Crystal structures of YeiE from Cronobacter sakazakii and the role of sulfite tolerance in gram-negative bacteria.

SignificanceYeiE has been identified as a master virulence factor of Cronobacter sakazakii. In this study, we determined the crystal structures of the regulatory domain of YeiE in complex with its physiological ligand sulfite ion (SO32-). The structure provides the basis for the molecular mechanisms for sulfite sensing and the ligand-dependent conformational changes of the regulatory domain. The genes under the control of YeiE in response to sulfite were investigated to reveal the functional roles of YeiE in the sulfite tolerance of the bacteria. We propose the molecular mechanism underlying the ability of gram-negative pathogens to defend against the innate immune response involving sulfite, thus providing a strategy to control the pathogenesis of bacteria.

ARBM101 (Methanobactin SB2) Drains Excess Liver Copper via Biliary Excretion in Wilson's Disease Rats.

BACKGROUND & AIMS: Excess copper causes hepatocyte death in hereditary Wilson's disease (WD). Current WD treatments by copper-binding chelators may gradually reduce copper overload; they fail, however, to bring hepatic copper close to normal physiological levels. Consequently, lifelong daily dose regimens are required to hinder disease progression. This may result in severe issues due to nonadherence or unwanted adverse drug reactions and also due to drug switching and ultimate treatment failures. This study comparatively tested bacteria-derived copper binding agents-methanobactins (MBs)-for efficient liver copper depletion in WD rats as well as their safety and effect duration. METHODS: Copper chelators were tested in vitro and in vivo in WD rats. Metabolic cage housing allowed the accurate assessment of animal copper balances and long-term experiments related to the determination of minimal treatment phases. RESULTS: We found that copper-binding ARBM101 (previously known as MB-SB2) depletes WD rat liver copper dose dependently via fecal excretion down to normal physiological levels within 8 days, superseding the need for continuous treatment. Consequently, we developed a new treatment consisting of repetitive cycles, each of ∼1 week of ARBM101 applications, followed by months of in-between treatment pauses to ensure a healthy long-term survival in WD rats. CONCLUSIONS: ARBM101 safely and efficiently depletes excess liver copper from WD rats, thus allowing for short treatment periods as well as prolonged in-between rest periods.

cDNA-Derived RNA Phage Assembly Reveals Critical Residues in the Maturation Protein of the Pseudomonas aeruginosa Leviphage PP7.

PP7 is a leviphage, with a single-stranded RNA genome, that infects Pseudomonas aeruginosa PAO1. A reverse genetic system for PP7 was previously created by using reverse-transcribed cDNA (PP7O) from a virion-derived RNA genome. Here, we have found that the PP7O cDNA contained 20 nucleotide differences from the PP7 genome sequence deposited in the database. We created another reverse genetic system exploiting chemically synthesized cDNA (PP7S) based on the database sequence. Unlike PP7O, which yielded infectious PP7 virions, PP7S-derived particles were incapable of plaque formation on PAO1 cells, which was restored in the PAO1 cells expressing the maturation protein (MP) from PP7O Using this reverse genetic system, we revealed two amino acid residues involved in the known roles of MP (i.e., adsorption and genome replication), fortuitously providing a lesson that the viral RNA genome sequencing needs functional verification, possibly by a reverse genetic system.IMPORTANCE The biological significance of RNA phages has been largely ignored, ironically, because few studies have focused on RNA phages. As an initial attempt to properly represent RNA phages in the phageome, we previously created, by using reverse-transcribed cDNA, a reverse genetic system for the small RNA phage PP7, which infects the opportunistic human pathogen Pseudomonas aeruginosa We report another system by using chemically synthesized cDNA based on the database genome that has 20 nucleotide differences from the previous cDNA. Investigation of those cDNA-derived phage virions revealed that two amino acids of the maturation protein are crucial for the normal phage lifecycle at different steps. Our study provides insight into the molecular basis for the RNA phage lifecycle and a lesson that the RNA genome sequencing needs to be carefully validated by cDNA-based phage assembly systems.

