Microbial Genomics and Infectious Diseases

Full text

Genomic Medicine
W. Gregory Feero, M.D., Ph.D., and Alan E. Guttmacher, M.D., Editors
Review article
n engl j med 365;4 nejm.org july 28, 2011
347
Microbial Genomics and Infectious Diseases
David A. Relman, M.D.
From the Departments of Medicine and
of Microbiology and Immunology, Stan-
ford University, Stanford; and the Veter-
ans Affairs (VA) Palo Alto Health Care
System, Palo Alto — both in California.
Address reprint requests to Dr. Relman at
VA Palo Alto Health Care System, 154T,
Bldg. 101, Rm. B4-185, 3801 Miranda
Ave., Palo Alto, CA 94304, or at relman@
stanford.edu.
N Engl J Med 2011;365:347-57.
Copyright © 2011 Massachusetts Medical Society.
T
he pace of technical advancement in microbial genomics has
been breathtaking. Since 1995, when the first complete genome sequence of a
free-living organism, Haemophilus influenzae, was published,
1
1554 complete
bacterial genome sequences (the majority of which are from pathogens) and 112 com-
plete archaeal genome sequences have been determined, and more than 4800 and
90, respectively, are in progress.
2
A total of 41 complete eukaryotic genome sequenc-
es have been determined (19 from fungi), and more than 1100 are in progress. Com-
plete reference genome sequences are available for 2675 viral species, and for some
of these species, a large number of strains have been completely sequenced. Nearly
40,000 strains of influenza virus
3
and more than 300,000 strains of human immuno-
deficiency virus (HIV) type 1 have been partially sequenced.
4
However, the selection
of microbes and viruses for genome sequencing is heavily biased toward the tiny mi-
nority that are amenable to cultivation in the laboratory, numerically dominant in
particular habitats of interest (e.g., the human body), and associated with disease.
In 2006, investigators reported in-depth metagenomic sequence data from a hu-
man mixed microbial community
5
; in 2007 more than 1000 genes from single cells
of cultivation-resistant bacteria were identified.
6
Since then, a flood of such data has
ensued (Fig. 1).
7-9
Individual investigators can now produce a draft sequence of a
bacterial genome containing 4 million base pairs in about a day.
10-12
The revolution
in DNA-sequencing technology has to a large extent democratized microbial geno-
mics and altered the way infectious diseases are studied.
11
However, gene annota-
tion and error correction still take time and effort. Today, the major challenges in
microbial genomics are to predict the function of gene products and the behavior of
organisms and communities from their sequences and to use genomic data to de-
velop improved tools for managing infectious diseases.
Genomic Div er sit y
The human body contains remarkable microbial taxonomic richness, with thousands
of symbiont species and strains per individual host. Of these, an estimated 90%
have not yet been cultivated in the laboratory.
13
Differences between closely related
strains and species are responsible for virulence, host-species adaptation, and other
aspects of lifestyle and account for the individualized nature of the human micro-
biota. For example, the gene content of pathogenic and nonpathogenic strains of
Escherichia coli, as well as different pathogenic types, varies by as much as 36%.
14,15
Comparisons of complete genome sequences from multiple strains of the same
bacterial species reveal a set of core genes that are common to all strains and a set
of dispensable genes that are absent in at least one strain.
16
The sum of these genes
(i.e., those represented in at least one strain) constitutes the species pangenome.
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As compared with the genomes of plants and
animals, genomes of microbes are small and usu-
ally contain one or two chromosomes, as well as
a variable number of plasmids (see Glossary). Yet,
approximately 90% of a typical microbial genome
encodes proteins or structural RNAs,
17
whereas
only about 1.1% of the human genome is coding
sequence.
18
As a result, some complex bacteria
have more genes than some simple eukaryotes.
Microbial diversification and adaptation have
been accompanied by gene loss and genome re-
duction, genome rearrangement, horizontal gene
transfer, and gene duplication.
