Biology subjects

Hatoum-Aslan, A.

Publications and source records attributed to Hatoum-Aslan, A..

6 recordsLinked to original sources

bioRxiv2023

Tandem mobilization of anti-phage defenses alongside SCCmec cassettes

Bacterial viruses (phages) and the immune systems targeted against them significantly impact bacterial survival, evolution, and the emergence of pathogenic strains. While recent research has made spectacular strides towards discovering and validating new defenses in a few model organisms1-3, the inventory of immune systems in clinically-relevant bacteria remains under-explored, and little is known about the mechanisms by which these systems horizontally spread. Such pathways not only impact the evolutionary trajectory of bacterial pathogens, but also threaten to undermine the effectiveness of phage-based therapeutics. Here, we investigate the battery of defenses in staphylococci, opportunistic pathogens that constitute leading causes of antibiotic-resistant infections. We show that these organisms harbor a variety of anti-phage defenses encoded within/near the infamous SCC (staphylococcal cassette chromosome) mec cassettes, mobile genomic islands that confer methicillin resistance. Importantly, we demonstrate that SCCmec-encoded recombinases mobilize not only SCCmec, but also tandem cassettes enriched with diverse defenses. Further, we show that phage infection potentiates cassette mobilization. Taken together, our findings reveal that beyond spreading antibiotic resistance, SCCmec cassettes play a central role in disseminating anti-phage defenses. This work underscores the urgent need for developing adjunctive treatments that target this pathway to save the burgeoning phage therapeutics from suffering the same fate as conventional antibiotics.

microbiology↗

bioRxiv2022

The structure of a Type III-A CRISPR-Cas effector complex reveals conserved and idiosyncratic contacts to target RNA and crRNA among Type III-A systems

Type III CRISPR-Cas systems employ multiprotein effector complexes bound to small CRISPR RNAs (crRNAs) to detect foreign RNA transcripts and elicit a complex immune response that leads to the destruction of invading RNA and DNA. Type III systems are among the most widespread in nature, and emerging interest in harnessing these systems for biotechnology applications highlights the need for detailed structural analyses of representatives from diverse organisms. We performed cryo-EM reconstructions of the Type III-A Cas10-Csm effector complex from S. epidermidis bound to an intact, cognate target RNA and identified two oligomeric states, a 276 kDa complex and a 318 kDa complex. 3.1 [A] density for the well-ordered 276 kDa complex allowed construction of atomic models for the Csm2, Csm3, Csm4 and Csm5 subunits within the complex along with the crRNA and target RNA. We also collected small-angle X-ray scattering data which was consistent with the 276 kDa Cas10-Csm architecture we identified. Detailed comparisons between the S. epidermidis Cas10-Csm structure and the well-resolved bacterial (S. thermophilus) and archaeal (T. onnurineus) Cas10-Csm structures reveal differences in how the complexes interact with target RNA and crRNA which are likely to have functional ramifications. These structural comparisons shed light on the unique features of Type III-A systems from diverse organisms and will assist in improving biotechnologies derived from Type III-A effector complexes.

molecular biology↗

bioRxiv2022

Structure and host specificity of Staphylococcus epidermidis bacteriophage Andhra

Staphylococcus epidermidis is an opportunistic pathogen of the human skin, often associated with infections of implanted medical devices. An increase in antibiotic resistance in S. epidermidis and other bacterial pathogens has led to renewed interest in the use of bacteriophages as an alternative to conventional antibiotics. Staphylococcal picoviruses are a group of strictly lytic, short-tailed bacteriophages with compact genomes that are attractive candidates for therapeutic use. Here, we report the structure of the complete virion of S. epidermidis-infecting phage Andhra, determined using high-resolution cryo-electron microscopy, allowing atomic modeling of the capsid and tail proteins, including twelve trimers of a unique receptor binding protein, the hexameric tail knob that acts as a gatekeeper for DNA ejection, and the tail tip, which is a heterooctamer of two different lytic proteins. Our findings elucidate critical features that enable host recognition and penetration, facilitating the development of this group of phages for therapeutic applications.

microbiology↗

bioRxiv2022

Critical roles for 'housekeeping' nucleases in Type III CRISPR-Cas immunity

CRISPR-Cas systems are a family of adaptive immune systems that use small CRISPR RNAs (crRNAs) and CRISPR-associated (Cas) nucleases to protect prokaryotes from invading plasmids and viruses (i.e. phages). Type III systems launch a multi-layered immune response that relies upon both Cas and non-Cas cellular nucleases, and although the functions of Cas components have been well described, the identities and roles of non-Cas participants remain poorly understood. Previously, we showed that the Type III-A CRISPR-Cas system in Staphylococcus epidermidis employs two degradosome-associated nucleases, PNPase and RNase J2, to promote crRNA maturation and eliminate invading nucleic acids (Chou-Zheng and Hatoum-Aslan, 2019). Here, we identify RNase R as a third housekeeping nuclease critical for immunity. We show that RNase R works in concert with PNPase to complete crRNA maturation, and identify specific interactions with Csm5, a member of the Type III effector complex, which facilitate nuclease recruitment/stimulation. Further, we demonstrate that RNase R and PNPase are required to maintain robust anti-plasmid immunity, particularly when targeted transcripts are sparse. Altogether, our findings expand the known repertoire of accessory nucleases required for Type III immunity and highlight the remarkable capacity of these systems to interface with diverse cellular pathways to ensure successful defense.

microbiology↗

bioRxiv2019

A unique mode of nucleic acid immunity performed by a single multifunctional enzyme

The perpetual arms race between bacteria and their viruses (phages) has given rise to diverse immune systems, including restriction-modification and CRISPR-Cas, which sense and degrade phage-derived nucleic acids. These complex systems rely upon production and maintenance of multiple components to achieve anti-phage defense. However, the prevalence and effectiveness of much simpler, single-component systems that cleave DNA remain unknown. Here, we describe a novel mode of nucleic acid immunity performed by a single enzyme with nuclease and helicase activities, herein referred to as Nhi. This enzyme provides robust protection against diverse staphylococcal phages and prevents phage DNA accumulation in cells stripped of all other known defenses. Our observations support a model in which Nhi acts as both the sensor and effector to degrade phage-specific replication intermediates. Importantly, Nhi homologs are distributed in diverse bacteria and exhibit functional conservation, highlighting the versatility of such compact weapons as major players in anti-phage defense.

microbiology↗

bioRxiv2019

A Type III-A CRISPR-Cas system employs degradosome nucleases to ensure robust immunity

CRISPR-Cas systems provide sequence-specific immunity against phages and mobile genetic elements using CRISPR-associated nucleases guided by short CRISPR RNAs (crRNAs). Type III systems exhibit a robust immune response that can lead to the extinction of a phage population, a feat coordinated by a multi-subunit effector complex that destroys invading DNA and RNA. Here, we demonstrate that a model Type III system in Staphylococcus epidermidis relies upon the activities of two degradosome-associated nucleases, PNPase and RNase J2, to mount a successful defense. Genetic, molecular, and biochemical analyses reveal that PNPase promotes crRNA maturation, and both nucleases are required for efficient clearance of phage-derived nucleic acids. Furthermore, functional assays show that RNase J2 is essential for immunity against diverse mobile genetic elements originating from plasmid and phage. Altogether, our observations reveal the evolution of a critical collaboration between two nucleic acid degrading machines which ensures cell survival when faced with phage attack.

microbiology↗