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503200.9519Hijacking a small plasmid to confer high-level resistance to aztreonam-avibactam and ceftazidime-avibactam. Acquired β-lactamase-encoding genes are typically carried by large plasmids in Gram-negative bacteria, which also commonly carry multi-copy small plasmids. This study found that mobile genetic elements carrying antimicrobial resistance genes are capable of hijacking small plasmids. This study focused on aztreonam-avibactam (ATM-AVI) as this combination can be used to effectively counter almost all β-lactamases produced by bacteria, and has been recommended against carbapenem-resistant Enterobacterales. A clinical strain (085003) of carbapenem-resistant Escherichia coli was investigated, and mutants (085003R32 and 085003R512) able to grow under 32/4 and 512/4 mg/L of ATM-AVI were obtained as representatives of low- and high-level resistance, respectively, by induction. Comparative genomics showed that 085003R32 and 085003R512 had a single nucleotide mutation of β-lactamase gene bla(CMY-2), encoding a novel CMY with a Thr319Ile substitution, assigned 'CMY-2R'. Cloning and enzyme kinetics were used to verify that CMY-2R conferred ATM-AVI resistance by compromising binding of AVI and subsequent protection of ATM. Mechanisms for the discrepant resistance between 085003R32 and 085003R512 were investigated. Three tandem copies of bla(CMY-2R) were identified on a self-transmissible IncP1 plasmid of 085003R32 due to IS1294 misrecognizing its end terIS and rolling-circle replication. 085003R512 had only a single copy of bla(CMY-2R) on the IncP1 plasmid, but possessed anther bla(CMY-2R) on an already present 4-kb small plasmid. IS1294-mediated mobilization on to this multi-copy small plasmid increased the copy number of bla(CMY-2R) significantly, rendering higher resistance. This study shows that bacteria can employ multiple approaches to accommodate selection pressures imposed by exposure to varied concentrations of antimicrobial agents.202337769749
11610.9518The ADEP Biosynthetic Gene Cluster in Streptomyces hawaiiensis NRRL 15010 Reveals an Accessory clpP Gene as a Novel Antibiotic Resistance Factor. The increasing threat posed by multiresistant bacterial pathogens necessitates the discovery of novel antibacterials with unprecedented modes of action. ADEP1, a natural compound produced by Streptomyces hawaiiensis NRRL 15010, is the prototype for a new class of acyldepsipeptide (ADEP) antibiotics. ADEP antibiotics deregulate the proteolytic core ClpP of the bacterial caseinolytic protease, thereby exhibiting potent antibacterial activity against Gram-positive bacteria, including multiresistant pathogens. ADEP1 and derivatives, here collectively called ADEP, have been previously investigated for their antibiotic potency against different species, structure-activity relationship, and mechanism of action; however, knowledge on the biosynthesis of the natural compound and producer self-resistance have remained elusive. In this study, we identified and analyzed the ADEP biosynthetic gene cluster in S. hawaiiensis NRRL 15010, which comprises two NRPSs, genes necessary for the biosynthesis of (4S,2R)-4-methylproline, and a type II polyketide synthase (PKS) for the assembly of highly reduced polyenes. While no resistance factor could be identified within the gene cluster itself, we discovered an additional clpP homologous gene (named clpP(ADEP)) located further downstream of the biosynthetic genes, separated from the biosynthetic gene cluster by several transposable elements. Heterologous expression of ClpP(ADEP) in three ADEP-sensitive Streptomyces species proved its role in conferring ADEP resistance, thereby revealing a novel type of antibiotic resistance determinant.IMPORTANCE Antibiotic acyldepsipeptides (ADEPs) represent a promising new class of potent antibiotics and, at the same time, are valuable tools to study the molecular functioning of their target, ClpP, the proteolytic core of the bacterial caseinolytic protease. Here, we present a straightforward purification procedure for ADEP1 that yields substantial amounts of the pure compound in a time- and cost-efficient manner, which is a prerequisite to conveniently study the antimicrobial effects of ADEP and the operating mode of bacterial ClpP machineries in diverse bacteria. Identification and characterization of the ADEP biosynthetic gene cluster in Streptomyces hawaiiensis NRRL 15010 enables future bioinformatics screenings for similar gene clusters and/or subclusters to find novel natural compounds with specific substructures. Most strikingly, we identified a cluster-associated clpP homolog (named clpP(ADEP)) as an ADEP resistance gene. ClpP(ADEP) constitutes a novel bacterial resistance factor that alone is necessary and sufficient to confer high-level ADEP resistance to Streptomyces across species.201931399403