19,20
The first two
of these processes are especially evident in human-
specific pathogens, such as Bordetella pertussis (the
causative agent of whooping cough),
21,22
Trophery-
ma whipplei (the agent of Whipple’s disease),
23
and
Yersinia pestis (the agent of bubonic plague). A total
of 3.7% of Y. pestis genes appear to be inactive,
especially those associated with enteropatho-
genicity.
24
The genome of Mycobacterium leprae, the
cause of leprosy, provides an even more dramatic
example of reductive evolution. Protein-coding
genes account for less than half of its genome,
whereas inactive and fragmented genes account
for most of the remainder.
25
Genomic islands are discrete clusters of con-
tiguous genes found in bacterial chromosomes
and plasmids, usually between 10,000 and 200,000
base pairs in length with features that suggest a
history and origin distinct from other segments
of the genome (see Glossary).
26,27
Some islands are
stably assimilated into the genome; others ap-
pear to have been acquired recently and may still
be mobile. Genomic islands enhance the fitness
of the recipient by providing new, accessory func-
tions, such as pathogenicity, drug resistance, or
catabolic functions.
One of the most dramatic examples of short-
term genome evolution can be seen in the CRISPR
(clustered regularly interspaced short palindrom-
ic repeat) loci of bacteria and archaea. CRISPRs
serve as a defense against invading phages and
plasmids, in a manner akin to adaptive immu-
nity.
28
These genomic loci contain segments of
phage and plasmid sequences captured from pre-
vious encounters. These segments are stored with-
in the CRISPR loci as spacer sequences and are
expressed as small RNAs, which then interfere
with replication of newly encountered phages and
plasmids that bear the same sequences.
Popu lation S truc tu re ,
Evolution, a nd Molecul ar
Epidemiolog y
Differences in the sequence and structure of ge-
nomes from members of a microbial population
reflect the composite effects of mutation, recom-
bination, and selection. With the increasing avail-
ability of genome sequences, these effects have
become better characterized and more effectively
exploited so as to understand the history and evo-
lution of microbes and viruses and their sometimes
No. of Projects
1000
100
500
10
5
1
50
0
1995 2000 2005 2010
Release Year
No. of Genomes
300
100
200
250
150
50
0
1995 1997 1999 2001 2003 2005
Year
2007 2011
(April)
2009
BCompleted Genomes
AGenome Projects
Microbes
Eukaryotes
Metagenomes
Figure 1. Genome Projects and Completed Genomes since 1995.
Panel A shows a cumulative plot of the number of genome projects —
involving microbial (bacterial and archaeal), eukaryotic, and viral genomes
— and metagenome projects, according to the release year at the National
Center for Biotechnology Information since 1995. Panel B shows the number
of completed microbial genome sequences according to year. (The most
recent data were collected on April 21, 2011.)
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349
intimate relationships with humans. The resulting
insights have practical importance for epidemio-
logic investigations, forensics, diagnostics, and vac-
cine development.
29
Y. pestis, the cause of the Black Death, arose
from a more genetically diverse ancestor that was
related to Y. pseudotuberculosis, through genome re-
duction and gene loss. By analyzing approximate-
ly 1200 single-nucleotide polymorphisms (SNPs)
and a worldwide collection of strains, the origins
of this monomorphic pathogen have been placed
between 2600 and 28,000 years ago in China, from
which it spread to other areas of the world, giving
rise to country-specific lineages.
30
All the Y. pestis
strains that are found in the United States today
are descendants of a single import that probably
arrived in San Francisco in 1899. As another ex-
ample, patterns of early human migration have
been traced by comparing genome sequences from
contemporary isolates of the chronic gastric patho-
gen, Helicobacter pylori.
31
Transmission of the patho-
gen is primarily from mother or other household
members to baby, and colonization is usually life-
long; thus, pathogen sequences are reasonable
markers of host ancestry and host migration. Se-
quence data for the H. pylori genome indicate the
sequential timing and directionality of two dis-
tinct waves of human migration into the Pacific
region.
