Bio 101 Lecture by

Seyed E. Hasnain

Evolution of Pathogenic Bacteria:Mycobacterium tuberculosis example

Why study Pathogen Evolution?

pathogengenome diversity

The composition of the prokaryotic genome. Bacterial genomes consist of a conserved “core gene pool” and a variable “flexible gene pool”. The latter consists of accessory and mobile genetic elements (modified after Morschhäuser et al., 2000).

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MicroevolutionMicroevolutionDevelopment of organisms in days and weeks

MacroevolutionMacroevolutionDevelopment of species and variants in long

term intervals

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MicroMicro--evolutionevolution

Point mutations Gene expression,Modulation

Plasmid,Phage transfer

Horizontal gene transfer

Phase, AntigenicVariation

Genetic rearrangements

DeletionsGenome reduction,Deletions

MacroMacro--evolutionevolution

PAI development

Development of new variants

Development of intracellular pathogens

Pathoadaptation

Genetic Genetic mechanismmechanism

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Why M. tuberculosis

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US 37.7%

Latin America 17.7%

Africa 9.6%

Asia 33.3%

Europe 2.1%

The morbidity and mortality statistics of TB is so extravagant that in the world someone dies of TB

every 15 seconds (WHO Report 2003 )

‘The Ticking Time Bomb’

TB Growth Rate - 2001

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Infection 9 Million cases/ yearDeath 2 Million cases / year2 Billion people are infected

in world

Special Feature: Tuberculosis: Nature Medicine : March 2007

Global Scenario of TB

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Magnitude of TB in India

40% of the Indian population is infected with the TB bacillus.

Every day, more than 20,000 people become infected with the TB bacillus and about 5000 develop the disease.

Every year 18 lakh (or 1.8 million) people inIndia develop TB, of which nearly 8 lakh(0.8 million) are infectious (sputum-positive).

Untreated pulmonary TB cases spread infection to others in the community—each infectious patient can infect 10-15 persons in a year unless effectively treated.

Despite being completely curable, TB claims the lives of >400,000 people in India every year

RNTPC report 2004

Tuberculosis in humans

INTRACELLULAR pathogen (facultative extra cellular)

ExposedInfected (2 billion, 8

million new cases per year)

Primary TB

Latent TB

Reactivation

30%

80-90%

5-10%

5-10%

Clearance

70%

Death

(2 million)

Problems of interventions against TB Lack of Epidemiological Data

Several genes with unknown function

Problems of Moon lighting

Persistence and Immune Evasion

Poor understanding of the pathogen-host-environment triangle

Emergence of MDR/XDR

Emergence of TB-IRIS

Emergence of TB-Diabetes synergy

Absence of Good Diagnostics: Tuberculin skin test >125 y

No new drug for the past 4 decades: 6 months MDT regime

No new vaccine (BCG : 75 y; M indicus pranii, a ray of hope)

No bio-marker for total sterilization

Two major paradigms govern evolution of persistent bacteria

Mycobacteria

Helicobacters

Emergence of ‘specialist’ lineages

Optimization of fitness

Vertical Genome Reduction

Lateral Genome Acquisition

Ahmed et al., 2008 Nature Rev Microbiol 6:387-394

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Evolution of GenomesEvolution of Genomes

Gene acquisition

Prophages

PlasmidsPAIs,Genomic islands (GEIs)Tn, IS, „Islets“,Integrons

TransformationTransductionConjugation

EvolvedGenome

RearrangementsMutations

Deletions

Genome reduction

• Genetic changes accumulate in the genome as a repertoire ofgene acquisition and loss, on an evolutionary time-scale

• Many human pathogens have such changes ascribed to rigorous selection against the host defenses and adaptation to different niches

• Genome wide analysis of such a repertoire in pathogens with different bio-geo-climatic history is a term coined by us as

“GEOGRAPHIC GENOMICS”

Geographic evolution: The concept of Geographic Genomics

Majeed et al., Bioinformatics 2004Hasnain and Ahmed LANCET Infect Dis 2004

Common ancestor M. leprae

M. canettii

RD9

TbD1

RD8 RD7

RD seal

RD12

RD14

M. tuberculosis (ancestral)

M. tuberculosis (modern)

M. africanumM. africanum

M. pinnipedae

M. microti

M. bovis

M. caprae

M. bovis BCG

decay (pseudogenization)

RD10

RD13

RD4

RD1

RD2

RD can

RD Mic

Reductional polymorphisms are the only major source of lineage diversity in pathogenic Mycobacteria

Genotype diversity is otherwise minimum, within the same geographical region

Reductive Evolution of the Mtb Complex genome

Brosch et al., 2002

Gen

ome

Gen

ome

size

size

•• Hos

t spe

cific

ityH

ost s

peci

ficity

•• Effe

ctiv

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vasi

onEf

fect

ive

inva

sion

•• Sur

viva

lSu

rviv

al

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Genomic Features of Ancient strains

