DSpace Repository :: Browsing by Author "Hatoum-Aslan, Asma"

Browsing by Author "Hatoum-Aslan, Asma"

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Complete Genome Sequence of Staphylococcus aureus Siphophage Lorac
(American Society of Microbiology, 2019) Marc, Antoine; Cater, Katie; Kongari, Rohit; Hatoum-Aslan, Asma; Young, Ryland F., III; Liu, Mei; Texas A&M University College Station; University of Alabama Tuscaloosa
Staphylococcus aureus is a leading cause of a wide range of clinical infections. Here, we announce the complete genome sequence of S. aureus si-phophage Lorac, a phiETA-like temperate phage that is similar at the nucleotide level to the previously described S. aureus prophage phiNM2.
Complete Genome Sequences of Staphylococcus epidermidis Myophages Quidividi, Terranova, and Twillingate
(American Society of Microbiology, 2019) Freeman, Miranda E.; Kenny, Sarah E.; Lanier, Amanda; Cater, Katie; Wilhite, Mary C.; Gamble, Paige; O'Leary, Chandler J.; Hatoum-Aslan, Asma; Young, Ryland F., III; Liu, Mei; Texas A&M University College Station; University of Alabama Tuscaloosa
Staphylococcus epidermidis is an opportunistic pathogen that commonly colonizes human skin and mucous membranes. We report here the complete genome sequences of three S. epidermidis phages, Quidividi, Terranova, and Twillingate, which are members of the Twort-like group of large myophages infecting Gram-positive hosts.
Draft Genome Sequences of Staphylococcus Podophages JBug18, Pike, Pontiff, and Pabna
(American Society of Microbiology, 2019) Culbertson, Emma K.; Bari, S. M. Nayeemul; Dandu, Vidya Sree; Kriznik, Jennie M.; Scopel, Samuel E.; Stanley, Samuel P.; Lackey, Kim; Hernandez, Adriana C.; Hatoum-Aslan, Asma; University of Alabama Tuscaloosa; Texas A&M University College Station
We report here the draft genome sequences of Staphylococcus bacteriophages JBug18, Pike, Pontiff, and Pabna, which infect and lyse S. epidermidis and S. aureus strains. All bacteriophages belong to the morphological family Podoviridae and constitute attractive candidates for use as whole-phage therapeutics due to their compact genomes and lytic lifestyles.
Investigation of the RNA-Protein Structure-Function Relationships in the CRISPR-Cas10 Complex and Erythromycin-Resistance RNA Methyltransferases
(University of Alabama Libraries, 2021) Nasef, Mohamed; Dunkle, Jack A.; University of Alabama Tuscaloosa
CRISPR-Cas systems are a class of adaptive immune systems in prokaryotes that use small CRISPR RNAs (crRNAs) in conjunction with CRISPR-associated (Cas) nucleases to recognize and degrade foreign nucleic acids. Recent studies have revealed that type III CRISPR-Cas systems synthesize second messenger molecules, previously unknown to exist in prokaryotes, known as cyclic oligoadenylates (cOA). These molecules activate the Csm6 nuclease to promote RNA degradation and may also coordinate additional cellular responses to foreign nucleic acids. Although cOA production has been reconstituted and characterized for a few bacterial and archaeal type III systems, cOA generation and its regulation have not been explored for the Gram-positive bacterium, Staphylococcus epidermidis, as well as the specific target RNA sequence requirements. In chapter 2, we demonstrate that this system performs Mg2+ dependent synthesis of 3-6 nt cOA. We show that activation of cOA synthesis is perturbed by single nucleotide mismatches between the crRNA and target RNA at discrete positions and synthesis is antagonized by Csm3-mediated target RNA cleavage. In chapter 3, we mechanistically investigated the effect of single and multiple mismatches on the activation of cOA synthesis of type III-A Cas10-Csm complex from S. epidermidis. We show that mismatches at specific positions in target RNA significantly reduced the amount of cOAs produced in a sequence context-dependent manner. We also show that the defects are not due to perturbations in binding efficiencies or in RNA cleavage.In chapter 4, we focus on an erythromycin resistance methyltransferases (Erm) found in many Gram-positive pathogens. Erm proteins are S-adenosyl methionine-dependent Rossmann-fold methyltransferases which convert A2058 of 23S rRNA to m62A2058. This modificationsterically blocks binding of several classes of antibiotics to 23S rRNA, resulting in a multi-drug resistant phenotype in bacteria expressing the enzyme. ErmC is an erythromycin resistance methyltransferase found in many Gram-positive pathogens, while ErmE is found in the soil bacterium that biosynthesizes erythromycin. Whether ErmC and ErmE, which possess only 24 % sequence identity, use similar structural elements for rRNA substrate recognition and positioning is not known. To investigate this question, we used structural data from related proteins to guide site-saturation mutagenesis of key residues and characterized selected variants by antibiotic susceptibility testing, single turnover kinetics, and RNA affinity binding assays. We demonstrate that residues in α4, α5 and the α5-α6 linker are essential for methyltransferase function including: an aromatic residue on α4 that likely forms stacking interactions with the substrate adenosine, and basic residues in α5 and the α5-α6 linker which likely mediate conformational rearrangements in the protein and cognate rRNA upon interaction. The functional studies led us to a new structural model for the ErmC or ErmE-rRNA complex.
