An essential and highly selective protein import pathway encoded by nucleus-forming phage.

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here we identify two components of this novel protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA, that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together our results allow us to propose a multistep model for the Protein Import Chimallivirus (PIC) pathway, where proteins are targeted to PicA by amino acids on their surface, and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely-related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts. Significance Statement: The phage nucleus is an enclosed replication compartment built by Chimalliviridae phages that, similar to the eukaryotic nucleus, separates transcription from translation and selectively imports certain proteins. This allows the phage to concentrate proteins required for DNA replication and transcription while excluding DNA-targeting host defense proteins. However, the mechanism of selective trafficking into the phage nucleus is currently unknown. Here we determine the region of a phage nuclear protein that targets it for nuclear import and identify a conserved, essential nuclear shell-associated protein that plays a key role in this process. This work provides the first mechanistic model of selective import into the phage nucleus.

An essential and highly selective protein import pathway encoded by nucleus-forming phage.

Targeting proteins to specific subcellular destinations is essential in prokaryotes, eukaryotes, and the viruses that infect them. Chimalliviridae phages encapsulate their genomes in a nucleus-like replication compartment composed of the protein chimallin (ChmA) that excludes ribosomes and decouples transcription from translation. These phages selectively partition proteins between the phage nucleus and the bacterial cytoplasm. Currently, the genes and signals that govern selective protein import into the phage nucleus are unknown. Here, we identify two components of this protein import pathway: a species-specific surface-exposed region of a phage intranuclear protein required for nuclear entry and a conserved protein, PicA (Protein importer of chimalliviruses A), that facilitates cargo protein trafficking across the phage nuclear shell. We also identify a defective cargo protein that is targeted to PicA on the nuclear periphery but fails to enter the nucleus, providing insight into the mechanism of nuclear protein trafficking. Using CRISPRi-ART protein expression knockdown of PicA, we show that PicA is essential early in the chimallivirus replication cycle. Together, our results allow us to propose a multistep model for the Protein Import Chimallivirus pathway, where proteins are targeted to PicA by amino acids on their surface and then licensed by PicA for nuclear entry. The divergence in the selectivity of this pathway between closely related chimalliviruses implicates its role as a key player in the evolutionary arms race between competing phages and their hosts.

Sequential membrane- and protein-bound organelles compartmentalize genomes during phage infection.

Eukaryotic viruses assemble compartments required for genome replication, but no such organelles are known to be essential for prokaryotic viruses. Bacteriophages of the family Chimalliviridae sequester their genomes within a phage-generated organelle, the phage nucleus, which is enclosed by a lattice of viral protein ChmA. Using the dRfxCas13d-based knockdown system CRISPRi-ART, we show that ChmA is essential for the E. coli phage Goslar life cycle. Without ChmA, infections are arrested at an early stage in which the injected phage genome is enclosed in a membrane-bound vesicle capable of gene expression but not DNA replication. Not only do we demonstrate that the phage nucleus is essential for genome replication, but we also show that the Chimalliviridae early phage infection (EPI) vesicle is a transcriptionally active, phage-generated organelle.

A mobile intron facilitates interference competition between co-infecting viruses.

Mobile introns containing homing endonucleases are widespread in nature and have long been assumed to be selfish elements that provide no benefit to the host organism. These genetic elements are common in viruses, but whether they confer a selective advantage is unclear. Here we studied a mobile intron in bacteriophage ΦPA3 and found its homing endonuclease gp210 contributes to viral competition by interfering with the virogenesis of co-infecting phage ΦKZ. We show that gp210 targets a specific sequence in its competitor ΦKZ, preventing the assembly of progeny viruses. This work reports the first demonstration of how a mobile intron can be deployed to engage in interference competition and provide a reproductive advantage. Given the ubiquity of introns, this selective advantage likely has widespread evolutionary implications in nature.

Bacterial cytological profiling reveals interactions between jumbo phage φKZ infection and cell wall active antibiotics in Pseudomonas aeruginosa.