Lysine acetylation of the housekeeping sigma factor enhances the activity of the RNA polymerase holoenzyme.

Protein lysine acetylation, one of the most abundant post-translational modifications in eukaryotes, occurs in prokaryotes as well. Despite the evidence of lysine acetylation in bacterial RNA polymerases (RNAPs), its function remains unknown. We found that the housekeeping sigma factor (HrdB) was acetylated throughout the growth of an actinobacterium, Streptomyces venezuelae, and the acetylated HrdB was enriched in the RNAP holoenzyme complex. The lysine (K259) located between 1.2 and 2 regions of the sigma factor, was determined to be the acetylated residue of HrdB in vivo by LC-MS/MS analyses. Specifically, the label-free quantitative analysis revealed that the K259 residues of all the HrdB subunits were acetylated in the RNAP holoenzyme. Using mutations that mimic or block acetylation (K259Q and K259R), we found that K259 acetylation enhances the interaction of HrdB with the RNAP core enzyme as well as the binding activity of the RNAP holoenzyme to target promoters in vivo. Taken together, these findings provide a novel insight into an additional layer of modulation of bacterial RNAP activity.

Structural details of the OxyR peroxide-sensing mechanism.

OxyR, a bacterial peroxide sensor, is a LysR-type transcriptional regulator (LTTR) that regulates the transcription of defense genes in response to a low level of cellular H2O2. Consisting of an N-terminal DNA-binding domain (DBD) and a C-terminal regulatory domain (RD), OxyR senses H2O2 with conserved cysteine residues in the RD. However, the precise mechanism of OxyR is not yet known due to the absence of the full-length (FL) protein structure. Here we determined the crystal structures of the FL protein and RD of Pseudomonas aeruginosa OxyR and its C199D mutant proteins. The FL crystal structures revealed that OxyR has a tetrameric arrangement assembled via two distinct dimerization interfaces. The C199D mutant structures suggested that new interactions that are mediated by cysteine hydroxylation induce a large conformational change, facilitating intramolecular disulfide-bond formation. More importantly, a bound H2O2 molecule was found near the Cys199 site, suggesting the H2O2-driven oxidation mechanism of OxyR. Combined with the crystal structures, a modeling study suggested that a large movement of the DBD is triggered by structural changes in the regulatory domains upon oxidation. Taken together, these findings provide novel concepts for answering key questions regarding OxyR in the H2O2-sensing and oxidation-dependent regulation of antioxidant genes.

A phage protein that inhibits the bacterial ATPase required for type IV pilus assembly.

Type IV pili (TFPs) are required for bacterial twitching motility and for phage infection in the opportunistic human pathogen Pseudomonas aeruginosa. Here we describe a phage-encoded protein, D3112 protein gp05 (hereafter referred to as Tip, representing twitching inhibitory protein), whose expression is necessary and sufficient to mediate the inhibition of twitching motility. Tip interacts with and blocks the activity of bacterial-encoded PilB, the TFP assembly/extension ATPase, at an internal 40-aa region unique to PilB. Tip expression results in the loss of surface piliation. Based on these observations and the fact that many P. aeruginosa phages require TFPs for infection, Tip-mediated twitching inhibition may represent a generalized strategy for superinfection exclusion. Moreover, because TFPs are required for full virulence, PilB may be an attractive target for the development of novel antiinfectives.

An outer membrane determinant for RNA phage genome entry in Pseudomonas aeruginosa.

Host range of a phage is determined at the various life cycle stages during phage infection. We reported the limited phage-receptor interaction between the RNA phage, PP7 and its host Pseudomonas aeruginosa strains: PAO1 has susceptible type IV pilus (TFP) pilin, whereas PA14 has resistant pilin. Here, we have created a PA14 derivative (PA14P) with the PAO1 pilin gene and found that other determinants than TFP pilin could limit PP7 infectivity in PA14P. Transposon mutant screens revealed that PP7 infectivity was restored in the PA14P mutants (htrB2) lacking a secondary acyltransferase in lipid A biosynthesis. The lack of this enzyme increased the RNA phage entry, which is deemed attributed to the loosened lipopolysaccharide (LPS) structure. Polymyxin B treatment also selectively increased the RNA phage entry. These results demonstrated that LPS structures could limit the entry stage of RNA phages, providing another determinant for the host range in diverse P. aeruginosa strains.

