Biology subjects

Piedra, F.-A.

Publications and source records attributed to Piedra, F.-A..

10 recordsLinked to original sources

bioRxiv2025

Functional genomics in a microbe that degrades and metabolizes PET plastic

Piscinibacter sakaiensis (formerly Ideonella sakaiensis) was the first bacterial species known to both completely degrade and assimilate polyethylene terephthalate (PET). However, the absence of efficient genetic tools has limited direct engineering of this organism, forcing most efforts to rely on heterologous expression of PET-degrading enzymes in model hosts. Here, we establish foundational genetic tools to engineer P. sakaiensis. We identify a functional plasmid origin of replication, multiple new selectable markers, and a transposon system for the strain. We use these tools to construct a genome-wide, barcoded transposon mutant library for pooled high-throughput functional screens. We apply this mutant library to growth on PET and identify metabolic and physiological genes that impact PET biodegradation. We also use this library to reveal mutants with improved DNA uptake for genome engineering. Together, these advances provide a platform for functional genomics in P. sakaiensis and positions this naturally evolved plastic-degrading bacterium as an engineerable chassis for synthetic biology and sustainable materials research.

microbiology↗

bioRxiv2024

Eco-Microbiology: Discovering Biochemical Enhancers of PET Biodegradation by Piscinibacter sakaiensis

The scale of plastic pollution boggles the mind. Nearly 400 megatons of virgin plastics are produced annually, with an environmental release rate of 80 percent; and plastic waste including micro- and nanoplastics are associated with a plethora of problems. The naturally evolved abilities of plastic-degrading and consuming microbes offer a starting point for generating sustainable and eco-centric solutions to plastic pollution. Here we developed an iterative discovery procedure coupling faster quasi-high-throughput polyethylene terephthalate (PET) dependent bioactivity screens with longer-term PET biodegradation assays to find small molecule and ionic boosters of PET consumption by the bacterium Piscinibacter sakaiensis. We discovered multiple hits supporting greater than 2-fold enhancement of PET biodegradation - with hits belonging to a small but heterogeneous set of compounds and mixtures, suggesting upregulation of PET consumption via multiple paths. This work has the potential to advance the creation of a fermentation-based process for solving PET plastic pollution. ImportancePlastic pollution is an urgent environmental issue. In addition, micro- and nanoplastics (MNPs) have become an acute source of worry with discoveries of the global distribution and transport of MNPs, their presence within a diversity of organisms including common foodstuffs and human tissues, and their potential association with declining fertility and various disease states. Solutions are needed and the microbial world offers abundant help via naturally evolved biodegraders of plastic waste. We created a non-genetic method to accelerate polyethylene terephthalate (PET) plastic biodegradation by Piscinibacter sakaiensis, a bacterium that evolved to slowly but completely consume PET. Our method entails a combination of plastic-dependent bioactivity screens and slower biodegradation tests to find extrinsic biochemical stimulators of PET biodegradation. The conditions we found boost PET biodegradation by over two-fold and provide a foundation for further studies to realize a fermentation-based process needed to solve PET plastic pollution.

microbiology↗

bioRxiv2023

Progress toward the development of an effective vaccine for Extraintestinal pathogenic E. coli (ExPEC): The application of the multiple-protein subunits vaccine in different murine models

Extraintestinal pathogenic E. coli (ExPEC) is the primary Gram-negative bacterial pathogen, and the leading cause of life-threatening sepsis and urinary tract infections (UTI) in adults. The emergence and increasing prevalence of multidrug-resistance (MDR) ExPEC strains have led to considerable treatment failures, increased hospitalization rates, morbidity, and mortality. A prophylactic vaccine against ExPEC has the potential to reduce severe infection-related morbidity and mortality, helping to address the escalating antimicrobial resistance (AMR) crisis worldwide. The -hemolysin (HlyA) is a critical, frequently detected secreted cytotoxic virulence factor in ExPEC, with HlyA-expressing ExPEC strains correlating with increased severity and infection dissemination in clinical levels. In this study, we assessed the protective efficacy of pro-HlyA (the inactive and immature precursor of HlyA) and Dual-Hit (a combination of pro-HlyA and SinH-3, the previously reported immunoglobulin-like domain-3 of the invasin-like autotransporter protein SinH), as ExPEC vaccine candidates. We demonstrated that immunizing mice with pro-HlyA or Dual-Hit significantly reduced bacterial burden and increased survival rates against pandemic ExPEC sequence type strains, ST73 (CFT073) and ST95 (UTI89), in the model of bacteremia and mortality. Both pro-HlyA or Dual-Hit immunizations also provided significant protection against UTI89 colonization in the bladder in the murine UTI model. Furthermore, vaccination with Dual-Hit provided enduring and robust protection against a mixture of ten typical high-virulent sequence types of ExPEC strains, resulting in a promising broad-spectrum vaccine candidate. These findings suggest that pro-HlyA and Dual-Hit might serve as highly effective vaccine targets and highlight the potential of these vaccine candidates for further development and evaluation.

