Biology subjects

Cardona, S. T.

Publications and source records attributed to Cardona, S. T..

7 recordsLinked to original sources

bioRxiv2025

Burkholderia Genes Involved in Extracellular Bioplastic Degradation Revealed by Tn-seq and CRISPR-Cas

Bioplastics represent promising alternatives to petroleum-based plastics, yet their biodegradation remains insufficiently understood. Identifying bacteria capable of degrading bioplastics extracellularly could enhance end-of-life management practices. To investigate Burkholderias capacities for the degradation of medium-chain-length polyhydroxyalkanoate (mcl-PHA), we screened a panel of Burkholderia strains and identified such capacity in strains of B. gladioli, B. multivorans, and B. vietnamiensis. To elucidate the genetic basis of this activity, we performed transposon mutagenesis followed by activity-based screening and Tn-seq on B. vietnamiensis LMG 16232. Disrupted genetic elements in transposon mutants with negative phenotypes were further investigated using a CRISPR-associated transposase (CAST) system. These included a lipase production gene cluster, encoding two putative triacylglycerol lipases and a chaperone, and genes coding for a a A24 family peptidase, a TetR/AcrR family transcriptional regulator and a type II secretion system (T2SS) protein. Complete loss or reduced extracellular mcl-PHA depolymerase activity was observed in the CAST mutants, validating their involvement in mcl-PHA degradation. Notably, only one of the two lipases encoded in the lipase production gene cluster was responsible for mcl-PHA degradation, suggesting that while lipases may show substrate promiscuity, lipase functional annotation does not necessarily imply mcl-PHA depolymerization. Docking experiments using the amino acid sequences of the two lipases supported these findings. Together, we identify a gene coding for an active mcl-PHA depolymerase in B. vietnamiensis and demonstrate the power of combining activity-based screening, Tn-seq, and CAST to rapidly establish gene-to-function links. ImportanceDue to their versatile metabolism, Burkholderia strains play critical roles in degradation of multiple compounds in the environment. Here we show that several Burkholderia species can extracellularly degrade medium-chain-length polyhydroxyalkanoates (mcl-PHAs), a promising class of bioplastics. By integrating transposon mutagenesis, Tn-seq, and CRISPR-associated transposase (CAST) technologies, we identify and validate key genetic determinants involved in mcl-PHA degradation in B. vietnamiensis. These genes encode a lipase, a secretion system component, and regulatory factors, underscoring the complexity and specificity of microbial bioplastic degradation pathways. These findings not only advance our understanding of PHA biodegradation but also identifies B. vietnamiensis as as a source of enzymes capable of degrading extracellular mcl-PHA.

microbiology↗

bioRxiv2024

Rationally Designed Pooled CRISPRi-seq Uncovers a Novel Inhibitor of Bacterial Peptidyl-tRNA Hydrolase

