microbial drug resistance: an old problem in need of …/media/files/activity files... · times...

Microbial Drug Resistance:

An Old Problem in Need of

New Solutions

“Barring geno-suicide, the human dominion is challenged

by only pathogenic microbes, for whom we remain the

prey; they the predator.”

Joshua Lederberg, May, 1993

Nicolas Poussin, The Plague of Ashdod, painted 1630, Louvre Museum

“Then the Philistines took the ark of God and broug ht it from Ebenezer to Ashdod.

. . . . But the hand of the Lord was heavy on the people of Ashdod, and He ravaged them and struck them with tumors, [*] both Ashdod and its territory.” -The Bible, New King James Version, 1 Samuel 5:1,6

[*] Septuagint and Vulgate texts add here “And in the m idst of their

A Plague of Biblical Proportions:

the Plague of the Philistines: ca. 11th century B.C.

During these times there was a pestilence, by which the whole human race came near to being annihilated. . . . It started fromthe Egyptians who dwell in Pelusium . . . . From the re it spread over the whole world, always moving forward and tra veling at times favorable to it. - Procopius, History of the Wars, II.xxii

Justinian’s Plague: ca. 540-543

The First Documented Plague Pandemic

QuickTime™ and aTIFF (Uncompressed) decompressor

are needed to see this picture.

Tentative chronology of the initial spread of plagu e in the mid-14th centuryWheelis, “Biological Warfare at the 1346 Siege of C affa,”

Emerging Infectious Diseases, 2002

Bubonic Plague: The Black Death: ca. 1345-1351



The Siege of Caffa: Biological Warfare in

the Fourteenth Century

Quoted in Wheelis, Emerging Infectious Diseases, 2002

The Seige of Caffa (1346)

But behold, the whole army was affected by a disease which overran the Tartars and killed thousands upon thousands every day. . . . The dying Tartars, stunned and stupefied by the immensity of the disaster brought about by the disease, and realizing that they had no hope of escape, lost interest in the siege. But they ordered corpses to be placed in catapults and lobbed into the city in the hope that the intolerable stench would kill everyone inside.What seemed like mountains of dead were thrown into the city. . . . And soon the rotting corpses tainted the air and poisoned the water supply, and the stench was so overwhelming that hardly one in several thousand was in a position to flee the remains of the Tartar army.

(now Feodosija, Ukraine)

Narrative of Gabriele De’ Mussi

1683VISUALIZATIONOF MICROBES

VAN LEEUWENHOEK 1870sGERM THEORY

PASTEUR,

KOCH

MICROBES AND DISEASE

“PRE-GERM”ERA OF

INFECTIOUSDISEASE

VACCINATIONFOR SMALLPOX

Chinese, Turks,Africans, JENNER

Louis Pasteur

(1822 - 1895)

Robert Koch

(1843 - 1910)



PASTEUR,

KOCH

1928TRANSFORMING

FACTORGRIFFITH

1944GENETIC

INFORMATIONRESIDES IN DNAAVERY, MACLEOD,

MCCARTY

1946CONJUGATION

AND RECOMBINATIONIN BACTERIA

LEDERBERG

AND TATUM



INFECTIOUSDISEASE



. . . These discussions have left a plethora of terms adrift: pangenes, bioblasts, plasmagenes, plastogenes, chondriogenes, cytogenes and proviruses, which have lost their original utility owing to the accretion of vague or contradictory connotations. At the risk of adding to this list, I propose plasmid as a generic term for any extra-chromosomal hereditary determinant. The plasmid itself may be genetically simple or complex. On occasion, the nuclear reference of the general term gene will be emphasized as chromogene.

Joshua LederbergPhysiological Reviews,Volume 31, Number 4, 403-30October, 1952

Electron-photomicrograph by J. Griffith

Cohen and Miller, 1968

Charles Brinton



PASTEUR,

KOCH

1928TRANSFORMING

FACTORGRIFFITH

1929DISCOVERY

OF PENICILLINFLEMING

Early 1940sUSE OF

ANTIBIOTICSAS TREATMENT

1943DISCOVERY OFSTREPTOMYCINWAKSMAN ET AL.

1944GENETIC

INFORMATIONRESIDES IN DNAAVERY, MACLEOD,

MCCARTY

1946CONJUGATIONIN BACTERIA

LEDERBERG

AND TATUM



INFECTIOUSDISEASE



WORLD-WIDE ERADICATION

OF BACTERIAL INFECTIONS USING

DRUGS THAT ATTACK MICROBES

EVOLUTION OF

DRUG RESISTANCE

IN PATHOGENS

INTELLIGENT DESIGN OF

Conventional Approach toAntimicrobial Therapy

- Antibacterials and antivirals nevertheless DO have toxic effects on host.

