Maternally transferred monoclonal antibodies protect neonatal mice from herpes simplex virus-induced mortality and morbidity

Neonatal herpes simplex virus (HSV) infections often result in significant mortality and neurological morbidity despite antiviral drug therapy. Maternally-transferred HSV-specific antibodies reduce the risk of clinically-overt neonatal HSV (nHSV), but this observation has not been translationally applied. Using a neonatal mouse model, we tested the hypothesis that passive transfer of HSV-specific human monoclonal antibodies (mAbs) can prevent mortality and morbidity associated with nHSV. The mAbs were expressed in vivo by vectored immunoprophylaxis, or administered in vivo following recombinant expression in vitro. Through these maternally-derived routes or through direct administration to pups, diverse mAbs to HSV glycoprotein D protected against neonatal HSV-1 and HSV-2 infection. Using in vivo bioluminescent imaging, both pre- and post-exposure mAb treatment significantly reduced viral load. Administration of mAb also reduced nHSV-induced behavioral morbidity, as measured by anxiety-like behavior. Together these studies support the notion that HSV-specific mAb-based therapies may prevent or improve HSV infection outcomes in neonates.

Molecular Characterization Revealed the Role of Thaumatin-Like Proteins of Bread Wheat in Stress Response

Thaumatin-like proteins (TLPs) are related to pathogenesis-related-5 (PR-5) family and involved in stress response. Herein, a total of 93 TLP genes were identified in the genome of Triticum aestivum. Further, we identified 26, 27, 39, and 37 TLP genes in the Brachypodium distachyon, Oryza sativa, Sorghum bicolor, and Zea mays genomes for comparative characterization, respectively. They could be grouped into small and long TLPs with conserved thaumatin signature motif. Tightly clustered genes exhibited conserved gene and protein structure. The physicochemical analyses suggested significant differences between small and long TLPs. Evolutionary analyses suggested the role of duplication events and purifying selection in the expansion of the TLP gene family. Expression analyses revealed the possible roles of TLPs in plant development and abiotic and fungal stress response. Recombinant expression of TaTLP2-B in Saccharomyces cerevisiae provided significant tolerance against cold, heat, osmotic, and salt stresses. The results depicted the importance of TLPs in cereal crops that would be highly useful in future crop improvement programs.

Engineering selectivity of Cutibacterium acnes phages by epigenetic imprinting

Cutibacterium acnes (C. acnes) is a gram-positive bacterium and a member of the human skin microbiome. Despite being the most abundant skin commensal, certain members have been associated with common inflammatory disorders such as acne vulgaris. The availability of the complete genome sequences from various C. acnes clades have enabled the identification of putative methyltransferases, some of them potentially belonging to restriction-modification (R-M) systems which protect the host of invading DNA. However, little is known on whether these systems are functional in the different C. acnes strains. To investigate the activity of these putative R-M and their relevance in host protective mechanisms, we analyzed the methylome of seven representative C. acnes strains by Oxford Nanopore Technologies (ONT) sequencing. We detected the presence of a 6-methyladenine modification at a defined DNA consensus sequence in strain KPA171202 and recombinant expression of this R-M system confirmed its methylation activity. Additionally, a R-M knockout mutant verified the loss of methylation properties of the strain. We studied the potential of one C. acnes bacteriophage (PAD20) in killing various C. acnes strains and linked an increase in its specificity to phage DNA methylation acquired upon infection of a methylation competent strain. We demonstrate a therapeutic application of this mechanism where phages propagated in R-M deficient strains selectively kill R-M deficient acne-prone clades while probiotic ones remain resistant to phage infection.

