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All sourcesNASA NTRSDOE OSTI
DOE OSTI2023

Moving Beyond Mortality: Development and Application of a Desirability of Outcome Ranking (DOOR) Endpoint for Hospital-Acquired Bacterial Pneumonia and Ventilator-Associated Bacterial Pneumonia

Abstract Background Hospital-acquired and ventilator-associated bacterial pneumonia (HABP/VABP) are frequently caused by multidrug-resistant organisms. Patient-centered endpoints in clinical trials are needed to develop new antibiotics for HABP/VABP. Desirability of outcome ranking (DOOR) is a paradigm for the design, analysis, and interpretation of clinical trials based on a patient-centered, benefit-risk evaluation. Methods A multidisciplinary committee created an infectious diseases DOOR endpoint customized for HABP/VABP, incorporating infectious complications, serious adverse events, and mortality. We applied this to 2 previously completed, large randomized controlled trials for HABP/VABP. ZEPHyR compared vancomycin to linezolid and VITAL compared linezolid to tedizolid. For each trial, we evaluated the DOOR distribution and probability, including DOOR component and partial credit analyses. We also applied DOOR in subgroup analyses. Results In both trials, the HABP/VABP DOOR demonstrated similar overall clinical outcomes between treatment groups. In ZEPHyR, the probability that a participant treated with linezolid would have a more desirable outcome than a participant treated with vancomycin was 50.2% (95% confidence interval [CI], 45.1%­−55.3%). In VITAL, the probability that a participant treated with tedizolid would have a more desirable outcome than a participant treated with linezolid was 48.7% (95% CI, 44.8%–52.6%). The DOOR component analysis revealed that participants treated with tedizolid had a less desirable outcome than those treated with linezolid when considering clinical response alone. However, participants with decreased renal function had improved overall outcomes with tedizolid. Conclusions The HABP/VABP DOOR provided more granular information about clinical outcomes than is typically presented in clinical trials. HABP/VABP trials would benefit from prospectively using DOOR.

Immunology↗
DOE OSTI2020

Current challenges and future opportunities of phage therapy

ABSTRACT Antibiotic resistance is a major public health challenge worldwide, whose implications for global health might be devastating if novel antibacterial strategies are not quickly developed. As natural predators of bacteria, (bacterio)phages may play an essential role in escaping such a dreadful future. The rising problem of antibiotic resistance has revived the interest in phage therapy and important developments have been achieved over the last years. But where do we stand today and what can we expect from phage therapy in the future? This is the question we set to answer in this review. Here, we scour the outcomes of human phage therapy clinical trials and case reports, and address the major barriers that stand in the way of using phages in clinical settings. We particularly address the potential of phage resistance to hinder phage therapy and discuss future avenues to explore the full capacity of phage therapy.

Pires, Diana P.↗
DOE OSTI2020

Linezolid-resistant (Tn 6246 :: fexB - poxtA ) Enterococcus faecium strains colonizing humans and bovines on different continents: similarity without epidemiological link

Abstract Objectives poxtA is the most recently described gene conferring acquired resistance to linezolid, a relevant antibiotic for treating enterococcal infections. We retrospectively screened for poxtA in diverse enterococci and aimed to characterize its genetic/genomic contexts. Methods poxtA was screened by PCR in 812 enterococci from 458 samples (hospitals/healthy humans/wastewater/animals/retail food) obtained in Portugal/Angola/Tunisia (1996–2019). Antimicrobial susceptibility testing was performed for 13 antibiotics (EUCAST/CLSI). poxtA stability (∼500 generations), transfer (filter mating), clonality (SmaI-PFGE) and location (S1-PFGE/hybridization) were tested. WGS (Illumina-HiSeq) was performed for clonal representatives. Results poxtA was detected in Enterococcus faecium from six samples (1.3%): a healthy human (rectal swab) in Porto, Portugal (ST32/2001); four farm cows (milk) in Mateur, Tunisia (ST1058/2015); and a hospitalized patient (faeces) in Matosinhos, Portugal (ST1058/2015). All expressed resistance to linezolid (MIC = 8 mg/L), chloramphenicol, tetracycline and erythromycin, with variable resistance to ciprofloxacin and streptomycin. ST1058-poxtA-carrying isolates from Tunisia and Portugal differed by two SNPs and had similar plasmid content. poxtA, located in an IS1216-flanked Tn6246-like element, co-hybridized with fexB on one or more plasmids per isolate (one to three plasmids of 30–100 kb), was stable after several generations and transferred only from ST1058. ST1058 strains carried resistance/virulence genes (Efmqnr/acm) possibly induced under selective quinolone treatment. Conclusions poxtA has been circulating in Portugal since at least 2001, corresponding to the oldest description worldwide to date. We also extend the reservoir of poxtA to bovines. The similar linezolid-resistant poxtA-carrying strains colonizing humans and livestock on different continents, and without a noticeable relationship, suggests a recent transmission event or convergent evolution of E. faecium populations in different hosts and geographic regions.

