1. Bacteriophages By Monique Hunter

2.  Bacteriophages or phages are viruses that infect bacteria.  In 1917, French-canadian microbiologist Felix d’Herelle observed an unknown substance killing bacteria as did his predecessor in 1915.  Called it bacteriophage meaning bacteria-eater.  Named after the bacterial species they infect. Example: Mycobacteriophages infect mycobacteria.  Estimated phage population is 10 31 making them the most abundant organisms in the biosphere. Introduction

3.  Pedulla: “If phages were the size of a beetle, they would cover the Earth and be many miles deep”.  Have different structures, but same features: a head or capsid and a tail.  The first mycobacteriophages were described in 1950s. Introduction

4. Figure1: Lambda phage or coliphage lambda

5. How Bacteriophages work?  Bacteriophages are viruses and can only replicate inside a host.  A phage attaches itself to bacterial’ cell wall by binding to specific receptors: adsorption.  Injects its DNA or RNA inside the host: penetration.  Duplication of genetic material and protein synthesis.  Maturation and release Figure 2: Phage adhesion to the host cell

6. Figure 3: Bacteriophages lytic and lysogenic cycles. Pierce BA. Genetics: A conceptual Approach. 3 rd edition Two alternative life cycles of Phages

7. Two alternative life cycles (Summary) 1. The lytic cycle (T4 and T7)  A phage attaches to a receptor on the bacterial cell wall and injects its DNA into the cell.  Inside the host cell, the phage will use the host cell’s chemical energy and biosynthetic machinery to replicate its DNA, transcribe and translate it, producing more phage DNA and phage proteins.  New phage particles are assembled from these components.  The phages produce enzyme that breaks open the host cell, releasing the new phages.  Virulence phages use only lytic cycle, always killing their host cells.

8. 2. The lysogenic cycle (Lambda)  The first 2 steps are identical to the lytic cycle.  However, inside the host, the phage DNA integrates into the bacterial chromosome, creating the prophage.  When the bacterium divides, the prophage is replicated along with the host DNA.  Certain conditions such as exposure to UV, desiccation can cause the prophage to enter the lytic cycle.

9. Phage Genomics  Highly diverse genome  All known mycobacteriophages are dsDNA (double strand DNA).  After infection, there is potentiality to recombine and generate new genomic arrangement.  Although L5, D29, Bxb1 and TM4 were isolated at different places and times, they have features in common.  Morphology, genome size (49.1-52.8kbp), arrangements of structure and assembly genes (many of which encode related genes products).

10.  Mycobacteriophage (L5): first genome to be sequenced in 1993  D29 second with complete genome isolated from soil sample in California. Figure 4: Mycobacteriophages virion morphologies. (Pedulla et al,.2003)

11. “Modular or mosaic evolution”  Postulated that phages evolve by genetic exchange at specific intergenic sites, either through homologous recombination or by site-specific mechanism.  Illegitimate recombination takes place quasi-randomly along the recombining genomes.  However, natural selection eliminates all but recombinants in which biological functions are intact.  Phages with mosaic joints that lies between genes.  Leading to population in which the sites of recombination are not random.

12. Figure 5: “Mosaicism in mycobacteriophage genomes. A segment of the Omega genome coding genes 159 to 171 is shown with homologues found in other mycobacteriophages indicated. Six of the genes (161, 165, 167, 168, 169 and 171) have no homologues. Omega gp163 and gp164 are a Clp protease and a DinG helicase, respectively, as shown, but the functions of the other genes are not known. The genome is characteristically mosaic, with individual genes representing modules that are present in other phage genomes”. (Cole et al, 2004)

13. Phage Genomics  Group according to:  Nucleotide sequence similarities  Gene content analyses  Arbitrary cutoff: any two genomes that have more than 50% nucleotide sequence similarity belong to the same cluster.