Engineering of the Recombinant Expression and PEGylation Efficiency of the Therapeutic Enzyme Human Thymidine Phosphorylase

Human thymidine phosphorylase (HsTP) is an enzyme with important implications in the field of rare metabolic diseases. Defective mutations of HsTP lead to mitochondrial neurogastrointestinal encephalomyopathy (MNGIE), a disease with a high unmet medical need that is associated with severe neurological and gastrointestinal complications. Current efforts focus on the development of an enzyme replacement therapy (ERT) using the Escherichia coli ortholog (EcTP). However, bacterial enzymes are counter-indicated for human therapeutic applications because they are recognized as foreign by the human immune system, thereby eliciting adverse immune responses and raising significant safety and efficacy risks. Thus, it is critical to utilize the HsTP enzyme as starting scaffold for pre-clinical drug development, thus de-risking the safety concerns associated with the use of bacterial enzymes. However, HsTP expresses very poorly in E. coli, whereas its PEGylation, a crucial chemical modification for achieving long serum persistence of therapeutic enzymes, is highly inefficient and negatively affects its catalytic activity. Here we focused on the engineering of the recombinant expression profile of HsTP in E. coli cells, as well as on the optimization of its PEGylation efficiency aiming at the development of an alternative therapeutic approach for MNGIE. We show that phylogenetic and structural analysis of proteins can provide important insights for the rational design of N’-terminus-truncation constructs which exhibit significantly improved recombinant expression levels. In addition, we developed and implemented a criteria-driven rational surface engineering strategy for the substitution of arginine-to-lysine and lysine-to-arginine residues to achieve more efficient, homogeneous and reproducible PEGylation without negatively affecting the enzymatic catalytic activity upon PEGylation. Collectively, our proposed strategies provide an effective way to optimize enzyme PEGylation and E. coli recombinant expression and are likely applicable for other proteins and enzymes.

Functional Trimeric SARS-CoV-2 Envelope Protein Expressed in Stable CHO Cells

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), a β-coronavirus, is the causative agent of the COVID-19 pandemic. One of the three membrane-bound envelope proteins is the spike protein (S), the one responsible for docking to the cellular surface protein ACE2 enabling infection with SARS-CoV-2. Although the structure of the S-protein has distinct similarities to other viral envelope proteins, robust and straightforward protocols for recombinant expression and purification are not described in the literature. Therefore, most studies are done with truncated versions of the protein, like the receptor-binding domain. To learn more about the interaction of the virus with the ACE2 and other cell surface proteins, it is mandatory to provide recombinant spike protein in high structural quality and adequate quantity. Additional mutant variants will give new insights on virus assembly, infection mechanism, and therapeutic drug development. Here, we describe the development of a recombinant CHO cell line stably expressing the extracellular domain of a trimeric variant of the SARS CoV-2 spike protein and discuss significant parameters to be considered during the expression and purification process.

Isolation and Characterization of Levoglucosan Metabolizing Bacteria

Bacteria were isolated from wastewater and soil containing charred wood remnants based on their ability to use levoglucosan as a sole carbon source and on their levoglucosan dehydrogenase (LGDH) activity. On the basis of their 16S rRNA gene sequences, these bacteria represented diverse genera of Microbacterium, Paenibacillus , Shinella , and Klebsiella . Genomic sequencing of the isolates verified that two isolates represented novel species, Paenibacillus athensensis MEC069 T and Shinella sumterensis MEC087 T , while the remaining isolates were closely related to either Microbacterium lacusdiani or Klebsiella pneumoniae . The genetic sequence of LGDH, lgdA , was found in the genomes of these four isolates as well as Pseudarthrobacter phenanthrenivorans Sphe3. The identity of the P. phenanthrenivorans LGDH was experimentally verified following recombinant expression in E. coli . Comparison of the putative genes surrounding lgdA in the isolate genomes indicated that several other gene products facilitate the bacterial catabolism of levoglucosan, including a putative sugar isomerase and several transport proteins. Importance Levoglucosan is the most prevalent soluble carbohydrate remaining after high temperature pyrolysis of lignocellulosic biomass, but it is not fermented by typical production microbes such as Escherichia coli and Saccharomyces cerevisiae . A few fungi metabolize levoglucosan via the enzyme levoglucosan kinase, while several bacteria metabolize levoglucosan via levoglucosan dehydrogenase. This study describes the isolation and characterization of four bacterial species which degrade levoglucosan. Each isolate is shown to contain several genes within an operon involved in levoglucosan degradation, furthering our understanding of bacteria which metabolize levoglucosan.