A TetR family regulator of an RND efflux system that directs artemisinin resistance in Vibrio cholerae.

Artemisinin (ARS) displayed bactericidal activity against Vibrio cholerae. To assess the mechanistic details of its antibacterial action, we have isolated V. cholerae mutants with enhanced ARS resistance and identified a gene (VCA0767) whose loss-of-function resulted in the ARS resistance phenotypes. This gene (atrR) encodes a TetR family transcriptional regulator, and its deletion mutant displayed the reduction in ARS-induced ROS formation and DNA damage. Transcriptomic analysis revealed that the genes encoding a resistance-nodulation-cell division (RND) efflux pump operon (vexRAB) and the outer membrane component (tolC) were highly upregulated in the artR mutant, suggesting that AtrR might act as a negative regulator of this operon and tolC. Gene deletion of vexR, vexB, or tolC abrogated the ARS resistance of the atrR mutant, and more importantly, the ectopic expression of VexAB-TolC was sufficient for the ARS resistance, indicating that the increased expression of the VexAB-TolC efflux system is necessary and sufficient for the ARS resistance of the atrR mutant. The cytoplasmic accumulation of ARS was compromised in the vexBtolC mutant, suggesting that the VexAB-TolC might be the primary efflux system exporting ARS to reduce its toxicity inside of the bacterial cells. The atrR mutant displayed resistance to erythromycin as well in a VexR-dependent manner. This result suggests that AtrR may act as a global regulator responsible for preventing intracellular accumulation of toxic chemicals by enhancing the RND efflux system.IMPORTANCEDrug efflux protein complexes or efflux pumps are considered as the major determinants of multiple antimicrobial resistance by exporting a wide range of structurally diverse antibiotics in bacterial pathogens. Despite the clinical significance of the increased expression of the efflux pumps, their substrate specificity and regulation mechanisms are poorly understood. Here, we demonstrated that VexAB-TolC, a resistance-nodulation-cell division (RND) efflux pump of V. cholerae, is responsible for the resistance to artemisinin (ARS), an antimalarial drug with bactericidal activity. Furthermore, we newly identified AtrR, a TetR family repressor, as a global regulator for VexRAB and the common outer membrane channel, TolC, where VexR functions as the pathway-specific regulator of the vexAB operon. Our findings will help improve our insight into a broad range of substrate specificity of the VexAB-TolC system and highlight the complex regulatory networks of the multiple RND efflux systems during V. cholerae pathogenesis.

Autolysis of Pseudomonas aeruginosa Quorum-Sensing Mutant Is Suppressed by Staphylococcus aureus through Iron-Dependent Metabolism.

Microorganisms usually coexist as a multifaceted polymicrobial community in the natural habitats and at mucosal sites of the human body. Two opportunistic human pathogens, Pseudomonas aeruginosa and Staphylococcus aureus commonly coexist in the bacterial infections for hospitalized and/or immunocompromised patients. Here, we observed that autolysis of the P. aeruginosa quorum-sensing (QS) mutant (lasRmvfR) was suppressed by the presence of the S. aureus cells in vitro. The QS mutant still displayed killing against S. aureus cells, suggesting the link between the S. aureus-killing activity and the autolysis suppression. Independent screens of the P. aeruginosa transposon mutants defective in the S. aureus-killing and the S. aureus transposon mutants devoid of the autolysis suppression revealed the genetic link between both phenotypes, suggesting that the iron-dependent metabolism involving S. aureus exoproteins might be central to both phenotypes. The autolysis was suppressed by iron treatment as well. These results suggest that the interaction between P. aeruginosa and S. aureus might be governed by mechanisms that necessitate the QS circuitry as well as the metabolism involving the extracellular iron resources during the polymicrobial infections in the human airway.