Pooled knockdown libraries of essential genes are useful tools for elucidating the mechanisms of action of antibacterial compounds, a pivotal step in antibiotic discovery. However, achieving genomic coverage of antibacterial targets poses a challenge due to the uneven proliferation of knockdown mutants during pooled growth, leading to the unintended loss of important targets. To overcome this issue, we describe the construction of CIMPLE (CRISPRi-mediated pooled library of essential genes), a rationally designed pooled knockdown library built in a model antibiotic-resistant bacteria, Burkholderia cenocepacia. By analyzing growth parameters of clonal knockdown populations of an arrayed CRISPRi library, we predicted strain depletion levels during pooled growth and adjusted mutant relative abundance, approaching genomic coverage of antibacterial targets during antibiotic exposure. We first benchmarked CIMPLE by chemical-genetic profiling of known antibacterials, then applied it to an uncharacterized bacterial growth inhibitor from a new class. CRISPRi-Seq with CIMPLE, followed by biochemical validation, revealed that the novel compound targets the peptidyl-tRNA hydrolase (Pth). Overall, CIMPLE leverages the advantages of arrayed and pooled CRISPRi libraries to uncover unexplored targets for antibiotic action. SummaryBacterial mutant libraries in which antibiotic targets are downregulated are useful tools to functionally characterize novel antimicrobials. These libraries are used for chemical-genetic profiling as target-compound interactions can be inferred by differential fitness of mutants during pooled growth. Mutants that are functionally related to the antimicrobial mode of action are usually depleted from the pool upon exposure to the drug. Although powerful, this method can fail when the unequal proliferation of mutant strains before exposure causes mutants to fall below the detection level in the library pool. To address this issue, we constructed an arrayed essential gene mutant library (EGML) in the antibiotic-resistant bacterium Burkholderia cenocepacia using CRISPR interference (CRISPRi) and analyzed the growth parameters of individual mutant strains. We then modelled depletion levels during pooled growth and used the model to rationally design an optimized CRISPR interference-mediated pooled library of essential genes (CIMPLE). By adjusting the initial inoculum of the knockdown mutants, we achieved coverage of the bacterial essential genome with mutant sensitization. We exposed CIMPLE to a recently discovered antimicrobial of a novel class and discovered it inhibits the peptidyl-tRNA hydrolase, an essential bacterial enzyme. In summary, we demonstrate the utility of CIMPLE and CRISPRi-Seq to uncover the mechanism of action of novel antimicrobial compounds. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/592284v2_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@b09cf9org.highwire.dtl.DTLVardef@1119fc9org.highwire.dtl.DTLVardef@116b9forg.highwire.dtl.DTLVardef@1b0ce3a_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Cell envelope structural and functional contributions to antibiotic resistance in Burkholderia cenocepacia

Antibiotic activity is limited by the physical construction of the Gram-negative cell envelope. Species of the Burkholderia cepacia complex (Bcc) are known as intrinsically multidrug-resistant opportunistic pathogens with low permeability cell envelopes. Here, we re-examined a previously performed chemical-genetic screen of barcoded transposon mutants in B. cenocepacia K56-2, focusing on cell envelope structural and functional processes. We identified structures mechanistically important for resistance to singular and multiple antibiotic classes. For example, the polymeric O-antigen was important for resistance to cationic antibiotics, while defects in peptidoglycan precursor synthesis specifically increased susceptibility to cycloserine and revealed a new putative amino acid racemase. Susceptibility to novobiocin, avibactam, and the LpxC inhibitor, PF-04753299, was linked to the BpeAB-OprB efflux pump, suggesting these drugs are substrates for this pump in B. cenocepacia. Additionally, disruption of the periplasmic disulfide bond formation system caused pleiotropic defects on outer membrane integrity and {beta}-lactamase activity. Our findings highlight the layering of resistance mechanisms in the structure and function of the cell envelope. Consequently, we point out processes that can be targeted for developing antibiotic potentiators. ImportanceThe Gram-negative cell envelope is a double-layered physical barrier that protects cells from extracellular stressors, such as antibiotics. The Burkholderia cell envelope is known to contain additional modifications that reduce permeability. We investigated Burkholderia cell envelope factors contributing to antibiotic resistance from a genome-wide view by re-examining data from a transposon mutant library exposed to an antibiotic panel. We identified susceptible phenotypes for defects in structures and functions in the outer membrane, periplasm, and cytoplasm. Overall, we show that resistance linked to the cell envelope is multifaceted and provides new targets for the development of antibiotic potentiators.

microbiology↗

bioRxiv2022

Profiling cell envelope-antibiotic interactions reveals vulnerabilities to β-lactams in a multidrug-resistant bacterium

