Biology subjects

Diaz, B.

Publications and source records attributed to Diaz, B..

8 recordsLinked to original sources

bioRxiv2026

Genome-wide overexpression screen of Pseudomonas putida phage Emajogi

Bacteriophage genomes contain large fractions of uncharacterized genes, motivating high-throughput functional screens to illuminate their roles in host interaction and cytotoxicity. Here, we performed a genome wide overexpression assay for a Pseudomonas putida KT2440 infecting phage, Emajogi, and identified eight cytotoxic genes that reduce host growth under both basal and induced expression. We show that these proteins are conserved across phages that infect a diverse set of hosts. The most cytotoxic gene identified was a hypothetical protein (gp18) with no predicted functional domains. Transcriptomic profiling revealed that overexpression of gp18 caused a pronounced metabolic remodeling of the cell, likely shifting cell resources to phage production. Overall, this genome wide screen provides information about phage genes that were previously unannotated that can support biotechnology and novel antimicrobial development.

microbiology↗

bioRxiv2026

Phage proofing Pseudomonas putida uncovers novel broad-spectrum phage resistance protein Psh

Broad-spectrum phage resistance offers an important layer of protection against bacterial fermenter crashes during biomanufacturing processes, yet the underlying mechanisms are often poorly defined or come at a fitness cost. Using experimental evolution, we generated two Pseudomonas putida strains that were resistant to at least six phage genera. Genome sequencing revealed a single frameshift deletion in each strain that restored functionality of a Type I secretion system (T1SS) ATPase. We also found strong transcriptional upregulation of a nearby protein, PP_1794, which we coin Phage shielding helix rich protein (Psh). Overexpression experiments show that Psh protein is secreted by this restored T1SS to confer complete phage resistance. When compared to wild-type P. putida KT2440, no growth or expression defects were evident, but pyoverdine production was reduced. The Psh gene and T1SS are widely distributed across Gram-negative bacteria. These findings uncover a previously uncharacterized phage defense system in P. putida based on secretion-mediated receptor masking.

microbiology↗

bioRxiv2026

Tuning T-cell immunological synapse by modular DNA-Nanobody engagers for precision immunotherapy

Bispecific T-cell engagers (TCEs) are a promising class of cancer immunotherapies, but their clinical use is limited by toxicity and insufficient specificity. Tuning the T cell- tumor interface through engager architecture may address these drawbacks. To this end, we engineered hybrid constructs composed of two nanobodies targeting CD3 and the model tumor antigen HER2, respectively, connected by rigid DNA linkers of variable length. Using cytotoxicity assays and hybrid biophysical platforms, we demonstrate a linker-length dependence of cell spreading on antigen, target killing and cytokine release, revealing a functional decoupling between killing and cytokine secretion, and implicating the glycocalyx as a key player. Through the addition of EGFR targeting, we also generate trispecific constructs implementing an "OR-gate" logic to address tumor heterogeneity and reduce resistance due to antigen loss. Overall, these versatile constructs show great therapeutic promise, and at the same time serve as platforms to test hypotheses on biophysical mechanisms.

synthetic biology↗

bioRxiv2026

HtPIP: High-Throughput Phage Isolation Platform increases diversity and reduces isolation time using multiple bacteria

Bacteriophages are ubiquitous in nature, but relatively few have been isolated and characterized compared to the number of bacterial strains. Phage biotechnology applications benefit from a diverse library of isolated phages to kill or transfer genetic material to a bacterium of interest. However, scaling phage discovery for diverse bacterial hosts can be time consuming and costly. We developed an approach to capture novel phages for multiple bacteria strains in parallel from an environmental sample using commercially available 0.2-micron filter plates. Using this High-throughput Phage Isolation Platform (HtPIP), we isolated twelve novel phages spanning nine diverse bacterial host genera. Eleven of the isolated phages define new phage species with nine also defining new genera. We show the HtPIP can discover both DNA and RNA phages; including a Tectiviridae infecting Pseudomonas putida mt-2 and a Leviviricetes infecting a Microbacterium isolate, which represents the first cultured RNA phage infecting a host outside of proteobacteria. Using a metagenomic approach, we demonstrate that the HtPIP captures a higher proportion of novel phages compared to traditional low-throughput methods.

microbiology↗

bioRxiv2022

Single-agent Foxo1 inhibition normalizes glycemia and induces gut β-like cells in streptozotocin-diabetic mice