Use of Cas9 Targeting and Red Recombination for Designer Phage Engineering.

Bacteriophages (phages) are natural antibiotics and biological nanoparticles, whose application is significantly boosted by recent advances of synthetic biology tools. Designer phages are synthetic phages created by genome engineering in a way to increase the benefits or decrease the drawbacks of natural phages. Here we report the development of a straightforward genome engineering method to efficiently obtain engineered phages in a model bacterial pathogen, Pseudomonas aeruginosa. This was achieved by eliminating the wild type phages based on the Streptococcus pyogenes Cas9 (SpCas9) and facilitating the recombinant generation based on the Red recombination system of the coliphage λ (λRed). The producer (PD) cells of P. aeruginosa strain PAO1 was created by miniTn7-based chromosomal integration of the genes for SpCas9 and λRed under an inducible promoter. To validate the efficiency of the recombinant generation, we created the fluorescent phages from a temperate phage MP29. A plasmid bearing the single guide RNA (sgRNA) gene for selectively targeting the wild type gp35 gene and the editing template for tagging the Gp35 with superfolder green fluorescent protein (sfGFP) was introduced into the PD cells by electroporation. We found that the targeting efficiency was affected by the position and number of sgRNA. The fluorescent phage particles were efficiently recovered from the culture of the PD cells expressing dual sgRNA molecules. This protocol can be used to create designer phages in P. aeruginosa for both application and research purposes.

Pleiotropic effects of N-acylhomoserine lactone synthase ExpI on virulence, competition, and transmission in Pectobacterium carotovorum subsp. carotovorum Pcc21.

BACKGROUND: Pectobacterium species are necrotrophic phytopathogenic bacteria that cause soft rot disease in economically important crops. The successful infection of host plants relies on interactions among virulence factors, competition, and transmission within hosts. Pectobacteria primarily produce and secrete plant cell-wall degrading enzymes (PCWDEs) for virulence. The regulation of PCWDEs is controlled by quorum sensing (QS). Thus, the QS system is crucial for disease development in pectobacteria through PCWDEs. RESULTS: In this study, we identified a Tn-insertion mutant, M2, in the expI gene from a transposon mutant library of P. carotovorum subsp. carotovorum Pcc21 (hereafter Pcc21). The mutant exhibited reduced production and secretion of PCWDEs, impaired flagellar motility, and increased sensitivity to hydrogen peroxide, resulting in attenuated soft rot symptoms in cabbage and potato tubers. Transcriptomic analysis revealed the down-regulation of genes involved in the production and secretion in the mutant, consistent with the observed phenotype. Furthermore, the Pcc21 wild-type transiently colonized in the gut of Drosophila melanogaster within 12 h after feeding, while the mutant compromised colonization phenotype. Interestingly, Pcc21 produces a bacteriocin, carocin D, to compete with other bacteria. The mutant exhibited up-regulation of carocin D-encoding genes (caroDK) and inhibited the growth of a closely related bacterium, P. wasabiae. CONCLUSION: Our results demonstrated the significance of ExpI in the overall pathogenic lifestyle of Pcc21, including virulence, competition, and colonization in plant and insect hosts. These findings suggest that disease outcome is a result of complex interactions mediated by ExpI across multiple steps. © 2023 Society of Chemical Industry.

Design, synthesis, and evaluation of N1,N3-dialkyldioxonaphthoimidazoliums as antibacterial agents against methicillin-resistant Staphylococcus aureus.

