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Diversity and disease pathogenesisin Mycobacterium tuberculosis

Digby F. Warner, Anastasia Koch, and Valerie MizrahiMRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DST/NRF Centre of Excellence for Biomedical TB Research, Institute of

Infectious Disease and Molecular Medicine and Department of Clinical Laboratory Sciences, University of Cape Town, Cape Town,

South

Africa

The increasing availability of whole-genome sequence

(WGS)

data

for

Mycobacterium

tuberculosis,

the

bacte-

rium that causes tuberculosis (TB), suggests that circu-

lating genotypes have been molded by three dominant

evolutionary forces: long-term persistence within the

human population, which requires a core programme

of infection, disease, and transmission; selective pres-

sure on specific genomic loci, which provides evidenceof lineage-specific

adaptation

to

host

populations;

and

drug exposure, which has driven the rapid emergence of

resistant isolates following theglobal implementation of

anti-TB

chemotherapy.

Here,

we

provide

an

overview

of

these factors in considering the implications of genotyp-

ic diversity for disease pathogenesis, vaccine efficacy,

and

drug

treatment.

Genetic

diversity

in

the

Mycobacterium

tuberculosis

complex

TB is a global problem, with recent reports estimating

approximately 8.6

million new cases and 1.3

million

deaths

annually

[1].

This

is

despite the existence

of

effective

front-line combination chemotherapy,

a

widely administeredvac-

cine, and the allocation

over

the past decade

of

massive

resources to develop improved interventions [2,3]. Co-infec-

tion

with

HIV, and the emergence of

drug

resistance

have

amplified the problem;

however, these represent

relatively

recent, or modern (Figure1), developments in theevolution

of

the causative

agent, Mycobacterium tuberculosis, as an

obligate human pathogen [4].

Modern

bacteriology

has

been

transformed

by

recent

advances in high-throughput DNA sequencing technology

[5] thathave

enabledthe democratization of

whole-genome

sequencing [6],

and the impact on

TB

genomics

has

been

profound [7].Mycobacterium tuberculosis isonememberofa

group of

closely related

bacteria known

as

the Mycobacteri-

um tuberculosis complex (MTBC), which comprises seven

closely related human lineages [8], animal-adapted strains

(including the TB

vaccine strain, Mycobacterium bovis;

BCG),

and themore

distantlyrelatedMycobacterium canet-

tii group, in which the smooth tubercle bacilli (STB) are

situated [9].Until recently, theMTBCwas considered clonal

or

monomorphic

[10].

As

a

result, the varied outcomes

following

exposure

of

an

individual

toM. tuberculosis were

reasonably assumed

to

depend

almost

exclusively

on

host

genetics and environmental

factors. Similarly, the efficacy

(and failure) of treatment and prophylaxis was in turn

understood

as

a

function

of the

host (and compliance).

Bacillary genotypic variation was considered unimportant.The

recent

availability

of

WGS

technologies

has

rejected

this

model:

increasing

evidence

of

strain

diversity

[7,11],

lineage-specific adaptation to host populations [12,13], and

microvariation

within

hosts

and

communities

[1416]

in-

stead

supports

the

idea

that

mycobacterial

genetics

and,

therefore, function, are a significant element in determin-

ing the

heterogeneous

outcomes

of

infection.

The M. tuberculosis infection cycle

As an obligate pathogen, the persistence ofM. tuberculosis

within the human population depends on the ability to

drive

successive

cycles

of

infection,

disease

(in

some

cases,

subclinical TB [17] followed by reactivation), and trans-mission.

The

reliance

on

a

single

host

species

necessarily

exposes

the

infecting

pathogen

to

multiple

potential

evo-

lutionary cul-de-sacs that might arise as a consequence of

the

elimination

of

the

bacillus

(clearance)

or

the

demise

of

the

organism

within

an

infected

individual

(controlled

subclinical

infection,

or

host

death)

before

it

is

able

to

ensure

transmission

to

a

new

host.

Moreover,

the

capacity

for the organism to remainviable during extended periods

of subclinical

TB

disease

means

that

the

infection

cycle

is

not defined by a uniform duration. For this reason, accu-

rate

dating

of

the

MTBC

remains

a

contentious

issue:

while

one

study

suggested

that

the

complex

emerged

approxi-

mately 70

000

years

ago

[8],

more

recent

work

estimatesthis

occurrence

at

approximately

5000

years

ago

[18]. Nev-

ertheless,

circulatingM. tuberculosis isolates represent the

genotypes that have successfully adapted to human colo-

nization

over

a

timescale

of

thousands

of

years

[4,8],

a

process that is marked by several historical events that

might

have

impacted

the

inferred

coevolution

of

host

and

pathogen

(Figure 1) [13].