The cell envelope of the Gram-negative 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, such as those living with cystic fibrosis. To systematically identify cell envelope-associated resistance and susceptibility determinants at the genome level, we constructed a high-density, randomly-barcoded transposon mutant library in the clinical isolate B. cenocepacia K56-2 and exposed it to a panel of more than twenty cell envelope-targeting antibiotics. By quantifying relative mutant fitness with BarSeq, followed by validation with CRISPR-interference, we profiled over a hundred new functional associations and identified novel mediators of antibiotic susceptibility in the Bcc cell envelope. We revealed new connections between {beta}-lactam susceptibility, peptidoglycan synthesis, and blockages in undecaprenyl phosphate metabolism, which highlight a vulnerability in sharing this lipid intermediate. We then show that the clinically relevant synergy of the {beta}-lactam/{beta}-lactamase inhibitor combination ceftazidime/avibactam is primarily mediated by inhibition of the PenB carbapenemase. Importantly, we found that avibactam more strongly potentiates the activity of aztreonam and meropenem than ceftazidime in a panel of Bcc clinical isolates. Finally, we characterize for first time in the Bcc the iron and receptor-dependent activity of the novel siderophore-cephalosporin antibiotic, cefiderocol. Overall, our work has implications for antibiotic target prioritization, and for using additional combinations of {beta}-lactam/{beta}-lactamase inhibitors that can extend the utility of our current clinical arsenal of antibacterial therapies. Author SummaryWhile the Gram-negative cell envelope is a major barrier to antibiotic action, we have an incomplete picture of how each component contributes to antibiotic resistance. To answer this question from a genome-wide perspective in a model of antibiotic-resistant bacteria, we constructed a library of randomly-barcoded transposon mutants in Burkholderia cenocepacia and exposed it to a panel of diverse cell envelope-targeting antibiotics. We identified individual genes and whole pathways associated with antibiotic resistance, including several novel players. Focusing on clinically relevant antibiotics, our strategy dissected how the synergy of the important {beta}-lactam/{beta}-lactamase inhibitor combination ceftazidime/avibactam primarily depends upon inhibition of a single {beta}-lactamase. Additionally, we are the first to characterise the molecular basis of the antibiotic mechanism of cefiderocol in Burkholderia, a new and very potent siderophore-cephalosporin conjugate drug. Encouragingly, we found that cefiderocol susceptibility was greatest at physiological iron concentrations. Overall, we highlight several avenues that can be taken to enhance or develop new therapeutic strategies against Burkholderia infection.

microbiology↗

bioRxiv2021

New auranofin analogs with antibacterial properties against Burkholderia clinical isolates

Bacteria of the genus Burkholderia include pathogenic Burkholderia mallei, Burkholderia pseudomallei and the Burkholderia cepacia complex (Bcc). These Gram-negative pathogens have intrinsic drug resistance, which makes treatment of infections difficult. Bcc affects individuals with cystic fibrosis (CF) and the species B. cenocepacia is associated with one of the worst clinical outcomes. Following the repurposing of auranofin as an antibacterial against Gram-positive bacteria, we previously synthetized auranofin analogs with activity against Gram-negatives. In this work, we show that two auranofin analogs, MS-40S and MS-40, have antibiotic activity against Burkholderia clinical isolates. The compounds are bactericidal against B. cenocepacia and kill stationary-phase cells and persisters without selecting for multistep resistance. Caenorhabditis elegans and Galleria mellonella tolerated high concentrations of MS-40S and MS-40, demonstrating that these compounds have low toxicity in these model organisms. In summary, we show that MS-40 and MS-40S have the potential to be effective therapeutic options to treat Burkholderia infections.

microbiology↗

bioRxiv2019

Phenylacetyl-CoA, not phenylacetic acid, mediates quorum sensing-regulated virulence in Burkholderia cenocepacia