11820.9445Trichlorination of a Teicoplanin-Type Glycopeptide Antibiotic by the Halogenase StaI Evades Resistance. Glycopeptide antibiotics (GPAs) include clinically important drugs used for the treatment of infections caused by Gram-positive pathogens. These antibiotics are specialized metabolites produced by several genera of actinomycete bacteria. While many GPAs are highly chemically modified, A47934 is a relatively unadorned GPA lacking sugar or acyl modifications, common to other members of the class, but which is chlorinated at three distinct sites. The biosynthesis of A47934 is encoded by a 68-kb gene cluster in Streptomyces toyocaensis NRRL 15009. The cluster includes all necessary genes for the synthesis of A47934, including two predicted halogenase genes, staI and staK In this study, we report that only one of the halogenase genes, staI, is necessary and essential for A47934 biosynthesis. Chlorination of the A47934 scaffold is important for antibiotic activity, as assessed by binding affinity for the target N-acyl-d-Ala-d-Ala. Surprisingly, chlorination is also vital to avoid activation of enterococcal and Streptomyces VanB-type GPA resistance through induction of resistance genes. Phenotypic assays showed stronger induction of GPA resistance by the dechlorinated compared to the chlorinated GPA. Correspondingly, the relative expression of the enterococcal vanA resistance gene was shown to be increased by the dechlorinated compared to the chlorinated compound. These results provide insight into the biosynthesis of GPAs and the biological function of GPA chlorination for this medically important class of antibiotic.201830275088
19230.9444N-Succinyltransferase Encoded by a Cryptic Siderophore Biosynthesis Gene Cluster in Streptomyces Modifies Structurally Distinct Antibiotics. The antibiotic desertomycin A and its previously undescribed inactive N-succinylated analogue, desertomycin X, were isolated from Streptomyces sp. strain YIM 121038. Genome sequencing and analysis readily identified the desertomycin biosynthetic gene cluster (BGC), which lacked genes encoding acyltransferases that would account for desertomycin X formation. Scouting the genome for putative N-acyltransferase genes led to the identification of a candidate within a cryptic siderophore BGC (csb) encoding a putative homologue of the N6'-hydroxylysine acetyltransferase IucB. Expression of the codon-optimized gene designated csbC in Escherichia coli yielded the recombinant protein that was able to N-succinylate desertomycin A as well as several other structurally distinct antibiotics harboring amino groups. Some antibiotics were rendered antibiotically inactive due to the CsbC-catalyzed succinylation in vitro. Unlike many known N-acyltransferases involved in antibiotic resistance, CsbC could not efficiently acetylate the same antibiotics. When expressed in E. coli, CsbC provided low-level resistance to kanamycin and ampicillin, suggesting that it may play a role in antibiotic resistance in natural habitats, where the concentration of antibiotics is usually low. IMPORTANCE In their natural habitats, bacteria encounter a plethora of organic compounds, some of which may be represented by antibiotics produced by certain members of the microbial community. A number of antibiotic resistance mechanisms have been described, including those specified by distinct genes encoding proteins that degrade, modify, or expel antibiotics. In this study, we report identification and characterization of an enzyme apparently involved in the biosynthesis of a siderophore, but also having the ability of modify and thereby inactivate a wide variety of structurally diverse antibiotics. This discovery sheds light on additional capabilities of bacteria to withstand antibiotic treatment and suggests that enzymes involved in secondary metabolism may have an additional function in the natural environment.202236040031
11740.9443Acyl depsipeptide (ADEP) resistance in Streptomyces. ADEP, a molecule of the acyl depsipeptide family, has an antibiotic activity with a unique mode of action. ADEP binding to the ubiquitous protease ClpP alters the structure of the enzyme. Access of protein to the ClpP proteolytic chamber is therefore facilitated and its cohort regulatory ATPases (ClpA, ClpC, ClpX) are not required. The consequent uncontrolled protein degradation in the cell appears to kill the ADEP-treated bacteria. ADEP is produced by Streptomyces hawaiiensis. Most sequenced genomes of Streptomyces have five clpP genes, organized as two distinct bicistronic operons, clpP1clpP2 and clpP3clpP4, and a single clpP5 gene. We investigated whether the different Clp proteases are all sensitive to ADEP. We report that ClpP1 is a target of ADEP whereas ClpP3 is largely insensitive. In wild-type Streptomyces lividans, clpP3clpP4 expression is constitutively repressed and the reason for the maintenance of this operon in Streptomyces has been elusive. ClpP activity is indispensable for survival of actinomycetes; we therefore tested whether the clpP3clpP4 operon, encoding an ADEP-insensitive Clp protease, contributes to a mechanism of ADEP resistance by target substitution. We report that in S. lividans, inactivation of ClpP1ClpP2 production or protease activity is indeed a mode of resistance to ADEP although it is neither the only nor the most frequent mode of resistance. The ABC transporter SclAB (orthologous to the Streptomyces coelicolor multidrug resistance pump SCO4959-SCO4960) is also able to confer ADEP resistance, and analysis of strains with sclAB deletions indicates that there are also other mechanisms of ADEP resistance.201121636652