1. Fewer than 6 copies of IS61102. Specific signature at MIRU Locus 43. Principle genetic group 14. Typical spoligotype5. TbD1 region is ‘intact’

spoligotype

isol

ate

no.

orig

in

PGG

TbD

1

MIRU-VNTRMIRU-VNTR dendrogram

EAI

Del

hi/C

AS

W/B

eijin

gLA

MT

X

Miru

02

VN

TR 4

24

VN

TR 5

77

Miru

04

Miru

40

Miru

10

Miru

16

VN

TR18

95

Miru

20

VN

TR23

47

VN

TR24

01

VN

TR24

61

Miru

23

Miru

24

Miru

26

Miru

27

VN

TR31

71

Miru

31

VN

TR36

90

VN

TR41

56

Miru

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Single, Double, Triple

Ancestral (40%)

Delhi (25%)

Beijing (8-10%)

Others (15-20%)

TbD1 region is present in about 36% isolates - ancient features

Ahmed et al. J Clin Microbiol.2004 42:3240-3247

TbD1/Rd9 analysis in the Indian isolates

Q

Do ‘ancestral’ lineages of Mycobacterium tuberculosis predominate in

India

If yes, does this denotes an ancient focus of tuberculosis in South Asia?

Does this provide any advantage for TB management in India?

Isolates from South India have been described to be of low virulenceand less disseminating?

A careful comparison of the virulence properties of ancient TbD1+ strains with those of the more modern strains, using the variety of animal models currently available, may thus provide novel insightsinto the evolutionary dynamics of this major pathogen.

Analysis of samples recovered from Egyptian mummies suggests that the modern lineages of M. tuberculosis diverged from the TbD1+ lineage thousands of years ago. Ancient Hindu scriptures also support the contention that this disease has been present as early as 10, 000 BC in India.

Therefore, the predominance of ancestral strains and the relatively poor representation of the most recent lineages in India, as apparent from this study, are consistent with the hypothesis that India is a historically ancient focus of tuberculosis.

Gutierrez + Ahmed et al., 2006 Emerging Infect Dis

Mycobacterium w genome program

Immune related disorders or infections are a result of changing lifestyles and thereby reduced exposure to certain bacteria that have been intricately associated as "old friends" during most of the mammalian evolution.

A very important group of bacteria among these organisms, are saprophytic mycobacteria, which are recognized by the innate immune system as biologically harmless. It is hypothesized that these "old friends" might be maintaining levels of regulatory immune cell populations (Rook et al., 2004), such as the cytokine secreting and antigen-presenting cells which are compromised in some allergies (asthma) and chronic infectious diseases (Crohn's disease due to M. avium complex).

These concepts are heralding the development of novel probiotic or immunomodulatory therapies for lifestyle diseases based on harmless organisms or their components.

One such ‘old friend’ is Mw !

Ahmed N et al., 2007 PLoS ONE

Evolution of Mycobacterial ‘Specialists’and ‘Generalists’

India is saved of the TB-time bomb (unlike South Africa) despite ~5.7 Million HIV cases

Why treatment success reaching ~90%? (Compare -> Russia=58%)Why no institutionalized outbreaks? (Compare -> Kwazulu Natal, SA)

India Russia

Bestowed with ancestral strains Crippled with Beijing strains

Source: WHO Report on TB, 2006, 2007 and 2008

India China RussiaOf new TB cases, % MDR-TB 2.8 4.0

13Of previously treated TB cases, % MDR-TB 17 26 49

India China RussiaOf new TB cases, % MDR-TB 2.8 4.0

13Of previously treated TB cases, % MDR-TB 17 26 49

Ancestral strains - Old is Gold?

“OLD is GOLD”: Issues to ponder

1. Ancestral lineages are widespread and perhaps do not allow spread of other genogroups

Host adaptation?Preferential colonization?Host Genetic resistance?

2. Are ancestral strains really advantageous for the TB control Program?

Slow disseminating types?Are these protective: Super infection?Are they less virulent: Less MDR/XDR?Are they more ‘cooperative’: Infection burden vs Disease burden?

3. How long this advantage sustains? Diabetes, HIV, Beijing, LAM

Ahmed et-al Inf Gen Evol 2009

Study of Evolutionary Dynamics and Molecular

Epidemiology not only permits tracking of a pathogen but

also enables the identification of new antigens of diagnostic

potential and also possible drug targets

“Humans and microbes are not ‘at war’. Rather, both parties are engaged in

amoral, self interested, co-evolutionary struggle.

We need to understand better, and therefore anticipate, the

dynamics of that process”

…and Until we Understand These Processes Mtb will Continue to Challenge Human Intelligence!!

A J McMichael. Phil. Trans. R. Soc. Lond. B (2004) 359, 1049–1058