During phenylalanine catabolism, phenylacetic acid (PAA) is converted to phenylacetyl-CoA (PAA-CoA) by a ligase, PaaK, and then epoxidized by a multicomponent monooxygenase, PaaABCDE, before further degradation to the TCA cycle. In the opportunistic pathogen Burkholderia cenocepacia, loss of paaABCDE attenuates virulence factor expression, which is under control of the LuxIR-like quorum sensing system, CepIR. To further investigate the link between CepIR-regulated virulence and PAA catabolism, we created knockout mutants of the first step of the pathway (PAA-CoA synthesis by PaaK) and characterized them in comparison to a paaABCDE mutant using liquid chromatography/mass spectrometry (LC-MS/MS) and virulence assays. We found that while loss of PaaABCDE decreased virulence, deletion of the paaK genes resulted in a more virulent phenotype than the wild type strain. Deletion of either paaK or paaABCDE led to higher levels of released PAA but no differences in internal accumulation, compared to wild type. While we found no evidence of direct cepIR downregulation by PAA-CoA or PAA, a low virulence cepR mutant reverted to a virulent phenotype upon removal of the paaK genes. On the other hand, removal of paaABCDE in the cepR mutant did not impact its attenuated phenotype. Together, our results suggest an indirect role for PAA-CoA in supressing B. cenocepacia CepIR-activated virulence.\n\nImportanceThe opportunistic pathogen Burkholderia cenocepacia uses a chemical signal process called quorum sensing (QS) to produce virulence factors. In B. cenocepacia, QS relies on the presence of the transcriptional regulator CepR, which upon binding QS signal molecules, activates virulence. In this work, we found that even in the absence of CepR, B. cenocepacia can elicit a pathogenic response if phenylacetyl-CoA, an intermediate of the phenylacetic acid degradation pathway, is not produced. Instead, accumulation of phenylacetyl-CoA appears to attenuate pathogenicity. Therefore, we have discovered that it is possible to trigger virulence in the absence of CepR, challenging the classical view of activation of virulence by this QS mechanism. Our work provides new insight into the relationship between metabolism and virulence in opportunistic bacteria. We propose that, in the event that QS signaling molecules cannot accumulate to trigger a pathogenic response, a metabolic signal can still activate virulence in B. cenocepacia.

microbiology↗

bioRxiv2019

A broad-host-range CRISPRi toolkit for silencing gene expression in Burkholderia

Genetic tools are critical to dissecting the mechanisms governing cellular processes, from fundamental physiology to pathogenesis. Members of the genus Burkholderia have potential for biotechnological applications but can also cause disease in humans with a debilitated immune system. The lack of suitable genetic tools to edit Burkholderia GC-rich genomes has hampered the exploration of useful capacities and the understanding of pathogenic features. To address this, we have developed CRISPR interference (CRISPRi) technology for gene silencing in Burkholderia, testing it in B. cenocepacia, B. multivorans and B. thailandensis. Tunable expression was provided by placing a codon-optimized dcas9 from Streptococcus pyogenes under control of a rhamnose-inducible promoter. As a proof of concept, the paaABCDE operon controlling genes necessary for phenylacetic acid degradation was targeted by plasmid-borne sgRNAs, resulting in near complete inhibition of growth on phenylacetic acid as the sole carbon source. This was supported by reductions in paaA mRNA expression. The utility of CRISPRi to probe other functions at the single cell level was demonstrated by knocking down phbC and fliF, which dramatically reduces polyhydroxybutyrate granule accumulation and motility, respectively. As a hallmark of the mini-CTX system is the broad host-range of integration, we putatively identified 67 genera of Proteobacteria that might be amenable to modification with our CRISPRi toolkit. Our CRISPRi tool kit provides a simple and rapid way to silence gene expression to produce an observable phenotype. Linking genes to functions with CRISPRi will facilitate genome editing with the goal of enhancing biotechnological capabilities while reducing Burkholderias pathogenic arsenal.\n\nAuthor contributionsSTC conceived the idea and design of the research; AMH designed and cloned the dCas9 constructs; AMH and ASMZ designed the sgRNAs, assessed knockdown phenotypes, processed data, and wrote and edited the manuscript; TJL performed RT-qPCR analysis and edited the manuscript; STC supervised the work and provided financial support.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC=\"FIGDIR/small/618413v3_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (22K):\norg.highwire.dtl.DTLVardef@b00aa5org.highwire.dtl.DTLVardef@18ae038org.highwire.dtl.DTLVardef@86af96org.highwire.dtl.DTLVardef@71bac4_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