843450.9443A potent and selective antimicrobial poly(amidoamine) dendrimer conjugate with LED209 targeting QseC receptor to inhibit the virulence genes of gram negative bacteria. The pandemic of multidrug-resistant Gram negative bacteria (GNB) is a worldwide healthcare concern, and very few antibiotics are being explored to match the clinical challenge. Recently, amino-terminated poly(amidoamine) (PAMAM) dendrimers have shown potential to function as broad antimicrobial agents. However, PAMAM displays a generation dependent cytotoxicity to mammalian cells and low selectivity on bacterial cells, which limits PAMAM to be developed as an antibacterial agent for systemic administration. We conjugated G3 PAMAM with LED209, a specific inhibitor of quorum sensor QseC of GNB, to generate a multifunctional agent PAMAM-LED209. Intriguingly, PAMAM-LED209 showed higher selectivity on GNB and lower cytotoxicity to mammalian cells, yet remained strong antibacterial activity. PAMAM-LED209 also inhibited virulence gene expression of GNB, and did not induce antibiotic-resistance. The present work firstly demonstrated that PAMAM-LED209 conjugate had a highly selective anti-GNB activity and low cytotoxicity, which offered a feasible strategy for combating multidrug-resistant GNB infections. FROM THE CLINICAL EDITOR: This research team demonstrated that a novel PAMAM-LED209 conjugate had highly selective activity against Gram-negative bacteria, coupled with low cytotoxicity, offering a potential strategy for combating multidrug-resistant infections.201525461286
300160.9440IS26 and the IS26 family: versatile resistance gene movers and genome reorganizers. SUMMARYIn Gram-negative bacteria, the insertion sequence IS26 is highly active in disseminating antibiotic resistance genes. IS26 can recruit a gene or group of genes into the mobile gene pool and support their continued dissemination to new locations by creating pseudo-compound transposons (PCTs) that can be further mobilized by the insertion sequence (IS). IS26 can also enhance expression of adjacent potential resistance genes. IS26 encodes a DDE transposase but has unique properties. It forms cointegrates between two separate DNA molecules using two mechanisms. The well-known copy-in (replicative) route generates an additional IS copy and duplicates the target site. The recently discovered and more efficient and targeted conservative mechanism requires an IS in both participating molecules and does not generate any new sequence. The unit of movement for PCTs, known as a translocatable unit or TU, includes only one IS26. TU formed by homologous recombination between the bounding IS26s can be reincorporated via either cointegration route. However, the targeted conservative reaction is key to generation of arrays of overlapping PCTs seen in resistant pathogens. Using the copy-in route, IS26 can also act on a site in the same DNA molecule, either inverting adjacent DNA or generating an adjacent deletion plus a circular molecule carrying the DNA segment lost and an IS copy. If reincorporated, these circular molecules create a new PCT. IS26 is the best characterized IS in the IS26 family, which includes IS257/IS431, ISSau10, IS1216, IS1006, and IS1008 that are also implicated in spreading resistance genes in Gram-positive and Gram-negative pathogens.202438436262
9570.9440NtPR1a regulates resistance to Ralstonia solanacearum in Nicotiana tabacum via activating the defense-related genes. Pathogenesis-related proteins (PRs) are associated with the development of systemic acquired resistance (SAR) against further infection enforced by fungi, bacteria and viruses. PR1a is the first PR-1 member that could be purified and characterized. Previous studies have reported its role in plants' resistance system against oomycete pathogens. However, the role of PR1a in Solanaceae plants against the bacterial wilt pathogen Ralstonia solanacearum remains unclear. To assess roles of NtPR1a in tobacco responding to R. solanacearum, we performed overexpression experiments in Yunyan 87 plants (a susceptible tobacco cultivar). The results illuminated that overexpression of NtPR1a contributed to improving resistance to R. solanacearum in tobacco Yunyan 87. Specifically speaking, NtPR1a gene could be induced by exogenous hormones like salicylic acid (SA) and pathogenic bacteria R. Solanacearum. Moreover, NtPR1a-overexpressing tobacco significantly reduced multiple of R. solanacearum and inhibited the development of disease symptoms compared with wild-type plants. Importantly, overexpression of NtPR1a activated a series of defense-related genes expression, including the hypersensitive response (HR)-associated genes NtHSR201 and NtHIN1, SA-, JA- and ET-associated genes NtPR2, NtCHN50, NtPR1b, NtEFE26, and Ntacc oxidase, and detoxification-associated gene NtGST1. In summary, our results suggested that NtPR1a-enhanced tobacco resistance to R. solanacearum may be mainly dependent on activation of the defense-related genes.201930545635
