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.

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Image source: http://www.medscape.com/viewarticle/715971_2

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Resistance

• Teixobactin is effective against many gram positive* pathogens, some of which are unresponsive to vancomycin (last resort)

Gram negative exception

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Image source: https://commons.wikimedia.org/wiki/File:Viral_entry_(Endocytosis_and_lysis).svg

Fig 2c

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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)

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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

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Fig 3a

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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

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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

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• 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

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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??

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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

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In vivo efficacy

Teixobactin stable in serum; low toxicity in mice

Extended Data; Fig 8a

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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

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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?

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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.