The emergence of antibiotic resistance in bacteria has led to the investigation of alternative treatments, such as phage therapy. In this study, we examined the interactions between the nucleus-forming jumbo phage ФKZ and antibiotic treatment against Pseudomonas aeruginosa. Using the fluorescence microscopy technique of bacterial cytological profiling, we identified mechanism-of-action-specific interactions between antibiotics that target different biosynthetic pathways and ФKZ infection. We found that certain classes of antibiotics strongly inhibited phage replication, while others had no effect or only mildly affected progression through the lytic cycle. Antibiotics that caused an increase in host cell length, such as the cell wall active antibiotic ceftazidime, prevented proper centering of the ФKZ nucleus via the PhuZ spindle at midcell, leading us to hypothesize that the kinetic parameters of the PhuZ spindle evolved to match the average length of the host cell. To test this, we developed a computational model explaining how the dynamic properties of the PhuZ spindle contribute to phage nucleus centering and why some antibiotics affect nucleus positioning while others do not. These findings provide an understanding of the molecular mechanisms underlying the interactions between antibiotics and jumbo phage replication.

Identifying the core genome of the nucleus-forming bacteriophage family and characterization of Erwinia phage RAY.

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were still to be determined. Here, we show that phages encoding the major phage nucleus protein chimallin share 72 conserved genes encoded within seven gene blocks. Of these, 21 core genes are unique to nucleus-forming phage, and all but one of these genes encode proteins of unknown function. We propose that these phages comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryoelectron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication are conserved among diverse chimalliviruses and reveal variations on this replication mechanism. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.

Identifying the core genome of the nucleus-forming bacteriophage family and characterization of Erwinia phage RAY.

We recently discovered that some bacteriophages establish a nucleus-like replication compartment (phage nucleus), but the core genes that define nucleus-based phage replication and their phylogenetic distribution were unknown. By studying phages that encode the major phage nucleus protein chimallin, including previously sequenced yet uncharacterized phages, we discovered that chimallin-encoding phages share a set of 72 highly conserved genes encoded within seven distinct gene blocks. Of these, 21 core genes are unique to this group, and all but one of these unique genes encode proteins of unknown function. We propose that phages with this core genome comprise a novel viral family we term Chimalliviridae. Fluorescence microscopy and cryo-electron tomography studies of Erwinia phage vB_EamM_RAY confirm that many of the key steps of nucleus-based replication encoded in the core genome are conserved among diverse chimalliviruses, and reveal that non-core components can confer intriguing variations on this replication mechanism. For instance, unlike previously studied nucleus-forming phages, RAY doesn't degrade the host genome, and its PhuZ homolog appears to form a five-stranded filament with a lumen. This work expands our understanding of phage nucleus and PhuZ spindle diversity and function, providing a roadmap for identifying key mechanisms underlying nucleus-based phage replication.

Subcellular organization of viral particles during maturation of nucleus-forming jumbo phage.

Many eukaryotic viruses assemble mature particles within distinct subcellular compartments, but bacteriophages are generally assumed to assemble randomly throughout the host cell cytoplasm. Here, we show that viral particles of Pseudomonas nucleus-forming jumbo phage PhiPA3 assemble into a unique structure inside cells we term phage bouquets. We show that after capsids complete DNA packaging at the surface of the phage nucleus, tails assemble and attach to capsids, and these particles accumulate over time in a spherical pattern, with tails oriented inward and the heads outward to form bouquets at specific subcellular locations. Bouquets localize at the same fixed distance from the phage nucleus even when it is mispositioned, suggesting an active mechanism for positioning. These results mark the discovery of a pathway for organizing mature viral particles inside bacteria and demonstrate that nucleus-forming jumbo phages, like most eukaryotic viruses, are highly spatially organized during all stages of their lytic cycle.

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.

Antimicrobials from a feline commensal bacterium inhibit skin infection by drug-resistant S. pseudintermedius.