Attack the Pathogen and Leave the Host Unaffected

Some problems with this approach:

- Pathogen can mutate and become insensitive to therapy.

- Pathogen can be altered intentionally to make it resistant.

Mutation of Pathogen Site Targeted by the therapy

= Population of Drug or Vaccine Resistant Pathogens

Pathogen

Drug or Vaccine

+ use of the therapeutic









Joshua Lederberg

at Stanford

This demonstration does not conflict with reversibleadaptive responses to a specific environment which

disappear after some generations of growth in an indifferentmedium. Directed, but nonheritable, responses have been

clearly demonstrated in adaptive enzyme formation

(Monod, 1947) and may be involved in the resistance phenomena investigated by Eagle (1951).

From J. Lederberg and E.M. Lederberg, 1952

ConventionalAntimicrobial Therapy

Pathogen DiseaseHost

+

“Infectious diseases is the result of

host screw up“

Bruce Levin May 20, 2008

Pathogen and host as adversaries: pathogen

inhibited by host defenses

vs

Pathogen recruitment of host functions

PATHOGEN-HOST INTERACTION

• The effects of infectious pathogens commonly require the cooperation of host cell genes.

Hypotheses Underlying Host-oriented Therapeutics

• Host cell genes and pathways are potential targets for countermeasures that thwart resistance.

• Some host genes may be required by multiple pathogens (i.e., potential for broad spectrum therapies).

The Road to Viral Pathogenicity

Disease

binding

genome internalization

gene expression

protein processing

viral replication

pathogenic event

morphogenesis & release

Virus

Host

The Road to Anthrax Toxicity

S. Leppla

Stan, the idea is interesting, but aren’t you worried that targeting

host genes instead of the pathogen will have toxic effects?

Well, maybe. But host genes and pathways are routinely

targeted in virtually other type of disease

How to find host genes required for pathogen

or toxin effects?

The Road to Mycobacterial Infection and Persistence

Collaboration with R. Chen, J. Koo, and C. Nathan

mutation

Phenotype

Phenotype-based Discoveryof Gene Function in Bacteria

1. Randomly mutagenize population2. Isolate individuals acquiring biological trait of interest3. Identify the gene(s) mutated in those individuals

mutation

Mutated chromosomal allele

Chromosomal allele lacking mutation

1. Normally requires mutations in both copies to produce biological effect

1. Random mutation frequency - 10-5 X 10-5 = 10-10

Obstacle to simple phenotypic selection of

mutations in mammalaian cells

Randomly insert lentiviral (GSV)into mammalian cell chromosomes.

Large cell population containsinserts in all or nearly all genes.

RHKO strategy for isolation of CGEPs(Cellular Genes Exploited by Pathogens)

Virus containing GSV

(Li and Cohen, 1996)

Inserted virus inactivates the genecopy at the insertion site but not

the other copy.

GSV

Chromosomal allele of Gene “A” containing

GSV

Chromosomal allele of Gene “A” lacking GSV

Regulated promoter inthe GSV initiates

transcripts extending intothe chromosome

GSV

Regulated

antisense

promoter

Regulated

antisense

promoter

Chromosomal

gene promoter(P)

Chromosomal

gene promoter(P)

Chromosomal transcript

Chromosomal

gene promoter(P1)

Chromosomal

gene promoter(P1)

Regulated

antisense

promoter

Regulated

antisense

promoter

Chromosomal allele lacking GSV


Resulting transcripts are complementary to mRNA from

sequences flanking the insertion site and also to transcripts

From the second copy of this gene. This accomplishes

HOMOZYGOUS silencing of gene function.

Chromosomal allele containing GSV

GSV

Chromosomal

gene promoter(P2)

Chromosomal

gene promoter(P2)


Select cells surviving

pathogen effects.

Demonstrates non-lethality of

Inactivation event

Gene of interest is ‘tagged’ by GSV

and can be identified and characterized.

Confirm phenotypic effects of

CGEP inactivation

Elucidation of genetic pathways by sequential inactivation

Other Possible Function-Based Approaches for

Global Gene Inactivation

• Antisense cDNA libraries

• Small interfering RNA libraries (siRNA; RNAi; shRNA)

• EST libraries

•• But….knockout limited to contents of constructs But….knockout limited to contents of constructs

present in library and is determined by the present in library and is determined by the

effectiveness of the cloned effectiveness of the cloned cDNA/siRNA/ESTcDNA/siRNA/EST

segment as gene segment as gene inactivatorinactivator..