32
Population mosaicism in H. pylori gene
sequences has been used to infer the history of
social interactions in human populations.
31
The power of full-genome sequencing to dis-
criminate between closely related strains and track
real-time evolution of disease-associated clonal
isolates offers the possibility of tracing person-to-
person transmission and identifying point sources
of outbreaks. Using this approach, investigators
established a previously unrecognized link among
five patients with the same clonal strain of methi-
cillin-resistant Staphylococcus aureus from a hospi-
tal in Thailand.
33
A study of Vibrio cholerae genome
sequences from the October 2010 cholera outbreak
Glossary
DNA microarray: A technology that is used to study many genes or other sequences at once. Thousands of sequences
are placed in known locations on a glass slide, silicon chip, or other surface. A sample containing DNA or RNA is
deposited on the slide, which is sometimes referred to as a gene chip. The binding of complementary base pairs
from the sample and the sequences on the chip can be measured with the use of fluorescence to detect the pres-
ence (and determine the amount) of specific sequences in the sample. In addition, when conserved sequences on
the chip are used to capture any member of a family of related sequences in the sample, the bound DNA can be
removed and its sequence determined.
Effector protein: A protein that is secreted by microbes directly into a host cell to alter physiological processes in the
host. Pathogens use these proteins to subvert host defenses and hence to enhance infection, promote their survival,
and produce disease.
Genome reduction: A decrease in the genome size during evolution of an organism, as measured by the total number
of nucleotides or by the number of genes. Genome reduction in pathogens and symbionts often results from the
deletion of genes that are no longer needed by or are disadvantageous to the microbe as it adapts to a host and
becomes restricted to fewer habitats.
Genomic islands: Regions of a genome with distinct nucleotide composition or with clusters of genes that encode spe-
cialized functions, such as virulence attributes. These discrete genomic regions are believed to be acquired from
other organisms by horizontal gene transfer and are flanked by direct repeats or phage attachment sites.
Horizontal (or lateral) gene transfer: The exchange of genetic material between contemporaneous, extant organisms,
as compared with vertical inheritance of genetic material from an ancestor. Mechanisms of horizontal gene transfer
include uptake of naked DNA (transformation), transfer mediated by plasmids or by pieces of DNA that promote
their own transposition to new sites in a genome (conjugation), and transfer mediated by viruses (transduction).
Metagenomic analysis: Genomic analysis that is performed directly on a mixture of heterogeneous organisms, ge-
nomes, or genes.
Plasmid: An extrachromosomal, self-replicating piece of DNA. Plasmids are usually circular and transferrable between
cells and sometimes carry genes that provide accessory functions, including drug resistance and virulence.
Pseudogene: A mutated form of a gene that is no longer functional, either because parts of the coding region are miss-
ing or altered or because it is no longer transcribed.
Ribosomal RNA: Noncoding ribonucleic acid that binds proteins to form the two subunits of the ribosome. All ribo-
somal RNAs (rRNAs) are named on the basis of their sedimentation rate, which is a reflection of their size and
shape. In bacteria, the small subunit rRNA is called the 16S rRNA.
Shotgun sequencing: An approach in which thousands or millions of short, random fragments of a DNA sample are
sequenced simultaneously and then reassembled with the use of computer algorithms on the basis of matching
overlapping ends. This technique can be applied to metagenomic analysis.
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in Haiti suggested that the Haitian strains were
clonal and more closely related to strains from
Bangladesh that were isolated in 2002 and 2008
than to strains isolated in Peru in 1991 and in
Mozambique in 2004. The authors concluded that
the Haitian outbreak may have originated with
the introduction of a V. cholerae strain from South
Asia as a result of human activity rather than
climatic events or other local environmental fac-
tors.
12
However, the source of this outbreak has
not been fully resolved; genome sequences of envi-
ronmental strains and additional clinical isolates
from Haiti may provide further insight.
