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Biochemistry Sep 2023Because purine nucleotides are essential for all life, differences between how microbes and humans metabolize purines can be exploited for the development of...
Because purine nucleotides are essential for all life, differences between how microbes and humans metabolize purines can be exploited for the development of antimicrobial therapies. While humans biosynthesize purine nucleotides in a 10-step pathway, most microbes utilize an additional 11th enzymatic activity. The human enzyme, aminoimidazole ribonucleotide (AIR) carboxylase generates the product 4-carboxy-5-aminoimidazole ribonucleotide (CAIR) directly. Most microbes, however, require two separate enzymes, a synthetase (PurK) and a mutase (PurE), and proceed through the intermediate, N-CAIR. Toward the development of therapeutics that target these differences, we have solved crystal structures of the N-CAIR mutase of the human pathogens (LpPurE) and (BcPurE) and used a structure-guided approach to identify inhibitors. Analysis of the structures reveals a highly conserved fold and active site architecture. Using this data, and three additional structures of PurE enzymes, we screened a library of FDA-approved compounds and identified a set of 25 candidates for further analysis. Among these, we identified several new PurE inhibitors with micromolar IC values. Several of these compounds, including the α-blocker Alfuzosin, inhibit the microbial PurE enzymes much more effectively than the human homologue. These structures and the newly described PurE inhibitors are valuable tools to aid in further studies of this enzyme and provide a foundation for the development of compounds that target differences between human and microbial purine metabolism.
Topics: Humans; Ribonucleotides; Escherichia coli; Intramolecular Transferases; Purine Nucleotides
PubMed: 37552766
DOI: 10.1021/acs.biochem.2c00705 -
Nature Communications Aug 2023The cell envelope of Gram-negative bacteria belonging to the Burkholderia cepacia complex (Bcc) presents unique restrictions to antibiotic penetration. As a consequence,...
The cell envelope of Gram-negative bacteria belonging to the Burkholderia cepacia complex (Bcc) presents unique restrictions to antibiotic penetration. As a consequence, Bcc species are notorious for causing recalcitrant multidrug-resistant infections in immunocompromised individuals. Here, we present the results of a genome-wide screen for cell envelope-associated resistance and susceptibility determinants in a Burkholderia cenocepacia clinical isolate. For this purpose, we construct a high-density, randomly-barcoded transposon mutant library and expose it to 19 cell envelope-targeting antibiotics. By quantifying relative mutant fitness with BarSeq, followed by validation with CRISPR-interference, we profile over a hundred functional associations and identify mediators of antibiotic susceptibility in the Bcc cell envelope. We reveal connections between β-lactam susceptibility, peptidoglycan synthesis, and blockages in undecaprenyl phosphate metabolism. The synergy of the β-lactam/β-lactamase inhibitor combination ceftazidime/avibactam is primarily mediated by inhibition of the PenB carbapenemase. In comparison with ceftazidime, avibactam more strongly potentiates the activity of aztreonam and meropenem in a panel of Bcc clinical isolates. Finally, we characterize in Bcc the iron and receptor-dependent activity of the siderophore-cephalosporin antibiotic, cefiderocol. Our work has implications for antibiotic target prioritization, and for using additional combinations of β-lactam/β-lactamase inhibitors that can extend the utility of current antibacterial therapies.
Topics: Humans; Anti-Bacterial Agents; Ceftazidime; beta-Lactamase Inhibitors; Meropenem; beta-Lactamases; Drug Combinations; Microbial Sensitivity Tests
PubMed: 37558695
DOI: 10.1038/s41467-023-40494-5 -
Journal of Bacteriology Oct 2023Antibiotic resistance in bacteria is a growing global concern and has spurred increasing efforts to find alternative therapeutics, such as the use of bacterial viruses,...
