RESEARCH ARTI CLE Open Access

Host response transcriptional profiling reveals

extracellular components and ABC (ATP-binding
cassette) transporters gene enrichment in
typhoid fever-infected Nigerian children
Sok Kean Khoo
1
, David Petillo
2
, Mrutyunjaya Parida
1
, Aik Choon Tan
3
, James H Resau
4
and Stephen K Obaro
5*
Abstract
Background: Salmonella enterica serovar Typhi (S. Typhi) is a human-specific pathogen that causes typhoid fever,
and remains a global health problem especially in developing countries. Its pathogenesis is complex and host
response is poorly understood. In Africa, typhoid fever can be a major cause of morbidity in young infected
children. The onset of the illness is insidious and clinical diagnosis is often unreliable. Gold standard blood culture
diagnostic services are limited, thus rapid, sensitive, and affordable diagnostic test is essential in poor-resourced
clinical settings. Routine typhoid fever vaccination is highly recommended but currently licensed vaccines provide
only 55-75% protection. Recent epidemiological studies also show the rapid emergence of multi-drug resistant S.
Typhi strains. High-throughput molecular technologies, such as microarrays, can dissect the molecular mechanisms
of host responses which are S. Typhi-specific to provide a comprehensive genomic component of immunological
responses and suggest new insights for diagnosis and treatment.
Methods: Global transcriptional profiles of S. Typhi-infected young Nigerian children were obtained from their
peripheral blood and compared with that of other bacteremic infections using Agilent gene expression
microarrays. The host-response profiles of the same patients in acute vs. convalescent phases were also
determined. The top 96-100 differentially-expressed genes were identified and four genes were validated by
quantitative real-time PCR. Gene clusters were obtained and functional pathways were predicted by DAVID
(Database for Annotation, Visualization and Integrated Discovery).
Results: Transcriptional profiles from S. Typhi-infected children could be distinguished from those of other
bacteremic infections. Enriched gene clusters included genes associated with extracellular peptides/components
such as lipocalin (LCN2) and systemic immune response which is atypical in bacterial invasion. Distinct gene
expression profiles can also be obtained from acute vs. convalescent phase during typhoid fever infection. We
found novel down-regulation of ABC (ATP-binding cassette) transporters genes such as ABCA7, ABCC5, and ABCD4
and ATPase activity as the highest enriched pathway.
Conclusions: We identified unique extracellular components and ABC transporters gene enrichments in typhoid
fever-infected Nigerian children, which have never been reported. These enriched gene clusters may represent
novel targeted pathways to improve diagnostic, prognostic, therapeutic and next-generation vaccine strategies for
typhoid fever in Africa.
* Correspondence: [email protected]
5
Department of Pediatrics and Human Development, College of Human
Medicine, Michigan State University, East Lansing, MI, USA
Full list of author information is available at the end of the article
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2011 Khoo et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons
Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in
any medium, provided the original work is properly cited.
Background
Salmonella enterica serovar Typhi (S. Typhi) is a Gram-
negative bacterium that causes typhoid fever. The
World Health Organization (WHO) recognizes S. Typhi
infection as a global health problem, with an estimated
21 million cases and between 200,000 and 600,000
deaths annually [1,2]. In Africa, typhoid fever affects
mainly school-age children and younger adults [3]. In
fact, in endemic and large outbreak areas, young chil-
dren represent a subgroup with the highest burden of
infection as well as high rates of morbidity and compli-
cations [4]. The clinical diagnosis is often unreliable and
confused with other febrile illness. Definitive diagnosis
through blood or bone-marrow culture is laborious,
expensive, and/or invasive, with sensitivity of only 40 to
70% [2,5]. Moreover, it takes several days for isolation
and identification that can cause a delay to initiate
proper antibiotic treatment. On the other hand, serolo-
gic diagnostic tests such as Widal tests lack sensitivity
and specificity [6,7]. Lack of precision in diagnosis often
implies non-targeted use of broad spectrum antibiotics
which leads to the rise of multi-drug resistant strains
[8]. A primary preventive strategy for disease prevention
is vaccination. However, protective efficacies of both
internationally licensed vaccines, Vi and Ty21, are mod-
est (< 75%) [9,10]. As long as the understanding of
pathogenesis of typhoid fever remains incomplete,
improvement on diagnosis, treatment and vaccine stra-
tegies will be subsequently delayed.
One of the approaches to elucidate the pathogenesis
of typhoid fever is to better understand the mechanisms
of virulence and susceptibility of hosts. Host responses
to bacterial infections are determined by specific struc-
tural and antigenic components of the bacteria and the
profile of immunological response should be unique to
different classes of bacteria or bacteria species. Global
transcriptional profiling of the peripheral blood using
microarrays has been proven feasible to identify distinct
host responses in various diseases [11-16]. In the field of
infectious diseases, a hosts immune response to a cer-
tain pathogen can be clearly defined and its unique
molecular signature identified using high-throughput
microarray technology, eliminating the limitation of
conventional diagnostic or microbiological techniques.
This is the first host response transcriptional profiling
study on young children with typhoid fever in Africa.
Our preliminary observations showed distinct transcrip-
tional profiles between S. Typhi vs. other bacteremic-
infected children, as well as acute vs. convalescent phase.
We identified novel sets of genes associated with extra-
cellular components which are specific to S. Typhi infec-
tion, as well as the ABC transporters gene cluster which
can discriminate acute from convalescence phases.