A major challenge is the prediction of patterns
of evolution and emergence of disease agents. The
antigenic evolution of influenza virus is known to
follow a punctuated equilibrium model in which
periods of relative virus stability around the globe
are followed by periods of rapid change, requiring
modification of the influenza vaccine. However, it
was not clear whether variants arise first in East
and Southeast Asia and then seed other geograph-
ic regions or whether strains persist locally and
evolve simultaneously in a similar fashion. An
analysis of the gene encoding hemagglutinin
(the major antigenic determinant) from more than
1000 human influenza A (H3N2) isolates that
were collected worldwide from 2002 through 2007
produced strong support for external seeding,
rather than local persistence, and suggested that
the source of seeding is East and Southeast Asia.
34
On the basis of whole-genome sequence analysis,
the novel 2009 human H1N1 influenza strain was
thought to have entered the human population in
January of that year after arising from multiple
swine virus progenitors that had probably been
circulating in swine populations undetected for at
least a decade.
35
Work of this type will help target
efforts regarding influenza virus surveillance more
effectively, refine the selection of vaccine strains,
and improve predictions of future antigenic char-
acteristics.
36
Similar approaches will assist in an-
ticipating the emergence and spread of antibiotic
and antiviral resistance.
Pa thogenesis a nd Sy mbiosis
Pathogens have received most of the attention in
microbial genomics, despite their relative rarity in
the microbial world.
17,19
As a result, we now have
a more complete and deeper understanding of
how microbes cause disease and of pathogen emer-
gence, host adaptation, and spread in human popu-
lations. The study of microbial genomes reveals
four themes with respect to virulence.
First, horizontal gene transfer (see Glossary)
has had a major role in the acquisition of genes
associated with virulence. Most genes that encode
virulence factors are physically segregated in clus-
ters and located within mobile genetic elements.
In S. aureus, these genes often occur within phage-
related chromosomal islands and encode a variety
of superantigens, including the toxin associated
with the toxic shock syndrome and staphylococcal
enterotoxin B, and encode factors that mediate
antibiotic resistance, biofilm induction, and other
virulence-associated properties (Fig. 2).
37
Genomic
islands with similar features occur in other gram-
positive bacteria, including streptococcus, entero-
coccus, and lactococcus species. The emergence
of the recent Shiga toxin–producing E. coli clone
in Germany was probably the result of horizon-
tal gene transfer, when a toxin-producing phage
infected an enteroaggregative E. coli strain.
38
Second, symbionts and avirulent relatives of
pathogens often contain many of the same viru-
lence-associated genes as do the microbes that
typically cause disease.
39
The genes that we com-
monly associate with virulence may have been se-
lected for the advantages they confer in promoting
colonization of animal and plant hosts, in avoid-
ing or surviving phagocytosis, and in enhancing
competition against symbionts.
40-42
For example,
the original role for bacterial toxins may have been
to protect the bacterium against predation by pro-
tozoa and nematodes. The legionella protein IcmT
facilitates the escape of the bacterium from human
macrophages and also from the far more ancient
predator, the free-living amoeba.
43
Virulence de-
pends on the choreographed expression of partic-
ular combinations of genes at the right place and
time in the right host. Commensals and other sym-
bionts also serve as reservoirs of antibiotic resis-
tance genes and genetic diversity.
44
A third theme is the surprising diversity of
genes associated with mechanisms of virulence.
In a study of four closely related fungal species,
all of which cause late blight disease but in dif-
ferent host plant species, investigators identified
specific regions of the fungal genomes with evi-
dence of accelerated rates of evolution, suggesting
that these regions have been under strong posi-
tive selective pressure.
45
The genes in these re-
gions produce effector molecules (see Glossary)
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that interact with host plant proteins and elicit
host cell death. One of these fungal pathogens,
the agent responsible for the 19th-century Irish
potato famine, expresses 196 related effectors of
unexpected complexity and diversity.
46
A fourth theme is genome reduction and pseu-
dogene formation (see Glossary), especially in
pathogens with a relatively specialized lifestyle
and with restricted numbers and types of habi-
tats, niches, and hosts.
