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[[File:Southern Blot.tiff|thumb|right|Figure 1: This image shows the [[southern blot]] of [[DNA]] extracted from Bacteriophage T12 infected bacteria.]]
[[File:Southern Blot.tiff|thumb|right|Figure 1: This image shows the [[southern blot]] of [[DNA]] extracted from Bacteriophage T12 infected bacteria.]]


The presence of lysogenic bacteriophage T12 can be tested through [[Plaque assay#Plaque assay|plaque assays]] if the indicator strain utilized is susceptible to the phage being detected. Plaque assays consist of pouring a soft agar solution with an indicator strain onto an agar plate. The indicator strain should be a strain of bacteria that can be infected by the phage that needs to be detected. After the soft agar is set the samples that are being tested for phage presence are then spread-plated onto the soft agar plates. The plates are then incubated overnight and checked for clearings (plaques) the next day. If the phage is present, indicator strains will become infected and go through the normal lysogenic cycle while the plates incubate, and then undergo [[lysis]]. The plaque that determines whether the phage is present or not is caused by the lysis of the indicator strains. [[Titer|Titers]] of plaques can be found by diluting the samples and counting plaque forming units (PFUs).<ref>{{cite web|last=Panec|first=Marie|title=Plaque Assay Protocols|url=http://www.microbelibrary.org/component/resource/laboratory-test/3073-plaque-assay-protocols|work=Microbe Library|publisher=American Society for Microbiology|accessdate=28 November 2012}}</ref>
The presence of lysogenic bacteriophage T12 can be tested through [[Plaque assay#Plaque assay|plaque assays]] if the indicator strain utilized is susceptible to the phage being tested. Plaque assays consist of pouring a soft agar solution with an indicator strain onto an agar plate. The indicator strain should be a strain of bacteria that can be infected by the phage that needs to be detected. After the soft agar is set the samples that are being tested for phage presence are then spread-plated onto the soft agar plates. The plates are then incubated overnight and checked for clearings (plaques) the next day. If the phage is present, indicator strains will become infected and go through the normal lysogenic cycle while the plates incubate, and then undergo [[lysis]]. The plaque that determines whether the phage is present or not is caused by the lysis of the indicator strains. [[Titer|Titers]] of plaques can be found by diluting the samples and counting plaque forming units (PFUs).<ref>{{cite web|last=Panec|first=Marie|title=Plaque Assay Protocols|url=http://www.microbelibrary.org/component/resource/laboratory-test/3073-plaque-assay-protocols|work=Microbe Library|publisher=American Society for Microbiology|accessdate=28 November 2012}}</ref>


Biochemical tests such as [[Southern Blot|southern blots]] can also be used to detect the speA that the phage produces from the ''spe''A gene. This was done in research by Johnson, Tomai and Schlievert in 1985 by isolating the DNA of Streptococcal strains and running a restriction digest using [[BglII]]. After the digest was complete, the DNA samples were run a DNA gel to separate the DNA. The DNA from this gel was then transferred to nitrocellulose paper and incubated with probes specific for speA. An image of this southern blot can be seen in Figure 1 of this article. <ref name="Johnson-Phage Involvement" />
Biochemical tests such as [[Southern Blot|southern blots]] can also be used to detect the speA that the phage produces from the ''spe''A gene. This was done in research by Johnson, Tomai and Schlievert in 1985 by isolating the DNA of Streptococcal strains and running a restriction digest using [[BglII]]. After the digest was complete, the DNA samples were run a DNA gel to separate the DNA. The DNA from this gel was then transferred to nitrocellulose paper and incubated with probes specific for speA. An image of this southern blot can be seen in Figure 1 of this article. <ref name="Johnson-Phage Involvement" />

Revision as of 03:28, 15 December 2012

Bacteriophage T12
Virus classification
Group:
Group I (dsDNA)
Order:
Unclassified