Many bacterial pathogens can accelerate evolution

through

the

enhanced

activity

of

their

DNA

repair

machin-

ery (e.g., recombination) or aggressive sampling of the

immediate

environment

(e.g.,

fratricide,

natural

compe-

tence,

and

conjugation).

Horizontal

gene

transfer

(HGT)

had an important role in the emergence of M. tuberculosis

Review

0966-842X/

2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tim.2014.10.005

Corresponding authors: Warner, D.F. (Digby.warner@uct.ac.za);

Mizrahi, V. (valerie.mizrahi@uct.ac.za).

Keywords: Mycobacterium tuberculosis;

genomics; epistasis; evolution; mutagenesis;

drugs; vaccine.

TIMI-1138;

No.

of

Pages

8

Trends in Microbiology xx (2014) 18 1

http://dx.doi.org/10.1016/j.tim.2014.10.005mailto:Digby.warner@uct.ac.zamailto:valerie.mizrahi@uct.ac.zamailto:valerie.mizrahi@uct.ac.zamailto:Digby.warner@uct.ac.zahttp://dx.doi.org/10.1016/j.tim.2014.10.005

7/26/2019 Tuberculosis Patogenesis Warner2014

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as an exquisitely human-adapted pathogen [19] and on-

going

recombination

has

been

suggested

as

a

source

of

genetic

variation

[20]. However,

little,

if any,

evidence

exists for a role of HGT in recent evolution in the MTBC

[4].

Instead,

the

modern

evolution

of M. tuberculosis has

been

driven

by

chromosomal

rearrangements

and

muta-

tions, features that result in part from the ecological

isolation

of

the

bacillus,

as

well

as

the

bottlenecks

that

occur

during

transmission

[12]. Contrary

to

some

other

bacterial

and

mycobacterial

species

[21], M. tuberculosis

does not have plasmids, and genetic drift is primarily

responsible

for

diversification

and

adaptation

of

this

group

of organisms [12]. However, evidence that the population

structure

of

human

MTBC

is

highly

subdivided,

both

geographically

and

within

the

lungs

of

infected

individuals

[12], suggests the capacity for diversification.

Evidence for microdiversity

Numerous studies have identified significant genotypic di-

versity

within bacilli isolated

from

single

hosts

[14,22

25].

In

some

cases, this has

been attributed to

mixed

infec-

tion with

two distinct strains [14,22], a

phenomenon that is

likely tooccur inhigh-burdensettingswith anelevated force

of infection

[26,27]

and, importantly, suggests

the potential

Lineage 4

(Europe)

MTB M

T

BC

Lineage 2

(East Asia)

Lineage 3

(Central Asia)

Lineage 1

(Indian Ocean)

Lineage 7

(Ethiopia)

Lineage 5 and 6

(West Africa)

BCG

vaccinaon (1920s)

Anbioc therapy

(1950s)

Migraon of

humans out of

Africa

(70 000 years ago)

HIV

(1970s)

Evoluon of

MTBC from

last commonancestor

Strain evoluon

to produce seven

main lineages of

MTB

Ongoing transmission

between hosts

(Local and global)

Diversity within

an individual

Diversity within

sites of infecon

in an individual

Informaon available from whole-

genome sequencing

Human evoluon

A

n

c

i

e

n

t

M

o

d

e

r

n

Host nutrion

Industrial revoluon

(19th century)

Evoluonary pressures

impacng MTB

TRENDS in Microbiology

Figure1 .

The impact of whole-genomesequencing on reconstructing the evolutionaryhistory ofMycobacterium tuberculosis(MTB). Abbreviation: MTBC,M. tuberculosis

complex.

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for

direct competition

between infecting

genotypes.

In

addi-

tion, there is

increasingevidence

of

microdiversitywithinM.

tuberculosis populationsthatdevelop froma single infecting

strain [23,24].Theseobservations reinforce earlierwork [28]

which

demonstrated

that different

drug-resistance

alleles

could

arise

in

discretepulmonarylesions from

a

single,

drug-

susceptible infecting genotype.