Molecular determinants for CRISPR RNA maturation in the Cas10-Csm complex and roles for non-Cas nucleases
(Oxford University Press, 2017) Walker, Forrest C.; Chou-Zheng, Lucy; Dunkle, Jack A.; Hatoum-Aslan, Asma; University of Alabama Tuscaloosa
CRISPR-Cas (Clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) is a prokaryotic immune system that destroys foreign nucleic acids in a sequence-specific manner using Cas nucleases guided by short RNAs (crRNAs). Staphylococcus epidermidis harbours a Type III-A CRISPR-Cas system that encodes the Cas10-Csm interference complex and crRNAs that are subjected to multiple processing steps. The final step, called maturation, involves a concerted effort between Csm3, a ruler protein in Cas10-Csm that measures six-nucleotide increments, and the activity of a nuclease(s) that remains unknown. Here, we elucidate the contributions of the Cas10-Csm complex toward maturation and explore roles of non-Cas nucleases in this process. Using genetic and biochemical approaches, we show that charged residues in Csm3 facilitate its self-assembly and dictate the extent of maturation cleavage. Additionally, acidic residues in Csm5 are required for efficient maturation, but recombinant Csm5 fails to cleave crRNAs in vitro. However, we detected cellular nucleases that co-purify with Cas10-Csm, and show that Csm5 regulates their activities through distinct mechanisms. Altogether, our results support roles for non-Cas nuclease(s) during crRNA maturation and establish a link between Type III-A CRISPR-Cas immunity and central nucleic acid metabolism.
A Novel Staphylococcus Podophage Encodes a Unique Lysin with Unusual Modular Design
(American Society of Microbiology, 2017) Cater, Katie; Dandu, Vidya Sree; Bari, S. M. Nayeemul; Lackey, Kim; Everett, Gabriel F. K.; Hatoum-Aslan, Asma; University of Alabama Tuscaloosa; Texas A&M University College Station
Drug-resistant staphylococci, particularly Staphylococcus aureus and Staphylococcus epidermidis, are leading causes of hospital-acquired infections. Bacteriophages and their peptidoglycan hydrolytic enzymes (lysins) are currently being explored as alternatives to conventional antibiotics; however, only a limited diversity of staphylococcal phages and their lysins has yet been characterized. Here, we describe a novel staphylococcal phage and its lysins. Bacteriophage Andhra is the first reported S. epidermidis phage belonging to the family Podoviridae. Andhra possesses an 18,546-nucleotide genome with 20 open reading frames. BLASTp searches revealed that gene product 10 (gp10) and gp14 harbor putative catalytic domains with predicted peptidase and amidase activities, characteristic functions of phage lysins. We purified these proteins and show that both Andhra_gp10 and Andhra_gp14 inhibit growth and degrade cell walls of diverse staphylococci, with Andhra_gp10 exhibiting more robust activity against the panel of cell wall substrates tested. Site-directed mutagenesis of its predicted catalytic residues abrogated the activity of Andhra_gp10, consistent with the presence of a catalytic CHAP domain on its C terminus. The active site location combined with the absence of an SH3b cell wall binding domain distinguishes Andhra_gp10 from the majority of staphylococcal lysins characterized to date. Importantly, close homologs of Andhra_gp10 are present in related staphylococcal podophages, and we propose that these constitute a new class of phage-encoded lysins. Altogether, our results reveal insights into the biology of a rare family of staphylococcal phages while adding to the arsenal of antimicrobials with potential for therapeutic use. IMPORTANCE The spread of antibiotic resistance among bacterial pathogens is inciting a global public health crisis. Drug-resistant Staphylococcus species, especially S. aureus and S. epidermidis, have emerged in both hospital and community settings, underscoring the urgent need for new strategies to combat staphylococcal infections. Bacterial viruses (phages) and the enzymes that they use to degrade bacterial cell walls (lysins) show promise as alternative antimicrobials; however, only a limited variety of staphylococcal phages and their lysins have yet been identified. Here, we report the discovery and characterization of a novel staphylococcal phage, Andhra. We show that Andhra encodes two lysins (Andhra_gp10 and Andhra_gp14) that inhibit growth and degrade the cell walls of diverse staphylococci, including S. aureus and S. epidermidis strains. Andhra and its unique lysins add to the arsenal of antimicrobials with potential for therapeutic use.