11980.9439Heterologous Expression Reveals Ancient Properties of Tei3—A VanS Ortholog from the Teicoplanin Producer Actinoplanes teichomyceticus. Glycopeptide antibiotics (GPAs) are among the most clinically successful antimicrobials. GPAs inhibit cell-wall biosynthesis in Gram-positive bacteria via binding to lipid II. Natural GPAs are produced by various actinobacteria. Being themselves Gram-positives, the GPA producers evolved sophisticated mechanisms of self-resistance to avoid suicide during antibiotic production. These self-resistance genes are considered the primary source of GPA resistance genes actually spreading among pathogenic enterococci and staphylococci. The GPA-resistance mechanism in Actinoplanes teichomyceticus—the producer of the last-resort-drug teicoplanin—has been intensively studied in recent years, posing relevant questions about the role of Tei3 sensor histidine kinase. In the current work, the molecular properties of Tei3 were investigated. The setup of a GPA-responsive assay system in the model Streptomyces coelicolor allowed us to demonstrate that Tei3 functions as a non-inducible kinase, conferring high levels of GPA resistance in A. teichomyceticus. The expression of different truncated versions of tei3 in S. coelicolor indicated that both the transmembrane helices of Tei3 are crucial for proper functioning. Finally, a hybrid gene was constructed, coding for a chimera protein combining the Tei3 sensor domain with the kinase domain of VanS, with the latter being the inducible Tei3 ortholog from S. coelicolor. Surprisingly, such a chimera did not respond to teicoplanin, but indeed to the related GPA A40926. Coupling these experimental results with a further in silico analysis, a novel scenario on GPA-resistance and biosynthetic genes co-evolution in A. teichomyceticus was hereby proposed.202236555354
11490.9436A novel enzyme conferring streptothricin resistance alters the toxicity of streptothricin D from broad-spectrum to bacteria-specific. Streptothricins (STs) produced by Streptomyces strains are broad-spectrum antibiotics. All STs consist of a carbamoylated D-gulosamine to which the beta-lysine homopolymer (1 to 7 residues) and the amide form of the unusual amino acid streptolidine (streptolidine lactam) are attached. Although many ST-resistance genes have been identified in bacteria, including clinically isolated pathogens and ST-producing Streptomyces strains, only one resistance mechanism has been identified to date. This mechanism involves the modification of the ST molecule by monoacetylation of the moiety of the beta-lysine(s). In this study, we successfully isolated a novel ST-resistance gene (sttH) from Streptomyces albulus, which is a known ST nonproducer. The in vitro analysis of SttH demonstrated that this enzyme catalyzes the hydrolysis of the amide bond of streptolidine lactam, thereby conferring ST resistance. Interestingly, the selective toxicity of ST-D possessing 3x beta-lysine moiety was altered from broad-spectrum to bacteria-specific by the hydrolysis of streptolidine lactam, although ST-F (1 x beta-lysine) was detoxified by SttH in both prokaryotes and eukaryotes (yeasts). STs have not been clinically developed due to their toxicities; however, in this study, we showed that hydrolyzed ST-D (ST-D-acid) exhibits potent antibacterial activity even when its toxicity against eukaryotic cells is reduced by SttH. This suggests that ST-D-acid is a potential candidate for clinical development or for use as a new lead compound for drug discovery.200616641084
9759100.9435Rapid emergence of resistance to broad-spectrum direct antimicrobial activity of avibactam. Avibactam (AVI) is a diazabicyclooctane (DBO) β-lactamase inhibitor used clinically in combination with ceftazidime. At concentrations higher than those typically achieved in vivo, it also has broad-spectrum direct antibacterial activity against Enterobacterales strains, including metallo-β-lactamase-producing isolates, mediated by inhibition of penicillin-binding protein 2 (PBP2). This activity has some mechanistic similarities to that of more potent novel DBOs (zidebactam and nacubactam) in late clinical development. We found that resistance to AVI emerged readily, with a mutation frequency of 2 × 10(-6) to 8 × 10(-5). Whole-genome sequencing of resistant isolates revealed a heterogeneous mutational target that permitted bacterial survival and replication despite PBP2 inhibition, in line with prior studies of PBP2-targeting drugs. While such mutations are believed to act by upregulating the bacterial stringent response, we found a similarly high mutation frequency in bacteria deficient in components of the stringent response, although we observed a different set of mutations in these strains. Although avibactam-resistant strains had increased lag time, suggesting a fitness cost that might render them less problematic in clinical infections, there was no statistically significant difference in growth rates between susceptible and resistant strains. The finding of rapid emergence of resistance to avibactam as the result of a large and complex mutational target adds to our understanding of resistance to PBP2-targeting drugs and has potential implications for novel DBOs with potent direct antibacterial activity, which are being developed with the goal of expanding cell wall-active treatment options for multidrug-resistant gram-negative infections.IMPORTANCEAvibactam (AVI) is the first in a class of novel β-lactamase inhibitor antibiotics called diazabicyclooctanes (DBOs). In addition to its ability to inhibit bacterial β-lactamase enzymes that can destroy β-lactam antibiotics, we found that AVI had direct antibacterial activity, at concentrations higher than those used clinically, against even highly multidrug-resistant bacteria. This activity is the result of inhibition of the bacterial enzyme penicillin-binding protein 2 (PBP2). Resistance to other drugs that inhibit PBP2 occurs through mutations that involve upregulation of the bacterial "stringent response" to stress. We found that bacteria developed resistance to AVI at a high rate, as a result of mutations in stringent response genes. We also found that bacteria with impairments in the stringent response could still develop resistance to AVI through different mutations. Our findings indicate the importance of studying how resistance will emerge to newer, more potent DBOs in development and early clinical use.202540503840
