Biology subjects

Hersch, S. J.

Publications and source records attributed to Hersch, S. J..

5 recordsLinked to original sources

bioRxiv2023

Manufacturing DNA in E. coli yields higher fidelity DNA than in vitro enzymatic synthesis

The rise of biotechnologies such as gene therapy have brought DNA vectors to the forefront of pharmaceutical development. The quality of the genetic starting material plays a pivotal role in determining the quality of the final product. In this study we examined the fidelity of DNA replication using enzymatic methods (in vitro) compared to plasmid DNA produced in vivo in E. coli. Next-generation sequencing approaches predominantly rely on in vitro polymerases, which have inherent limitations in sensitivity. To address this challenge, we introduce a novel assay based on loss-of-function (LOF) mutations in the conditionally toxic sacB gene. Our findings show that DNA production in E. coli results in significantly fewer LOF mutations (approximately 80-to 3000-fold less) compared to various enzymatic DNA synthesis methods. This includes the most accurate PCR polymerase (Q5) and a commonly employed rolling circle amplification (RCA) DNA polymerase (Phi29). These results suggest that using low-fidelity starting material DNA synthesized in vitro by PCR or RCA may introduce a substantial number of impurities, potentially affecting the quality and yield of final pharmaceutical products. In summary, our study underscores that DNA synthesized in vitro has a significantly higher mutation rate than DNA produced traditionally in E. coli. Therefore, utilizing in vitro enzymatically-produced DNA in biotechnology and biomanufacturing may entail considerable fidelity-related risks, while DNA starting material derived from E. coli substantially mitigates this risk, enhancing overall quality in the production processes.

synthetic biology↗

bioRxiv2021

High throughput identification of genes conferring resistance or sensitivity to toxic effectors delivered by the type VI secretion system

The type six secretion system (T6SS) is a prevalent bacterial weapon delivering toxic effector proteins into nearby competitors. In addition to immunity genes that protect against a particular effector, alternate yet crucial nonspecific defences have also recently been identified. To systematically identify genes influencing T6SS susceptibility in numerous species, we designed a Tn-Seq-based competition assay. Combined with follow-up analyses using E. coli and V. cholerae gene knockout collections, we demonstrate that our Tn-Seq competition technique can be used to identify both immunity and non-immunity defences against the T6SS. We also identify E. coli proteins that facilitate T6SS-mediated cell death, including metabolic genes such as cyaA and gltA, where mutant strains were resistant to attack. Our findings act as a proof-of-concept for the technique while also illuminating novel genes of interest. Since Tn-Seq can be applied in numerous species, our method has broad potential for identifying diverse T6SS defence genes across genomes in a high-throughput manner. ImportanceThe type six secretion system (T6SS) is a molecular poison-tipped spear that bacteria use to kill nearby competitors. To prevent self killing, they use antitoxins called immunity genes that specifically neutralize the poisons. Beyond immunity genes, multiple additional defences have recently been discovered but there are likely many more across the genomes of diverse species. To help discover these novel mechanisms, we designed a high-throughput method that can be used in numerous different species to rapidly identify genes involved in sensitivity to T6SS attacks. Using T6SS killers delivering individual poisons and two commonly studied prey bacteria, we show proof-of-principle that the technique can discover proteins that make the prey cells more resistant or sensitive to particular poisons. This will greatly improve the speed at which genes influencing the T6SS can be identified and selected for further study in follow-up analyses.

microbiology↗

bioRxiv2021

Engineered type six secretion systems deliver active exogenous effectors and Cre recombinase

Genetic editing has revolutionized biotechnology but delivery of endonuclease genes as DNA can lead to aberrant integration or overexpression, leading to off-target effects. Here we develop a mechanism to deliver Cre recombinase as a protein by engineering the bacterial type six secretion system (T6SS). Using multiple T6SS fusion proteins, Aeromonas dhakensis or attenuated Vibrio cholerae donor strains, and a gain-of-function cassette for detecting Cre recombination, we demonstrate successful delivery of active Cre directly into recipient cells. Most efficient transfer was achieved using a truncated version of PAAR2 from V. cholerae, resulting in a relatively small (118 amino acid) delivery tag. We further demonstrate the versatility of this system by delivering an exogenous effector, TseC, enabling V. cholerae to kill Pseudomonas aeruginosa. This implicates that P. aeruginosa is naturally resistant to all native effectors of V. cholerae and that the TseC chaperone protein is not required for its activity. Moreover, it demonstrates that the engineered system can improve T6SS efficacy against specific pathogens, proposing future application in microbiome manipulation or as a next-generation antimicrobial. Inexpensive and easy to produce, this protein delivery system has many potential applications ranging from studying T6SS effectors to genetic editing. ImportanceDelivery of protein-based drugs, antigens, and gene-editing agents has broad applications. The type VI protein secretion system (T6SS) can target both bacteria and eukaryotic cells and deliver proteins of diverse size and function. Here we harness the T6SS to successfully deliver Cre recombinase to genetically edit bacteria without requiring the introduction of exogenous DNA into the recipient cells. This demonstrates a promising advantage over current genetic editing tools that require transformation or conjugation of DNA. The engineered secretion tag can also deliver a heterologous antimicrobial toxin that kills an otherwise unsusceptible pathogen, Pseudomonas aeruginosa. These results demonstrate the potential of T6SS-mediated delivery in areas including genome editing, killing drug-resistant pathogens, and studying toxin functions.