Methicillin-resistant Staphylococcus pseudintermedius (MRSP) is an important emerging zoonotic pathogen that causes severe skin infections. To combat infections from drug-resistant bacteria, the transplantation of commensal antimicrobial bacteria as a therapeutic has shown clinical promise. We screened a collection of diverse staphylococcus species from domestic dogs and cats for antimicrobial activity against MRSP. A unique strain (S. felis C4) was isolated from feline skin that inhibited MRSP and multiple gram-positive pathogens. Whole genome sequencing and mass spectrometry revealed several secreted antimicrobials including a thiopeptide bacteriocin micrococcin P1 and phenol-soluble modulin beta (PSMß) peptides that exhibited antimicrobial and anti-inflammatory activity. Fluorescence and electron microscopy revealed that S. felis antimicrobials inhibited translation and disrupted bacterial but not eukaryotic cell membranes. Competition experiments in mice showed that S. felis significantly reduced MRSP skin colonization and an antimicrobial extract from S. felis significantly reduced necrotic skin injury from MRSP infection. These findings indicate a feline commensal bacterium that could be utilized in bacteriotherapy against difficult-to-treat animal and human skin infections.

Bacterial Cytological Profiling Identifies Rhodanine-Containing PAINS Analogs as Specific Inhibitors of Escherichia coli Thymidylate Kinase In Vivo.

In this study, we sought to determine whether an in vivo assay for studying antibiotic mechanisms of action could provide insight into the activity of compounds that may inhibit multiple targets. Thus, we conducted an activity screen of 31 structural analogs of rhodanine-containing pan-assay interference compounds (PAINS). We identified nine active molecules against Escherichia coli and classified them according to their in vivo mechanisms of action. The mechanisms of action of PAINS are generally difficult to identify due to their promiscuity. However, we leveraged bacterial cytological profiling, a fluorescence microscopy technique, to study these complex mechanisms. Ultimately, we found that although some of our molecules promiscuously inhibit multiple cellular pathways, a few molecules specifically inhibit DNA replication despite structural similarity to related PAINS. A genetic analysis of resistant mutants revealed thymidylate kinase (essential for DNA synthesis) as an intracellular target of some of these rhodanine-containing antibiotics. This finding was supported by in vitro activity assays, as well as experiments utilizing a thymidylate kinase overexpression system. The analog that demonstrated the half-maximal inhibitory concentration in vitro and MIC in vivo displayed the greatest specificity for inhibition of the DNA replication pathway, despite containing a rhodamine moiety. Although it is thought that PAINS cannot be developed as antibiotics, this work showcases novel inhibitors of E. coli thymidylate kinase. Moreover, perhaps more importantly, this work highlights the utility of bacterial cytological profiling for studying the in vivo specificity of antibiotics and demonstrates that bacterial cytological profiling can identify multiple pathways that are inhibited by an individual molecule. IMPORTANCE We demonstrate that bacterial cytological profiling is a powerful tool for directing antibiotic discovery efforts because it can be used to determine the specificity of an antibiotic's in vivo mechanism of action. By assaying analogs of PAINS, molecules that are notoriously intractable and nonspecific, we (surprisingly) identify molecules with specific activity against E. coli thymidylate kinase. This suggests that structural modifications to PAINS can confer stronger inhibition by targeting a specific cellular pathway. While in vitro inhibition assays are susceptible to false-positive results (especially from PAINS), bacterial cytological profiling provides the resolution to identify molecules with specific in vivo activity.

Asymmetric localization of the cell division machinery during Bacillus subtilis sporulation.

The Gram-positive bacterium Bacillus subtilis can divide via two modes. During vegetative growth, the division septum is formed at the midcell to produce two equal daughter cells. However, during sporulation, the division septum is formed closer to one pole to yield a smaller forespore and a larger mother cell. Using cryo-electron tomography, genetics and fluorescence microscopy, we found that the organization of the division machinery is different in the two septa. While FtsAZ filaments, the major orchestrators of bacterial cell division, are present uniformly around the leading edge of the invaginating vegetative septa, they are only present on the mother cell side of the invaginating sporulation septa. We provide evidence suggesting that the different distribution and number of FtsAZ filaments impact septal thickness, causing vegetative septa to be thicker than sporulation septa already during constriction. Finally, we show that a sporulation-specific protein, SpoIIE, regulates asymmetric divisome localization and septal thickness during sporulation.