20
This is illustrated by an
unusual multidrug resistant strain of Salmonella
enterica serovar Typhimurium that emerged in sub-
Saharan Africa in the early 1990s to become the
most common cause of invasive bacterial disease
in some regions of that continent. Bacteremia and
meningitis are common features of this disease,
as they are for typhoid and paratyphoid fevers.
The genome sequence of this strain reveals a
large number of partially degraded and deleted
genes, many of which are also degraded or de-
leted in the genomes of salmonella serovars Typhi
and Paratyphi A.
47
S. aureus
Pathogenicity
Islands 12345678910111213141516kb
Integrase
Phage DNA
replication
genes
(enzyme that
mediates
integration of
phage DNA into
S. aureus
chromosome) Toxic shock
syndrome toxin
Enterotoxins
Phage packaging
genes
Cause food
poisoning
and shock
Int pri rep terS Unknownori pifstl str xis
Packaging genes
SaPI4
SaPI1028
SaPIbov1
SaPImw2
SaPIm4
ShPI2
SaPI3
SaPI5
SaPIm1/n1
SaPI2
SaPI122
SaPIbov2
SaPI1
2
Phimister
7/08/11
AUTHOR PLEASE NOTE:
Figure has been redrawn and type has been reset
Please check carefully
Author
Fig #
Title
ME
DE
Artist
Issue date
COLOR FIGURE
Draft 6
Relman
Knoper
07/14/11
Figure 2. Unexpected Diversity of Virulence Factors for Staphylococcus aureus, as Shown by Comparative Genome Analysis.
Shown are phage-related, virulence-associated genomic islands from different strains of S. aureus (listed to the left of the genomes),
with their genes colored according to functional categories. The islands are located at specific phage integration sites in the S. aureus
genome and are responsible for the production of toxins by these strains. The numbers at the top are distance markers along the
genomic islands. Data are adapted from Novick et al.
37
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The Hum an Microb iome
and Me tagenom ics
The role of the human indigenous microbiota in
human heath and disease has received a great
deal of attention in the past 5 years.
7,8,13,48
Sur-
veys of bacterial phylogenetic diversity that are
based on comparative analyses of ribosomal RNA
gene sequences recovered directly from clinical
specimens have confirmed habitat- and individual-
specific patterns in healthy persons.
49,50
Yet, core
features of the indigenous microbial communities
are conserved in healthy persons.
51
A metagenomic analysis (see Glossary) of fecal
samples from 124 healthy European subjects iden-
tified an average of 536,112 unique genes in each
of these samples, 99.1% of which were bacterial
and 0.8% of which were archaeal — and a total of
3.3 million unique genes overall, or 150 times the
number of genes in the human genome.
9
Approxi-
mately 38% of an individual’s fecal gene pool is
shared by at least half of all other individuals.
The shared gene products are predicted to mediate
degradation of complex sugars, such as pectin
and sorbitol, and of glycans harvested from the
host diet or intestinal lining, as well as fermen-
tation of mannose, fructose, cellulose, and sucrose
(to short-chain fatty acids) and vitamin biosynthe-
sis. These conserved genes constitute an accessory
human genome that facilitates dietary energy har-
vest and nutrition. Alterations in the human micro-
biome are associated with a number of diseases in
which no single organism seems to explain either
the presence or the absence of disease. For these
diseases (of which Crohn’s disease is a leading
example), the concept of community as pathogen
has been proposed.
52
Elucidation of the role played
by altered microbial communities in such condi-
tions and the associated mechanisms are likely to
emerge from the application of genomic approach-
es during the next decade.
Pa thogen Discov er y
and Diagnostics
Genomic approaches have introduced a new era
in the discovery and detection of microbial patho-
gens. The robustness, reliability, and portability of
molecular sequence-based data for phylogenetic
assessments and for characterization of previously
unrecognized pathogens, coupled with technology
developments, recommend genomic approaches
for both research and routine clinical applica-
tions
53-63
(
Table 1
and Fig. 3; interactive graphic,
available with the full text of this article at NEJM
.org). Broad-range molecular methods for microbi-
al discovery were introduced two decades ago.