Freitas, Ana R.↗
DOE OSTI2021

Impact of PrsA on membrane lipid composition during daptomycin-resistance-mediated β-lactam sensitization in clinical MRSA strains

Abstract Background The cyclic anionic lipopeptide daptomycin is used in the treatment of severe infections caused by Gram-positive pathogens, including MRSA. Daptomycin resistance, although rare, often results in treatment failure. Paradoxically, in MRSA, daptomycin resistance is usually accompanied by a concomitant decrease in β-lactam resistance in what is known as the ‘see-saw effect’. This resensitization is extensively used for the treatment of MRSA infections, by combining daptomycin and a β-lactam antibiotic, such as oxacillin. Objectives We aimed: (i) to investigate the combined effects of daptomycin and oxacillin on the lipid composition of the cellular membrane of both daptomycin-resistant and -susceptible MRSA strains; and (ii) to assess the involvement of the post-translocational protein PrsA, which plays an important role in oxacillin resistance in MRSA, in membrane lipid composition and remodelling during daptomycin resistance/β-lactam sensitization. Results The combination of microbiological and biochemical studies, with fluorescence microscopy using lipid probes, showed that the lipid composition and surface charge of the daptomycin-resistant cells exposed to daptomycin/oxacillin were dependent on antibiotic concentration and directly associated with PrsA, which influenced cardiolipin remodelling/relocation. Conclusions Our findings show that PrsA, in addition to its post-transcriptional role in the maturation of PBP 2a, is a key mediator of cell membrane remodelling connected to the see-saw effect and may have a key role in the resensitization of daptomycin-resistant strains to β-lactams, such as oxacillin.

de Carvalho, Carla C. C. R.↗
DOE OSTI2022

Expanding the genetic engineering toolbox for the metabolically flexible acetogen Eubacterium limosum

Abstract Acetogenic bacteria are an increasingly popular choice for producing fuels and chemicals from single carbon (C1) substrates. Eubacterium limosum is a promising acetogen with several native advantages, including the ability to catabolize a wide repertoire of C1 feedstocks and the ability to grow well on agar plates. However, despite its promise as a strain for synthetic biology and metabolic engineering, there are insufficient engineering tools and molecular biology knowledge to leverage its native strengths for these applications. To capitalize on the natural advantages of this organism, here we extended its limited engineering toolbox. We evaluated the copy number of three common plasmid origins of replication and devised a method of controlling copy number and heterologous gene expression level by modulating antibiotic concentration. We further quantitatively assessed the strength and regulatory tightness of a panel of promoters, developing a series of well-characterized vectors for gene expression at varying levels. In addition, we developed a black/white colorimetric genetic reporter assay and leveraged the high oxygen tolerance of E. limosum to develop a simple and rapid transformation protocol that enables benchtop transformation. Finally, we developed two new antibiotic selection markers—doubling the number available for this organism. These developments will enable enhanced metabolic engineering and synthetic biology work with E. limosum.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Protoplast fusion as a strategy to increase ploidy in Rhodotorula toruloides for strain development