Bacteriophage T12 is a bacteriophage that infects the bacterial species Streptococcus pyogenes, and converts a harmless strain of bacteria into a virulent strain. It carries the speA gene which codes for erythrogenic toxin A.[1] speA is also known as streptococcal pyrogenic exotoxin A, scarlet fever toxin A, or even scarlatinal toxin.[2][3] Note that when the term 'spe'A is italicized, the reference is to the gene. In contrast, when the term 'spe'A is not italicized, the toxin itself is being referred to. Erythrogenic toxin A converts a harmless, nonvirulent strain of Streptococcus pyogenes to a virulent strain through lysogeny, a life cycle which is characterized by the ability of the genome to become a part of and be stably maintained in the host cell for generations.[4] Phages with a lysogenic life cycle are also called temperate phages.[1] A virulent strain of bacteria is one that is "extremely infective" and causes medical, clinical symptoms. [5] Bacteriophage T12, a member of a family of related speA-carrying bacteriophages, is also a prototypic phage for all the speA-containing phages of Streptococcus pyogenes, meaning that its genome is the prototype for the genomes of all such phages of S.pyogenes.[6] It is the main suspect as the cause of Scarlet Fever, an infectious disease that affects small children.[4]

Research and Discovery

The possibility of bacteriophage involvement in speA production was first introduced in 1926 when Cantacuzene and Boncieu reported that nonvirulent strains of S.pyogenes were transformed to virulent strains through some transferable element. Frobisher and Brown reported similar results in 1927, and in 1949, the reports were confirmed by Bingel [7][8] Later, in 1964, Zabriskie reported that phage T12 could cause speA production by lysogeny in strains that it became a part of.[9] In 1980, Johnson, Schlievert and Watson were able to confirm this and show that the gene for speA production was transferred from toxigenic strains of bacteria to non-toxigenic strains through lysogeny. In their experiment, every transformed, toxin-producing bacterial colony was lysogenic, i.e. contained the T12 gene. In addition, none of the colonies containing the T12 genome was negative for speA, and therefore, the conclusion was drawn that all lysogens produced the toxin. [10] However, McKane and Ferretti reported, in 1981, that a mutant of phage T12 induced speA production virulently. This mutant, the bacteriophage T253, entered the lytic cycle, a life cycle in which the host cell is destroyed.[11] In 1983, Johnson and Schlievert published a map of the T12 genome, revealing also that three rounds of packaging occur in the genome.[9] The very next year, Johnson and Schlievert and Weeks and Ferreti also found, independently, that the bacteriophage T12 carries the structural gene for speA.[8][12] In 1986, Johson, Tomai and Schlievert mapped the attachment site (attP) for T12 adjacent to the speA gene, and established that all bacterial strains producing the toxin carry either phage T12 itself, or a closely related bacteriophage.[4] And finally, in 1997, McShan and Ferretti published that they had found the second attachment site (attR) for T12, while also revealing in another publication, which was also credited to Tang, that bacteriophage T12 inserts into a gene that encodes a serine tRNA in the host.[1][6]

Genome

This image shows how known genes of bacteriophage T12 are arranged once the phage chromosome has integrated into the chromosome of S. pyogenes. The green box represents the phage chromosome, while the black line represents the bacterial chromosome into which T12 integrates. The arrows in the diagram show the direction in which the genes are transcribed. The red arrows show the arrangement of the speA and int genes. The pink arrows indicate only the orientation of the serine tRNA gene into which the phage integrates. The coding region of the serine tRNA gene remains intact even after the phage integrates.

The physical map of the T12 genome was found to be circular with a total length of 36.0kb.[9] The phage genome is reported to carry the speA gene,[12] which is a 1.7kb segment of the phage T12 genome flanked by SalI and HindIII sites.[8]

The phage integrase gene (int) and the phage attachment site (attp) are located just upstream of the speA gene in the phage genome. The bacteriophage T12 integrates into S. pyogenes chromosome by site-specific recombination into the anticodon loop of a gene that codes for serine tRNA. The bacterial attachment site (attB) has a 96 base pair sequence homologous to the phage attachment site and is located at the 3’ end of the tRNA gene such that the coding sequence of the tRNA gene remains intact after integration of the prophage. Phage T12 is the first example of a phage from a gram-positive, low G-C content host that uses this kind of integration site.[1] [6]