Phage Genetic Engineering Using CRISPR-Cas Systems
(MDPI, 2018) Hatoum-Aslan, Asma; University of Alabama Tuscaloosa
Since their discovery over a decade ago, the class of prokaryotic immune systems known as CRISPR-Cas have afforded a suite of genetic tools that have revolutionized research in model organisms spanning all domains of life. CRISPR-mediated tools have also emerged for the natural targets of CRISPR-Cas immunity, the viruses that specifically infect bacteria, or phages. Despite their status as the most abundant biological entities on the planet, the majority of phage genes have unassigned functions. This reality underscores the need for robust genetic tools to study them. Recent reports have demonstrated that CRISPR-Cas systems, specifically the three major types (I, II, and III), can be harnessed to genetically engineer phages that infect diverse hosts. Here, the mechanisms of each of these systems, specific strategies used, and phage editing efficacies will be reviewed. Due to the relatively wide distribution of CRISPR-Cas systems across bacteria and archaea, it is anticipated that these immune systems will provide generally applicable tools that will advance the mechanistic understanding of prokaryotic viruses and accelerate the development of novel technologies based on these ubiquitous organisms.
Regulation of cyclic oligoadenylate synthesis by the Staphylococcus epidermidis Cas10-Csm complex
(Cold Spring Harbor Lab Press, 2019) Nasef, Mohamed; Muffly, Mary C.; Beckman, Andrew B.; Rowe, Sebastian J.; Walker, Forrest C.; Hatoum-Aslan, Asma; Dunkle, Jack A.; University of Alabama Tuscaloosa
CRISPR-Cas systems are a class of adaptive immune systems in prokaryotes that use small CRISPR RNAs (crRNAs) in conjunction with CRISPR-associated (Cas) nucleases to recognize and degrade foreign nucleic acids. Recent studies have revealed that Type III CRISPR-Cas systems synthesize second messenger molecules previously unknown to exist in prokaryotes, cyclic oligoadenylates (cOA). These molecules activate the Csm6 nuclease to promote RNA degradation and may also coordinate additional cellular responses to foreign nucleic acids. Although cOA production has been reconstituted and characterized for a few bacterial and archaeal Type III systems, cOA generation and its regulation have not been explored for the Staphylococcus epidermidis Type III-A CRISPR-Cas system, a longstanding model for CRISPR-Cas function. Here, we demonstrate that this system performs Mg2+-dependent synthesis of 3-6 nt cOA. We show that activation of cOA synthesis is perturbed by single nucleotide mismatches between the crRNA and target RNA at discrete positions, and that synthesis is antagonized by Csm3-mediated target RNA cleavage. Altogether, our results establish the requirements for cOA production in a model Type III CRISPR-Cas system and suggest a natural mechanism to dampen immunity once the foreign RNA is destroyed.