microbiology↗

bioRxiv2023

Examining intra-host genetic variation of RSV by short read high-throughput sequencing

Every viral infection entails an evolving population of viral genomes. High-throughput sequencing technologies can be used to characterize such populations, but to date there are few published examples of such work. In addition, mixed sequencing data are sometimes used to infer properties of infecting genomes without discriminating between genome-derived reads and reads from the much more abundant, in the case of a typical active viral infection, transcripts. Here we apply capture probe-based short read high-throughput sequencing to nasal wash samples taken from a previously described group of adult hematopoietic cell transplant (HCT) recipients naturally infected with respiratory syncytial virus (RSV). We separately analyzed reads from genomes and transcripts for the levels and distribution of genetic variation by calculating per position Shannon entropies. Our analysis reveals a low level of genetic variation within the RSV infections analyzed here, but with interesting differences between genomes and transcripts in 1) average per sample Shannon entropies; 2) the genomic distribution of variation hotspots; and 3) the genomic distribution of hotspots encoding alternative amino acids. In all, our results suggest the importance of separately analyzing reads from genomes and transcripts when interpreting high-throughput sequencing data for insight into intra-host viral genome replication, expression, and evolution.

genomics↗

bioRxiv2022

Modeling nonsegmented negative-strand RNA virus (NNSV) transcription with ejective polymerase collisions and biased diffusion

Infections by nonsegmented negative-strand RNA viruses (NNSV) are widely thought to entail gradient gene expression from the well-established existence of a single promoter at the 3 end of the viral genome and the assumption of constant transcriptional attenuation between genes. But multiple recent studies show viral mRNA levels in infections by respiratory syncytial virus (RSV), a major human pathogen and member of NNSV, that are inconsistent with a simple gradient. Here we integrate known and newly predicted phenomena into a biophysically reasonable model of NNSV transcription. Our model succeeds in capturing published observations of RSV and vesicular stomatitis virus (VSV) mRNA levels. We therefore propose a novel understanding of NNSV transcription based on the possibility of ejective polymerase-polymerase collisions and, in the case of RSV, biased polymerase diffusion.

biophysics↗

bioRxiv2021

The human nose organoid respiratory virus model: an ex-vivo human challenge model to study RSV and SARS-CoV-2 pathogenesis and evaluate therapeutics

There is an unmet need for pre-clinical models to understand the pathogenesis of human respiratory viruses; and predict responsiveness to immunotherapies. Airway organoids can serve as an ex-vivo human airway model to study respiratory viral pathogenesis; however, they rely on invasive techniques to obtain patient samples. Here, we report a non-invasive technique to generate human nose organoids (HNOs) as an alternate to biopsy derived organoids. We made air liquid interface (ALI) cultures from HNOs and assessed infection with two major human respiratory viruses, respiratory syncytial virus (RSV) and severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Infected HNO-ALI cultures recapitulate aspects of RSV and SARS-CoV-2 infection, including viral shedding, ciliary damage, innate immune responses, and mucus hyper-secretion. Next, we evaluated the feasibility of the HNO-ALI respiratory virus model system to test the efficacy of palivizumab to prevent RSV infection. Palivizumab was administered in the basolateral compartment (circulation) while viral infection occurred in the apical ciliated cells (airways), simulating the events in infants. In our model, palivizumab effectively prevented RSV infection in a concentration dependent manner. Thus, the HNO-ALI model can serve as an alternate to lung organoids to study respiratory viruses and testing therapeutics.

microbiology↗

bioRxiv2021

Multiple RSV strains infecting HEp-2 and A549 cells reveal cell line-dependent differences in resistance to RSV infection.

Respiratory syncytial virus (RSV) is a leading cause of pediatric acute respiratory infection worldwide. There are currently no approved vaccines or antivirals to combat RSV disease. A few transformed cell lines and two historic strains have been extensively used to study RSV. Here we report a thorough molecular and cell biological characterization of HEp-2 and A549 cells infected with four strains of RSV representing both major subgroups as well as historic and more contemporaneous genotypes -- [RSV/A/Tracy (GA1), RSV/A/Ontario (ON), RSV/B/18537 (GB1), RSV/B/Buenos Aires (BA)] -- via measurements of viral replication kinetics and viral gene expression, immunofluorescence-based imaging of gross cellular morphology and cell-associated RSV, and measurements of host response including transcriptional changes and levels of secreted cytokines and growth factors. Our findings strongly suggest 1) the existence of a conserved difference in gene expression between RSV subgroups A and B; 2) the A549 cell line is a more stringent and natural host of replicating RSV than the HEp-2 cell line; and 3) consistent with previous studies, determining the full effects of viral genetic variation in RSV pathogenesis requires model systems as tractable as transformed cell lines but better representative of the human host. IMPORTANCEInfection with respiratory syncytial virus (RSV) early in life is essentially guaranteed and can lead to severe disease. In vitro data from two historic RSV/A strains and two cell lines, HEp-2 and A549, constitute most of our knowledge; but RSV contains ample variation from two evolving subgroups (A and B) showing recent convergent evolution. Here we measure viral action and host response in HEp-2 and A549 cells infected with four RSV strains from both subgroups and representing both historic and more contemporaneous strains. We discover a subgroup-dependent difference in viral gene expression and find A549 cells are more potently antiviral and more sensitive, albeit subtly, to viral variation. Our findings reveal important differences between RSV subgroups and two widely used cell lines and provide baseline data for experiments with model systems better representative of natural RSV infection.