54,64
Approaches for targeting differentially abundant
or phylogenetically informative molecules have
now been joined by less efficient but more pow-
erful methods for broad sequence surveys of clin-
ical and environmental samples with the use of
high-density DNA microarrays
55,65
and shotgun
sequencing
56,66
(see Glossary). The advantages of
DNA microarrays include the simultaneous de-
tection of diverse sequences with widely varying
relative abundance and recovery of captured se-
quences of interest directly from the microarray.
A panviral DNA microarray with oligonucleotides
designed from all known viral genera was used
to characterize the novel causative agent of the
severe acute respiratory syndrome (SARS)
55
and
has been used to detect viruses in nasopharyngeal
aspirates from children with a variety of acute re-
spiratory syndromes.
65
The disadvantages of DNA
microarrays include their insensitivity to rare mi-
crobial sequences in the presence of highly abun-
dant host sequences (i.e., those obtained from host
tissues) and their reliance on previous knowledge
of microbial sequence diversity for oligonucle-
otide design.
High-throughput shotgun sequencing offers
important new opportunities for the detection and
discovery of microbial pathogens. This approach
has revealed both previously known viruses (e.g.,
rotavirus, adenovirus, calicivirus, and astrovirus)
and unknown viruses (e.g., novel types of pico-
birnavirus, enterovirus, TT virus, and norovirus)
in fecal samples from children with unexplained
acute diarrhea
66
and a novel Old World arenavi-
rus that caused fatal disease in three recipients
of organs from a single donor.
56
Dramatic ad-
vances in sequencing technology highlight the
need to understand the diversity of microbial se-
quences in healthy subjects and to develop better
methods for distinguishing rare, genuine micro-
bial sequences from sequencing errors.
Sequence-based characterization of pathogens
enables the design and development of sensitive
and specific diagnostic assays and, in some cases,
methods for cultivation of the pathogen. Charac-
terization of the 16S ribosomal RNA gene from
the agent of Whipple’s disease, T. whipplei, led to a
molecular diagnostic assay for this disease agent.
67
An interactive
graphic showing
microbial genomics
and tool development
is available at
NEJM.org
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Subsequent determination of its complete genome
sequence
23,68
provided additional potential tar-
get sequences and the basis for a more sensitive
diagnostic test.
61
It also provided insight into the
metabolic defects of this bacterium, such that
cell-free growth medium could be designed to
include missing, needed growth factors.
69
Ther apeu tics a nd Drug
Dis cov ery
Genome sequences provide the blueprint for es-
sential microbial and viral components, the dis-
ruption of which can lead to growth inhibition
and death. These same sequences can sometimes
indicate resistance of the microbe or virus to a
particular drug. Although drug susceptibility
and resistance are often governed by multiple ge-
netic components, some drug-resistance traits are
encoded by single genes and can therefore be eas-
ily predicted by detecting or sequencing such genes.
Examples include rifampin resistance in M. tuber-
culosis, methicillin resistance in S. aureus, trimeth-
o prim–sulfamethoxazole resistance in T. whipplei,
70
and resistance to some antiretroviral drugs in HIV.
Genome sequences have also provided new tar-
gets and leads for the development of new anti-
microbials.
The standard of care for the management of
HIV infection now includes targeted drug selec-
tion with the use of a profile for HIV-drug sus-
ceptibility that is derived from the sequence of the
infecting HIV species.
59
Testing for genotypic resis-
tance is recommended for patients with HIV in-
fection when they enter care and when there is a
suboptimal reduction in viral load while they are
receiving first- or second-line antiretroviral regi-
mens. Clinically important resistance mutations
occur in HIV genes encoding the reverse-tran-
scriptase, protease, envelope, and integrase pro-
teins. Interpretation of these mutant genotypes
is facilitated by several databases, including those
maintained by the International Antiviral Society–
USA
71
and a research group at Stanford Univer-
sity.