In some

respects,

it

has

been

difficult

to

reconcile

thelevels of intrapatient diversity with the lower levels of

genotypic

variation

detected

within

transmission

clusters.

For example,

while

epidemiologically

clustered

strains

might differ by as few as five SNPs [16], as many as seven

SNPs

separated

strains

isolated

from

the

lungs

of

an

individual patient [24]. Therefore, it appears that the same

degree

of

heterogeneity

can

characterize

intra-

and

inter-

patient

diversity.

By

contrast,

a

separate

report

identified

only two SNPs (in katG, encoding the catalase-peroxidase

enzyme

that

activates

the

frontline

anti-TB

drug,

isonia-

zid,

and

in rpoB, which encodes the b subunit of the RNA

polymerase complex, the target of another key frontline

agent, rifampicin)

in

serial

isolates

that

developed

sequen-

tial resistance to isoniazid and rifampicin over 12 years inanoncompliant

patient

[29]. As

noted

elsewhere

[30], the

use

in this case of a reference genome comprising pooled data

from

all

the

serial

isolates

might

have

obscured

SNPs

present

at

low

frequencies,

highlighting

the

potential

im-

pact of the method used for genome assembly on the

interpretation

of

sequence

data

[19]. The

same

caveat

is

likely

to

apply

generally

to

studies

that

rely

on

the

use

of

reference genomes for sequence alignment and assembly:

as noted

recently

[6], improved

methods

to

detect

larger

genomic

deletions

and

alterations

are

required

to

provide

better insight into the dynamics that might underlie mi-

croevolution.

It is

also

possible

that

diversity

is

lost

in

sample

collec-

tion,

or

during

downstream

manipulations

required

for

strain

propagation

and

DNA

isolation

for

WGS

(Figure 2). Clinical isolates are usually cultured from

sputum

samples,

which

may

not

harbor

bacteria

that

are

representative

of

the

entire

population

residing

within

the host, and might instead contain only a subset of the

phenotypic

(and

genetic)

variants.

Some

strains

might

notgrow in laboratory media, while others might grow so well

as

to

dominate

cultured

bacillary

populations

[26]. More-

over,

important

new

evidence

suggests

that

propagation

in

laboratory media induces genomic changes in cultured

isolates:

a

recent

study

reported

a

strong

selective

advan-

tage for a large genomic duplication (approximately

350 kb)

that

arose

in

the

bacillary

population

after

only

five

rounds

of

passage

in

broth

media,

and

was

associated

with attenuated virulence in mice [31]. In addition to the

implications

for

genotypephenotype

analyses,

this

result

reinforces

the

potential

to

select

inadvertently

for

labora-

tory-adapted variants, a possibility that is generally not

considered

in

genomic

studies

and

might

influence

epide-

miological inferences of strain prevalence and fitness [32].

What are the implications of genotypic diversity for

pathogenesis?

The

natural

lifecycle

of M. tuberculosis suggests a further

explanation for the apparent discrepancy between the

relative

genetic

stability

of

transmitted

strains

and

the

potential

intrapatient

diversity.Mycobacterium tuberculo-

sis is transmitted in infectious aerosols, which are inhaled

deep

into

the

lung

where

the

bacilli

lodge

in

alveoli

and

are

engulfed

by

resident

macrophages.

Although

the

precise

details remain to be determined, it is assumed that suc-

cessful

transmission

from

a

prevalent

TB

case

to

a

new

How many bacilli are required to

establish an infecon?

How many bacilli are present in a

lesion and how does the lesional

bacillary burden change with

disease progression?

How many bacilli are available for

transmission to a new host?

Infecon Replicaon within the host Transmission

Technical limitaons and biases

Sampling

Only a (small) fracon of bacilli present in the clinical

specimen are culturable and/or selected for sequencing

Wgs and data analysis

Sequencing methodology and/or data analysis

Potenal

sources of

genotoxic

stress

Key quesons

Diagnosis

Symptoms

Smear microscopy

Culture

Gene Xpert RIF/MTB

Whole-genome sequencing

To what extent are bacilli cultured from a

clinical specimen representave of the

bacillary populaon within the host?

Do bacilli that establish infecon

have specific genotypic and/or

epigenec characteriscs?

How does the anatomical locaon

of a lesion impact genotoxic

stress on the bacilli?