9053110.9435Nordihydroguaiaretic acid reverses the antibacterial activity of colistin against MCR-1-positive bacteria in vivo/in vitro by inhibiting MCR-1 activity and injuring the bacterial cell membrane. BACKGROUND: Colistin (polymyxin E) is an effective antibiotic for the treatment of most multidrug-resistant Gram-negative bacteria. However, some bacteria, including bacterial spp. belonging to the Enterobacteriaceae family, have an acquired resistance against polymyxins, which is attributed to they possess plasmid-carried resistance genes (mcr-1 and its variants). So, there is an urgent need to develop new therapeutic strategies to target broad spectrum resistant spp. from Enterobacteriaceae family in response to the loss of the protective barrier of last-line antibiotics. Here, we report the adjuvant capacity of nordihydroguaiaretic acid (NDGA) for restoring the antibacterial activity of colistin against MCR-1-positive E. coli ZJ487 in vivo/in vitro. METHODS: A checkerboard assay, time-killing analysis, isobolograms, growth curves and inducible resistance test showed the effect of NDGA combined with colistin in vitro. TLC was used to detect the inhibitory effect of NDGA on MCR-1. Colony determination and hematoxylin and eosin (HE) staining were used to assess the synergistic effect of NDGA and colistin in mice. RESULTS: Our results showed that NDGA in combination with colistin showed a synergistic bactericidal action without inducing resistance. NDGA directly inhibited MCR-1 activity and resulted in measurable injury to the bacterial cell membrane to recover the antibacterial effect of colistin. Most importantly, NDGA in combination with colistin exhibited an in vivo synergistic effect in murine peritonitis infection models, as evidenced by the survival rate of MCR-1-positive E. coli ZJ487-infected mice which increased from 6.67 to 50.0%. CONCLUSION: Our study demonstrated that NDGA effectively rescues the efficiency of colistin against MCR-positive E. coli ZJ487 by simultaneously inhibiting both, the MCR activity and the injury to the cell membrane of bacteria.202235158237
1535120.9435Complete Genome Sequencing of Acinetobacter baumannii AC1633 and Acinetobacter nosocomialis AC1530 Unveils a Large Multidrug-Resistant Plasmid Encoding the NDM-1 and OXA-58 Carbapenemases. Carbapenem-resistant Acinetobacter spp. are considered priority drug-resistant human-pathogenic bacteria. The genomes of two carbapenem-resistant Acinetobacter spp. clinical isolates obtained from the same tertiary hospital in Terengganu, Malaysia, namely, A. baumannii AC1633 and A. nosocomialis AC1530, were sequenced. Both isolates were found to harbor the carbapenemase genes bla(NDM-1) and bla(OXA-58) in a large (ca. 170 kb) plasmid designated pAC1633-1 and pAC1530, respectively, that also encodes genes that confer resistance to aminoglycosides, sulfonamides, and macrolides. The two plasmids were almost identical except for the insertion of ISAba11 and an IS4 family element in pAC1633-1, and ISAba11 along with relBE toxin-antitoxin genes flanked by inversely orientated pdif (XerC/XerD) recombination sites in pAC1530. The bla(NDM-1) gene was encoded in a Tn125 composite transposon structure flanked by ISAba125, whereas bla(OXA-58) was flanked by ISAba11 and ISAba3 downstream and a partial ISAba3 element upstream within a pdif module. The presence of conjugative genes in plasmids pAC1633-1/pAC1530 and their discovery in two distinct species of Acinetobacter from the same hospital are suggestive of conjugative transfer, but mating experiments failed to demonstrate transmissibility under standard laboratory conditions. Comparative sequence analysis strongly inferred that pAC1633-1/pAC1530 was derived from two separate plasmids in an IS1006-mediated recombination or transposition event. A. baumannii AC1633 also harbored three other plasmids designated pAC1633-2, pAC1633-3, and pAC1633-4. Both pAC1633-3 and pAC1633-4 are cryptic plasmids, whereas pAC1633-2 is a 12,651-bp plasmid of the GR8/GR23 Rep3-superfamily group that encodes the tetA(39) tetracycline resistance determinant in a pdif module.IMPORTANCE Bacteria of the genus Acinetobacter are important hospital-acquired pathogens, with carbapenem-resistant A. baumannii listed by the World Health Organization as the one of the top priority pathogens. Whole-genome sequencing of carbapenem-resistant A. baumannii AC1633 and A. nosocomialis AC1530, which were isolated from the main tertiary hospital in Terengganu, Malaysia, led to the discovery of a large, ca. 170-kb plasmid that harbored genes encoding the New Delhi metallo-β-lactamase-1 (NDM-1) and OXA-58 carbapenemases alongside genes that conferred resistance to aminoglycosides, macrolides, and sulfonamides. The plasmid was a patchwork of multiple mobile genetic elements and comparative sequence analysis indicated that it may have been derived from two separate plasmids through an IS1006-mediated recombination or transposition event. The presence of such a potentially transmissible plasmid encoding resistance to multiple antimicrobials warrants vigilance, as its spread to susceptible strains would lead to increasing incidences of antimicrobial resistance.202133504662