The Many Faces of The Many Faces of

Tumor Susceptibility Gene 101Tumor Susceptibility Gene 101

Paradigm for Host-Oriented Treatment of Infectious Diseases

Tumor susceptibilityand cell proliferation

Transcription Ubiquitination

Endocytictrafficking

Viral budding

Tumor susceptibilityCell proliferation

Transcription Ubiquitination

Endocytictrafficking

Viral budding

Proteins That Interact with TSG101 inYeast Two-Hybrid Screens

Gene name Clones Strength HitsGene name Clones Strength Hits

HrsHrs

Tsg101Tsg101

Sug1Sug1

Swi/SNFSwi/SNF

TPRTPR

SODD1SODD1

UnknownsUnknowns

AOE14b, 28a, 37aAOE14b, 28a, 37a

AOE4cAOE4c

AOE8a, 32cAOE8a, 32c

AOE9aAOE9a

AOE34cAOE34c

AOE19aAOE19a

++++++

++++++

++++

++++

++++

++++++

33

11

22

11

11

11

66

Endocytosis of Cell Surface Receptors

EGFR

J. Marx, J. Marx, ScienceScience, 2002, 2002

Tsg101 and the Vacuolar Protein Sorting PathwayAre Essential for HIV-1 Budding

Garrus et al., 2001

TSG101 B inds V i r a l L a t e Domain TSG101 B inds V i r a l L a t e Domain Pro t e i nsPro t e i ns

UEVUEV PP--RichRich Coiled CoilCoiled Coil SS--BoxBox11 146146 240240 311311 390390330330215215

UEVUEV PP--RichRich Coiled CoilCoiled Coil SS--BoxBox11 146146 240240 311311 390390330330215215

Ebo la

Vi rus LD P ro t e i n

VP40

St ruc tu r e Mot i f

7PTAP107 10

HIV p6 Gag 7PTAP107 10

In f luenzaM1

RSV M pro t e in ( ? )

PIV M Pro t e in ( ? )

16PSPG19

19YNVL22

9PTSP13

16 19

19 22

9 13

TSG101 r e l evance t o mu l t i p l e t ypes TSG101 r e l evance t o mu l t i p l e t ypes o f v i ru s e so f v i ru s e s

HIVHIV--11 Re t rov i ru sRe t rov i ru s VerP l ank e t a l ( 2001 ) VerP l ank e t a l ( 2001 ) PNASPNAS

Ebo laEbo la F i l ov i ru s F i l ov i ru s Mar t i nMar t i n -- S e r r ano e t a l ( 2 0 01 ) S e r r ano e t a l ( 2 0 01 )

Na tu r e Med .Na tu r e Med .

HIVHIV--22 Re t rov i ru sRe t rov i ru s Myer s and A l l en ( 2 0 02 ) Myer s and A l l en ( 2 0 02 ) J J

Vi r o l .Vi r o l .

HTLVHTLV--11 Re t rov i ru sRe t rov i ru s Bouamr e t a l ( 2 0 03 ) Bouamr e t a l ( 2 0 03 ) J V i r o lJ V i r o l . .

MPMVMPMV Re t rov i ru sRe t rov i ru s Got twe in e t a l , ( 2 0 03 ) Got twe in e t a l , ( 2 0 03 ) J J

Vi r o lVi r o l ..

Arenav i rusArenav i rus Arenav i ru sArenav i ru s Pe r ez e t a l ( 2 0 0 3 ) Pe r ez e t a l ( 2 0 0 3 )

PNASPNAS ..

MLVMLV Re t rov i ru sRe t rov i ru s Camus e t a l ( 2 0 0 7 ) Camus e t a l ( 2 0 0 7 ) Mo l B i o l Mo l B i o l

Ce l lCe l l

EIAVEIAV Re t rov i ru s Re t rov i ru s ShehuShehu --X i l aga e t a l . ( 2 0 03 ) X i l aga e t a l . ( 2 0 03 ) J J


In f luenza In f luenza –– Seasona lSeasona l Or thomyxov i ru sOr thomyxov i ru s Func t i ona l Gene t i c s , Func t i ona l Gene t i c s ,

unpub l i shedunpub l i shed

In f luenza In f luenza –– Pandemic Pandemic Or thomyxov i ru sOr thomyxov i ru s Func t i ona l Gene t i c s , Func t i ona l Gene t i c s ,

unpub l i shedunpub l i shed

J apanese Enceph .J apanese Enceph . F l av iv i ru sF l av iv i ru s Ch iou e t a l . ( 2 0 03 ) Ch iou e t a l . ( 2 0 03 ) J G en J G en


Foamy Foamy Re t rov i ru sRe t rov i ru s Pa t t on e t a l . ( 2 005 ) Pa t t on e t a l . ( 2 005 ) J V i r o l .J V i r o l .