How does the anatomical locaon

of a lesion impact genotypic diver-

sity within the lesion?

To what extent are cultured bacilli

representave of transmied bacilli?Do transmied bacilli have parcu-

lar genotypic and/or epigenec

characteriscs?

Immune effectors

Phagosomal acidificaon

Oxidave stress

Nitrosave stress

Nutrient starvaon

Hypoxia

Anbioc treatment

Innate immunity Adapve immunity Tissue damage and cavitaon

TRENDS in Microbiology

Figure 2.

Genetic diversification ofMycobacterium tuberculosis within a host: key questions, technical limitations, and biases. Abbreviation: WGS, whole-genome

sequencing.

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host

requires

that

multiple

conditions

be

met.

First,

bacilli

must

escape

in

sufficient

numbers

and

in

a

physiological

state(s)

that

will

ensure

transient

survival

in

the

environ-

ment before inhalation by the new host. Inhaled organisms

must

then

overcome

(or

subvert)

the

barrier

defence

sys-

tems

of

the

host

to

gain

access

to

the

alveoli.

Again,

the

details are not clear, but it is assumed that at least a single

M. tuberculosis

bacillus

must

then

establish

infection

and,subsequently, overcome immune defences to replicate and

produce

TB

disease

capable

of

driving

a

new

infection

cycle

[33].

Even in high-burden settings, the probability of TB

infection

is

relatively

rare

(approximately

45%

per

annum [27]), which, as noted above, reflects the multiple

obstacles

at

which

a

potential

infection

is

thwarted.

These

inherent

bottlenecks

are

likely

to

have

significant

implica-

tions for the apparent monomorphism of M. tuberculosis

[10]. Given that the infecting (transmitted) bacillus must

replicate

until

a

population

size

is

reached

that

is

suffi-

ciently large to establish a foothold in the new host, the

founder

genotype

will

necessarily

dominate

the

expand-

ing population. Moreover, the M. tuberculosis lifecycle isnot

dependent

on

achieving

maximal

bacillary

numbers

withina givenmicroenvironment: recent evidence from the

nonhuman

primate

model

indicates

that,

in

immune

com-

petent

hosts,

bacterial

populations

within

individual

lesions consistently achieve a maximum size of approxi-

mately 2105 bacilli

per

lesion

before

onset

of

the

adaptive

immune

response

and,

following

depletion

owing

to

im-

mune-mediated killing, stabilize at approximately 102

bacilli

per

lesion

during

active

disease

[34]. Permissive

lesions

that

exceed

this

carrying

capacity

and

spread

locally, or result in TB pneumonia, are rare. Therefore,

the

microenvironmental

and

molecular

mechanisms

that

might

enable

small

bacillary

populations

to

accumulatethe

mutational

diversity

suggested

by

inferred in vivo

mutation rates (reviewed in [30]) require elucidation.

The bottlenecks described above imply

that any muta-

tions that are generated

during

host infection

are

likely

to

achieve relatively low frequencies within discrete popula-

tions. That

is,

while

numerous

SNPs

might

arise

during the

course

of

infection,

a

complex

interplay

of

factors

will

deter-

mine whether specific individual mutations ultimately be-

come

fixed

alleles

that are transmitted as

distinct strains

and sublineages. Critically, allelic fixation will depend on

theabilityof thebacillus to overcomethosesamebarriers as

the infection cycle progresses. However, there are two im-

portant

exceptions:first,becausedrug

treatment

represents

an

immediate

threat to

survival,

resistance-conferring

mutations will be rapidly fixed in any bacillary population

exposed

to

extended

therapy. Accordingly,

comparative ge-

nomics studies

are united

in

identifying

drug

resistance

polymorphisms regardless of strain diversity or geographic

region [15,35,36]. Paradoxically, selection

of

resistance

mutations

is

exacerbated in

the presence

of

functioning

TB control programs, which allow even low-fitness drug-

resistant

strains to

outcompete

both drug-sensitive

and

other,

less-fit

drug-resistant

strains [37,38]. Where strains

acquire compensatory

mutations,

fixation in

the circulating

population is accelerated [39]. As a result of their close

association

with drug

resistance,

compensatory

mutations

constitute

the second exception: whether

preceding

(en-

abling)

or

following

(modifying)

the acquisition of

the drug

resistance mutation,

this form

of

epistasis

is

the subject

of

intense research to understand the development and prop-

agation

of

resistance and,

potentially,

to

identifyalternative

counteracting therapies and interventions.