microbiology↗

bioRxiv2020

Oligonucleotide Capture Sequencing of the SARS-CoV-2 Genome and Subgenomic Fragments from COVID-19 Individuals

The newly emerged and rapidly spreading SARS-CoV-2 causes coronavirus disease 2019 (COVID-19). To facilitate a deeper understanding of the viral biology we developed a capture sequencing methodology to generate SARS-CoV-2 genomic and transcriptome sequences from infected patients. We utilized an oligonucleotide probe-set representing the full-length genome to obtain both genomic and transcriptome (subgenomic open reading frames [ORFs]) sequences from 45 SARS-CoV-2 clinical samples with varying viral titers. For samples with higher viral loads (cycle threshold value under 33, based on the CDC qPCR assay) complete genomes were generated. Analysis of junction reads revealed regions of differential transcriptional activity and provided evidence of expression of ORF10. Heterogeneous allelic frequencies along the 20kb ORF1ab gene suggested the presence of a defective interfering viral RNA species subpopulation in one sample. The associated workflow is straightforward, and hybridization-based capture offers an effective and scalable approach for sequencing SARS-CoV-2 from patient samples.

genomics↗

bioRxiv2020

Oligonucleotide capture sequencing of the SARS-CoV-2 genome and subgenomic fragments from COVID-19 individuals

microbiology↗

bioRxiv2019

Genotype-dependent and non-gradient patterns of RSV gene expression

Respiratory syncytial virus (RSV) is a nonsegmented negative-strand (NNS) RNA virus and a leading cause of severe lower respiratory tract illness in infants and the elderly. Transcription of the ten RSV genes proceeds sequentially from the 3 promoter and requires conserved gene start (GS) and gene end (GE) signals. Previous studies using the prototypical GA1 genotype Long and A2 strains have indicated a gradient of gene transcription. However, recent reports show data that appear inconsistent with a gradient. To better understand RSV transcriptional regulation, mRNA abundances from five RSV genes were measured by quantitative real-time PCR (qPCR) in three cell lines and cotton rats infected with virus isolates belonging to four different genotypes (GA1, ON, GB1, BA). Relative mRNA levels reached steady-state between four and 24 hours post-infection. Steady-state patterns were genotype-specific and non-gradient, where mRNA levels from the G (attachment) gene exceeded those from the more promoter-proximal N (nucleocapsid) gene across isolates. Transcript stabilities could not account for the non-gradient patterns observed, indicating that relative mRNA levels more strongly reflect transcription than decay. While the GS signal sequences were highly conserved, their alignment with N protein in the helical ribonucleocapsid, i.e., N-phase, was variable, suggesting polymerase recognition of GS signal conformation affects transcription initiation. The effect of GS N-phase on transcription efficiency was tested using dicistronic minigenomes. Ratios of minigenome gene expression showed a switch-like dependence on N-phase with a period of seven nucleotides. Our results indicate that RSV gene expression is in part sculpted by polymerases that initiate transcription with a probability dependent on GS signal N-phase.\n\nAuthor SummaryRSV is a major viral pathogen that causes significant morbidity and mortality, especially in young children. Shortly after RSV enters a host cell, transcription from its nonsegmented negative-strand (NNS) RNA genome starts at the 3 promoter and proceeds sequentially. Transcriptional attenuation is thought to occur at each gene junction, resulting in a gradient of gene expression. However, recent studies showing non-gradient levels of RSV mRNA suggest that transcriptional regulation may have additional mechanisms. We show using RSV isolates belonging to four different genotypes that gene expression is genotype-dependent and one gene (the G or attachment gene) is consistently more highly expressed than an upstream neighbor. We hypothesize that variable alignment of highly conserved gene start (GS) signals with nucleoprotein (i.e., variable GS N-phase) can affect transcription and give rise to non-gradient patterns of gene expression. We show using dicistronic RSV minigenomes wherein the reporter genes differ only in the N-phase of one GS signal that GS N-phase affects gene expression. Our results suggest the existence of a novel mechanism of transcriptional regulation that might play a role in other NNS RNA viruses.

molecular biology↗