Rhodotorula toruloides is a red oleaginous yeast with growing commercial interest because of its hardiness and exceptional lipid production capacity. Because it is a basidiomycete yeast with a complex life cycle, many of the classical breeding methods used with ascomycetes are unavailable for strain improvement. However, we have been able to construct polyploid yeast by fusing protoplasts of parents with the same mating type. Fusing of Y-6985 (A2) and Y-48190 (A2), which had been transformed with complementary antibiotic markers, led to the recovery of two diploids and one triploid. The stability of the fusion yeasts was tested by plating them on non-selective medium after several growth cycles under antibiotics and then testing five colonies per strain for nuclear DNA contents using flow cytometry and standard cell cycle analysis: the triploid and one diploid were stable. Fusants inherited their mitochondria from a single parent, which was demonstrated using restriction fragment length polymorphism (RFLP) of mitochondrial DNA. The phenotypic properties of the parents and fusants were compared in glucose fed-batch bioreactor studies and cellulosic sugar batch cultures. The final lipid titers for the fed-batch cultures were 24.9–39.7 g/L with Y-6985 and the diploid and triploid performing the best and worst, respectively. The fusants demonstrated intermediate hardiness for growth on hydrolysate prepared with dilute-acid pretreated switchgrass and were outperformed by Y-48190. Unlike one of the haploid parents, the fusants grew in 70% v/v concentrated hydrolysate. Furthermore, they did not grow as fast as the other haploid. In this study, a modernized protoplast fusion method is resurrected a useful tool for strain development in this yeast, which is complementary with other available methods.

Lignocellulose↗
DOE OSTI2022

Structural basis for the inability of chloramphenicol to inhibit peptide bond formation in the presence of A-site glycine

Ribosome serves as a universal molecular machine capable of synthesis of all the proteins in a cell. Small-molecule inhibitors, such as ribosome-targeting antibiotics, can compromise the catalytic versatility of the ribosome in a context-dependent fashion, preventing transpeptidation only between particular combinations of substrates. Classic peptidyl transferase center inhibitor chloramphenicol (CHL) fails to inhibit transpeptidation reaction when the incoming A site acceptor substrate is glycine, and the molecular basis for this phenomenon is unknown. Here, we present a set of high-resolution X-ray crystal structures that explain why CHL is unable to inhibit peptide bond formation between the incoming glycyl-tRNA and a nascent peptide that otherwise is conducive to the drug action. Our structures reveal that fully accommodated glycine residue can co-exist in the A site with the ribosome-bound CHL. Moreover, binding of CHL to a ribosome complex carrying glycyl-tRNA does not affect the positions of the reacting substrates, leaving the peptide bond formation reaction unperturbed. These data exemplify how small-molecule inhibitors can reshape the A-site amino acid binding pocket rendering it permissive only for specific amino acid residues and rejective for the other substrates extending our detailed understanding of the modes of action of ribosomal antibiotics.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Applying a polysaccharide lyase from Stenotrophomonas maltophilia to disrupt alginate exopolysaccharide produced by Pseudomonas aeruginosa clinical isolates

Pseudomonas aeruginosa is considered one of the most challenging, drug-resistant, opportunistic pathogens partly due to its ability to synthesize robust biofilms. Biofilm is a mixture of extracellular polymeric substances (EPS) that encapsulates microbial cells, leading to immune evasion, antibiotic resistance, and thus higher risk of infection. In the cystic fibrosis lung environment, P. aeruginosa undergoes a mucoid transition, defined by overproduction of the exopolysaccharide alginate. Alginate encapsulation results in bacterial resistance to antibiotics and the host immune system. Given its role in airway inflammation and chronic infection, alginate is an obvious target to improve treatment for P. aeruginosa infection. Previously, we demonstrated polysaccharide lyase Smlt1473 from Stenotrophomonas maltophilia strain k279a can catalyze the degradation of multiple polyuronides in vitro, including D-mannuronic acid (poly-ManA). Poly-ManA is a major constituent of P. aeruginosa alginate, suggesting that Smlt1473 could have potential application against multidrug-resistant P. aeruginosa and perhaps other microbes with related biofilm composition. In this study, we demonstrate that Smlt1473 can inhibit and degrade alginate from P. aeruginosa. Additionally, we show that tested P. aeruginosa strains are dominant in acetylated alginate and that all but one have similar M-to-G ratios. These results indicate that variation in enzyme efficacy among the isolates is not primarily due to differences in total EPS or alginate chemical composition. Overall, these results demonstrate Smlt1473 can inhibit and degrade P. aeruginosa alginate and suggest that other factors including rate of EPS production, alginate sequence/chain length, or non-EPS components may explain differences in enzyme efficacy.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Bacterial Secondary Metabolite Biosynthetic Potential in Soil Varies with Phylum, Depth, and Vegetation Type