In combination, these observations suggest that the

intrapatient

diversity

might

be

greater

than

expected.They also imply that the capacity to generate diversity

might

be

critical

to

disease

progression

within

individual

hosts,

even

though

the

resulting

SNPs

are

not

necessarily

broadly selected (transmitted) within a population. Some

evidence

to

support

this

hypothesis

stems

from

the

obser-

vation that MTBC strains are associated with a high

proportion

of

nonsynonymous

SNPs

within

the

3R

genes

involved

in

DNA

replication,

repair,

and

recombination

[40]. Therefore, it is tempting to speculate that the identi-

fied

polymorphisms

result

in

a

relaxation

of

3R

function

and

fidelity

that

facilitates

the

rapid

generation

of

genetic

diversity during host infection, perhaps as a strategy to

enable

adaptation

to

allopatric

hosts.

That

is,

while M.

tuberculosis maintains a core gene set that enables infec-tion

and

transmission,

it

retains

the

capacity

for

micro-

diversity through mutations in other genes. The

epidemiological

success

of

modern

strains

might

indicate

the exploitation

of

this

capacity

to

develop

increased

viru-

lence against a background of greater host population

density

and

comorbidities,

such

as

HIV

(Figure

1).

Evidence for a conserved interaction between host and

pathogen

The contention that coevolution might have resulted in a

core M. tuberculosishost interaction is supported by

several

observations that

derive

from

independent anal-

yses of both bacillary and host

genotypes

and functions.For

example, a

key study[41]

showed

that

T

cell epitopes

are highly conserved across M. tuberculosis lineages,

suggesting

that

selective pressure acts

against sequence

diversity

in immunogenic regions.

This

is reinforced by

a

more recent analysis [42] that revealed that sequence

variation in pe_pgrs genes (thought to be involved in

antigenic variation) is restricted

to

regions

that are dis-

tinct from the known T cell epitopes, suggesting instead

that another selective

pressure drives

sequence

variation

in these loci.

At a functional level, evidence that macrophage infec-

tion triggers a conserved, coremycobacterial transcription-

al

response

[43]

(with

some

scope

for

lineage-specific

effects)

appears

to

have

a

corollary

in

the

corresponding

host response, which has elements consistent with both

conserved

and

lineage-dependent

function

[4446].

More-

over,

the

observation

that

hypervirulent

mutants

often

contain causal mutations in structural and regulatory

genes

[47]

perhaps

indicates

that

virulence

in M. tubercu-

losis is under tight control [48]. An additional line of

support is provided by the specific hostpathogen interac-

tions

that

occur

in

the

different

members

of the

MTBC:

despite

close

phylogenetic

relations,

human

TB

is

caused

almost

exclusively

by M. tuberculosis and Mycobacterium

africanum, with little evidence of zoonotic transmission of

any

of

the

other

MTBC

members.

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Genotypephenotype variability in a host-adapted

pathogen

Given

the significant bottlenecks to

allelic

fixation within

theM. tuberculosis population,what factors drive thespread

ofSNPs

not associated

with

drug

resistance?

A

recent study

conducted

in

a

low-density

setting indicated that there is

a

sympatric relation between specificM. tuberculosis strains

and

cognatehosts

[49],

suggesting

thathostgenotypes

havesome influence on bacillary diversity. However, in high-

density

settingswith

significant

bacterial andhostgenomic

diversity, there is

likely to

be

less selective pressure:

al-

though interstrain competition may be strong, the popula-

tion of

susceptible

hosts

is

large andso

able

to

accommodate

reduced fitness variants, including multidrug-resistant

strains (reviewed in

[50]).

This

effect

will be

exacerbated

if

infection

with

one bacillary

genotype

favours re-infection

withanother different genotype [14], and could result in an

explosion

of

genotypic

variation, as

suggested

by recent

evidence

of

significant

strain and lineage diversity

within

a well-defined setting in an endemic region [51]. Of course,

frequent

bottlenecks imposed

by

transmission raise

the

possibility that chance, not selection, is a major factordetermining

circulating genotypes

[52],

which

is

again

con-

sistent with observation thatmost SNPs in MTBC occur as

singletons.

