Biology subjects

Azam, A. H.

Publications and source records attributed to Azam, A. H..

10 recordsLinked to original sources

bioRxiv2026

Antimicrobial-resistance plasmids encode Evangelion, a widespread DUF4062 anti-phage defence family

Mobile genetic elements have profoundly shaped the evolution of bacterial anti-phage immunity, yet much of this defence repertoire remains undiscovered. Antimicrobial-resistance plasmids are well known for disseminating antibiotic-resistance genes, but whether they also serve as reservoirs of previously unrecognised antiviral immunity remains poorly understood. Here, we identify Evangelion, a widespread family of single-gene anti-phage defence systems, through phenotype-guided interrogation of a naturally occurring methicillin-resistant Staphylococcus aureus plasmid. Eva01, the founding member of the family, protects against lytic Silviavirus phages and is built around a conserved DUF4062 core coupled to a highly diversified auxiliary region that is essential for defence activity. Evangelion systems are enriched on antimicrobial-resistance plasmids but are distributed across diverse bacterial hosts and mobile genetic elements, revealing an evolutionarily conserved defence framework that has diversified through horizontal gene transfer and adaptation to distinct phage environments. Genetic, structural and functional analyses support a model in which Eva01 senses intracellular phage replication-associated processes and couples activation of a DUF4062 effector to NAD depletion and abortive infection. Together, our findings define a previously unrecognised family of mobile anti-phage defence systems, establish DUF4062 proteins as a new component of the bacterial anti-phage repertoire, and demonstrate that phenotype-guided interrogation of mobile genetic elements provides a powerful strategy for discovering defence systems beyond the reach of current computational approaches.

molecular biology↗

bioRxiv2024

Systematic Discovery of Phage Genes that Inactivate Bacterial Immune Systems

Bacteria have developed numerous defense systems to counter phage infections. However, the extent to which phages possess countermeasures against these defense systems remains unclear. In this study, we combined a phage gene knockout library with a defense system library to analyze the mechanisms by which phages counteract bacterial defense systems. After attempting gene deletions of 105 open reading frames (ORFs) in the DruSM1 phage ({Phi}DruSM1), we successfully generated 73 different ORF knockout phages. By infecting this library with bacteria harboring defense system expression plasmids, we identified inactivators of Druantia type I (Druad1), Brex type I, AVAST type III, Sir2+HerA, DUF4297+HerA, and hhe, as well as an activator of Retron Ec86, in a single phage genome. Synthetic phages incorporating Druad1 effectively eradicated Escherichia coli harboring the robust Druantia type I defense system by altering DNA methylation at m6A sites of the phage. This study highlighted the prevalence of various antidefense mechanisms employed by phages to overcome bacterial defense systems.

microbiology↗

bioRxiv2023

Viruses encode tRNA and anti-retron to evade bacterial immunity

Retrons are bacterial genetic retroelements that encode reverse transcriptase capable of producing multicopy single-stranded DNA (msDNA) and function as antiphage defense systems. Phages employ several strategies to counter the host defense systems, but no mechanisms for evading retrons are known. Here, we show that tRNATyr and Rad (retron anti-defense) of T5 phage family inhibit the defense activity of retron 78 and a broad range of retrons, respectively. The effector protein of retron 78, ptuAB, specifically degraded tRNATyr leading abortive infection, but phage countervailed this defense by supplying tRNATyr. Rad inhibited retron function by degrading noncoding RNA, the precursor of msDNA. In summary, we demonstrated that viruses encode at least two independent strategies for overcoming bacterial defense systems: anti-defense, such as Rad, and defense canceler, like tRNA.

microbiology↗

bioRxiv2023

Selective bacteriophages reduce the emergence of resistant bacteria in the bacteriophage-antibiotic combination therapy

Escherichia coli O157:H7 is a globally important foodborne pathogen that affects food safety. Antibiotic administration against O157:H7 may contribute to the exacerbation of hemolytic uremic syndrome (HUS) and antibiotic-resistant strains increase; therefore, bacteriophage therapy (phage therapy) is considered a useful alternative. In the treatment of resistant bacterial infections, combination therapy with bacteriophages and antibiotics, taking advantage of the benefits of both agents, has been suggested to be effective in inhibiting the emergence of antimicrobial-resistant strains; however, its effectiveness against O157:H7 is not well understood. In this study, we isolated SP015, a phage that infects O157:H7, and compared the combined effect of the bacteriophage and fosfomycin (FOM) with that of the PP01 phage. Genomic analysis revealed that FOM exerts its antibacterial activity through glycerol-3-phosphate transporter (GlpT) and hexose phosphate transporter (UhpT) proteins, and the receptors of PP01 and SP015 phages are the outer membrane protein C (OmpC) and ferrichrome outer membrane transporter protein (FhuA), respectively. Experiments with knockout strains have suggested that FOM also uses OmpC, the receptor for PP01, as a transporter. This may explain why the combination treatment with PP01 resulted in a faster emergence of resistance than the combination treatment with SP015. We propose that phage-antibiotic combination therapy requires careful selection of the phage to be used.