Microbes produce specialized compounds to compete or communicate with one another and their environment. Some of these compounds, such as antibiotics, are also useful in medicine and biotechnology. Historically, most antibiotics have come from soil bacteria which can be isolated and grown in the lab. Though the vast majority of soil bacteria cannot be isolated, we can extract their genetic information and search it for genes which produce these specialized compounds. These understudied soil bacteria offer a wealth of potential for the discovery of new and important microbial products. Here, we identified the ability to produce these specialized compounds in diverse and novel bacteria in a range of soil environments. This information will be useful to other researchers who wish to isolate certain products. Beyond their use to humans, understanding the distribution and function of microbial products is key to understanding microbial communities and their effects on biogeochemical cycles.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Physiological roles of an Acinetobacter -specific σ factor

ABSTRACT The Gram-negative pathogen Acinetobacter baumannii is considered an “urgent threat” to human health due to its propensity to become antibiotic resistant. Understanding the distinct regulatory paradigms used by A. baumannii to mitigate cellular stresses may uncover new therapeutic targets. Many γ-proteobacteria use the extracytoplasmic function (ECF) σ factor, RpoE, to invoke envelope homeostasis networks in response to stress. Acinetobacter species contain the poorly characterized ECF “SigAb”; however, it is unclear if SigAb has the same physiological role as RpoE. Here, we show that SigAb is a metal stress-responsive ECF that appears unique to Acinetobacter species and distinct from RpoE-like ECFs. We combine promoter mutagenesis, motif scanning, and chromatin immunoprecipitation-sequencing (ChIP-seq) to define the direct SigAb regulon, which consists of genes encoding SigAb itself, the stringent response mediator, RelA, and the uncharacterized small RNA, “SabS.” However, RNA-seq of strains overexpressing SigAb revealed a large, indirect regulon containing hundreds of genes. Metal resistance genes are key elements of the indirect regulon, as CRISPRi knockdown of sigAb or sabS resulted in increased copper sensitivity and excess copper-induced SigAb-dependent transcription. Furthermore, we found that two uncharacterized genes in the sigAb operon, “ aabA ” and “ aabB ,” have anti-SigAb activity. Finally, employing a targeted Tn-seq approach that uses CRISPR-associated transposons, we show that sigAb , aabA , and aabB are important for fitness even during optimal growth conditions. Our work reveals new physiological roles for SigAb and SabS, provides a novel approach for assessing gene fitness, and highlights the distinct regulatory architecture of A. baumannii . IMPORTANCE Acinetobacter baumannii is a hospital-acquired pathogen, and many strains are resistant to multiple antibiotics. Understanding how A. baumannii senses and responds to stress may uncover novel routes to treat infections. Here, we examine how the Acinetobacter -specific transcription factor, SigAb, mitigates stress. We find that SigAb directly regulates only a small number of genes, but indirectly controls hundreds of genes that have substantial impacts on cell physiology. We show that SigAb is required for maximal growth, even during optimal conditions, and is acutely required during growth in the presence of elevated copper. Given that copper toxicity plays roles in pathogenesis and on copper-containing surfaces in hospitals, we speculate that SigAb function may be important in clinically relevant contexts.