Mining marine metagenomes revealed a quorum-quenching lactonase with improved biochemical properties that inhibits the food spoilage bacteria Pseudomonas fluorescens

The marine environment presents great potential as a source of microorganisms that possess novel enzymes with unique activities and biochemical properties. Examples of such are the quorum-quenching (QQ) enzymes that hydrolyze bacterial quorum-sensing (QS) signaling molecules, such as N-acyl-homoserine lactones (AHLs). QS is a form of cell-to-cell communication that enables bacteria to synchronize gene expression in correlation with population density. Searching marine metagenomes for sequences homologous to an AHL lactonase from the phosphotriesterase-like lactonase (PLL) family, we identified new putative AHL lactonases (sharing 30-40% amino acid identity to a thermostable PLL member). Phylogenetic analysis indicated that these putative AHL lactonases comprise a new clade of marine enzymes in the PLL family. Following recombinant expression and purification, we verified the AHL lactonase activity for one of these proteins, named marine originated Lactonase Related Protein (moLRP). This enzyme presented greater activity and stability at a broad range of temperatures and pH, and tolerance to high salinity levels (up to 5M NaCl), as well as higher durability in bacterial culture, compared to another PLL member. The addition of purified moLRP to cultures of Pseudomonas fluorescens inhibited its extracellular protease activity, expression of the protease encoding gene, biofilm formation, and the sedimentation process in milk-based medium. These findings suggest that moLRP is adapted to the marine environment, and can potentially serve as an effective QQ enzyme, inhibiting the QS process in gram-negative bacteria involved in food spoilage. Importance Our results emphasize the potential of sequence and structure-based identification of new quorum-quenching (QQ) enzymes from environmental metagenomes, such as from the ocean, with improved stability or activity. The findings also suggest that purified QQ enzymes can present new strategies against food spoilage, in addition to their recognized involvement in inhibiting bacterial pathogen virulence factors. Future studies on the delivery and safety of enzymatic QQ strategy against bacterial food spoilage should be performed.

Rational domestication of a plant-based recombinant expression system expands its biosynthetic range.

Plant molecular farming aims to provide a green, flexible, and rapid alternative to conventional recombinant expression systems, capable of producing complex biologics such as enzymes, vaccines, and antibodies. Historically, the recombinant expression of therapeutic peptides in plants has proven difficult, largely due to their small size and instability. However, some plant species harbour the capacity for peptide backbone cyclization, a feature inherent in stable therapeutic peptides. One obstacle to realizing the potential of plant-based therapeutic peptide production is the proteolysis of the precursor before it is matured into its final stabilized form. Here we demonstrate the rational domestication of Nicotiana benthamiana within two generations to endow this plant molecular farming host with an expanded repertoire of peptide sequence space. The in planta production of molecules including an insecticidal peptide, a prostate cancer therapeutic lead and an orally active analgesic are demonstrated.

Novel Cysteine Protease Inhibitor Derived from the Haementeria vizottoi Leech: Recombinant Expression, Purification, and Characterization

Cathepsin L (CatL) is a lysosomal cysteine protease primarily involved in the terminal degradation of intracellular and endocytosed proteins. More specifically, in humans, CatL has been implicated in cancer progression and metastasis, as well as coronary artery diseases and others. Given this, the search for potent CatL inhibitors is of great importance. In the search for new molecules to perform proteolytic activity regulation, salivary secretions from hematophagous animals have been an important source, as they present protease inhibitors that evolved to disable host proteases. Based on the transcriptome of the Haementeria vizzotoi leech, the cDNA of Cystatin-Hv was selected for this study. Cystatin-Hv was expressed in Pichia pastoris and purified by two chromatographic steps. The kinetic results using human CatL indicated that Cystatin-Hv, in its recombinant form, is a potent inhibitor of this protease, with a Ki value of 7.9 nM. Consequently, the present study describes, for the first time, the attainment and the biochemical characterization of a recombinant cystatin from leeches as a potent CatL inhibitor. While searching out for new molecules of therapeutic interest, this leech cystatin opens up possibilities for the future use of this molecule in studies involving cellular and in vivo models.