microbiology↗

bioRxiv2022

Identification of inosine monophosphate dehydrogenase as a potential target for anti-monkeypox virus agents

Monkeypox virus (MPXV) is a neglected zoonotic pathogen that caused a worldwide outbreak in May 2022. Given the lack of an established therapy, the development of an anti-MPXV strategy is of vital importance. To identify drug targets for the development of anti-MPXV agents, we screened a chemical library using an MPXV infection cell assay and found that gemcitabine, trifluridine, and mycophenolic acid (MPA) inhibited MPXV propagation. These compounds showed broad-spectrum anti-orthopoxvirus activities and presented lower 90% inhibitory concentrations (0.032-1.40 M) than brincidofovir, an approved anti-smallpox agent. These three compounds have been suggested to target the post-entry step to reduce the intracellular production of virions. Knockdown of inosine monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme of guanosine biosynthesis and a target of MPA, dramatically reduced MPXV DNA production. Moreover, supplementation with guanosine recovered the anti-MPXV effect of MPA, suggesting that IMPDH and its guanosine biosynthetic pathway regulate MPXV replication. By targeting IMPDH, we identified a series of compounds with stronger anti-MPXV activity than MPA. These evidences propose that IMPDH is a potential target for the development of anti-MPXV agents. ImportanceMonkeypox is a zoonotic disease caused by infection with the monkeypox virus, and a worldwide outbreak occurred in May 2022. The smallpox vaccine has recently been approved for clinical use against monkeypox in the United States. Although brincidofovir and tecovirimat are drugs approved for the treatment of smallpox by the U.S. Food and Drug Administration, their efficacy against monkeypox has not been established. Moreover, these drugs may present negative side effects. Therefore, new anti-monkeypox virus agents are needed. This study revealed that gemcitabine, trifluridine, and mycophenolic acid inhibited monkeypox virus propagation, exhibited broad-spectrum anti-orthopoxvirus activities. We also suggested inosine monophosphate dehydrogenase as a potential target for the development of anti-monkeypox virus agents. By targeting this molecule, we identified a series of compounds with stronger anti-monkeypox virus activity than mycophenolic acid.

microbiology↗

bioRxiv2022

Potential anti-monkeypox virus activity of atovaquone, mefloquine, and molnupiravir, and their potential use as treatments

Monkeypox virus (MPXV) is a zoonotic orthopoxvirus that causes smallpox-like symptoms in humans and caused an outbreak in May 2022 that led the WHO to declare global health emergency. In this study, from a screening of approved-drug libraries using an MPXV infection cell system, atovaquone, mefloquine, and molnupiravir exhibited anti-MPXV activity, with 50% inhibitory concentrations of 0.51-5.2 M, which is more potent than cidofovir. Whereas mefloquine was suggested to inhibit viral entry, atovaquone and molnupiravir targeted post-entry process to impair intracellular virion accumulation. Inhibitors of dihydroorotate dehydrogenase, an atovaquones target enzyme, showed conserved anti-MPXV activities. Combining atovaquone with tecovirimat enhanced the anti-MPXV effect of tecovirimat. Quantitative mathematical simulations predicted that atovaquone can promote viral clearance in patients by seven days at clinically relevant drug concentrations. Moreover, atovaquone and molnupiravir exhibited pan-Orthopoxvirus activity against vaccinia and cowpox viruses. These data suggest that atovaquone would be potential candidates for treating monkeypox.

microbiology↗

bioRxiv2019

Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria

Emergence of antimicrobial-resistant bacteria is an increasingly serious threat to global health, necessitating the development of innovative antimicrobials. We established a series of CRISPR-Cas13a-based antibacterial nucleocapsid, termed CapsidCas13a(s), capable of sequence-specific killing of carbapenem-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus through promiscuous RNA cleavage after recognizing corresponding antimicrobial resistance genes. CapsidCas13a constructs were generated by packaging CRISPR-Cas13a into a bacteriophage capsid to target antimicrobial resistance genes. Contrary to Cas9-based antimicrobials that lack bacterial killing capacity when the target genes are located on a plasmid, the CapsidCas13a(s) exhibited strong bacterial killing activities upon recognizing target genes regardless of their location. The antimicrobials treatment efficacy was confirmed using a Galleria mellonella larvae model. Further, we demonstrated that the CapsidCas13a(s) can assist in bacterial gene detection without employing nucleic acid amplification and optical devices.

