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A new antibiotic kills pathogens without detectable resistance
Losee L. Ling, Tanja Schneider2, Aaron J. Peoples, Amy L. Spoering, Ina Engels, Brian P. Conlon, Anna Mueller,Till F. Scha¨berle, Dallas E. Hughes, Slava Epstein, Michael Jones, Linos Lazarides, Victoria A. Steadman, Douglas R. Cohen,
Cintia R. Felix, K. Ashley Fetterman, William P. Millett, Anthony G. Nitti, Ashley M. Zullo, Chao Chen & Kim Lewis
Saman Amirzadegan & Krista Jastrzembski
BINF 704
Antibiotic Resistance
Overview
• A Short History of Antibiotics
• Development of Antibiotic Resistance
• Teixobactin• Cultivation
• Identification
• Mechanism of Action
• Discussion
The Antibiotic Era1909 – Paul Ehrlich et. al. discover a drug effective
against syphilis
Discovery of PenicillinSeptember 3, 1928
Alexander Fleming returns to his lab after a lengthy vacation to discover Penicillin
Antibiotic Mechanisms of Action
Superbugs from Super-fast Evolution
1940 Penicillin
introduced – Resistant
bacteria uncommon
1950’s Penicillin resistant S. aureus
common in hospital settings
1961 – Methicillin introduced
1962 – Methicillin resistant A. aureus
strains begin to appear
2002 – Vancomyci
n resistant
strain of S. aureus isolated
from patient in Michigan
Bacteria’s evolutionary advantages Vertical Transmission
Generation time
Population Size
Horizontal Transmission
No need to wait for random mutations
Multiple resistance genes
Mechanisms of Antibiotic Resistance
• Modification of the Antibiotic
• Removal from the cell
• Modification of the target site
The costs of Antibiotic resistance
• 2 million yearly infections in the US
• 23,000 deaths directly related to antibiotic resistant infections
• $20 billion lost to excessive healthcare costs
• $35 billion due to lost productivity
Antibiotic Discovery tapers after 1960Difficulty of new discovery combined with increasing antibiotic resistance results in potential public
health crisis
iChip device for growing uncultured microorganisms
Eleftheria terrae • New species of b-proteobacteria
• New genus related to the genus Aquabacteria
• 16S sequencing
• DNA/DNA hybridization
Teixobactin
• Molecular Mass: 1,242 Da
• Depsipeptide containing enduracididine, methylphenylalanine and four D-amino acids.
19
Image source: http://www.medscape.com/viewarticle/715971_2
20
Resistance
• Teixobactin is effective against many gram positive* pathogens, some of which are unresponsive to vancomycin (last resort)
Gram negative exception
21
Image source: https://commons.wikimedia.org/wiki/File:Viral_entry_(Endocytosis_and_lysis).svg
Fig 2c
22
Resistance
• No mutants (S.aureus or M.tuberculosis) observed when plated on teixobactin – NO RESISTANCE
• No S.aureus mutants observed, even after 27 day repeated exposure to teixobactin – STILL NO RESISTANCE
• Indicates “non – specific” mechanism & toxicity• BUT no toxicity observed in mammalian cells (highest possible
dose)
23
Mechanism of Action
• Teixobactin is non – haemolytic* and doesn’t bind DNA
• How did the research team figure out specifically where teixobactin is active?
• Rate of label* incorporation into S.aureus’ main biosynthetic pathways• Teixobactin inhibits peptidoglycan* synthesis
• Teixobactin has no observed effect on label incorporation into DNA, RNA, or protein
24
Fig 3a
25
Mechanism of Action
• In vancomycin, the absence of resistance suggests that the mechanism target is not a protein. Could the same be true for teixobactin?
• Vancomycin binds lipid II* – does teixobactin bind this also?
• S.aureus built-up a peptidoglycan precursor (UDP-MurNAc-pentapeptide), when exposed to teixobactin in concentrations of 1x – 5x the MIC* • Vancomycin control does the same; indicates inhibition of a peptidoglycan
biosynthesis step
• In test tubes, teixobactin blocked peptidoglycan synthesis reactions: lipid I, lipid II, or undecaprenyl - pyrophosphate
26
Mechanism of Action
• What we know so far: Teixobactin is specifically involved with peptidoglycan precursors, as opposed to the other enzymes involved in its biosynthetic pathway
• The follow – up question: What specific part of these precursors is the is teixobactin targeting?
• How to investigate: Study direct involvement with peptidoglycan precursors coupled in pairs
27
• Paired peptidoglycan precursors:• purified and incubated
with teixobactin at various molar ratios
• extracted and analyzed by thin – layer chromatography
• Teixobactin bound if amount of lipid intermediates were reduced
Image Source: http://www.waters.com/waters/en_US/HPLC---High-Performance-Liquid-Chromatography-Beginner's-Guide/nav.htm?cid=10048919&locale=en_US
28
Mechanism of Action
• What did teixobactin bind? All peptidoglycan precursors and wall teichoic acid*
• Fun fact! Lipid I and lipid II form a stable complex impermeable to the effects of teixobactin
• Teixobactin > Vancomycin??
29
Mechanism of Action
• WTA is not needed for the organism to survive, inhibition of wall teichoic acid biosynthesis has lethal toxic intermediates (late stage)
• Teichoic acids bind autolysins*; complex prevents uncontrolled peptidoglycan break down.
• Inhibition of teichoic acid synthesis by teixobactin releases autolysins; lytic/killing activity of the bacteria increases
30
In vivo efficacy
Teixobactin stable in serum; low toxicity in mice
Extended Data; Fig 8a
31
In vivo efficacy
• Teixobactin also observed to be effective in mice with:• MRSA* septicemia* dose of 90% lethality, if administered with as
little as 1mg/kg intravenously within an hour
• In a follow up experiment the PD50* was observed to be 0.2 mg/kg; better than vancomycin* (PD50 of 2.75 mg/kg)
• Thigh model of MRSA infection*
• Streptococcus pneumonie infections
32
Summary
• Antibiotics are an invaluable medical advancement that have greatly reduced suffering and death
• Bacteria have evolutionary advantages when it comes to evading antibiotic activity
• New techniques are necessary to advance the discovery of novel antibiotics
• Teixobactin is a strong and promising antibiotic with efficacy better than that of the last resort antibiotic - Vancomycin
• Further research must be done, in terms of clinical trials – in vitro human trials?
33
References
• Clin Orthop Relat Res. 2005 Oct;439:23-6. THE CLASSIC: penicillin as a chemotherapeutic agent. 1940. Chain E, Florey HW, Gardner AD, Heatley NG, Jennings MA, Orr-Ewing J, Sanders AG.
• Cell. 2007 Sep 7;130(5):797-810. A common mechanism of cellular death induced by bactericidal antibiotics. Kohanski MA1, Dwyer DJ, Hayete B, Lawrence CA, Collins JJ.
• Lancet Infect Dis. 2010 Dec;10(12):816-8. doi: 10.1016/S1473-3099(10)70259-0. VRSA-doomsday superbug or damp squib? Gould IM.