Rous Sa r coma Rous Sa r coma Re t rov i ru sRe t rov i ru s Med ina e t a l . ( 2 005 ) Med ina e t a l . ( 2 005 ) T r a f f i cT r a f f i c

LassaLassa Arenav i ru sArenav i ru s Ura t a e t a l . ( 2 0 06 ) Ura t a e t a l . ( 2 0 06 ) J V i r o l .J V i r o l .

Hepa t i t i s EHepa t i t i s E Hepadnav i ru sHepadnav i ru s Sur j i tSur j i t e t a l . ( 2 006 ) e t a l . ( 2 006 ) J B i o l J B i o l

Chem .Chem .

Eps t e inEps t e in --Ba r rBa r r Herpe sv i ru sHerpe sv i ru s Chua e t a l . , ( 2 007 ) Chua e t a l . , ( 2 007 ) J V i r o l .J V i r o l .

THERAPEUTIC APPROACHES TARGETING TSG101:

SMALL MOLECULES (including cyclic peptides) THAT INTERFERE WITH

TSG101 INTERACTION WITH VIRAL PROTEINS

ANTIBODY THERAPIES: TSG101 BROUGHT TO CELL SURFACE

BY HIV, EBOLA, FLU VIRUS

Some Pathogens Potentially Amenable to

Host-Oriented Therapies

Viruses Toxins Bacteria• Smallpox • Ricin • B. anthracis

• Ebola • Anthrax

Neurotoxins

• Shigellosis

• Marburg • Mycotoxins • Typhus

• Encephalitis • Botulinum • Cholera

• livestock and

plant viruses

• C. perfringens • Tularemia

• Influenza • Staph.

enterotoxins

• Plague

• HIV • Tuberculosis

• Herpes

viruses

• Others

The Three Most Important Factors in

Function-Based Target Discovery

1.1. AssayAssay

2.2. AssayAssay

3.3. AssayAssay

The Road to Anthrax Toxicity

S. Leppla

Other genes…….

Other toxins…….

0%

20%

40%

60%

80%

100%

1.E-13 1.E-12 1.E-11 1.E-10 1.E-09 1.E-08 1.E-07

PA (M)

Cell

Via

bil

ity (

% o

f C

on

tro

l)

M2182tTA

ATR43

(100 x magnification)

M2182

M2182/EST

M2182/pATR43

No Toxin PA/FP59

Clone ATR43 Resistance to PA + FP59Clone ATR43 Resistance to PA + FP59

LRP6--co-receptor for Wnt signaling

Wnt signalling: embryogenesis, differentiation,

colorectal cancer, adipogenesis, osteogenesis

Deficiency of LRP6 by siRNAs in naïve cells

reconstitutes toxin resistance phenotype

LRP6

LRP6

LRP6

LRP6

LRP6

Internalization of LRP6 Triggered by PA

Red (LRP6) and green

(PA) co-localized at cell

surface at 4oC

Red (LRP6) at cell surface at

4oC

In presence of PA, LRP6

is internalized upon

temperature shift

In absence of PA, LRP6

remains on cell surface on

T\temperature shift

Antibodies Against LRP6 Inhibit Anthrax

Toxicity

Selection of RHKO Macrophages that survive M. tuberculosis

infection

Library of 107 RHKO Macrophages

Infect with MtbA B

Add antibiotics to kill Mtb

Grow for 14 days

Collect surviving Macrophages and repeat selection

MOI 10 MOI 3Almost complete killing

of Macrophages

V. Koo and C. Nathan

Gene Connections on the Road to Mtb Lethality

PARTICIPANTS IN VIRAL PATHOGEN

AND TOXIN PROJECTS

STANFORD UNIVERSITY

S.N. Cohen Lab

Wensheng Wei

Yanan Feng

Quan Lu

Paul Kowalski

Ran Chen

Annie C.Y. Chang

Ronen Mosseri

MARC

Will Laegreid Lab

UNIVERSITY OF NM

Rick Lyons Lab

NIH

Stephen H. Leppla Lab

G. Jilani Chaudry

PLUM ISLAND (USDHS)

Daniel Rock Lab

Laszlo Zsak

Maria Piccone

John G. Neilan

Claudio Afonso

Weill Cornell Medical College

Jovanka Koo,

Carl Nathan

Research support from NIH, DTRA and DARPA

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