Increasing antibiotic resistance of bacterial pathogens poses a serious threat to human health worldwide. Methicillin-resistant Staphylococcus aureus (MRSA) is among the most deleterious bacterial pathogens owing to its multidrug resistance, necessitating the development of new antibacterial agents against it. We previously identified a novel dioxonaphthoimidazolium agent, c5, with moderate antibacterial activity against MRSA from an anticancer clinical candidate, YM155. In this study, we aimed to design and synthesize several novel cationic amphiphilic N1,N3-dialkyldioxonaphthoimidazolium bromides with enhanced lipophilicity of the two side chains in the imidazolium scaffold and improved antibacterial activities compared to those of c5 against gram-positive bacteria in vitro and in vivo. Our new antibacterial lead, N1,N3-n-octylbenzyldioxonaphthoimidazolium bromide (11), exhibited highly potent antibacterial activities against various gram-positive bacterial strains (MICs: 0.19-0.39 µg/mL), including MRSA, methicillin-sensitive S. aureus, and Bacillus subtilis. Moreover, antibacterial mechanism of 11 against MRSA based on the generation of reactive oxygen species (ROS) was evaluated. Although compound 11 exhibited cytotoxic effects in vitro and lacked a therapeutic index against the HEK293 and HDFa mammalian cell lines, it exhibited low toxicity in the Drosophila animal model. Remarkably, 11 exhibited better in vivo antibacterial efficacy than c5 and the clinically used antibiotic, vancomycin, in SA3-infected Drosophila model. Moreover, the development of bacterial resistance to 11 was not observed after 16 consecutive passages. Therefore, rational design of antibacterial cationic amphiphiles based on ROS-generating pharmacophores with optimized lipophilicity can facilitate the identification of potent antibacterial agents against drug-resistant infections.

Complete genome sequences of two Pseudomonas aeruginosa temperate phages, MP29 and MP42, which lack the phage-host CRISPR interaction.

We report the complete genome sequence of two Pseudomonas aeruginosa phages MP29 and MP42. Their genomes are similar to those of P. aeruginosa temperate phages DMS3 and MP22, whose lysogens are impaired in swarming motilities, involving the host CRISPR loci. Both MP29 and MP42 lysogens, however, were proficient in swarming, suggesting the absence of the phage-host CRISPR interaction.

Complete genome sequence of Pseudomonas aeruginosa siphophage MP1412.

We report the complete genome sequence of Pseudomonas aeruginosa siphophage MP1412, which displays synteny to those of P. aeruginosa phages M6 and YuA. However, the presence of two homing endonucleases of the GIY-YIG family is unique to MP1412, suggesting their unique role in the phage life cycle of the bacterial host.

Chemokine CCL6 Plays Key Role in the Inhibitory Effect of Vitamin A on Norovirus Infection.

Norovirus (NoV) is the most common viral cause of acute gastroenteritis worldwide. Vitamin A has demonstrated the potential to protect against gastrointestinal infections. However, the effects of vitamin A on human norovirus (HuNoV) infections remain poorly understood. This study aimed to investigate how vitamin A administration affects NoV replication. We demonstrated that treatment with retinol or retinoic acid (RA) inhibited NoV replication in vitro based on their effects on HuNoV replicon-bearing cells and murine norovirus-1 (MNV-1) replication in murine cells. MNV replication in vitro showed significant transcriptomic changes, which were partially reversed by retinol treatment. RNAi knockdown of CCL6, a chemokine gene that was downregulated by MNV infection but upregulated by retinol administration, resulted in increased MNV replication in vitro. This suggested a role of CCL6 in the host response to MNV infections. Similar gene expression patterns were observed in the murine intestine after oral administration of RA and/or MNV-1.CW1. CCL6 directly decreased HuNoV replication in HG23 cells, and might indirectly regulate the immune response against NoV infection. Finally, relative replication levels of MNV-1.CW1 and MNV-1.CR6 were significantly increased in CCL6 knockout RAW 264.7 cells. This study is the first to comprehensively profile transcriptomes in response to NoV infection and vitamin A treatment in vitro, and thus may provide new insights into dietary prophylaxis and NoV infections.

Antibacterial efficacy of temperate phage-mediated inhibition of bacterial group motilities.