5034130.9433Resensitizing carbapenem- and colistin-resistant bacteria to antibiotics using auranofin. Global emergence of Gram-negative bacteria carrying the plasmid-borne resistance genes, bla(MBL) and mcr, raises a significant challenge to the treatment of life-threatening infections by the antibiotics, carbapenem and colistin (COL). Here, we identify an antirheumatic drug, auranofin (AUR) as a dual inhibitor of metallo-β-lactamases (MBLs) and mobilized colistin resistance (MCRs), two resistance enzymes that have distinct structures and substrates. We demonstrate that AUR irreversibly abrogates both enzyme activity via the displacement of Zn(II) cofactors from their active sites. We further show that AUR synergizes with antibiotics on killing a broad spectrum of carbapenem and/or COL resistant bacterial strains, and slows down the development of β-lactam and COL resistance. Combination of AUR and COL rescues all mice infected by Escherichia coli co-expressing MCR-1 and New Delhi metallo-β-lactamase 5 (NDM-5). Our findings provide potential therapeutic strategy to combine AUR with antibiotics for combating superbugs co-producing MBLs and MCRs.202033067430
748140.9432Contact-dependent growth inhibition toxins exploit multiple independent cell-entry pathways. Contact-dependent growth inhibition (CDI) systems function to deliver toxins into neighboring bacterial cells. CDI+ bacteria export filamentous CdiA effector proteins, which extend from the inhibitor-cell surface to interact with receptors on neighboring target bacteria. Upon binding its receptor, CdiA delivers a toxin derived from its C-terminal region. CdiA C-terminal (CdiA-CT) sequences are highly variable between bacteria, reflecting the multitude of CDI toxin activities. Here, we show that several CdiA-CT regions are composed of two domains, each with a distinct function during CDI. The C-terminal domain typically possesses toxic nuclease activity, whereas the N-terminal domain appears to control toxin transport into target bacteria. Using genetic approaches, we identified ptsG, metI, rbsC, gltK/gltJ, yciB, and ftsH mutations that confer resistance to specific CdiA-CTs. The resistance mutations all disrupt expression of inner-membrane proteins, suggesting that these proteins are exploited for toxin entry into target cells. Moreover, each mutation only protects against inhibition by a subset of CdiA-CTs that share similar N-terminal domains. We propose that, following delivery of CdiA-CTs into the periplasm, the N-terminal domains bind specific inner-membrane receptors for subsequent translocation into the cytoplasm. In accord with this model, we find that CDI nuclease domains are modular payloads that can be redirected through different import pathways when fused to heterologous N-terminal "translocation domains." These results highlight the plasticity of CDI toxin delivery and suggest that the underlying translocation mechanisms could be harnessed to deliver other antimicrobial agents into Gram-negative bacteria.201526305955
3003150.9431IS26-Mediated Formation of Transposons Carrying Antibiotic Resistance Genes. The IS26 transposase, Tnp26, catalyzes IS26 movement to a new site and deletion or inversion of adjacent DNA via a replicative route. The intramolecular deletion reaction produces a circular molecule consisting of a DNA segment and a single IS26, which we call a translocatable unit or TU. Recently, Tnp26 was shown to catalyze an additional intermolecular, conservative reaction between two preexisting copies of IS26 in different plasmids. Here, we have investigated the relative contributions of homologous recombination and Tnp26-catalyzed reactions to the generation of a transposon from a TU. Circular TUs containing the aphA1a kanamycin and neomycin resistance gene or the tet(D) tetracycline resistance determinant were generated in vitro and transformed into Escherichia coli recA cells carrying R388::IS26. The TU incorporated next to the IS26 in R388::IS26 forms a transposon with the insertion sequence (IS) in direct orientation. Introduction of a second TU produced regions containing both the aphA1a gene and the tet(D) determinant in either order but with only three copies of IS26. The integration reaction, which required a preexisting IS26, was precise and conservative and was 50-fold more efficient when both IS26 copies could produce an active Tnp26. When both ISs were inactivated by a frameshift in tnp26, TU incorporation was not detected in E. coli recA cells, but it did occur in E. coli recA (+) cells. However, the Tnp-catalyzed reaction was 100-fold more efficient than RecA-dependent homologous recombination. The ability of Tnp26 to function in either a