However,

an

important consequence is

that

elucidating genotypephenotype

associations becomesdiffi-

cult:allelessuggestingconvergentevolutionacrossdifferent

lineagesoffer

rare

glimpses

into functional

adaptations [53].

Implications of genotypic diversity: transmission of

hypervirulent strains

The

conserved

hostpathogen

interaction

proposed

above

assumes that M. tuberculosis is primarily infecting im-

mune

competent

individuals.

As

noted

elsewhere

[54], a

functional

adaptive

immune

response

is

essential

for

M.tuberculosis to complete its lifecycle. When infection and

disease occur against a background of compromised immu-

nity,

TB

disease

manifestation

and,

therefore,

the

infection

cycle, are

corrupted,

consistent

with

the

finding

that

HIV-

positive individuals are poor TB transmitters [55]. The

dependence

on

an

immune-competent

host

for

optimal

transmission

also

suggests

that

the

ability

to

cause

active

disease (i.e., strain virulence) will directly impact trans-

missibility.

Where

a

positive

correlation

exists

between

pathogen virulence and transmission, selection acts to

increase virulence and reduce latency to maximize expo-

sure to potential new hosts (reviewed in [13]). For M.

tuberculosis,

whose

natural

evolution

has

occurred

in

the

context

of

increasing

human

population

density

[8], the

selective pressure for transmission is likely to be associat-

edwith

an

increase

in

strain

virulence.

The

inevitable,

and

concerning,

consequence

is

that

the

combination

in

high-

burden TB settings of elevated strain diversity, direct

competition

between

genotypes,

and

strong

drug

pressure

will

drive

the

emergence

of

increasingly

virulent

drug-

resistant isolates with low to no short-term fitness costs.

In

some

respects,

the

recent

expansion

of

the

Beijing

family

of

strains

(a

sublineage

of

Lineage

2)

in

diverse

geographic

settings,

and

their

association

with

drug

resistance

and

hypervirulence in animal models, provides a cogent reali-

zation

of

these

combined

selective

pressures

[56].

What are the processes underlying genome dynamics in

M.

tuberculosis?

The

observed

intrapatient

microdiversity

implies

that

the

M. tuberculosis mutation rate might be elevated during

host

infection,

a

possibility

that

has

also

been

invoked

to

explain

the

emergence

of

multidrug

resistance

in the

pres-

ence of combination therapy (reviewed in [30]). To date,

however,

evidence

from

both

animal

[57]

and

human

stud-ies [58] suggests that, during active disease, mutations

accumulate

at

rates

that

are

within

the

ranges

calculated

in vitro. Determining the mutation rate during latent

infection is more complex, as reflected by the fact that

values

calculated

using M. tuberculosis isolates obtained

from patients presenting with reactivation disease [58]

differ

from

those

predicted

in

a

nonhuman

primate

model

of infection

[57]. Importantly,

a

recent

clinical

study

esti-

mated that the mutation rate during extended latency

(>20 years)

is

at

least

30

times

lower

than

the

rate

that

occurs

during

active

disease,

a

result

that

is

consistent

with separate analyses reporting apparent genetic stasis

in

a

well-characterized

panel

of

reactivation

TB

isolates

[59]. While this may indicate that there is little hostpressure

on

the

organism

during

latent

or

subclinical

infection, fixation of mutations requires chromosomal rep-

lication

which

in

turn,

raises

important

questions

regard-

ing

the

assumed

rate

at

which

bacilli

divide

during

host

infection.

Various

lines

of

evidence

led

to

the

assumption

that

the

bacillary

population

remains

stable

during

chronic

TB,

and

comprises slow or nonreplicating organisms. However, the

application

of

a

clock

plasmid

that

is

lost

from

daughter

cells

during

division

has

established

that,

during

chronic

infection in the mouse model, a stable balance is estab-

lished

between

bacillary

replication

and

death

[60]. Pro-

found

differences

in

TB

pathology,

particularly

withrespect

to

the

formation

of

hypoxic

microenvironments

within granulomatous lesions [61], mean that extrapola-

tion

of

findings

from

the

chronic

mouse

model

to

humans

must

be

made

with

caution.

Nonetheless,

in addition

to

suggesting that the bacilli are under constant immune

surveillance,

these

observations

imply

that

bacilli

may

be

replicating

at

a

rate

higher

than

previously

thought,

a possibility that is consistent with current models that

propose

that

a

continuum

of

mycobacterial

growth

states

prevails during host infection [62]. A compelling mathe-

matical model [63] utilized the possibility of an elevated

bacillary replication rate to demonstrate that the likeli-

hood of emergence

of

drug

resistance

before

initiation

of

anti-TB

therapy

is

higher

than

previously

expected,

even

when based on established in vitro mutation rates.

