Missense 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.

Exploring Cluster-Dependent Antibacterial Activities and Resistance Pathways of NOSO-502 and Colistin against Enterobacter cloacae Complex Species.

The Enterobacter cloacae complex (ECC) is a group of diverse environmental and clinically relevant bacterial species associated with a variety of infections in humans. ECC have emerged as one of the leading causes of nosocomial infections worldwide. The purpose of this paper is to evaluate the activity of NOSO-502 and colistin (CST) against a panel of ECC clinical isolates, including different Hoffmann's clusters strains, and to investigate the associated resistance mechanisms. NOSO-502 is the first preclinical candidate of a novel antibiotic class, the odilorhabdins (ODLs). MIC50 and MIC90 of NOSO-502 against ECC are 1 µg/mL and 2 µg/mL, respectively, with a MIC range from 0.5 µg/mL to 32 µg/mL. Only strains belonging to clusters XI and XII showed decreased susceptibility to both NOSO-502 and CST while isolates from clusters I, II, IV, and IX were only resistant to CST. To understand this phenomenon, E. cloacae ATCC 13047 from cluster XI was chosen for further study. Results revealed that the two-component system ECL_01761-ECL_01762 (ortholog of CrrAB from Klebsiella pneumoniae) induces NOSO-502 hetero-resistance by expression regulation of the ECL_01758 efflux pump component (ortholog of KexD from K. pneumoniae) which could compete with AcrB to work with the multidrug efflux pump proteins AcrA and TolC. In E. cloacae ATCC 13047, CST-hetero-resistance is conferred via modification of the lipid A by addition of 4-amino-4-deoxy-l-arabinose controlled by PhoPQ. We identified that the response regulator ECL_01761 is also involved in this resistance pathway by regulating the expression of the ECL_01760 membrane transporter.

The Odilorhabdin Antibiotic Biosynthetic Cluster and Acetyltransferase Self-Resistance Locus Are Niche and Species Specific.

Antibiotic resistance is an increasing threat to human health. A direct link has been established between antimicrobial self-resistance determinants of antibiotic producers, environmental bacteria, and clinical pathogens. Natural odilorhabdins (ODLs) constitute a new family of 10-mer linear cationic peptide antibiotics inhibiting bacterial translation by binding to the 30S subunit of the ribosome. These bioactive secondary metabolites are produced by entomopathogenic bacterial symbiont Xenorhabdus (Morganellaceae), vectored by the soil-dwelling nematodes. ODL-producing Xenorhabdus nematophila symbionts have mechanisms of self-protection. In this study, we cloned the 44.5-kb odl biosynthetic gene cluster (odl-BGC) of the symbiont by recombineering and showed that the N-acetyltransferase-encoding gene, oatA, is responsible for ODL resistance. In vitro acetylation and liquid chromatography-tandem mass spectrometry (LC-MS/MS) analyses showed that OatA targeted the side chain amino group of ODL rare amino acids, leading to a loss of translation inhibition and antibacterial properties. Functional, genomic, and phylogenetic analyses of oatA revealed an exclusive cis-link to the odilorhabdin BGC, found only in X. nematophila and a specific phylogenetic clade of Photorhabdus. This work highlights the coevolution of antibiotic production and self-resistance as ancient features of this unique tripartite complex of host-vector-symbiont interactions without odl-BGC dissemination by lateral gene transfer. IMPORTANCE Odilorhabdins (ODLs) constitute a novel antibiotic family with promising properties for treating problematic multidrug-resistant Gram-negative bacterial infections. ODLs are 10-mer linear cationic peptides inhibiting bacterial translation by binding to the small subunit of the ribosome. These natural peptides are produced by Xenorhabdus nematophila, a bacterial symbiont of entomopathogenic nematodes well known to produce large amounts of specialized secondary metabolites. Like other antimicrobial producers, ODL-producing Xenorhabdus nematophila has mechanisms of self-protection. In this study, we cloned the ODL-biosynthetic gene cluster of the symbiont by recombineering and showed that the N-acetyltransferase-encoding gene, oatA, is responsible for ODL resistance. In vitro acetylation and LC-MS/MS analyses showed that OatA targeted the side chain amino group of ODL rare amino acids, leading to a loss of translation inhibition and antibacterial properties. Functional, genomic, and phylogenetic analyses of oatA revealed the coevolution of antibiotic production and self-resistance as ancient feature of this particular niche in soil invertebrates without resistance dissemination.

Structure-activity relationship studies on the inhibition of the bacterial translation of novel Odilorhabdins analogues.