replicative or conservative mode is likely to explain the prominence of IS26-bounded transposons in the resistance regions found in Gram-negative bacteria. IMPORTANCE In Gram-negative bacteria, IS26 recruits antibiotic resistance genes into the mobile gene pool by forming transposons carrying many different resistance genes. In addition to replicative transposition, IS26 was recently shown to use a novel conservative movement mechanism in which an incoming IS26 targets a preexisting one. Here, we have demonstrated how IS26-bounded class I transposons can be produced from translocatable units (TUs) containing only an IS26 and a resistance gene via the conservative reaction. TUs were incorporated next to an existing IS26, creating a class I transposon, and if the targeted IS26 is in a transposon, the product resembles two transposons sharing a central IS26, a configuration observed in some resistance regions and when a transposon is tandemly duplicated. Though homologous recombination could also incorporate a TU, Tnp26 is far more efficient. This provides insight into how IS26 builds transposons and brings additional transposons into resistance regions.201627303727
115160.9431Genome mining reveals uncommon alkylpyrones as type III PKS products from myxobacteria. Type III polyketide synthases (PKSs) are comparatively small homodimeric enzymes affording natural products with diverse structures and functions. While type III PKS biosynthetic pathways have been studied thoroughly in plants, their counterparts from bacteria and fungi are to date scarcely characterized. This gap is exemplified by myxobacteria from which no type III PKS-derived small molecule has previously been isolated. In this study, we conducted a genomic survey of myxobacterial type III PKSs and report the identification of uncommon alkylpyrones as the products of type III PKS biosynthesis from the myxobacterial model strain Myxococcus xanthus DK1622 through a self-resistance-guided screening approach focusing on genes encoding pentapetide repeat proteins, proficient to confer resistance to topoisomerase inhibitors. Using promoter-induced gene expression in the native host as well as heterologous expression of biosynthetic type III PKS genes, sufficient amounts of material could be obtained for structural elucidation and bioactivity testing, revealing potent topoisomerase activity in vitro.201930506464
3004170.9429IS26-Mediated Precise Excision of the IS26-aphA1a Translocatable Unit. We recently showed that, in the absence of RecA-dependent homologous recombination, the Tnp26 transposase catalyzes cointegrate formation via a conservative reaction between two preexisting IS26, and this is strongly preferred over replicative transposition to a new site. Here, the reverse reaction was investigated by assaying for precise excision of the central region together with a single IS26 from a compound transposon bounded by IS26. In a recA mutant strain, Tn4352, a kanamycin resistance transposon carrying the aphA1a gene, was stable. However, loss of kanamycin resistance due to precise excision of the translocatable unit (TU) from the closely related Tn4352B, leaving behind the second IS26, occurred at high frequency. Excision occurred when Tn4352B was in either a high- or low-copy-number plasmid. The excised circular segment, known as a TU, was detected by PCR. Excision required the IS26 transposase Tnp26. However, the Tnp26 of only one IS26 in Tn4352B was required, specifically the IS26 downstream of the aphA1a gene, and the excised TU included the active IS26. The frequency of Tn4352B TU loss was influenced by the context of the transposon, but the critical determinant of high-frequency excision was the presence of three G residues in Tn4352B replacing a single G in Tn4352. These G residues are located immediately adjacent to the two G residues at the left end of the IS26 that is upstream of the aphA1a gene. Transcription of tnp26 was not affected by the additional G residues, which appear to enhance Tnp26 cleavage at this end. IMPORTANCE: Resistance to antibiotics limits treatment options. In Gram-negative bacteria, IS26 plays a major role in the acquisition and dissemination of antibiotic resistance. IS257 (IS431) and IS1216, which belong to the same insertion sequence (IS) family, mobilize resistance genes in staphylococci and enterococci, respectively. Many different resistance genes are found in compound transposons bounded by IS26, and multiply and extensively antibiotic-resistant Gram-negative bacteria often include regions containing several antibiotic resistance genes and multiple copies of IS26. We recently showed that in addition to replicative transposition, IS26 can use a conservative movement mechanism in which an incoming IS26 targets a preexisting one, and this reaction can create these regions. This mechanism differs from that of all the ISs examined in detail thus far. Here, we have continued to extend understanding of the reactions carried out by IS26 by examining whether the reverse precise excision reaction is also catalyzed by the IS26 transposase.201526646012
