Evolution of Pathogenic Bacteria: Mycobacterium tuberculosis example

Bio 101 Lecture

by

Seyed E. Hasnain

Why study Pathogen Evolution?

pathogen genome 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 Morschhuser et al., 2000).

Microevolution
Development of organisms in days and weeks

Macroevolution
Development of species and variants in long term intervals
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Genetic mechanism
Horizontal gene transfer

Microevolution
Plasmid, Phage transfer

Macroevolution
PAI development

Genome reduction, Deletions

Deletions

Development of intracellular pathogens

Genetic rearrangements

Phase, Antigenic Variation

Development of new variants Pathoadaptation

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Point mutations

Gene expression, Modulation

Why M. tuberculosis

The Ticking Time Bomb

The morbidity and mortality statistics of TB is so extravagant that in the world someone dies of TB every 15 seconds (WHO Report 2003 )
Europe 2.1% US 37.7% Asia 33.3%

Africa 9.6% Latin America 17.7%

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TB Growth Rate - 2001

Global Scenario of TB Infection 9 Million cases/ year Death 2 Million cases / year 2 Billion people are infected in world

Special Feature: Tuberculosis: Nature Medicine : March 2007

Despite being completely curable, TB claims the lives of >400,000 people in India every year
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 in India develop TB, of which nearly 8 lakh (0.8 million) are infectious (sputum-positive). Untreated pulmonary TB cases spread infection to others in the communityeach infectious patient can infect 10-15 persons in a year unless effectively treated.
RNTPC report 2004
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Tuberculosis in humans
INTRACELLULAR pathogen (facultative extra cellular)
Primary TB Latent TB
5-10%

Death (2 million)

5-10% 30%

Infected
(2 billion, 8 million new cases per year)

Exposed
70%

80-90%

Reactivation

Clearance

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

Lateral Genome Acquisition

Helicobacters
Optimization of fitness

Vertical Genome Reduction

Mycobacteria
Emergence of specialist lineages Ahmed et al., 2008 Nature Rev Microbiol 6:387-394

Evolution of Genomes
Gene acquisition
Transformation Transduction Conjugation
Plasmids PAIs, Genomic islands (GEIs) Tn, IS, Islets,Integrons Prophages

Rearrangements Mutations

Deletions

Evolved Genome

Genome reduction

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Geographic evolution: The concept of Geographic Genomics Genetic changes accumulate in the genome as a repertoire of
gene 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

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

Reductional polymorphisms are the only major source of lineage diversity in pathogenic Mycobacteria
M. canettii M. tuberculosis (ancestral)
RD can TbD1 decay (pseudogenization)

Common ancestor
RD10 RD8

RD9

M. leprae

M. tuberculosis (modern)
RD7 RD seal RD12 RD13 RD Mic

M. africanum M. pinnipedae

M. africanum

M. caprae
RD4

M. microti M. bovis
RD1 RD2 RD14
Genotype diversity is otherwise minimum, within the same geographical region

M. bovis BCG

Reductive Evolution of the Mtb Complex genome

Host specificity Effective invasion Survival

Genome size

Brosch et al., 2002

Genomic Features of Ancient strains

1. Fewer than 6 copies of IS6110 2. Specific signature at MIRU Locus 4 3. Principle genetic group 1 4. Typical spoligotype 5. TbD1 region is intact

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X LAMW/Beijing Delhi/CAS
MIRU-VNTR dendrogram

T EAI

spoligotype
Miru 02 VNTR 424 VNTR 577 Miru 04 Miru 40 Miru 10 Miru 16 VNTR1895

Delhi (25%)

Beijing (8-10%)

Ancestral (40%)

Others (15-20%)
Single, Double, Triple

Miru 20

VNTR2347 VNTR2401

MIRU-VNTR
VNTR2461 Miru 23 Miru 24 Miru 26 Miru 27 VNTR3171 Miru 31 VNTR3690 VNTR4156 Miru 39

isolate no.

origin

PGG TbD1

TbD1/Rd9 analysis in the Indian isolates

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

Q
Ahmed et al. J Clin Microbiol.2004 42:32403247

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?

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. Isolates from South India have been described to be of low virulence and 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 insights into the evolutionary dynamics of this major pathogen.

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

Ancestral strains - Old is Gold?

India Of new TB cases, % MDR-TB 13 Of previously treated TB cases, % MDR-TB China 2.8 26 Russia 4.0 49

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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

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
A J McMichael. Phil. Trans. R. Soc. Lond. B (2004) 359, 10491058

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