A structure-activity relationship (SAR) study of NOSO-95179, a nonapeptide from the Odilorhabdin class of antibacterials, was performed by systematic variations of amino acids in positions 2 and 5 of the peptide. A series of non-proteinogenic amino acids was synthesized in high enantiomeric purity from Williams' chiral diphenyloxazinone by highly diastereoselective alkylation or by aldol-type reaction. NOSO-95179 analogues for SAR studies were prepared using solid-phase peptide synthesis. Inhibition of bacterial translation by each of the synthesized Odilorhabdin analogues was measured using an in vitro test. For the most efficient analogues, antibacterial efficacy was measured against two wild-type Enterobacteriaceae (Escherichia coli and Klebsiella pneumoniae) and against an efflux defective E. coli strain (ΔtolC) to evaluate the impact of efflux on the antibacterial activity.

Total Synthesis and Structure-Activity Relationships Study of Odilorhabdins, a New Class of Peptides Showing Potent Antibacterial Activity.

The spread of antibiotic-resistant pathogens is a growing concern, and new families of antibacterials are desperately needed. Odilorhabdins are a new class of antibacterial compounds that bind to the bacterial ribosome and kill bacteria through inhibition of the translation. NOSO-95C, one of the first member of this family, was synthesized for the first time, and then a structure-activity relationships study was performed to understand which groups are important for antibacterial activity and for inhibition of the bacterial translation. Based on this study an analogue showing improved properties compared to the parent compound was identified and showed promising in vitro and in vivo efficacy against Enterobacteriaceae.

In Vitro and In Vivo Characterization of NOSO-502, a Novel Inhibitor of Bacterial Translation.

Antibacterial activity screening of a collection of Xenorhabdus strains led to the discovery of the odilorhabdins, a new antibiotic class with broad-spectrum activity against Gram-positive and Gram-negative pathogens. Odilorhabdins inhibit bacterial translation by a new mechanism of action on ribosomes. A lead optimization program identified NOSO-502 as a promising candidate. NOSO-502 has MIC values ranging from 0.5 to 4 µg/ml against standard Enterobacteriaceae strains and carbapenem-resistant Enterobacteriaceae (CRE) isolates that produce KPC, AmpC, or OXA enzymes and metallo-ß-lactamases. In addition, this compound overcomes multiple chromosome-encoded or plasmid-mediated resistance mechanisms of acquired resistance to colistin. It is effective in mouse systemic infection models against Escherichia coli EN122 (extended-spectrum ß-lactamase [ESBL]) or E. coli ATCC BAA-2469 (NDM-1), achieving a 50% effective dose (ED50) of 3.5 mg/kg of body weight and 1-, 2-, and 3-log reductions in blood burden at 2.6, 3.8, and 5.9 mg/kg, respectively, in the first model and 100% survival in the second, starting with a dose as low as 4 mg/kg. In a urinary tract infection (UTI) model with E. coli UTI89, urine, bladder, and kidney burdens were reduced by 2.39, 1.96, and 1.36 log10 CFU/ml, respectively, after injection of 24 mg/kg. There was no cytotoxicity against HepG2, HK-2, or human renal proximal tubular epithelial cells (HRPTEpiC), no inhibition of hERG-CHO or Nav 1.5-HEK current, and no increase of micronuclei at 512 µM. NOSO-502, a compound with a new mechanism of action, is active against Enterobacteriaceae, including all classes of CRE, has a low potential for resistance development, shows efficacy in several mouse models, and has a favorable in vitro safety profile.

Odilorhabdins, Antibacterial Agents that Cause Miscoding by Binding at a New Ribosomal Site.

Growing resistance of pathogenic bacteria and shortage of antibiotic discovery platforms challenge the use of antibiotics in the clinic. This threat calls for exploration of unconventional sources of antibiotics and identification of inhibitors able to eradicate resistant bacteria. Here we describe a different class of antibiotics, odilorhabdins (ODLs), produced by the enzymes of the non-ribosomal peptide synthetase gene cluster of the nematode-symbiotic bacterium Xenorhabdus nematophila. ODLs show activity against Gram-positive and Gram-negative pathogens, including carbapenem-resistant Enterobacteriaceae, and can eradicate infections in animal models. We demonstrate that the bactericidal ODLs interfere with protein synthesis. Genetic and structural analyses reveal that ODLs bind to the small ribosomal subunit at a site not exploited by current antibiotics. ODLs induce miscoding and promote hungry codon readthrough, amino acid misincorporation, and premature stop codon bypass. We propose that ODLs' miscoding activity reflects their ability to increase the affinity of non-cognate aminoacyl-tRNAs to the ribosome.