Antibiotic resistance in bacteria is a growing global concern and has spurred increasing efforts to find alternative therapeutics, such as the use of bacterial viruses, or bacteriophages. One promising approach is to use phages that not only kill pathogenic bacteria but also select phage-resistant survivors that are newly sensitized to traditional antibiotics, in a process called "phage steering." Members of the bacterial genus , which includes various human pathogens, are highly resistant to most antimicrobial agents, including serum immune components, antimicrobial peptides, and polymixin-class antibiotics. However, the application of phages in combination with certain antibiotics can produce synergistic effects that more effectively kill pathogenic bacteria. Herein, we demonstrate that serum resistance is due to intact lipopolysaccharide (LPS) and membranes, and phage-induced resistance altering LPS structure can enhance bacterial sensitivity not only to immune components in serum but also to membrane-associated antibiotics such as colistin. IMPORTANCE Bacteria frequently encounter selection pressure from both antibiotics and lytic phages, but little is known about the interactions between antibiotics and phages. This study provides new insights into the evolutionary trade-offs between phage resistance and antibiotic sensitivity. The creation of phage resistance through changes in membrane structure or lipopolysaccharide composition can simultaneously be a major cause of antibiotic sensitivity. Our results provide evidence of synergistic therapeutic efficacy in phage-antibiotic interactions and have implications for the future clinical use of phage steering in phage therapy applications.
Topics: Humans; Bacteriophages; Burkholderia cenocepacia; Anti-Bacterial Agents; Lipopolysaccharides; Virulence
PubMed: 37791751
DOI: 10.1128/jb.00196-23 -
Applied Microbiology and Biotechnology Jun 2023Small non-coding RNAs (sRNAs) are key regulators of post-transcriptional gene expression in bacteria. Despite the identification of hundreds of bacterial sRNAs, their...
Small non-coding RNAs (sRNAs) are key regulators of post-transcriptional gene expression in bacteria. Despite the identification of hundreds of bacterial sRNAs, their roles on bacterial physiology and virulence remain largely unknown, as is the case of bacteria of the Burkholderia cepacia complex (Bcc). Bcc is a group of opportunistic pathogens with relatively large genomes that can cause lethal lung infections amongst cystic fibrosis (CF) patients. To characterise sRNAs expressed by Bcc bacteria when infecting a host, the nematode Caenorhabditis elegans was used as an infection model by the epidemic CF strain B. cenocepacia J2315. A total of 108 new and 31 previously described sRNAs with a predicted Rho independent terminator were identified, most of them located on chromosome 1. RIT11b, a sRNA downregulated under C. elegans infection conditions, was shown to directly affect B. cenocepacia virulence, biofilm formation, and swimming motility. RIT11b overexpression reduced the expression of the direct targets dusA and pyrC, involved in biofilm formation, epithelial cell adherence, and chronic infections in other organisms. The in vitro direct interaction of RIT11b with the dusA and pyrC messengers was demonstrated by electrophoretic mobility shift assays. To the best of our knowledge this is the first report on the functional characterization of a sRNA directly involved in B. cenocepacia virulence. KEY POINTS: • 139 sRNAs expressed by B. cenocepacia during C. elegans infection were identified • The sRNA RIT11b affects B. cenocepacia virulence, biofilm formation, and motility • RIT11b directly binds to and regulates dusA and pyrC mRNAs.
Topics: Animals; Humans; Burkholderia cenocepacia; Caenorhabditis elegans; Burkholderia cepacia complex; RNA, Small Untranslated; Burkholderia Infections
PubMed: 37097504
DOI: 10.1007/s00253-023-12530-3 -
Microbiology Spectrum May 2024Across the Burkholderia genus -linked protein glycosylation is highly conserved. While the inhibition of glycosylation has been shown to be detrimental for virulence in...
UNLABELLED
Across the Burkholderia genus -linked protein glycosylation is highly conserved. While the inhibition of glycosylation has been shown to be detrimental for virulence in complex species, such as , little is known about how specific glycosylation sites impact protein functionality. Within this study, we sought to improve our understanding of the breadth, dynamics, and requirement for glycosylation across the glycoproteome. Assessing the glycoproteome across different culture media using complementary glycoproteomic approaches, we increase the known glycoproteome to 141 glycoproteins. Leveraging this repertoire of glycoproteins, we quantitively assessed the glycoproteome of using Data-Independent Acquisition (DIA) revealing the glycoproteome is largely stable across conditions with most glycoproteins constitutively expressed. Examination of how the absence of glycosylation impacts the glycoproteome reveals that the protein abundance of only five glycoproteins (BCAL1086, BCAL2974, BCAL0525, BCAM0505, and BCAL0127) are altered by the loss of glycosylation. Assessing Δ (ΔBCAL0525), Δ (ΔBCAL0127), and ΔBCAM0505 strains, we demonstrate the loss of FliF, and to a lesser extent MotB, mirror the proteomic effects observed in the absence of glycosylation in Δ. While both MotB and FliF are essential for motility, we find loss of glycosylation sites in MotB or FliF does not impact motility supporting these sites are dispensable for function. Combined this work broadens our understanding of the glycoproteome supporting that the loss of glycoproteins in the absence of glycosylation is not an indicator of the requirement for glycosylation for protein function.