Insulin treatment remains the sole effective intervention for Type 1 Diabetes. Here, we investigated the therapeutic potential of converting intestinal epithelial cells to insulin-producing, glucose-responsive {beta}-like cells by targeted inhibition of Foxo1. We have shown that this can be achieved by genetic ablation in gut Neurogenin3 progenitors, adenoviral or shRNA-mediated inhibition in human gut organoids, and chemical inhibition in Akita mice, a model of insulin-deficient diabetes. In the present study, we provide evidence that two novel Foxo1 inhibitors, FBT432 and FBT374 have glucose-lowering and gut {beta}-like cell-inducing properties in mice rendered insulin-deficient by administration of streptozotocin. FBT432 is also highly effective in combination with a Notch inhibitor in this model. The data add to a growing body of evidence suggesting that Foxo1 inhibition be pursued as an alternative treatment to insulin administration in diabetes.

pharmacology and toxicology↗

bioRxiv2022

Triple Notch/Tgfβ/FoxO1 blockade converts multiple intestinal sub-lineages into β-like cells and lowers glycemia in diabetic animals

Insulin is the essential treatment of Type 1 (T1D) and is often used in Type 2 Diabetes. For nearly five decades, efforts have been focused on replenishing {beta}-cells in T1D patients as a more durable treatment. Gut endocrine cells can be converted into insulin-producing cells, but their numbers are limited. In this study we report that insulin-immunoreactive cells with Paneth/goblet cell features are present in human fetal intestine, in addition to enteroendocrine cells. Accordingly, lineage tracing experiments show that, besides enterochromaffin cells, the Paneth/goblet lineage can undergo conversion to the insulin lineage upon genetic or pharmacologic Foxo1 ablation in mice. We leveraged these data to design a screening platform in organoids to accurately quantitate {beta}-like cell reprogramming and fine-tune a combination treatment to increase the efficiency of the conversion process by expanding the intestinal secretory lineage. We identified a triple blockade of FoxO1, Notch, and Tgf{beta} that, when tested in insulin-deficient diabetic animals resulted in a near-normalization of glucose levels, associated with the appearance of gut insulin-producing cells. The findings illustrate a therapeutic approach to replace insulin treatment in diabetes.

cell biology↗

bioRxiv2021

Chemical induction of gut β-like-cells by combined FoxO1/Notch inhibition as a glucose-lowering treatment for diabetes

Lifelong insulin replacement remains the mainstay of type 1 diabetes treatment. Genetic FoxO1 ablation promotes enteroendocrine cell (EECs) conversion into glucose-responsive {beta}-like cells. Here, we tested whether chemical FoxO1 inhibitors can generate {beta}-like gut cells. Pan-intestinal epithelial FoxO1 ablation expanded the EEC pool, induced {beta}-like cells, and improved glucose tolerance in Ins2Akita/+ mice. This genetic effect was phenocopied by small molecule FoxO1 inhibitor, Cpd10. Cpd10 induced {beta}-like cells that released insulin in response to glucose in mouse gut organoids, and this effect was strengthened by the Notch inhibitor, DBZ. In Ins2Akita/+ mice, a five-day course of either Cpd10 or DBZ induced insulin-immunoreactive {beta}-like cells in the gut, lowered glycemia, and increased plasma insulin levels without apparent adverse effects. These results provide proof of principle of gut cell conversion into {beta}-like cells by a small molecule FoxO1 inhibitor, paving the way for clinical applications. One Sentence SummaryOrally available small molecule FoxO1 inhibitor phenocopied genetic FoxO1 ablation in generating gut {beta}-like cells

developmental biology↗

bioRxiv2020

Heterologous expression of cryptomaldamide in a cyanobacterial host

Filamentous marine cyanobacteria make a variety of bioactive molecules that are produced by polyketide synthases, non-ribosomal peptide synthetases, and hybrid pathways that are encoded by large biosynthetic gene clusters. These cyanobacterial natural products represent potential drugs leads; however, thorough pharmacological investigations have been impeded by the limited quantity of compound that is typically available from the native organisms. Additionally, investigations of the biosynthetic gene clusters and enzymatic pathways have been difficult due to the inability to conduct genetic manipulations in the native producers. Here we report a set of genetic tools for the heterologous expression of biosynthetic gene clusters in the cyanobacteria Synechococcus elongatus PCC 7942 and Anabaena (Nostoc) PCC 7120. To facilitate the transfer of gene clusters in both strains, we engineered a strain of Anabaena that contains S. elongatus homologous sequences for chromosomal recombination at a neutral site and devised a CRISPR-based strategy to efficiently obtain segregated double recombinant clones of Anabaena. These genetic tools were used to express the large 28.7 kb cryptomaldamide biosynthetic gene cluster from the marine cyanobacterium Moorena (Moorea) producens JHB in both model strains. S. elongatus did not produce cryptomaldamide, however high-titer production of cryptomaldamide was obtained in Anabaena. The methods developed in this study will facilitate the heterologous expression of biosynthetic gene clusters isolated from marine cyanobacteria and complex metagenomic samples. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/267179v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@660caforg.highwire.dtl.DTLVardef@1cab871org.highwire.dtl.DTLVardef@130de4org.highwire.dtl.DTLVardef@f50c64_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