Diseases

Diseases like Scarlet Fever and Toxic Shock-like Syndrome are caused by lysogenized streptococcal strains that produce speA. The diseases are systemic responses to the speA circulating within the body.[13]

Scarlet fever

Scarlet fever, also known as scarletina, is so called because of the characteristic bright red rash it causes. It is most common in children between four and eight years of age.[14]

Signs and Symptoms

The first stage of scarlet fever is typically strep throat (streptococcal pharyngitis) characterized by sore throat, fever, headache and sometimes nausea and vomiting. In two to three days, this is followed by the appearance of a diffuse erythematous rash that has a sandpaper texture. The rash first appears on the neck, then spreads to the chest, back and body extremities. A yellowish white coating covers the tongue, and is later shed, leaving the tongue with a strawberry appearance and swollen papillae. The rash fades away after five to six days of the onset of the disease, and is followed by peeling of skin, particularly over the hands and feet.[14] [15] [16]

Treatment

Penicillin, an antibiotic, is the drug of choice for the treatment of scarlet fever as for any other S. pyogenes infection. For those who are allergic to penicillin, the antibiotics erythromycin or clindamycin can be used. However, occasional resistance to these drugs has been reported.[17]

Streptococcal Toxic Shock Syndrome

In Streptococcal toxic shock syndrome (StrepTSS), speA produced by infected streptococcal strains acts as a superantigen and interacts with human monocytes and T lymphocytes, inducing T-cell proliferation and production of monokines (e.g. tumor necrosis factor α, interleukin 1, interleukin 6), and lymphokines (e.g. tumor necrosis factor β, interleukin 2, and gamma-interferon).These cytokines(TNFα, TNFβ) so produced seems to mediate the fever, shock and organ failure characteristic of the disease.[18][19] [13]

Signs and Symptoms

Strep TSS is an acute, febrile illness that begins with a mild viral-like syndrome characterized by fever, chills, myalgia, diarrhea, vomiting and nausea and involves minor soft-tissue infection that may progress to shock, multi-organ failure, and death. [19]

Treatment

While penicillin is an effective treatment of mild infection, it is less effective in a severe case. Emerging treatments for strep TSS include clindamycin and intravenous gamma-globulin. [19]

Bacteriophage T12 Control

Bacteriophages are very robust organisms, very hard to kill[20] and very easily spread.[21] Ultraviolet light can enhance the production of phage T12 and speA, both.[3] However, this is only to a point. UV light stresses lysogenic bacteria, causing them to propagate and burst the host bacterial cells.[22] In the case of T12, exposure to UV light increases the propagation of bacteriophage T12 at 20 seconds of exposure. After 20 seconds of exposure the UV light starts to kill the bacteriophage by damaging it's genome.[23]

Detection Assays

Figure 1: This image shows the southern blot of DNA extracted from Bacteriophage T12 infected bacteria.

The presence of lysogenic bacteriophage T12 can be tested through plaque assays if the indicator strain utilized is susceptible to the phage being tested. Plaque assays consist of pouring a soft agar solution with an indicator strain onto an agar plate. The indicator strain should be a strain of bacteria that can be infected by the phage that needs to be detected. After the soft agar is set the samples that are being tested for phage presence are then spread-plated onto the soft agar plates. The plates are then incubated overnight and checked for clearings (plaques) the next day. If the phage is present, indicator strains will become infected and go through the normal lysogenic cycle while the plates incubate, and then undergo lysis. The plaque that determines whether the phage is present or not is caused by the lysis of the indicator strains. Titers of plaques can be found by diluting the samples and counting plaque forming units (PFUs).[24]

Biochemical tests such as southern blots can also be used to detect the speA that the phage produces from the speA gene. This was done in research by Johnson, Tomai and Schlievert in 1985 by isolating the DNA of Streptococcal strains and running a restriction digest using BglII. After the digest was complete, the DNA samples were run a DNA gel to separate the DNA. The DNA from this gel was then transferred to nitrocellulose paper and incubated with probes specific for speA. An image of this southern blot can be seen in Figure 1 of this article. [4]