IMPORTANCE
is an opportunistic pathogen of concern within the Cystic Fibrosis community. Despite a greater appreciation of the unique physiology of gained over the last 20 years a complete understanding of the proteome and especially the O-glycoproteome, is lacking. In this study, we utilize systems biology approaches to expand the known glycoproteome as well as track the dynamics of glycoproteins across growth phases, culturing media and in response to the loss of glycosylation. We show that the glycoproteome of is largely stable across conditions and that the loss of glycosylation only impacts five glycoproteins including the motility associated proteins FliF and MotB. Examination of MotB and FliF shows, while these proteins are essential for motility, glycosylation is dispensable. Combined this work supports that glycosylation can be dispensable for protein function and may influence protein properties beyond stability.
PubMed: 38709084
DOI: 10.1128/spectrum.00346-24 -
Annals of Medicine Dec 2024complex () is a bacterial group with 'natural' multi-antimicrobial resistance. This complex has generated epidemic outbreaks across the world. In people with cystic... (Review)
Review
complex () is a bacterial group with 'natural' multi-antimicrobial resistance. This complex has generated epidemic outbreaks across the world. In people with cystic fibrosis (CF), can cause severe lung infections that lead to accelerated lung damage, which can be complicated by necrotizing pneumonia accompanied by high fevers, leucocytosis, and bacteraemia, which commonly causes fatal outcomes. Specifically, infection by is considered an exclusion criterion for lung transplantation. The species of exhibit both genetic and phenotypic hypervariability that complicate their accurate microbiological identification. Automated methods such as MALDI-TOF can err in the determination of species. Their slow growth even in selective agars and the absence of international consensuses on the optimal conditions for their isolation make early diagnosis a difficult challenge to overcome. The absence of correlations between antibiograms and clinical results has resulted in the absence of standardized cut-off values of antimicrobial susceptibility, a fact that brings a latent risk since incorrect antibiotic therapy can induce the selection of more aggressive variants that worsen the clinical picture of the host, added to the absence of a clear therapeutic guide for the eradication of pulmonary infections by in patients with CF, resulting in frequently ineffective treatments. There is an urgent need to standardize methods and diagnostic tools that would allow an early and accurate diagnosis, as well as to perform clinical studies of the effectiveness of available antibiotics to eradicate infections, which would allow us to establish standardized therapeutic schemes for -infected patients.
Topics: Humans; Cystic Fibrosis; Burkholderia cepacia complex; Lung Transplantation; Anti-Bacterial Agents; Bacteremia
PubMed: 38261606
DOI: 10.1080/07853890.2024.2307503 -
Applied and Environmental Microbiology Oct 2023Outer membrane vesicle (OMV)-delivered quinolone signal (PQS) plays a critical role in cell-cell communication in . However, the functions and mechanisms of...
Outer membrane vesicle (OMV)-delivered quinolone signal (PQS) plays a critical role in cell-cell communication in . However, the functions and mechanisms of membrane-enclosed PQS in interspecies communication in microbial communities are not clear. Here, we demonstrate that PQS delivered by both OMVs from and liposome reduces the competitiveness of , which usually shares the same niche in the lungs of cystic fibrosis patients, by interfering with quorum sensing (QS) in through the LysR-type regulator ShvR. Intriguingly, we found that ShvR regulates the production of the QS signals cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactone (AHL) by directly binding to the promoters of signal synthase-encoding genes. Perception of PQS influences the regulatory activity of ShvR and thus ultimately reduces QS signal production and virulence in . Our findings provide insights into the interspecies communication mediated by the membrane-enclosed QS signal among bacterial species residing in the same microbial community.IMPORTANCEQuorum sensing (QS) is a ubiquitous cell-to-cell communication mechanism. Previous studies showed that mainly employs cis-2-dodecenoic acid (BDSF) and N-acyl homoserine lactone (AHL) QS systems to regulate biological functions and virulence. Here, we demonstrate that quinolone signal (PQS) delivered by outer membrane vesicles from or liposome attenuates virulence by targeting the LysR-type regulator ShvR, which regulates the production of the QS signals BDSF and AHL in a. Our results not only suggest the important roles of membrane-enclosed PQS in interspecies and interkingdom communications but also provide a new perspective on the use of functional nanocarriers loaded with QS inhibitors for treating pathogen infections.
