Antibiotic-resistant strains of Staphylococcus aureus pose a significant threat in healthcare, demanding urgent therapeutic solutions. Combining bacteriophages with conventional antibiotics, an innovative approach termed phage-antibiotic synergy, presents a promising treatment avenue. However, to enable new treatment strategies, there is a pressing need for methods to assess their efficacy reliably and rapidly. Here, we introduce a novel approach for real-time monitoring of pathogen lysis dynamics employing the piezoelectric quartz crystal microbalance (QCM) with dissipation (QCM-D) technique. The sensor, a QCM chip modified with the bacterium S. aureus RN4220 ΔtarM, was utilized to monitor the activity of the enzyme lysostaphin and the phage P68 as model lytic agents. Unlike conventional QCM solely measuring resonance frequency changes, our study demonstrates that dissipation monitoring enables differentiation of bacterial growth and lysis caused by cell-attached lytic agents. Compared to reference turbidimetry measurements, our results reveal distinct alterations in the growth curve of the bacteria adhered to the sensor, characterized by a delayed lag phase. Furthermore, the dissipation signal analysis facilitated the precise real-time monitoring of phage-mediated lysis. Finally, the QCM-D biosensor was employed to evaluate the synergistic effect of subinhibitory concentrations of the antibiotic amoxicillin with the bacteriophage P68, enabling monitoring of the lysis of P68-resistant wild-type strain S. aureus RN4220. Our findings suggest that this synergy also impedes the formation of bacterial aggregates, the precursors of biofilm formation. Overall, this method brings new insights into phage-antibiotic synergy, underpinning it as a promising strategy against antibiotic-resistant bacterial strains with broad implications for treatment and prevention.

• Keywords
• Staphylococcus aureus, Antimicrobial treatment, Multidrug-resistant bacteria, Phage therapy, Phage-antibiotic synergy, Piezoelectric biosensor,
• MeSH
• Anti-Bacterial Agents pharmacology   MeSH
• Bacteriophages physiology   MeSH
• Bacteriolysis * drug effects   MeSH
• Biosensing Techniques * methods   instrumentation   MeSH
• Lysostaphin pharmacology   MeSH
• Quartz Crystal Microbalance Techniques * methods   MeSH
• Staphylococcus aureus * drug effects   virology   growth & development   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Anti-Bacterial Agents MeSH
• Lysostaphin MeSH

Bacteriophages are the most abundant biological entities on Earth, but our understanding of many aspects of their lifecycles is still incomplete. Here, we have structurally analysed the infection cycle of the siphophage Casadabanvirus JBD30. Using its baseplate, JBD30 attaches to Pseudomonas aeruginosa via the bacterial type IV pilus, whose subsequent retraction brings the phage to the bacterial cell surface. Cryo-electron microscopy structures of the baseplate-pilus complex show that the tripod of baseplate receptor-binding proteins attaches to the outer bacterial membrane. The tripod and baseplate then open to release three copies of the tape-measure protein, an event that is followed by DNA ejection. JBD30 major capsid proteins assemble into procapsids, which expand by 7% in diameter upon filling with phage dsDNA. The DNA-filled heads are finally joined with 180-nm-long tails, which bend easily because flexible loops mediate contacts between the successive discs of major tail proteins. It is likely that the structural features and replication mechanisms described here are conserved among siphophages that utilize the type IV pili for initial cell attachment.

• Keywords
• Pseudomonas aeruginosa, Cryo-EM, Phage, Pili, Structure,
• MeSH
• Fimbriae, Bacterial metabolism   ultrastructure   virology   MeSH
• DNA, Viral metabolism   genetics   MeSH
• Cryoelectron Microscopy * MeSH
• Pseudomonas Phages * ultrastructure   genetics   metabolism   physiology   MeSH
• Pseudomonas aeruginosa * virology   metabolism   MeSH
• Virus Replication * MeSH
• Siphoviridae genetics   ultrastructure   physiology   metabolism   MeSH
• Capsid Proteins metabolism   chemistry   genetics   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Viral MeSH
• Capsid Proteins MeSH

The revolution in cryo-electron microscopy has resulted in unprecedented power to resolve large macromolecular complexes including viruses. Many methods exist to explain density corresponding to proteins and thus entire protein capsids have been solved at the all-atom level. However methods for nucleic acids lag behind, and no all-atom viral double-stranded DNA genomes have been published at all. We here present a method which exploits the spiral winding patterns of DNA in icosahedral capsids. The method quickly generates shells of DNA wound in user-specified, idealized spherical or cylindrical spirals. For transition regions, the method allows guided semiflexible fitting. For the kuravirus SU10, our method explains most of the density in a semiautomated fashion. The results suggest rules for DNA turns in the end caps under which two discrete parameters determine the capsid inner diameter. We suggest that other kuraviruses viruses may follow the same winding scheme, producing a discrete rather than continuous spectrum of capsid inner diameters. Our software may be used to explain the published density maps of other double-stranded DNA viruses and uncover their genome packaging principles.