References

  1. ^ a b c d McShan, WM; Tang, YF; Ferretti, JJ (1997). "Bacteriophage T12 of Streptococcus pyogenes integrates into the gene encoding a serine tRNA". Molecular Microbiology. 23 (4): 719–28. doi:10.1046/j.1365-2958.1997.2591616.x. PMID 9157243. {{cite journal}}: Cite has empty unknown parameter: |author-name-separator= (help); Unknown parameter |author-separator= ignored (help)
  2. ^ Stevens, Dennis L. (6 July 1989). "Severe Group A Streptococcal Infections Associated with a Toxic Shock-like Syndrome and Scarlet Fever Toxin A". New England Journal of Medicine. 321 (1): 1–7. doi:10.1056/NEJM198907063210101. {{cite journal}}: Unknown parameter |coauthors= ignored (|author= suggested) (help); Unknown parameter |month= ignored (help)CS1 maint: date and year (link)
  3. ^ a b Wagner, PL; Waldor, MK (2002). "Bacteriophage control of bacterial virulence". Infection and Immunity. 70 (8): 3985–93. doi:10.1128/IAI.70.8.3985-3993.2002. PMC 128183. PMID 12117903. {{cite journal}}: Cite has empty unknown parameter: |author-name-separator= (help); Unknown parameter |author-separator= ignored (help)
  4. ^ a b c d Johnson, LP; Tomai, MA; Schlievert, PM (1986). "Bacteriophage involvement in group a streptococcal pyrogenic exotoxin a production". Journal of Bacteriology. 166 (2): 623–7. PMC 214650. PMID 3009415. {{cite journal}}: Cite has empty unknown parameter: |author-name-separator= (help); Unknown parameter |author-separator= ignored (help)
  5. ^ "Dictionary.com Collins English Dictionary - Complete & Unabridged 10th Edition". HarperCollins Publishers. Retrieved 11 December 2012.
  6. ^ a b c McShan, WM (1997 Oct). "Genetic diversity in temperate bacteriophages of Streptococcus pyogenes: identification of a second attachment site for phages carrying the erythrogenic toxin A gene". Journal of bacteriology. 179 (20): 6509–11. PMID 9335304. {{cite journal}}: Check date values in: |date= (help); Unknown parameter |coauthors= ignored (|author= suggested) (help)
  7. ^ Ferretti, Joseph (1982). "Phage Influence on the Synthesis of Extracellular Toxins in Group A Streptococci". Infection and Immunity. 36: 745–750. PMID PMC351293. {{cite journal}}: |access-date= requires |url= (help); Check |pmid= value (help); Unknown parameter |coauthors= ignored (|author= suggested) (help); Unknown parameter |month= ignored (help)
  8. ^ a b c Weeks, CR; Ferretti, JJ (1984). "The gene for type a streptococcal exotoxin (erythrogenic toxin) is located in bacteriophage T12". Infection and Immunity. 46 (2): 531–6. PMC 261567. PMID 6389348. {{cite journal}}: Cite has empty unknown parameter: |author-name-separator= (help); Unknown parameter |author-separator= ignored (help)
  9. ^ a b c Johnson, LP (1983). "A physical map of the group A streptococcal pyrogenic exotoxin bacteriophage T12 genome". Molecular & general genetics : MGG. 189 (2): 251–5. doi:10.1007/BF00337813. PMID 6304466,. {{cite journal}}: Check |pmid= value (help); Unknown parameter |coauthors= ignored (|author= suggested) (help)CS1 maint: extra punctuation (link)
  10. ^ Johnson, LP (1980 Apr). "Transfer of group A streptococcal pyrogenic exotoxin production to nontoxigenic strains of lysogenic conversion". Infection and immunity. 28 (1): 254–7. PMID 6991440. {{cite journal}}: Check date values in: |date= (help); Unknown parameter |coauthors= ignored (|author= suggested) (help)