72
Genotypic analysis is cheaper and faster than
phenotypic analysis for HIV-drug resistance and is
often more sensitive for detecting resistant strains
within mixtures of drug-susceptible viruses.
73
Table 1. Examples of the Use of Microbial Genomics to Enhance the Management of Infectious Diseases.*
Application and Disease or Pathogen Approach Reference
Epidemiology
Methicillin-resistant Staphylococcus aureus Whole-genome sequencing Harris et al.
33
Tuberculosis Whole-genome sequencing Gardy et al.
53
Cholera Whole-genome sequencing Chin et al.
12
Influenza Whole-genome sequencing Smith et al.
35
Pathogen discovery
Bartonella henselae Broad-range PCR Relman et al.
54
Severe acute respiratory syndrome coronavirus Viral microarray Wang et al.
55
Novel arenavirus Deep sequencing Palacios et al.
56
Diagnostic testing
Tuberculosis Rapid PCR Boehme et al.
57
HIV and HIV resistance RT-PCR Li et al.
58
and Panel on Antiretroviral
Guidelines for Adults and
Adolescents
59
H1N1 influenza A RT-PCR Wang et al.
60
Whipple’s disease PCR Fenollar et al.
61
Therapeutic use
Schistosoma mansoni High-throughput screening Sayed et al.
62
Preventive use
Meningococcus B vaccine Reverse vaccinology Giuliani et al.
63
* HIV denotes human immunodeficiency virus, PCR polymerase chain reaction, and RT reverse transcriptase.
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However, commercial assays of both types do not
routinely detect resistant viruses when they are less
than 10 to 20% of the overall circulating virus
population. With newer sequencing techniques,
less abundant strains are easier to detect and char-
acterize. Although the clinical relevance of rare
resistant variants is not fully understood, the
pretreatment detection of such variants has been
shown to have clinical value.
58
Traditional phe-
notypic testing (measuring the ability of the virus
to replicate in the presence of the antiviral drug)
is still recommended for patients in whom viruses
are suspected of having complex drug-resistance
mutation patterns.
Schistosomiasis is a chronic and debilitating
disease that affects approximately 210 million
people in 76 countries around the globe and re-
sults in some 280,000 deaths per year in sub-
Saharan Africa alone. Praziquantel has been the
drug of choice for the treatment of schistoso-
miasis but is in danger of losing efficacy because
of parasite resistance. Schistosoma mansoni is one of
three helminths for which there is now a draft
genome sequence available to the public.
74
Besides
enabling the study of gene and protein expres-
sion,
75
the nuclear genome of S. mansoni and its
approximately 11,800 putative genes point to
critical compounds and processes on which the
worm depends to survive in its host. These com-
pounds and processes reveal potential new drug
3
Phimister
6/29/11
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Title
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Genome
sequence Population Structure,
Evolutionary History
Insights into Growth
Requirements
Protein
Expression
Insights into
Virulence
Specific Sequences for Strain Detection
and Outbreak Investigation
Specific Sequences
for Strain Detection
Outbreak
Investigation
Diagnostics
Proteins and
Immunoresponse
Proteins and
Drug Design
Comparative analysis of genome sequences from multiple
strains of the same species allows for the creation of a tree
showing lineage and relationships among the strains
(e.g., strains of Yersinia pestis in the United States were
probably introduced in San Francisco in 1899).
The determination of Bacillus
anthracis genome sequences
enables the development of tests
specific to the Ames strains found
in the 2001 mailing of anthrax-
containing letters and greatly
aids in the federal investigation.
Drug-susceptibility profiles make it easier for
laboratories to detect and diagnose infection.
Identification of
virulence factors as
new targets for
therapeutics leads
to development of
new preventive
strategies to minimize
occurrence or effect of
outbreaks (e.g., emergence
of strains of Salmonella
enterica serovar Typhimurium
that has taken on features
of serogroup Typhi).