• MeSH
• DNA, Viral genetics   metabolism   MeSH
• Cryoelectron Microscopy MeSH
• Capsid * metabolism   MeSH
• Podoviridae * MeSH
• Virus Assembly genetics   MeSH
• Capsid Proteins genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• DNA, Viral MeSH
• Capsid Proteins MeSH

Both temperate and obligately lytic phages have crucial roles in the biology of staphylococci. While superinfection exclusion among closely related temperate phages is a well-characterized phenomenon, the interactions between temperate and lytic phages in staphylococci are not understood. Here, we present a resistance mechanism toward lytic phages of the genus Kayvirus, mediated by the membrane-anchored protein designated PdpSau encoded by Staphylococcus aureus prophages, mostly of the Sa2 integrase type. The prophage accessory gene pdpSau is strongly linked to the lytic genes for holin and ami2-type amidase and typically replaces genes for the toxin Panton-Valentine leukocidin (PVL). The predicted PdpSau protein structure shows the presence of a membrane-binding α-helix in its N-terminal part and a cytoplasmic positively charged C terminus. We demonstrated that the mechanism of action of PdpSau does not prevent the infecting kayvirus from adsorbing onto the host cell and delivering its genome into the cell, but phage DNA replication is halted. Changes in the cell membrane polarity and permeability were observed from 10 min after the infection, which led to prophage-activated cell death. Furthermore, we describe a mechanism of overcoming this resistance in a host-range Kayvirus mutant, which was selected on an S. aureus strain harboring prophage 53 encoding PdpSau, and in which a chimeric gene product emerged via adaptive laboratory evolution. This first case of staphylococcal interfamily phage-phage competition is analogous to some other abortive infection defense systems and to systems based on membrane-destructive proteins. IMPORTANCE Prophages play an important role in virulence, pathogenesis, and host preference, as well as in horizontal gene transfer in staphylococci. In contrast, broad-host-range lytic staphylococcal kayviruses lyse most S. aureus strains, and scientists worldwide have come to believe that the use of such phages will be successful for treating and preventing bacterial diseases. The effectiveness of phage therapy is complicated by bacterial resistance, whose mechanisms related to therapeutic staphylococcal phages are not understood in detail. In this work, we describe a resistance mechanism targeting kayviruses that is encoded by a prophage. We conclude that the defense mechanism belongs to a broader group of abortive infections, which is characterized by suicidal behavior of infected cells that are unable to produce phage progeny, thus ensuring the survival of the host population. Since the majority of staphylococcal strains are lysogenic, our findings are relevant for the advancement of phage therapy.

• Keywords
• Kayvirus, Staphylococcus aureus, abortive infection, bacteriophage evolution, bacteriophage therapy, bacteriophages, cell death, lysogeny, phage resistance, phage therapy,
• MeSH
• Humans MeSH
• Lysogeny MeSH
• Membrane Proteins genetics   MeSH
• Prophages * genetics   MeSH
• Staphylococcus Phages genetics   MeSH
• Staphylococcal Infections * microbiology   MeSH
• Staphylococcus aureus genetics   MeSH
• Staphylococcus MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Membrane Proteins MeSH

Escherichia coli phage SU10 belongs to the genus Kuravirus from the class Caudoviricetes of phages with short non-contractile tails. In contrast to other short-tailed phages, the tails of Kuraviruses elongate upon cell attachment. Here we show that the virion of SU10 has a prolate head, containing genome and ejection proteins, and a tail, which is formed of portal, adaptor, nozzle, and tail needle proteins and decorated with long and short fibers. The binding of the long tail fibers to the receptors in the outer bacterial membrane induces the straightening of nozzle proteins and rotation of short tail fibers. After the re-arrangement, the nozzle proteins and short tail fibers alternate to form a nozzle that extends the tail by 28 nm. Subsequently, the tail needle detaches from the nozzle proteins and five types of ejection proteins are released from the SU10 head. The nozzle with the putative extension formed by the ejection proteins enables the delivery of the SU10 genome into the bacterial cytoplasm. It is likely that this mechanism of genome delivery, involving the formation of the tail nozzle, is employed by all Kuraviruses.