microbiology↗

bioRxiv2019

Composition and Diversity of CRISPR-Cas13a systems in the genus Leptotrichia

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, previously known as CRISPR-C2c2, is the most recently identified RNA-guided RNA-targeting CRISPR-Cas system and has the unique characteristics of both targeted and collateral single-stranded RNA (ssRNA) cleavage activities, which was first identified in Leptotrichia shahii CRISPR-Cas13a. Here, the complete whole genome sequences of 11 Leptotrichia strains were determined and compared with 18 publicly available Leptotrichia genomes in regard to the composition, occurrence and diversity of the CRISPR-Cas13a and other CRISPR-Cas systems. Various types of CRISPR-Cas systems, I-B, II-C, III-A, III-D, III-like and VI-A, were unevenly distributed among the Leptotrichia genomes, accounting for 10 (34.4%), 1 (2.6%), 6 (15.4%), 6 (15.4%), 3 (7.7%) and 11 (37.9%) of the 29 strains, respectively, while 8 (20.5%) strains had no CRISPR-Cas system. The Cas13a effectors were highly divergent with amino acid sequence similarities ranging from 61% to 90% to that of L. shahii and their collateral ssRNA cleavage activities, resulting in cytotoxicity to host cell, were found to be maintained. CRISPR-Cas spacers represent a sequential achievement of former intruder encounters, and the retained spacers reflect the evolutionary phylogeny or relatedness of strains. Analysis of spacer contents and numbers among Leptotrichia species showed considerable diversity with only 2 (0.5%) of 400 spacers through CRIPSR-Cas I-VI were shared by two strains. The organisation and distribution of CRISPR-Cas systems (type I-VI) encoded by all registered Leptotrichia species revealed that effector or spacer sequences of the CRISPR-Cas systems were very divergent, and the prevalence of types I, III and VI was almost equal. There was only one strain carrying type II, while none carried type IV or V. These results provide new insights into the characteristics and divergences of CRISPR-Cas systems among Leptotrichia species.

microbiology↗

bioRxiv2019

Analysis host-recognition mechanism of staphylococcal kayvirus phiSA039 reveals a novel strategy that protects Staphylococcus aureus against infection by Staphylococcus pseudintermedius Siphoviridae phages

Following the emergence of antibiotic resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP), phage therapy has attracted significant attention as an alternative to antibiotic treatment. Bacteriophages belonging to kayvirus (previously known as Twort-like phages) have broad host range and are strictly lytic in Staphylococcus spp. Previous work revealed that kayvirus {phi}SA039 has a host-recognition mechanism distinct from those of other known kayviruses: most of kayviruses use the backbone of wall teichoic acid (WTA) as their receptor; by contrast, {phi}SA039 uses the {beta}-N-acetylglucosamine ({beta}-GlcNAc) residue in WTA. In this study, we found that {phi}SA039 could switch its receptor to be able to infect S. aureus lacking the {beta}-GlcNAc residue by acquiring a spontaneous mutation in open reading frame (ORF) 100 and ORF102. Moreover, {phi}SA039 could infect S. pseudintermedius, which has a different WTA structure than S. aureus. By comparison with newly isolated S. pseudintermedius-specific phage (SP phages), we determined that glycosylation in WTA of S. pseudintermedius is essential for adsorption of SP phages, but not {phi}SA039. Finally, we describe a novel strategy of S. aureus which protects the bacteria from infection of SP phages. Notably, glycosylation of ribitol phosphate (RboP) WTA by TarM or/and TarS prevents infection of S. aureus by SP phages. These findings could help to establish a new strategy for treatment of S. aureus and S. pseudintermedius infection, as well as provide valuable insights into the biology of phage-host interactions.

microbiology↗

bioRxiv2018

Analysis of phage-resistant mechanisms in Staphylococcus aureus SA003 reveals a different binding mechanism for the closely related Twort-like phages phiSA012 and phiSA039

We have previously generated strains of Staphylococcus aureus SA003 resistant to its specific phage {phi}SA012 through long-term coevolution experiment. However, the DNA mutations responsible for the phenotypic change of phage resistance are unknown. Whole-genome analysis revealed six genes that acquired unique point mutations: five missense mutations and one nonsense mutation. Moreover, one deletion, 1.779-bp, resulted in the deletion of the genes encoding glycosyltransferase, TarS, and iron-sulfure repair protein, ScdA. The deletion occurred from the second round of coculture (SA003R2) and remained through the last round. The {phi}SA012 infection toward SA003R2 had decreased to 79.77{+/-}7.50% according to plating efficiency. Complementation of the phage-resistant strain by the wild-type allele showed two mutated host genes were linked to the inhibition of post-adsorption, and five genes were linked to phage adsorption of {phi}SA012. Unlike {phi}SA012, infection by {phi}SA039, a close relative of {phi}SA012, onto SA003R2 was impaired drastically. Complementation of SA003R2 by wild-type tarS restores the infectivity of {phi}SA039. Thus, we concluded that {phi}SA039 requires {beta}-GlcNAc in Wall Teichoic Acid (WTA) for its binding. In silico analysis of the {phi}SA039 genome revealed that several proteins in the tail and baseplate region were different from {phi}SA012; notably the partial deletion of orf96 of {phi}SA039, a homolog of orf99 of {phi}SA012. Orf100 of {phi}SA039, a homolog of Orf103 of {phi}SA012, a previously reported receptor binding protein (RBP), had low similarity (86%) to that of {phi}SA012. The difference in tail and baseplate proteins might be the factor for specificity difference between {phi}SA012 and {phi}SA039.

microbiology↗