Metabolic differentiation and intercellular nurturing underpin bacterial endospore formation.

Despite intensive research, the role of metabolism in bacterial sporulation remains poorly understood. Here, we demonstrate that Bacillus subtilis sporulation entails a marked metabolic differentiation of the two cells comprising the sporangium: the forespore, which becomes the dormant spore, and the mother cell, which dies as sporulation completes. Our data provide evidence that metabolic precursor biosynthesis becomes restricted to the mother cell and that the forespore becomes reliant on mother cell-derived metabolites for protein synthesis. We further show that arginine is trafficked between the two cells and that proposed proteinaceous channels mediate small-molecule intercellular transport. Thus, sporulation entails the profound metabolic reprogramming of the forespore, which is depleted of key metabolic enzymes and must import metabolites from the mother cell. Together, our results provide a bacterial example analogous to progeny nurturing.

Bacterial-fungal interactions revealed by genome-wide analysis of bacterial mutant fitness.

Microbial interactions are expected to be major determinants of microbiome structure and function. Although fungi are found in diverse microbiomes, their interactions with bacteria remain largely uncharacterized. In this work, we characterize interactions in 16 different bacterial-fungal pairs, examining the impacts of 8 different fungi isolated from cheese rind microbiomes on 2 bacteria (Escherichia coli and a cheese-isolated Pseudomonas psychrophila). Using random barcode transposon-site sequencing with an analysis pipeline that allows statistical comparisons between different conditions, we observed that fungal partners caused widespread changes in the fitness of bacterial mutants compared to growth alone. We found that all fungal species modulated the availability of iron and biotin to bacterial species, which suggests that these may be conserved drivers of bacterial-fungal interactions. Species-specific interactions were also uncovered, a subset of which suggested fungal antibiotic production. Changes in both conserved and species-specific interactions resulted from the deletion of a global regulator of fungal specialized metabolite production. This work highlights the potential for broad impacts of fungi on bacterial species within microbiomes.

Shaping an Endospore: Architectural Transformations During Bacillus subtilis Sporulation.

Endospore formation in Bacillus subtilis provides an ideal model system for studying development in bacteria. Sporulation studies have contributed a wealth of information about the mechanisms of cell-specific gene expression, chromosome dynamics, protein localization, and membrane remodeling, while helping to dispel the early view that bacteria lack internal organization and interesting cell biological phenomena. In this review, we focus on the architectural transformations that lead to a profound reorganization of the cellular landscape during sporulation, from two cells that lie side by side to the endospore, the unique cell within a cell structure that is a hallmark of sporulation in B. subtilis and other spore-forming Firmicutes. We discuss new insights into the mechanisms that drive morphogenesis, with special emphasis on polar septation, chromosome translocation, and the phagocytosis-like process of engulfment, and also the key experimental advances that have proven valuable in revealing the inner workings of bacterial cells.

Group A Streptococcal S Protein Utilizes Red Blood Cells as Immune Camouflage and Is a Critical Determinant for Immune Evasion.

Group A Streptococcus (GAS) is a human-specific pathogen that evades the host immune response through the elaboration of multiple virulence factors. Although many of these factors have been studied, numerous proteins encoded by the GAS genome are of unknown function. Herein, we characterize a biomimetic red blood cell (RBC)-captured protein of unknown function-annotated subsequently as S protein-in GAS pathophysiology. S protein maintains the hydrophobic properties of GAS, and its absence reduces survival in human blood. S protein facilitates GAS coating with lysed RBCs to promote molecular mimicry, which increases virulence in vitro and in vivo. Proteomic profiling reveals that the removal of S protein from GAS alters cellular and extracellular protein landscapes and is accompanied by a decrease in the abundance of several key GAS virulence determinants. In vivo, the absence of S protein results in a striking attenuation of virulence and promotes a robust immune response and immunological memory.