Strategies for Editing Virulent Staphylococcal Phages Using CRISPR-Cas10
(American Chemical Society, 2017) Bari, S. M. Nayeemul; Walker, Forrest C.; Cater, Katie; Aslan, Barbaros; Hatoum-Aslan, Asma; University of Alabama Tuscaloosa
Staphylococci are prevalent skin-dwelling bacteria that are also leading causes of antibiotic-resistant infections. Viruses that infect and lyse these organisms (virulent staphylococcal phages) can be used as alternatives to conventional antibiotics and represent promising tools to eliminate or manipulate specific species in the microbiome. However, since over half their genes have unknown functions, virulent staphylococcal phages carry inherent risk to cause unknown downstream side effects. Further, their swift and destructive reproductive cycle make them intractable by current genetic engineering techniques. CRISPR-Cas10 is an elaborate prokaryotic immune system that employs small RNAs and a multisubunit protein complex to detect and destroy phages and other foreign nucleic acids. Some staphylococci naturally possess CRISPR-Cas10 systems, thus providing an attractive tool already installed in the host chromosome to harness for phage genome engineering. However, the efficiency of CRISPR-Cas10 immunity against virulent staphylococcal phages and corresponding utility as a tool to facilitate their genome editing has not been explored. Here, we show that the CRISPR-Cas10 system native to Staphylococcus epidermidis exhibits robust immunity against diverse virulent staphylococcal phages. On the basis of this activity, a general two-step approach was developed to edit these phages that relies upon homologous recombination machinery encoded in the host. Variations of this approach to edit toxic phage genes and access phages that infect CRISPR-less staphylococci are also presented. This versatile set of genetic tools enables the systematic study of phage genes of unknown functions and the design of genetically defined phage-based antimicrobials that can eliminate or manipulate specific Staphylococcus species.
Structural investigations and determination of biocatalyst potential of Pseudomonas putida CBB5
(University of Alabama Libraries, 2020) Mills, Shelby Brooks; Summers, Ryan M.; University of Alabama Tuscaloosa
Pseudomonas putida CBB5 has evolved the ability to metabolize caffeine and other methylxanthines to xanthine using a set of five enzymes, NdmABCDE. NdmABC are N1-, N3-, and N7-specific N-demethylases, respectively, that are part of the multicomponent Rieske oxygenase family. Structural investigations of NdmA, NdmB, and NdmD were conducted along with determining the applicability of harnessing this N-demethylation system for the bioproduciton of methylxanthines. Protein co-expression and purification of NdmA and NdmB confirmed the presence of an NdmAB complex that is constructed to perform N-demethylation. The interactions of NdmD and NdmAB were then elucidated because NdmD serves as the sole Rieske reductase for this set of enzymes and transfers electrons to each catalytic site. NdmD is unique amongst Rieske reductases because it contains an extra Rieske [2Fe-2S] cluster at the N-terminal end. The hypothesis is the Rieske [2Fe-2S] cluster on NdmD is used in conjunction with the Rieske-less NdmC enzyme and structural subunit NdmE to carry out the N7-demethylation of 7-methylxanthine to xanthine, but is not required for activity with the NdmA and NdmB enzymes. The results support the hypothesis and expand it by suggesting that the extra Rieske [2Fe-2S] cluster can be used as a secondary electron transport pathway to NdmAB. NdmA converts caffeine to theobromine, which is further N3-demethylated by NdmB, resulting in 7-methylxanthine. However, NdmA exhibits a slight promiscuity toward the N3-methyl group, resulting in 1.5% of caffeine being convertedto paraxanthine. Analysis of the NdmA and NdmB structures identified that only two of the nine amino acids in the binding pocket differ between NdmA and NdmB. Mutation of the two unique amino acids in NdmA to mimic the NdmB active site produced a mutant enzyme with a paraxanthine:theobromine ratio of at least 3:1, over a 100-fold improvement from the wild-type ratio (1:39). Additionally, a peptide loop near the active sites also differs between NdmA and NdmB. Mutation of the NdmA loop sequence to match that of the NdmB loop further increased the yield of paraxanthine. This research confirms that biocatalytic production of paraxanthine from caffeine is achievable and begins to optimize the starting reaction conditions.
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
(PLOS, 2023) Paraan, Mohammadreza; Nasef, Mohamed; Chou-Zheng, Lucy; Khweis, Sarah A. A.; Schoeffler, Allyn J. J.; Hatoum-Aslan, Asma; Stagg, Scott M. M.; Dunkle, Jack A. A.; University of Alabama Tuscaloosa; University of Illinois Urbana-Champaign; Loyola University New Orleans; Florida State University
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 & ANGS; 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.
A type III-A CRISPR-Cas System employs degradosome necleases to ensure robust immunity
(eLife Sciences Publications, 2019) Chou-Zheng, Lucy; Hatoum-Aslan, Asma; University of Alabama Tuscaloosa
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.

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