Bacon, Emily E. (ORCID:0000000180907689)↗
DOE OSTI2023

A previously uncharacterized divisome-associated lipoprotein, DalA, is needed for normal cell division in Rhodobacterales

ABSTRACT The bacterial cell envelope is a key subcellular compartment with important roles in antibiotic resistance, nutrient acquisition, and cell morphology. We seek to gain a better understanding of proteins that contribute to the function of the cell envelope in Alphaproteobacteria . Using Rhodobacter sphaeroides , we show that a previously uncharacterized protein, RSP_1200, is an outer membrane (OM) lipoprotein that non-covalently binds peptidoglycan (PG). Using a fluorescently tagged version of this protein, we find that RSP_1200 undergoes a dynamic repositioning during the cell cycle and is enriched at the septum during cell division. We show that the position of RSP_1200 mirrors the location of FtsZ rings, leading us to propose that RSP_1200 is a newly identified component of the R. sphaeroides ’ divisome. Additional support for this hypothesis includes the co-precipitation of RSP_1200 with FtsZ, the Pal protein, and several predicted PG L,D-transpeptidases. We also find that a ∆ RSP_1200 mutation leads to defects in cell division, sensitivity to PG-active antibiotics, and results in the formation of OM protrusions at the septum during cell division. Based on these results, we propose to name RSP_1200 DalA (for division-associated lipoprotein A) and postulate that DalA serves as a scaffold to position or modulate the activity of PG transpeptidases that are needed to form envelope invaginations during cell division. We find that DalA homologs are present in members of the Rhodobacterales order within Alphaproteobacteria . Therefore, we propose that further analysis of this and related proteins will increase our understanding of the macromolecular machinery and proteins that participate in cell division in Gram-negative bacteria. IMPORTANCE Multi-protein complexes of the bacterial cell envelope orchestrate key processes like growth, division, biofilm formation, antimicrobial resistance, and production of valuable compounds. The subunits of these protein complexes are well studied in some bacteria, and differences in their composition and function are linked to variations in cell envelope composition, shape, and proliferation. However, some envelope protein complex subunits have no known homologs across the bacterial phylogeny. We find that Rhodobacter sphaeroides RSP_1200 is a newly identified lipoprotein (DalA) and that loss of this protein causes defects in cell division and changes the sensitivity to compounds, affecting cell envelope synthesis and function. We find that DalA forms a complex with proteins needed for cell division, binds the cell envelope polymer peptidoglycan, and colocalizes with enzymes involved in the assembly of this macromolecule. The analysis of DalA provides new information on the cell division machinery in this and possibly other Alphaproteobacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Synthetic overlapping genes stabilize genetic systems

Overlapping genes—wherein two different proteins are translated from alternative reading frames of the same DNA sequence—provide a means to stabilize an engineered gene by directly linking its evolutionary fate with that of an overlapping gene. However, creating overlapping gene pairs is challenging, as it requires redesigning both protein products to accommodate overlap constraints. Here, we present a new “overlapping, alternate-frame insertion” (OAFI) method for creating synthetic overlapping genes by inserting an “inner” gene, encoded in an alternate frame, into a flexible region of an “outer” gene. Using OAFI, we create new overlapping gene pairs of genetic reporters and bacterial toxins within an antibiotic resistance gene. We show that both the inner and outer genes retain function despite redesign, with translation of the inner gene influenced by its overlap position in the outer gene. Importantly, we show that, despite these inner gene sequences not contributing to outer gene function, selection for the outer gene alters the permitted inactivating mutations in the inner gene, and that overlapping toxins can restrict horizontal gene transfer of the antibiotic resistance gene. Overall, OAFI offers a versatile tool for synthetic biology, expanding the applications of overlapping genes in gene stabilization and biocontainment.

Biological and medical sciences↗
DOE OSTI2021

Competitive Exclusion and Metabolic Dependency among Microorganisms Structure the Cellulose Economy of an Agricultural Soil