5097180.9428Comparing Graph Sample and Aggregation (SAGE) and Graph Attention Networks in the Prediction of Drug-Gene Associations of Extended-Spectrum Beta-Lactamases in Periodontal Infections and Resistance. INTRODUCTION: Gram-negative bacteria exhibit more antibiotic resistance than gram-positive bacteria due to their cell wall structure and composition differences. Porins, or protein channels in these bacteria, can allow small, hydrophilic antibiotics to diffuse, affecting their susceptibility. Mutations in porin protein genes can also impair antibiotic entry. Predicting drug-gene associations of extended-spectrum beta-lactamases (ESBLs) is crucial as they confer resistance to beta-lactam antibiotics, challenging the treatment of infections. This aids clinicians in selecting suitable treatments, optimizing drug usage, enhancing patient outcomes, and controlling antibiotic resistance in healthcare settings. Graph-based neural networks can predict drug-gene associations in periodontal infections and resistance. The aim of the study was to predict drug-gene associations of ESBLs in periodontal infections and resistance. METHODS: The study focuses on analyzing drug-gene associations using probes and drugs. The data was converted into graph language, assigning nodes and edges for drugs and genes. Graph neural networks (GNNs) and similar algorithms were implemented using Google Colab and Python. Cytoscape and CytoHubba are open-source software platforms used for network analysis and visualization. GNNs were used for tasks like node classification, link prediction, and graph-level prediction. Three graph-based models were used: graph convolutional network (GCN), Graph SAGE, and graph attention network (GAT). Each model was trained for 200 epochs using the Adam optimizer with a learning rate of 0.01 and a weight decay of 5e-4. RESULTS: The drug-gene association network has 57 nodes, 79 edges, and a 2.730 characteristic path length. Its structure, organization, and connectivity are analyzed using the GCN and Graph SAGE, which show high accuracy, precision, recall, and an F1-score of 0.94. GAT's performance metrics are lower, with an accuracy of 0.68, precision of 0.47, recall of 0.68, and F1-score of 0.56, suggesting that it may not be as effective in capturing drug-gene relationships. CONCLUSION: Compared to ESBLs, both GCN and Graph SAGE demonstrate excellent performance with accuracy, precision, recall, and an F1-score of 0.94. These results indicate that GCN and Graph SAGE are highly effective in predicting drug-gene associations related to ESBLs. GCN and Graph SAGE outperform GAT in predicting drug-gene associations for ESBLs. Improvements include data augmentation, regularization, and cross-validation. Ethical considerations, fairness, and open-source implementations are crucial for future research in precision periodontal treatment.202439347119
661190.9428A Putative Bacterial ABC Transporter Circumvents the Essentiality of Signal Peptidase. The type I signal peptidase of Staphylococcus aureus, SpsB, is an attractive antibacterial target because it is essential for viability and extracellularly accessible. We synthesized compound 103, a novel arylomycin-derived inhibitor of SpsB with significant potency against various clinical S. aureus strains (MIC of ~1 µg/ml). The predominant clinical strain USA300 developed spontaneous resistance to compound 103 with high frequency, resulting from single point mutations inside or immediately upstream of cro/cI, a homolog of the lambda phage transcriptional repressor cro These cro/cI mutations led to marked (>50-fold) overexpression of three genes encoding a putative ABC transporter. Overexpression of this ABC transporter was both necessary and sufficient for resistance and, notably, circumvented the essentiality of SpsB during in vitro culture. Mutation of its predicted ATPase gene abolished resistance, suggesting a possible role for active transport; in these bacteria, resistance to compound 103 occurred with low frequency and through mutations in spsB Bacteria overexpressing the ABC transporter and lacking SpsB were capable of secreting a subset of proteins that are normally cleaved by SpsB and instead were cleaved at a site distinct from the canonical signal peptide. These bacteria secreted reduced levels of virulence-associated proteins and were unable to establish infection in mice. This study reveals the mechanism of resistance to a novel arylomycin derivative and demonstrates that the nominal essentiality of the S. aureus signal peptidase can be circumvented by the upregulation of a putative ABC transporter in vitro but not in vivo IMPORTANCE: The type I signal peptidase of Staphylococcus aureus (SpsB) enables the secretion of numerous proteins by cleavage of the signal peptide. We synthesized an SpsB inhibitor with potent activity against various clinical S. aureus strains. The predominant S. aureus strain USA300 develops resistance to this inhibitor by mutations in a novel transcriptional repressor (cro/cI), causing overexpression of a putative ABC transporter. This mechanism promotes the cleavage and secretion of various proteins independently of SpsB and compensates for the requirement of SpsB for viability in vitro However, bacteria overexpressing the ABC transporter and lacking SpsB secrete reduced levels of virulence-associated proteins and are unable to infect mice. This study describes a bacterial resistance mechanism that provides novel insights into the biology of bacterial secretion.201627601569