Phage therapy against bacterial pathogens has been resurrected as an alternative and supplementary anti-infective modality. Here, we observed that bacterial group motilities were impaired in Pseudomonas aeruginosa strain PA14 lysogens for some temperate siphophages; the PA14 lysogens for DMS3 and MP22 were impaired in swarming motility, whereas the PA14 lysogen for D3112 was impaired in twitching motility. The swarming and twitching motilities of PA14 were also affected in the presence of MP22 and D3112, respectively. The in vitro killing activities of D3112 and MP22 toward PA14 did not differ, and neither did their in vivo persistence in the absence of bacterial infections in mice as well as in flies. Nevertheless, administration of D3112, not MP22, significantly reduced the mortality and the bacterial burdens in murine peritonitis-sepsis and Drosophila systemic infection caused by PA14. Taken together, we suggest that a temperate phage-mediated twitching motility inhibition might be comparably effective to control the acute infections caused by P. aeruginosa.

Elevated post-transfusion serum transaminase values associated with a highly significant trend for increasing prevalence of anti-Vesivirus antibody in Korean patients.

A highly significant increase in anti-Vesivirus (family Caliciviridae) antibody prevalence, along the axis from healthy blood donors; donors with elevated transaminase; patients with clinical hepatitis; and patients with post-transfusion/dialysis hepatitis, has been reported in human sera from the USA and Europe. Asian samples have now been tested retrospectively using serology and enzyme immunoassay (EIA) with a Vesivirus partial-capsid antigen expressed as a fusion protein. Anti-vesiviral antibodies were measured by optical densities (OD(650)) and compared in patients separated by age, gender and Groups A-F as follows: Control Group A, an Experimental Group B, which was divided further into Group C, patients with elevated enzymes (alanine transaminase (ALT), aspartate transaminase (AST), and γ-glutamyl transpeptidase (γ-GT); Group D, patients receiving transfused blood; Group E, patients with high enzyme levels after transfusion; and Group F, hepatitis B and C positive patients. Using multivariate logistic regression analyses, a significantly greater proportion of patients receiving transfusion(s), were positive for anti-Vesivirus antibody compared with non-transfused patients (P = 0.008; OR: 3.86, 95% CI: 1.43-10.43). Also, anti-Vesivirus antibody was significantly associated with elevated biochemical liver function tests: ALT ≥ 20 IU or AST ≥ 120 IU (P = 0.017; OR: 4.23, 95% CI: 1.30-13.80). In the blood transfusion group, anti-Vesivirus antibody was significantly correlated with high enzyme levels (ALT, P = 0.018; AST, P = 0.010; γ-GT, P = 0.020). These data provide serologic evidence of vesiviral transfusion-transmission-associated disease, which could include infection of any organ system where cytopathology resulted in high levels of either ALT or AST.

Pyocyanin and 1-Hydroxyphenazine Promote Anaerobic Killing of Pseudomonas aeruginosa via Single-Electron Transfer with Ferrous Iron.

Previously, it was reported that natural phenazines are able to support the anaerobic survival of Pseudomonas aeruginosa PA14 cells via electron shuttling, with electrodes poised as the terminal oxidants (Y. Wang, S. E. Kern, and D. K. Newman, J Bacteriol 192:365-369, 2010, https://doi.org/10.1128/JB.01188-09). The present study shows that both pyocyanin (PYO) and 1-hydroxyphenazine (1-OHPHZ) promoted the anaerobic killing of PA14 Δphz cells presumably via a single-electron transfer reaction with ferrous iron. However, phenazine-1-carboxylic acid (PCA) did not affect anaerobic survival in the presence of ferrous iron. Anaerobic cell death was alleviated by the addition of antioxidant compounds, which inhibit electron transfer via DNA damage. Neither superoxide dismutase (SOD) nor catalase was able to alleviate P. aeruginosa cell death, ruling out the possibility of reactive oxygen species (ROS)-induced killing. Further, the phenazine degradation profile and the redox state-associated color changes suggested that phenazine radical intermediates are likely generated by single-electron transfer. In this study, we showed that the phenazines 1-OHPHZ and PYO anaerobically killed the cell via single-electron transfer with ferrous iron and that the killing might have resulted from phenazine radicals. IMPORTANCE Pseudomonas aeruginosa is an opportunistic human pathogen which infects patients with burns, immunocompromised individuals, and in particular, the mucus that accumulates on the surface of the lung in cystic fibrosis (CF) patients. Phenazines as redox-active small molecules have been reported as important compounds for the control of cellular functions and virulence as well as anaerobic survival via electron shuttles. We show that both pyocyanin (PYO) and 1-hydroxyphenazine (1-OHPHZ) generate phenazine radical intermediates via presumably single-electron transfer reaction with ferrous iron, leading to the anaerobic killing of Pseudomonas cells. The recA mutant defect in the DNA repair system was more sensitive to anaerobic conditions. Our results collectively suggest that both phenazines anaerobically kill cells via DNA damage during electron transfer with iron.