Microorganisms that degrade cellulose utilize extracellular reactions that yield free by-products which can promote interactions with noncellulolytic organisms. We hypothesized that these interactions determine the ecological and physiological traits governing the fate of cellulosic carbon (C) in soil. We performed comparative genomics with genome bins from a shotgun metagenomic-stable isotope probing experiment to characterize the attributes of cellulolytic and noncellulolytic taxa accessing 13 C from cellulose. We hypothesized that cellulolytic taxa would exhibit competitive traits that limit access, while noncellulolytic taxa would display greater metabolic dependency, such as signatures of adaptive gene loss. We tested our hypotheses by evaluating genomic traits indicative of competitive exclusion or metabolic dependency, such as antibiotic production, growth rate, surface attachment, biomass degrading potential, and auxotrophy. The most 13 C-enriched taxa were cellulolytic Cellvibrio (Gammaproteobacteria) and Chaetomium (Ascomycota), which exhibited a strategy of self-sufficiency (prototrophy), rapid growth, and competitive exclusion via antibiotic production. Auxotrophy was more prevalent in cellulolytic Actinobacteria than in cellulolytic Proteobacteria, demonstrating differences in dependency among cellulose degraders. Noncellulolytic taxa that accessed 13 C from cellulose (Planctomycetales, Verrucomicrobia, and Vampirovibrionales) were also more dependent, as indicated by patterns of auxotrophy and 13 C labeling (i.e., partial labeling or labeling at later stages). Major 13 C-labeled cellulolytic microbes (e.g., Sorangium, Actinomycetales, Rhizobiales, and Caulobacteraceae) possessed adaptations for surface colonization (e.g., gliding motility, hyphae, attachment structures) signifying the importance of surface ecology in decomposing particulate organic matter. Our results demonstrated that access to cellulosic C was accompanied by ecological trade-offs characterized by differing degrees of metabolic dependency and competitive exclusion.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Characterization of Pseudomonas aeruginosa and Acinetobacter calcoaceticus-baumannii complex traumatic wound isolates

Healing of traumatic wounds is complicated by infecting pathogens, with Pseudomonas aeruginosa and members of the Acinetobacter calcoaceticus-baumannii complex among the most common infectious agents. However, a full understanding of genotypic and phenotypic differences between circulating wound isolates is lacking. To address this, traumatic wounds were sampled at Emory University Hospital, and 24 isolates were characterized; we focused on P. aeruginosa and Acinetobacter due to their prevalence and tendency for antibiotic resistance. Even though these species are renowned for antibiotic resistance, only two of the tested isolates could be classified as multidrug resistant. Whole-genome sequencing and analysis revealed that isolates from different patients were genetically distinct; however, longitudinal isolates from the same patient were closely related and appeared to represent chronic colonization by the same strain. Phylogenetic analysis revealed that laboratory strains (PAO1 and AB5075) that were isolated decades previously and from different locations grouped closely to subsets of the recent wound isolates. Given the importance of biofilm formation in infection, the ability of the isolates to form biofilms was assessed; all isolates formed biofilms but showed temporal and magnitude differences. Analysis of a subset of isolates revealed that planktonic P. aeruginosa was resistant to human serum-mediated killing. While the same was true for the majority of Acinetobacter isolates, one showed planktonic sensitivity that was abrogated when grown in a biofilm. Taken together, these data reveal genotypic and phenotypic differences in circulating isolates of P. aeruginosa and A. baumannii.

Acinetobacter↗
DOE OSTI2024

Characterization of two affinity matured Anti-Yersinia pestis F1 human antibodies with medical countermeasure potential

Yersinia pestis , the causative agent of plague and a biological threat agent, presents an urgent need for novel medical countermeasures due to documented cases of naturally acquired antibiotic resistance and potential person-to-person spread during a pneumonic infection. Immunotherapy has been proposed as a way to circumvent current and future antibiotic resistance. Here, we describe the development and characterization of two affinity matured human antibodies (αF1Ig AM2 and αF1Ig AM8) that promote survival of mice after exposure to aerosolized Y . pestis . We share details of the error prone PCR and yeast display technology-based affinity maturation process that we used. The resultant matured antibodies have nanomolar affinity for Y . pestis F1 antigen, are produced in high yield, and are resilient to 37°C stress for up to 6 months. Importantly, in vitro assays using a murine macrophage cell line demonstrated that αF1Ig AM2 and αF1Ig AM8 are opsonic. Even more importantly, in vivo studies using pneumonic plague mouse models showed that 100% of the mice receiving 500 μg of IgGs αF1Ig AM2 and αF1Ig AM8 survived lethal challenge with aerosolized Y . pestis CO92. Combined, these results provide evidence of the quality and robustness of αF1Ig AM2 and αF1Ig AM8 and support their development as potential medical countermeasures against plague.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Diversified Therapeutic Phage Cocktails from Close Relatives of the Target Bacterium

