Y34N, W140R, N141I, P151A, P151L, P151S, P151T, F303Y) were selected. Full susceptibility to NOSO-502 was restored in crrA or crrB deleted K. pneumoniae NCTC 13442 CrrB(P151L) mutants, confirming the role of CrrAB in controlling this resistance pathway. Deletion of kexD (but no other neighboring genes) in the same mutant also restored NOSO-502-susceptibility. Upregulation of the kexD gene expression was observed for all CrrB mutants. Finally, plasmid expression of kexD in a K. pneumoniae strain missing the locus crrABC and kexD significantly increased resistance to NOSO-502.202333685902