An iron-chelating sulfonamide identified from Drosophila-based screening for antipathogenic discovery.

We exploited bacterial infection assays using the fruit fly Drosophila melanogaster to identify anti-infective compounds that abrogate the pathological consequences in the infected hosts. Here, we demonstrated that a pyridine-3-N-sulfonylpiperidine derivative (4a) protects Drosophila from the acute infections caused by bacterial pathogens including Pseudomonas aeruginosa. 4a did not inhibit the growth of P. aeruginosa in vitro, but inhibited the production of secreted toxins such as pyocyanin and hydrogen cyanide, while enhancing the production of pyoverdine and pyochelin, indicative of iron deprivation. Based on its catechol moiety, 4a displayed iron-chelating activity in vitro toward both iron (II) and iron (III), more efficiently than the approved iron-chelating drugs such as deferoxamine and deferiprone, concomitant with more potent antibacterial efficacy in Drosophila infections and unique transcriptome profile. Taken together, these results delineate a Drosophila-based strategy to screen for antipathogenic compounds, which interfere with iron uptake crucial for bacterial virulence and survival in host tissues.

Iron-Induced Respiration Promotes Antibiotic Resistance in Actinomycete Bacteria.

The bacterial response to antibiotics eliciting resistance is one of the key challenges in global health. Despite many attempts to understand intrinsic antibiotic resistance, many of the underlying mechanisms still remain elusive. In this study, we found that iron supplementation promoted antibiotic resistance in Streptomyces coelicolor. Iron-promoted resistance occurred specifically against bactericidal antibiotics, irrespective of the primary target of antibiotics. Transcriptome profiling revealed that some genes in the central metabolism and respiration were upregulated under iron-replete conditions. Iron supported the growth of S. coelicolor even under anaerobic conditions. In the presence of potassium cyanide, which reduces aerobic respiration of cells, iron still promoted respiration and antibiotic resistance. This suggests the involvement of a KCN-insensitive type of respiration in the iron effect. This phenomenon was also observed in another actinobacterium, Mycobacterium smegmatis. Taken together, these findings provide insight into a bacterial resistance strategy that mitigates the activity of bactericidal antibiotics whose efficacy accompanies oxidative damage by switching the respiration mode. IMPORTANCE A widely investigated mode of antibiotic resistance occurs via mutations and/or by horizontal acquisition of resistance genes. In addition to this acquired resistance, most bacteria exhibit intrinsic resistance as an inducible and adaptive response to different classes of antibiotics. Increasing attention has been paid recently to intrinsic resistance mechanisms because this may provide novel therapeutic targets that help rejuvenate the efficacy of the current antibiotic regimen. In this study, we demonstrate that iron promotes the intrinsic resistance of aerobic actinomycetes Streptomyces coelicolor and Mycobacterium smegmatis against bactericidal antibiotics. A surprising role of iron to increase respiration, especially in a mode of using less oxygen, appears a fitting strategy to cope with bactericidal antibiotics known to kill bacteria through oxidative damage. This provides new insights into developing antimicrobial treatments based on the availability of iron and oxygen.