Is

there any evidence

of

mutator

strains of

M.

tuberculo-

sis? For an obligate pathogen, the benefits of a mutator

phenotype for the development of drug resistance are likely

to

be

outweighed by

negative effects

on

virulence and the

susceptibility

of

mutators

to

extinction

as

a

result

of

bottle-

necks. Nevertheless, the existence of strain-specific muta-

tion

rateswas

suggested

in a

recent study

[64] thatreported

that M.

tuberculosis

strains from

the East

Asian lineage

acquire drug

resistance

SNPs

more rapidly

compared

with strains from the Euro-American lineage under the

same

conditions in

vitro.

Importantly, these experiments

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eliminated the possibility that this effect

results

from

an

increasedabilityto

adapt

to

antibioticpressure, andinstead

indicated

that East

Asian

lineage strains are associated

with an elevated mutation rate in the absence of antibiotic

pressure,

although the causative

mechanism

is

unknown.

Linking strain genotypes with disease phenotypes

The

complex

genotypes

associated

with

drug

resistance[35,36], as well as emerging evidence of the impact of

compensatory

mutations

on

the

acquisition

and

mainte-

nance of

resistance

alleles

[32], highlight

the

importance

of

determining epistatic interactions. For those mutations

that

occur

in

the

absence

of

drug

resistance,

it

is

even

more challenging to determine the functional conse-

quences

of

different

mutations:

as

noted

elsewhere

[11],

the

absence

of

HGT

means

that

all

SNPs

in

an

individual

M. tuberculosis genome are in linkage disequilibrium.

Therefore,

while

low-level

homoplasy

means

that

SNPs

can

be

usefully

applied

to

measure

evolutionary

distances

among isolates, determining their impact on bacillary

function

and

pathogenesis

is

not

trivial.

For

this

reason,

despite themassive increase in sequence data, there is stilla

need

to

obtain

additional

genomic

information

for

care-

fully selected panels of clinical M. tuberculosis isolates as

well

as

related

nontuberculous

mycobacteria

and

other

Actinobacteria [9,19,65]. As discussed below, alternative

approaches to sampling bacilli from different microenvir-

onments

and

anatomical

loci

will

be

critical

to

future

efforts

to

determine

the

degree

of

heterogeneity

within

bacillary subpopulations, as well the impact of the pan

genome

on

bacterial

pathogenesis

and

disease

outcome.

A

powerful

example

of

the

utility

of

diverse

genomes

for

comparative analyses was recently provided by the dem-

onstration

that

SNPs

in

the

two-component

regulator,

PhoPR,

contribute

directly

to

the

reduced

virulence

andtransmissibility

of

animal-adapted

and M. africanum

strains by reducing the export of virulence factors, such

as

the

major

secreted

antigen,

ESAT-6,

and

decreasing

the

synthesis

of

polyacyltrehalose

lipids

and

sulfolipids

[53]. Given recent evidence implicating PhoPR in the

metabolic

adaptation

of M. tuberculosis to low pH [66], it

is

likely

that

a

compromised

ability

to

cope

with

this

important antimicrobial defence exacerbates the pheno-

type

of phoPR mutants, a possibility that is reinforced by

the observation that the PhoPR regulon also includes the

pH-responsive aprABC locus [67] which is limited to mem-

bers of the MTBC.

In addition to

known

drug-resistance

alleles

[15,35,36],

some

nonsynonymous

mutations

and insertion-deletion

events are likely to be inactivating [68]; however, for most

genomic

mutations and rearrangements, predicting the

impact

of

specific

polymorphisms

on

gene

expression,

pro-

tein function, and strain fitness remains a major challenge

[69,70], and is

exacerbated where multiple

mutations

dif-

ferentiate

the strain of

interest

from

the parental isolate.

Moreover, evidence that synonymous SNPs can influence

function

[69,71] suggests

that, for

many

genomic

mutations,

inferring

the potential

impact from

sequence

data

alone is

not

possible

and will require

experimental investigation

by means of allelic exchange mutagenesis [72] as well as

additional

multiletter

acronym

or

MLA-seq

applications

(e.g.,

RNA-seq)

that can provide

a

comprehensive inventory

of

the consequences

of

specific mutations

for

information

pathway

function

[6].