molecular biology↗

bioRxiv2019

Salmonella stress response mediates growth repression in succinate media

Bacteria have evolved to sense and respond to their environment by altering gene expression and metabolism to promote growth and survival. In this work we demonstrate that Salmonella displays an extensive (>30 hour) lag in growth when subcultured into media where dicarboxylates such as succinate are the sole carbon source. This growth lag is regulated in part by RpoS, the RssB anti-adaptor IraP, translation elongation factor P, and to a lesser degree the stringent response. We also show that small amounts of proline or citrate can trigger early growth in succinate media and that, at least for proline, this effect requires the multifunctional enzyme/regulator PutA. We demonstrate that activation of RpoS results in the repression of dctA, encoding the primary dicarboxylate importer, and that constitutive expression of dctA induced growth. This dicarboxylate growth lag phenotype is far more severe across multiple Salmonella isolates than in its close relative E. coli. Replacing 200 nt of the Salmonella dctA promoter region with that of E. coli was sufficient to eliminate the observed lag in growth. We hypothesize that this cis-regulatory divergence might be an adaptation to Salmonellas virulent lifestyle where levels of phagocyte-produced succinate increase in response to bacterial LPS. We found that impairing dctA repression had no effect on Salmonellas survival in acidified succinate or in macrophage but propose alternate hypotheses of fitness advantages acquired by repressing dicarboxylate uptake. ImportanceBacteria have evolved to sense and respond to their environment to maximize their chance of survival. By studying differences in the responses of pathogenic bacteria and closely related non-pathogens, we can gain insight into what environments they encounter inside of an infected host. Here we demonstrate that Salmonella diverges from its close relative E. coli in its response to dicarboxylates such as the metabolite succinate. We show that this is regulated by stress response proteins and ultimately can be attributed to Salmonella repressing its import of dicarboxylates. Understanding this phenomenon may reveal a novel aspect of the Salmonella virulence cycle, and our characterization of its regulation yields a number of mutant strains that can be used to further study it.

microbiology↗

bioRxiv2019

Salmonella ItaR responds to itaconate in macrophage

Itaconate is a dicarboxylic acid that inhibits the isocitrate lyase enzyme of the bacterial glyoxylate shunt. Activated macrophages have been shown to produce itaconate, suggesting that these immune cells may employ this metabolite as a weapon against invading bacteria. Here we demonstrate that, in vitro, itaconate can exhibit bactericidal effects under acidic conditions similar to the pH of a macrophage phagosome. In parallel, successful pathogens including Salmonella have acquired a genetic operon encoding itaconate degradation proteins, which are induced heavily in macrophages. We characterize the regulation of this operon by the neighbouring gene, ripR, in specific response to itaconate. Moreover, we develop an itaconate biosensor based on the operon promoter that can detect itaconate in a semi-quantitative manner and, when combined with the ripR gene, is sufficient for itaconate-regulated expression in E. coli. Using this biosensor with fluorescence microscopy, we observe bacteria responding to itaconate in the phagosomes of macrophage and provide additional evidence that interferon-{gamma} stimulates macrophage itaconate synthesis and that J774 mouse macrophages produce substantially more itaconate than the human THP-1 monocyte cell line. In summary, we examine the role of itaconate as an antibacterial metabolite in mouse and human macrophage, characterize the regulation of Salmonellas defense against it, and develop it as a convenient itaconate biosensor and inducible promoter system. ImportanceIn response to invading bacteria, immune cells can produce a molecule called itaconate, which can inhibit microbial metabolism. Here we show that itaconate can also directly kill Salmonella when combined with moderate acidity, further supporting itaconates role as an antibacterial weapon. We also discover how Salmonella recognizes itaconate and activates a defense to degrade it, and we harness this response to make a biosensor that detects the presence of itaconate. This biosensor is versatile, working in Salmonella enterica or lab strains of Escherichia coli, and can detect itaconate quantitatively in the environment and in immune cells. By understanding how immune cells kill bacteria and how the microbes defend themselves, we can better develop novel antibiotics to inhibit pathogens such as Salmonella.

microbiology↗