Purified protein can be used to determine its 3D structure, and from the structure drugs that might
inhibit the activity of the protein can be designed. Libraries of possible drug compounds can be screened
to see whether they inhibit or bind the protein directly (e.g., new drugs for treating schistosomiasis).
Vaccine development
3D structure Drug design
Hospitals are now using real-time
PCR assays to screen patients for colonization
by Staphylococcus aureus, including
methicillin-resistant strains.
Serologic tests
Real-time PCR assays
DNA microarrays
Figure 3. Microbial Genomics and Tool Development.
A genome sequence facilitates the development of a variety of tools and approaches for understanding, manipulating, and mitigating
the overall effect of a microbe. The sequence provides insight into the population structure and evolutionary history of a microbe for ep-
idemiologic investigation, information with which to develop new diagnostic tests and cultivation methods, new targets of drug develop-
ment, and antigens for vaccine development.
The New England Journal of Medicine
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GENOMIC MEDICI NE
n engl j med 365;4 nejm.org july 28, 2011
355
targets, one of which is a redox enzyme, thiore-
doxin–glutathione reductase.
74
Quantitative high-
throughput screening of small-molecule libraries
for compounds with activity against the S. man-
soni thioredoxin–glutathione reductase has already
identified some candidate drugs.
62
Microbes produce a wealth of druglike mole-
cules, the vast majority of which remain unchar-
acterized.
76,77
Because many of these molecules
are not expressed under typical laboratory con-
ditions, they often escape detection when labo-
ratory culture filtrates are screened for druglike
properties. Some of these molecules can now be
identified by recognizing the relevant genes in
the parent organism’s genome with the use of
computational tools and detecting the molecules
with mass spectroscopy techniques.
78,79
Deriva-
tive compounds can be designed and tested.
Vaccines
In the same way that genome sequences reveal
drug-resistance profiles, vulnerabilities, and syn-
thetic capabilities of microbes and viruses, these
sequences also provide clues about antigenic rep-
ertoire. This information can be exploited for vac-
cine design and other immunoprophylactic inter-
ventions. Genome-based antigen discovery has
also been undertaken for more complex pathogens.
One approach, known as reverse vaccinology, in-
volves cloning and expressing all proteins that are
predicted (from the organism’s complete genome)
to be secreted or surface-associated, starting with
the complete genome sequence (Fig. 3).
80
After im-
munizing mice with each of the proteins, each of
the corresponding antiserum samples is tested for
its ability to neutralize or kill the original target
organism. On the basis of this approach, a small
group of proteins from group B meningococcus,
81
a pathogen that has so far eluded vaccine develop-
ment, has shown promise as a candidate multiva-
lent subunit vaccine. A similar approach has been
taken with group B streptococcus
82
and extrain-
testinal pathogenic E. coli.
83
Protective antigens
that are discovered through these sorts of meth-
ods may have been previously ignored because they
are not immunogenic during natural infections.
Fut ur e Dire ct ions
Without question, the techniques for microbial
and viral genome sequencing are becoming in-
creasingly rapid and less expensive. Genome se-
quencing of a microbe or virus will soon be easier
than characterization of its growth-based behav-
ior in the laboratory. In the next 3 to 5 years, di-
rect shotgun sequencing of the DNA and RNA in
a clinical sample may become a routine matter.
What is less clear is how clinically relevant infor-
mation will be most effectively extracted from
the ensuing massive amounts of data. In the near
term, genomic and metagenomic analyses of mi-
crobes are most likely to be useful in areas such
as the cataloguing and understanding of micro-
bial and viral diversity in the human body, the
identification of molecular determinants of viru-
lence and symbiosis, and real-time tracking of
particular strains of pathogens. Such analyses
will also provide a deeper understanding of how
pathogens spread and cause disease and will
identify new targets for therapies and antigens
for vaccines. Thoughtfully designed clinical and
epidemiologic studies will be required to see the
full realization of these benefits.
Disclosure forms provided by the author are available with the
full text of this article at NEJM.org.
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