14. Figure 6: Diversity of mycobacteriophages. Sequenced genomes for 471mycobacteriophages compared ac cording to their shared gene contents. Colored circles are clusters (A-T), grey circles are singleton genome s with no close relatives. 10 of the clusters are divided into subclusters. (Hatfull et al, 2014)

15. Phage Genomics  Phages acquire genes from, and contribute genes to, not only other phages genomes but also bacterial genomes.  Therefore are powerful forces in the evolution, physiology and pathogenecity of their hosts. Figure 7: Genes can be transferred from one bacterium to another through generalized transd uction. Pierce BA. Genetics: A conceptual Approach. 3 rd edition

16. Do phages contribute to bacterial pathogenesis?  They help certain hosts become more virulent.  Examples: Vibrio cholerae, Clostridium botulinum, Salmonella enterica serovar Typhimurium, and Escherichia coli.  Mycobacterium tuberculosis’s pathogenesis is not due to mycobacteriophages, unlike other species of mycobacteria.

17.  For diagnosis of mycobacterial infection such as tuberculosis  In the development of tools for mycobacterial genetics  Therapeutics to cure patients with tuberculosis  Detect drug resistant tuberculosis  Better genetic engineering of a vaccine to prevent tuberculosis Potential Bacteriophage Applications

18.  Treat diseases: In 1921, phages were used to successfully treat staphylococcal skin infections.  Despite controversial results, Russia, Poland continued using phage therapy.  Antibiotic resistant bacteria problem rekindling the practical applications of bacteriophages.  Phages are highly specific against only a specific bacterial species, and cannot infect eukaryotic cells.

19.  In 2006 FDA approved a phage cocktail that targets Listeria monocytogenes contaminants in meat and poultry products.  In 2008, FDA approved first phage phase I clinical trial. Cocktail containing 8 phages targeting Staphylococcus aureus, Pseudomonas aeruginosa and E. coli.  Phages usage in water treatment in USA is being evaluated  They are abundant in the environment meaning that we are exposed to them. So they are safe. Bacteriophage Applications

20. Limitations and concerns  Since phages are very specific, it is important to precisely determine the targeted pathogen before phage therapy.  In vivo elimination of bacteriophages (several dose of phages can be given).  Bacteriophages neutralizing antibodies (phages persist long enough to sustain their therapeutic effects).  Phage-resistant mutants.  The efficacy of phage therapy  phages have enzymes: lysins

21. Applications: Mycobacteriophages as delivery vehicles  Since bacteriophages infect bacteria, they can be used in the delivery of genetic material.  Examples: use of mycobacteriophages for the delivery of transposons and for gene replacement.  Use for delivery of reporter genes to mycobacteria, to monitor their physiological status.

22. Conclusion  The study of bacteriophages will help understand their host. ( We still don’t understand M. tuberculosis).  Very efficient in delivering genetic material, useful tool in genetics.  Phages would target pathogens without damaging beneficial bacterial flora.

23. In this Lab  Isolate phages that infect Mycobacterium smegmatis, a fast- growing, non-pathogenic soil bacterium.  Collect soil samples  Follow protocol (Schwebach et al., 2006)

24. References  Cole ST et al. Tuberculosis and the Tubercle Bacillus. ASM Press. 2005.  Hatfull GF. Mycobacteriophages: Genes and Genomes. Annu. Rev. Microbiol. 2010.64:331-356.  Hatfull GF. Mycobacteriophages: Windows into Tuberculosis. PLoS Pathog. 2014.10(3): e1003953. doi:10.1371/journal.ppat.1003953  Phages.org/bacteriophage/  Pedulla ML et al. Origins of Highly Mosaic Mycobacteriophages Genomes. Cell. 2003. 113: 171-182.  Pierce BA. Genetics a conceptual approach. W.H. Freeman and Campany New York. 3 rd edition.  Potera C. Phage Renaissance: New Hope against Antibiotic Resistance. EHP. 2015. ehp.niehs.nih.gov/121-a48/

25. References  Schwebach RJ and Jacobs RW Jr. Phage-Finding Using Mycobacteria: A Secondary School or Undergraduate Research Module with the Potential To Gain Scientific Authorship. The American Biology Teacher. 2006. 68:8  Sulakvelidze A. Bacteriaphages offer opportunities for safety managing bacterial infections. Microbe. 2011  Travis J. All the World’s a Phage Viruses that eat bacteria abound and surprise. Science News online. www.phschool.com/science/science_news/articles/all_words_phage.html www.phschool.com/science/science_news/articles/all_words_phage.html