Isolation and characterization of Streptomyces bacteriophages and Streptomyces strains encoding biosynthetic arsenals.

The threat to public health posed by drug-resistant bacteria is rapidly increasing, as some of healthcare's most potent antibiotics are becoming obsolete. Approximately two-thirds of the world's antibiotics are derived from natural products produced by Streptomyces encoded biosynthetic gene clusters. Thus, to identify novel gene clusters, we sequenced the genomes of four bioactive Streptomyces strains isolated from the soil in San Diego County and used Bacterial Cytological Profiling adapted for agar plate culturing in order to examine the mechanisms of bacterial inhibition exhibited by these strains. In the four strains, we identified 104 biosynthetic gene clusters. Some of these clusters were predicted to produce previously studied antibiotics; however, the known mechanisms of these molecules could not fully account for the antibacterial activity exhibited by the strains, suggesting that novel clusters might encode antibiotics. When assessed for their ability to inhibit the growth of clinically isolated pathogens, three Streptomyces strains demonstrated activity against methicillin-resistant Staphylococcus aureus. Additionally, due to the utility of bacteriophages for genetically manipulating bacterial strains via transduction, we also isolated four new phages (BartholomewSD, IceWarrior, Shawty, and TrvxScott) against S. platensis. A genomic analysis of our phages revealed nearly 200 uncharacterized proteins, including a new site-specific serine integrase that could prove to be a useful genetic tool. Sequence analysis of the Streptomyces strains identified CRISPR-Cas systems and specific spacer sequences that allowed us to predict phage host ranges. Ultimately, this study identified Streptomyces strains with the potential to produce novel chemical matter as well as integrase-encoding phages that could potentially be used to manipulate these strains.

Development of a VRC01-class germline targeting immunogen derived from anti-idiotypic antibodies.

An effective HIV-1 vaccine will likely need to elicit broadly neutralizing antibodies (bNAbs). Broad and potent VRC01-class bNAbs have been isolated from multiple infected individuals, suggesting that they could be reproducibly elicited by vaccination. Several HIV-1 envelope-derived germline-targeting immunogens have been designed to engage naive VRC01-class precursor B cells. However, they also present off-target epitopes that could hinder development of VRC01-class bNAbs. We characterize a panel of anti-idiotypic monoclonal antibodies (ai-mAbs) raised against inferred-germline (iGL) VRC01-class antibodies. By leveraging binding, structural, and B cell sorting data, we engineered a bispecific molecule derived from two ai-mAbs; one specific for VRC01-class heavy chains and one specific for VRC01-class light chains. The bispecific molecule preferentially activates iGL-VRC01 B cells in vitro and induces specific antibody responses in a murine adoptive transfer model with a diverse polyclonal B cell repertoire. This molecule represents an alternative non-envelope-derived germline-targeting immunogen that can selectively activate VRC01-class precursors in vivo.

Bacterial Cytological Profiling as a Tool To Study Mechanisms of Action of Antibiotics That Are Active against Acinetobacter baumannii.

An increasing number of multidrug-resistant Acinetobacter baumannii (MDR-AB) infections have been reported worldwide, posing a threat to public health. The establishment of methods to elucidate the mechanism of action (MOA) of A. baumannii-specific antibiotics is needed to develop novel antimicrobial therapeutics with activity against MDR-AB We previously developed bacterial cytological profiling (BCP) to understand the MOA of compounds in Escherichia coli and Bacillus subtilis Given how distantly related A. baumannii is to these species, it was unclear to what extent it could be applied. Here, we implemented BCP as an antibiotic MOA discovery platform for A. baumannii We found that the BCP platform can distinguish among six major antibiotic classes and can also subclassify antibiotics that inhibit the same cellular pathway but have different molecular targets. We used BCP to show that the compound NSC145612 inhibits the growth of A. baumannii via targeting RNA transcription. We confirmed this result by isolating and characterizing resistant mutants with mutations in the rpoB gene. Altogether, we conclude that BCP provides a useful tool for MOA studies of antibacterial compounds that are active against A. baumannii.

Rapid Inhibition Profiling Identifies a Keystone Target in the Nucleotide Biosynthesis Pathway.

Understanding the mechanism of action (MOA) of new antimicrobial agents is a critical step in drug discovery but is notoriously difficult for compounds that appear to inhibit multiple cellular pathways. We recently described image-based approaches [bacterial cytological profiling and rapid inducible profiling (RIP)] for identifying the cellular pathways targeted by antibiotics. Here we have applied these methods to examine the effects of proteolytically degrading enzymes involved in pyrimidine nucleotide biosynthesis, a pathway that produces intermediates for transcription, DNA replication, and cell envelope synthesis. We show that rapid removal of enzymes directly involved in deoxyribonucleotide synthesis blocks DNA replication. However, degradation of cytidylate kinase (CMK), which catalyzes reactions involved in the synthesis of both ribonucleotides and deoxyribonucleotides, blocks both DNA replication and wall teichoic acid biosynthesis, producing cytological effects identical to those created by simultaneously inhibiting both processes with the antibiotics ciprofloxacin and tunicamycin. Our results suggest that RIP can be used to identify and characterize potential keystone enzymes like CMK whose inhibition dramatically affects multiple pathways, thereby revealing important metabolic connections. Identifying and understanding the role of keystone targets might also help to determine the MOAs of drugs that appear to inhibit multiple targets.