• MeSH
• Bacteriophages * genetics   metabolism   MeSH
• DNA, Viral genetics   MeSH
• Phosmet * MeSH
• Genome, Viral genetics   MeSH
• Podoviridae * genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Viral MeSH
• Phosmet * MeSH

The Czech Republic, a part of the former Czechoslovakia, has been at the forefront of several research directions in virology, genetics and physiology [...].

• MeSH
• Virology * MeSH
• Publication type
• Research Support, Non-U.S. Gov't MeSH
• Editorial MeSH
• Geographicals
• Czech Republic MeSH

Leishmania parasites cause a variety of symptoms, including mucocutaneous leishmaniasis, which results in the destruction of the mucous membranes of the nose, mouth, and throat. The species of Leishmania carrying Leishmania RNA virus 1 (LRV1), from the family Totiviridae, are more likely to cause severe disease and are less sensitive to treatment than those that do not contain the virus. Although the importance of LRV1 for the severity of leishmaniasis was discovered a long time ago, the structure of the virus remained unknown. Here, we present a cryo-electron microscopy reconstruction of the virus-like particle of LRV1 determined to a resolution of 3.65 Å. The capsid has icosahedral symmetry and is formed by 120 copies of a capsid protein assembled in asymmetric dimers. RNA genomes of viruses from the family Totiviridae are synthetized, but not capped at the 5' end, by virus RNA polymerases. To protect viral RNAs from degradation, capsid proteins of the L-A totivirus cleave the 5' caps of host mRNAs, creating decoys to overload the cellular RNA quality control system. Capsid proteins of LRV1 form positively charged clefts, which may be the cleavage sites for the 5' cap of Leishmania mRNAs. The putative RNA binding site of LRV1 is distinct from that of the related L-A virus. The structure of the LRV1 capsid enables the rational design of compounds targeting the putative decapping site. Such inhibitors may be developed into a treatment for mucocutaneous leishmaniasis caused by LRV1-positive species of LeishmaniaIMPORTANCE Twelve million people worldwide suffer from leishmaniasis, resulting in more than 30 thousand deaths annually. The disease has several variants that differ in their symptoms. The mucocutaneous form, which leads to disintegration of the nasal septum, lips, and palate, is caused predominantly by Leishmania parasites carrying Leishmania RNA virus 1 (LRV1). Here, we present the structure of the LRV1 capsid determined using cryo-electron microscopy. Capsid proteins of a related totivirus, L-A virus, protect viral RNAs from degradation by cleaving the 5' caps of host mRNAs. Capsid proteins of LRV1 may have the same function. We show that the LRV1 capsid contains positively charged clefts that may be sites for the cleavage of mRNAs of Leishmania cells. The structure of the LRV1 capsid enables the rational design of compounds targeting the putative mRNA cleavage site. Such inhibitors may be used as treatments for mucocutaneous leishmaniasis.

• Keywords
• CAP-4, LRV1, Leishmania, RNA, Totiviridae, Viannia, capsid, cryo-electron microscopy, decapping, genome, leishmaniasis, mRNA, parasite, structure, uncoating, virion, virus,
• MeSH
• Cryoelectron Microscopy MeSH
• Genome, Viral MeSH
• Capsid chemistry   metabolism   MeSH
• Leishmaniavirus chemistry   genetics   metabolism   MeSH
• RNA, Viral genetics   metabolism   MeSH
• Capsid Proteins chemistry   genetics   metabolism   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• RNA, Viral MeSH
• Capsid Proteins MeSH

Alphaproteobacteria, which are the most abundant microorganisms of temperate oceans, produce phage-like particles called gene transfer agents (GTAs) that mediate lateral gene exchange. However, the mechanism by which GTAs deliver DNA into cells is unknown. Here we present the structure of the GTA of Rhodobacter capsulatus (RcGTA) and describe the conformational changes required for its DNA ejection. The structure of RcGTA resembles that of a tailed phage, but it has an oblate head shortened in the direction of the tail axis, which limits its packaging capacity to less than 4,500 base pairs of linear double-stranded DNA. The tail channel of RcGTA contains a trimer of proteins that possess features of both tape measure proteins of long-tailed phages from the family Siphoviridae and tail needle proteins of short-tailed phages from the family Podoviridae. The opening of a constriction within the RcGTA baseplate enables the ejection of DNA into bacterial periplasm.

• MeSH
• Bacteriophages genetics   physiology   ultrastructure   MeSH
• DNA, Bacterial genetics   MeSH
• Cryoelectron Microscopy MeSH
• Gene Transfer, Horizontal MeSH
• Gene Expression Regulation, Bacterial MeSH
• Rhodobacter capsulatus genetics   virology   MeSH
• Siphoviridae genetics   physiology   ultrastructure   MeSH
• Gene Transfer Techniques * MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Bacterial MeSH