  11. ^ McKane, L (1981 Dec). "Phage-host interactions and the production of type A streptococcal exotoxin in group A streptococci". Infection and immunity. 34 (3): 915–9. PMID 7037644. {{cite journal}}: Check date values in: |date= (help); Unknown parameter |coauthors= ignored (|author= suggested) (help)
  12. ^ a b Johnson, LP; Schlievert, PM (1984). "Group a streptococcal phage T12 carries the structural gene for pyrogenic exotoxin type A". Molecular & General Genetics. 194 (1–2): 52–6. doi:10.1007/BF00383496. PMID 6374381. {{cite journal}}: Cite has empty unknown parameter: |author-name-separator= (help); Unknown parameter |author-separator= ignored (help)
  13. ^ a b Musser, JM (1991 Apr 1). "Streptococcus pyogenes causing toxic-shock-like syndrome and other invasive diseases: clonal diversity and pyrogenic exotoxin expression". Proceedings of the National Academy of Sciences of the United States of America. 88 (7): 2668–72. PMID 1672766. {{cite journal}}: Check date values in: |date= (help); Unknown parameter |coauthors= ignored (|author= suggested) (help)
  14. ^ a b "Scarlet fever".
  15. ^ Stevens, Dennis. "Streptococcus pyogenes (Group A β-hemolytic Streptococcus)".
  16. ^ "Streptococcal Infections (S. pyogenes — Group A streptococci)".
  17. ^ "Streptococcal Infections (Invasive Group A Strep)".
  18. ^ Hackett, SP (1992 May). "Streptococcal toxic shock syndrome: synthesis of tumor necrosis factor and interleukin-1 by monocytes stimulated with pyrogenic exotoxin A and streptolysin O." The Journal of infectious diseases. 165 (5): 879–85. PMID 1569337. {{cite journal}}: Check date values in: |date= (help); Unknown parameter |coauthors= ignored (|author= suggested) (help)
  19. ^ a b c Stevens, DL (2000). "Streptococcal toxic shock syndrome associated with necrotizing fasciitis". Annual review of medicine. 51: 271–88. PMID 10774464.
  20. ^ Broxmeyer, L (2004). "Bacteriophages: Antibacterials with a future?". Medical Hypotheses. 62 (6): 889–93. doi:10.1016/j.mehy.2004.02.002. PMID 15142642. {{cite journal}}: Cite has empty unknown parameter: |author-name-separator= (help); Unknown parameter |author-separator= ignored (help)
  21. ^ Ramirez, E; Carbonell, X; Villaverde, A (2001). "Phage spread dynamics in clonal bacterial populations is depending on features of the founder cell". Microbiological Research. 156 (1): 35–40. doi:10.1078/0944-5013-00087. PMID 11372651. {{cite journal}}: Cite has empty unknown parameter: |author-name-separator= (help); Unknown parameter |author-separator= ignored (help)
  22. ^ Atsumi, S; Little, JW (2006). "Role of the lytic repressor in prophage induction of phage lambda as analyzed by a module-replacement approach". Proceedings of the National Academy of Sciences of the United States of America. 103 (12): 4558–63. doi:10.1073/pnas.0511117103. PMC 1450210. PMID 16537413. {{cite journal}}: Cite has empty unknown parameter: |author-name-separator= (help); Unknown parameter |author-separator= ignored (help)
  23. ^ Zabriskie, JB (1964). "The Role of Temperate Bacteriophage in the Production of Erythrogenic Toxin by Group a Streptococci". The Journal of Experimental Medicine. 119 (5): 761–80. doi:10.1084/jem.119.5.761. PMC 2137738. PMID 14157029. {{cite journal}}: Cite has empty unknown parameter: |author-name-separator= (help); Unknown parameter |author-separator= ignored (help)
  24. ^ Panec, Marie. "Plaque Assay Protocols". Microbe Library. American Society for Microbiology. Retrieved 28 November 2012.
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