This project tackles the antibiotic resistance crisis, developing a new method for discovering numerous efficacious bacteriophages for therapeutic cocktails against bacterial pathogens. The phage therapy approach to infectious disease, recently rekindled in U.S. medicine, requires numerous phages for each bacterial pathogen. Our approach 1) uses Sandia-unique software to identify dormant phages (prophages) integrated into bacterial chromosomes, 2) identifies prophage-laden bacteria that are close relatives of the target pathogenic strain to be killed, and 3) engineers away properties of these phages that are undesirable for therapy. We have perfected our phage-finding software, implemented our phage therapy strategy by targeting the pathogen Pseudomonas aeruginosa, and prepared new software to assist the phage engineering. We then turned toward Burkholderia pathogens, aiming to overcome the difficulty to transform these bacteria with a novel phage conjugation approach. Our work demonstrates the validity of a new approach to phage therapy for killing antibiotic resistant pathogens.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Enabling generalized phage therapy to treat secondary infections in COVID-19 (CRADA Final Report)

COVID-19 is a global health threat, with 32 millions infected and more than 500,000 deaths in the U.S. alone. A key aspect of COVID-19 deaths is bacterial secondary infections, with Staphylococcus aureus, Acinetobacter baumannii, and Pseudomonas aeruginosa identified as some of the major causative pathogens. Phage therapy, i.e., the administration of bacteriophages (viruses infecting these bacterial pathogens) to patients offers a complementary solution to antibiotics with multiple key advantages. Felix Biotechnology’s is developing a high-throughput platform to identify phages that not only kill pathogens, but also drive targeted trade-off of pathogenic phenotypes such as reduced virulence and antibiotic resistance, at a fraction of the time and R&D cost of any competitor. In this project, collaboration with scientists from the Lawrence Berkeley National Laboratory enabled Felix Biotechnology to establish robust analysis pipeline to investigate phage-host interactions in complex samples. These should enable a quick and robust identification of promising phages and key genetic determinants for therapy applications, paving the way towards a streamlined and safe use of phages in the clinic.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Omics-Based Comparison of Fungal Virulence Genes, Biosynthetic Gene Clusters, and Small Molecules in Penicillium expansum and Penicillium chrysogenum

Penicillium expansum is a ubiquitous pathogenic fungus that causes blue mold decay of apple fruit postharvest, and another member of the genus, Penicillium chrysogenum, is a well-studied saprophyte valued for antibiotic and small molecule production. While these two fungi have been investigated individually, a recent discovery revealed that P. chrysogenum can block P. expansum-mediated decay of apple fruit. To shed light on this observation, we conducted a comparative genomic, transcriptomic, and metabolomic study of two P. chrysogenum (404 and 413) and two P. expansum (Pe21 and R19) isolates. Global transcriptional and metabolomic outputs were disparate between the species, nearly identical for P. chrysogenum isolates, and different between P. expansum isolates. Further, the two P. chrysogenum genomes revealed secondary metabolite gene clusters that varied widely from P. expansum. This included the absence of an intact patulin gene cluster in P. chrysogenum, which corroborates the metabolomic data regarding its inability to produce patulin. Additionally, a core subset of P. expansum virulence gene homologues were identified in P. chrysogenum and were similarly transcriptionally regulated in vitro. Molecules with varying biological activities, and phytohormone-like compounds were detected for the first time in P. expansum while antibiotics like penicillin G and other biologically active molecules were discovered in P. chrysogenum culture supernatants. Our findings provide a solid omics-based foundation of small molecule production in these two fungal species with implications in postharvest context and expand the current knowledge of the Penicillium-derived chemical repertoire for broader fundamental and practical applications.

Bartholomew, Holly P. (ORCID:0000000292726399)↗