The molecular architecture of engulfment during Bacillus subtilis sporulation.

The study of bacterial cell biology is limited by difficulties in visualizing cellular structures at high spatial resolution within their native milieu. Here, we visualize Bacillus subtilis sporulation using cryo-electron tomography coupled with cryo-focused ion beam milling, allowing the reconstruction of native-state cellular sections at molecular resolution. During sporulation, an asymmetrically-positioned septum generates a larger mother cell and a smaller forespore. Subsequently, the mother cell engulfs the forespore. We show that the septal peptidoglycan is not completely degraded at the onset of engulfment. Instead, the septum is uniformly and only slightly thinned as it curves towards the mother cell. Then, the mother cell membrane migrates around the forespore in tiny finger-like projections, whose formation requires the mother cell SpoIIDMP protein complex. We propose that a limited number of SpoIIDMP complexes tether to and degrade the peptidoglycan ahead of the engulfing membrane, generating an irregular membrane front.

Viral Capsid Trafficking along Treadmilling Tubulin Filaments in Bacteria.

Cargo trafficking along microtubules is exploited by eukaryotic viruses, but no such examples have been reported in bacteria. Several large Pseudomonas phages assemble a dynamic, tubulin-based (PhuZ) spindle that centers replicating phage DNA sequestered within a nucleus-like structure. Here, we show that capsids assemble on the membrane and then move rapidly along PhuZ filaments toward the phage nucleus for DNA packaging. The spindle rotates the phage nucleus, distributing capsids around its surface. PhuZ filaments treadmill toward the nucleus at a constant rate similar to the rate of capsid movement and the linear velocity of nucleus rotation. Capsids become trapped along mutant static PhuZ filaments that are defective in GTP hydrolysis. Our results suggest a transport and distribution mechanism in which capsids attached to the sides of filaments are trafficked to the nucleus by PhuZ polymerization at the poles, demonstrating that the phage cytoskeleton evolved cargo-trafficking capabilities in bacteria.

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.

SCH79797 improves outcomes in experimental bacterial pneumonia by boosting neutrophil killing and direct antibiotic activity.

Objectives: The role of protease-activated receptor 1 (PAR1) in the pathogenesis of pneumonia and sepsis is ambiguous given the existing literature. As PAR1 is classically activated by the coagulation-based protease thrombin and leads to vascular leakage, our hypothesis was that PAR1 blockade with SCH79797 would be therapeutically beneficial in an experimental model of murine Gram-negative pneumonia. Methods: In this study, we administered SCH79797 via the intrapulmonary route 6 h after the establishment of Escherichia coli pneumonia and observed a significant improvement in survival, lung injury, bacterial clearance and inflammation. We focused on neutrophils as a potential target of the PAR1 antagonist, since they are the predominant inflammatory cell type in the infected lung. Results: Neutrophils appear to express PAR1 at low levels and the PAR1 antagonist SCH79797 enhanced neutrophil killing. Part of this effect may be explained by alterations in the generation of reactive oxygen species (ROS). SCH79797 also led to robust neutrophil extracellular trap (NET) generation and cathelicidin-related antimicrobial peptide (CRAMP) release by neutrophils. Surprisingly, SCH79797 also had a potent, direct antibiotic effect with disruption of the E. coli cell membrane. However, the newer-generation PAR1 antagonist, vorapaxar (SCH530348), had no appreciable effect on neutrophil activity or direct bacterial killing, which suggests the effects seen with SCH79797 may be PAR1 independent. Conclusions: In summary, we observed that intrapulmonary treatment with SCH79797 has significant therapeutic effects in a model of E. coli pneumonia that appear to be due, in part, to both neutrophil-stimulating and direct antibacterial effects of SCH79797.