Topics: Humans; Quorum Sensing; Virulence; Acyl-Butyrolactones; Liposomes; Bacterial Proteins; Burkholderia cenocepacia; Pseudomonas aeruginosa; Gene Expression Regulation, Bacterial
PubMed: 37796010
DOI: 10.1128/aem.01184-23 -
British Journal of Biomedical Science 2023is an opportunistic pathogen that is primarily associated with severe respiratory infections in people with cystic fibrosis. These bacteria have significant intrinsic...
is an opportunistic pathogen that is primarily associated with severe respiratory infections in people with cystic fibrosis. These bacteria have significant intrinsic resistance to antimicrobial therapy, and there is a need for more effective treatments. Bacterial zinc uptake and homeostasis systems are attractive targets for new drugs, yet our understanding of how bacteria acquire and utilise zinc remains incomplete. Here we have used RNA-sequencing and differential gene expression analysis to investigate how H111 is able to survive in zinc poor environments, such as those expected to be encountered within the host. The data shows that 201 genes are significantly differentially expressed when zinc supply is severely limited. Included in the 85 upregulated genes, are genes encoding a putative ZnuABC high affinity zinc importer, two TonB-dependent outer membrane receptors that may facilitate zinc uptake across the outer cell membrane, and a COG0523 family zinc metallochaperone. Amongst the 116 downregulated genes, are several zinc-dependent enzymes suggesting a mechanism of zinc sparring to reduce the cells demand for zinc when bioavailability is low.
Topics: Humans; Burkholderia cenocepacia; Zinc; Gene Expression Regulation, Bacterial; Burkholderia Infections
PubMed: 37822354
DOI: 10.3389/bjbs.2023.11597 -
Microbiology Spectrum Dec 2023causes severe infections in cystic fibrosis (CF) patients. CF patients are prone to reoccurring infections due to the accumulation of mucus in their lungs, where...
causes severe infections in cystic fibrosis (CF) patients. CF patients are prone to reoccurring infections due to the accumulation of mucus in their lungs, where bacteria can adhere and grow. Some of the antibiotics that inhibit in the laboratory are not effective for CF patients. A major contributor to poor clinical outcomes is that antibiotic testing in laboratories occurs under conditions that are different from those of sputum. CF sputum may be acidic and have increased concentrations of iron and zinc. Here, we used a medium that mimics CF sputum and found that acidic pH decreased the activity of many of the antibiotics used against . In addition, we assessed susceptibility to more than 500 antibiotics and found four active compounds against . Our findings give a better understanding of the lack of a relationship between susceptibility testing and the clinical outcome when treating infections.
Topics: Humans; Burkholderia cenocepacia; Cystic Fibrosis; Burkholderia Infections; Anti-Bacterial Agents; Hydrogen-Ion Concentration
PubMed: 37966209
DOI: 10.1128/spectrum.02731-23 -
Journal of Proteome Research Jun 2023The process of O-linked protein glycosylation is highly conserved across the genus and mediated by the oligosaccharyltransferase PglL. While our understanding of...
The process of O-linked protein glycosylation is highly conserved across the genus and mediated by the oligosaccharyltransferase PglL. While our understanding of glycoproteomes has increased in recent years, little is known about how species respond to modulations in glycosylation. Utilizing CRISPR interference (CRISPRi), we explored the impact of silencing of O-linked glycosylation across four species of ; K56-2, MSMB375, ATCC17616, and E264. Proteomic and glycoproteomic analyses revealed that while CRISPRi enabled inducible silencing of PglL, this did not abolish glycosylation, nor recapitulate phenotypes such as proteome changes or alterations in motility that are associated with glycosylation null strains, despite inhibition of glycosylation by nearly 90%. Importantly, this work also demonstrated that CRISPRi induction with high levels of rhamnose leads to extensive impacts on the proteomes, which without appropriate controls mask the impacts specifically driven by CRISPRi guides. Combined, this work revealed that while CRISPRi allows the modulation of O-linked glycosylation with reductions up to 90% at a phenotypic and proteome levels, appears to demonstrate a robust tolerance to fluctuations in glycosylation capacity.
Topics: Glycosylation; Proteome; Proteomics; Clustered Regularly Interspaced Short Palindromic Repeats; Burkholderia
PubMed: 36995114
DOI: 10.1021/acs.jproteome.2c00790