Concluding remarks: approaching a systems biology of

TB

Mycobacterium tuberculosis has a 4.4-Mb genome that

harbors

evidence

of

the reductive

evolution

characteristicofanobligatepathogen[4,65]; however,the bacillus remains

a

formidable

prototroph

capable

of

colonizing

diverse host

environments and resisting the associated stresses. We

have argued here that at least part of the success of the

organism

appears

to

reside

in thestable interaction with its

obligate human host while retaining the capacity to gener-

atephenotypic

diversity.

Specifically,armingdiscrete

bacil-

lary

subpopulations

withgenotypicvariability

might

enable

a small infecting population to explore a huge fitness

landscape.Althoughbeyond thescope

of

thecurrent review,

increasing

evidence

of

stochastic behavior [73] as

well

as

potential for epigenetic modifications, such as DNA meth-

ylation,

to

alter

bacillary physiology

[74],

further

supports

the application of novel sampling methods and sequencingtechnologies[75] to

catalogthe fulldiversity

of

physiological

states in clinicaland experimentalTB infection. The recent

use

of

shotgun

metagenomics to

detect

and characterize

M. tuberculosis in clinical samples [76] might herald the

widespread application of culture-free techniques to this

Box

1.

Outstanding

questions

Transmission

When is Mycobacterium tuberculosis transmitted during the

infection cycle?

Howmany bacilli are transmitted?

What is the anatomical and microenvironmental origin

of

transmitted bacilli?

Colonization

What factors determine lineage-specific immune responses?

What is the impact of the host microbiome on M. tuberculosis

infection?

How sterile is the M. tuberculosisniche?

What is the impact of mixed M. tuberculosisinfection?

Disease

What is the size of the infecting M. tuberculosispopulation?

Howmuch diversity is there within individual lesions?

What are the correlates of host specificity?

How do host and pathogen genotypes interact?

Latent and/or subclinical TB infection

How big is theM. tuberculosisreservoir?

Does reactivation occur in only approximately 10% of cases

because these are the only individuals who harbor viable bacilli?

Do bacilli replicate throughout clinical latency?

Microdiversity

What is the impact of intrapatient diversity on disease progres-

sion?

What determines lineage-specific mutation rates?

What factors determine strain success?

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end and, critically, offers one approach

to

avoid the biases

inherent in

strain sampling

and propagation.

Understanding

the evolutionary processes that have

shaped andenabled the exquisite adaptation ofM. tubercu-

losis may provide clues to biological processes that are

important

for

pathogenesisand,therefore,

potential

targets

for novel therapeutics [77]. Similarly, the influence on the

adaptive

immune

response of

exposure

to

complex

circulat-ing genotypes in endemic settings suggests that future

vaccinedesigns

and studies

will

have

to

consider

thepoten-

tial

impact of

strain diversity

on

efficacy.

From

a

diagnostic

perspective, advances in the application of metabolomics

techniques

to

analyze

sputum

for

both mycobacterial and

host markers of disease [78] suggest that further refine-

ments

will

enable

the differentiation

of

major

lineages by

analogy with

recent

reports

from Salmonella [79].

Finally, the model presented here is consistent with

emerging

evidence

from

several

other

bacterial

systems

in which

the

application

of

advanced

genomics

techniques

has revealed a similarly unexpected ability of a founding

strain

to

drive

high

levels

of

genotypic

and

phenotypic

diversification (reviewed in [5]). Further research will berequired

to

ascertain

the

implications

of

diversity

for

M.

tuberculosis pathogenesis and future interventions (Box 1).

However,

there

is

an

urgent

need

to

develop

systems

biology

approaches

to

determine

the

emergent

properties

of discrete, genotypically diverse bacterial populations on

the single

infected

host.

Acknowledgments

Weapologize to all those authors whose workwas not cited owing to space

limitations. We acknowledge funding from the South African Medical

Research Council (SAMRC), the National Research Foundation of South

Africa, and the Howard Hughes Medical Institute (Senior International

Research Scholars grant to V.M.). Work in our laboratory on TB

transmission is funded by the SA MRC with funds from NationalTreasury under the Economic Competitiveness and Support Package

(MRC-RFA-UFSP-01-2013/CCAMP).

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