Methods
Subject recruitment
This is a sub study for our CABSYNC (Community-
Acquired Bacteremic Syndrome in Young Nigerian Chil-
dren) surveillance program for children aged 2 months
to 14 years in central Nigeria. Children with fever (tem-
perature 38.5C) and any of the following: respiratory
distress, convulsion or diarrhea, were eligible for enroll-
ment. Since the use of antibiotics could modify the tran-
scriptional profile, only treatment-nave patients were
recruited. Blood was drawn using aseptic technique for
culture into an aerobic Pediatric plus Bactec culture bot-
tle (Becton Dickinson (BD)) which contains antibiotic
resins. The bottles were incubated for no longer than 5
days in the Bactec 9050 instrumented blood culture sys-
tems (BD). Positive specimens were Gram stained and
sub-cultured on appropriate agar plates. After primary
isolation and characterization, secondary confirmation
was performed at the Medical Research Council Labora-
tories, Gambia, or Sparrow Regional Laboratories, East
Lansing, Michigan, USA. Salmonella isolates were
further characterized at the Sanger Institute, Oxford,
United Kingdom. Antibiotic susceptibility testing was
performed using standard methods. Antimicrobial activ-
ity in serum was detected using the Micrococcus luteus
assay. Blood from acute (before initiating antibiotics)
and convalescent (4 weeks after acute presentation)
phases of the same patients were sampled. Parental con-
sents were obtained from all children in this study. This
study was approved by the Institutional Review Board of
Michigan State University and the National Hospital,
and the Ethics committee of the Federal Capital Terri-
tory Abuja, Nigeria.
RNA extraction and purification
Two to five milliliters of peripheral blood were drawn
from each subject into a PAXgene blood RNA tube
(BD). PAXgene tubes contain a proprietary reagent that
immediately stabilizes intracellular RNA for 3 days at
18-25C and 5 days at 2-8C, and thereafter at -70C for
long-term storage. The PAXgene blood RNA system is a
product approved by the US FDA for collection, storage,
and transport of blood for molecular diagnostic testing.
Extraction and purification protocols were performed
according to PAXgene blood RNA kit (Qiagen). The
quality and quantity of RNA was measured on a RNA
nano microfluidic chip using the BioAnalyzer (Agilent
Technologies).
Microarray
Whole human genome 4 44K gene expression 2-color
microarrays from Agilent were used in this study. In
brief, 200 ng of total RNA was amplified, fluorescently
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labeled, and hybridized onto the arrays according to the
manufacturers protocols. RNA from acute phase was
labeled with Cy3 (green dye) and convalescence phase
with Cy5 (red dye). After hybridization, the arrays were
washed and scanned with an Agilent scanner. Probe fea-
tures were extracted from the microarray scan data
using Feature Extraction software v.9.5.3.1 (Agilent).
Microarray data were deposited in Gene Expression
Omnibus (GEO) with the accession number #GSE28658.
Gene expression analysis
The gene expression of each sample was loaded into the
R environment and the Limma package was applied for
analysis. Data were background corrected in order to
obtain accurate intensity values while red and green
channels were separated and quantile normalization was
applied to remove systemic/technical variations before
statistical analysis. A linear model was applied before
eBayes test and top up- or down-regulated genes were
generated. The statistic used for significance analysis
was the moderated t-test. This has the same interpreta-
tion as a standard t-test except that the standard errors
were moderated across genes, i.e., shrunk towards a
common value, using a simple Bayesian model. As the
number of samples is small, we chose to use the moder-
ated t-test in this pilot study as an exploratory approach
to identify differentially- expressed genes.
Pathway analysis
The Database for Annotation, Visualization and Integrated
Discovery (DAVID) Bioinformatics Resources v.6.7 from
the National Institute of Allergy and Infectious Diseases
(NIAID/NIH) http://david.abcc.ncifcrf.gov/, a gene-cen-
tered database integrating heterogeneous gene annotation
resources, was applied to facilitate high-throughput gene
functional/pathway analysis [17,18]. The group enrich-
ment score, the geometric mean (in -log scale) of mem-
bers P-values in a corresponding annotation cluster, is
used to rank their functional significance.
Quantitative Real time PCR (Q-PCR)
To validate the microarray results, four genes with dif-
ferential expression (MYPOP, PSAP, IL11RA, and
MUC20) were evaluated using Q-PCR (Table 1). All
RNA samples were diluted to the same concentration
(500 ng/l), and the same quantity, 5 l, (2.5 g total
RNA) was used for reverse transcription to cDNA,
according to standard ABI protocols, using the TaqMan
PCR (Applied Biosystems (ABI)). cDNA samples were
then quantitated with a NanoDrop spectrophotometer
(Thermo-Scientific), using the same total cDNA quanti-
ties for each sample for Q-PCR assay on an ABI Ste-
pOne Plus instrument (ABI). Single-plex PCR reactions
were carried out in triplicate using TaqMan Universal
PCR Master Mix with no UNG (ABI). Q-PCR data were
initially collected and a preliminary analysis was per-
formed using the StepOne Software version 2.1. Con-
trols included human beta-actin (endogenous gene
reference), an ABI Control Total RNA (human positive
control) and nuclease-free water (non-template negative
control). Further Q-PCR analyses were performed with
the comparative 2-C
T
quantitation method [19].
Results and discussion
Clinical data of patients and feasibility of recruitment
During this pilot study, 12 patients were recruited. Six
patients were excluded for the microarray study due to
unavailability of convalescent phase sample, no bacteria
growth from blood cultures, or insufficient or low qual-
ity RNA. Three patients with S. Typhi infection and one
each of Acinetobacter, Klebsiella, and non-typhoidal sal-
monella infection of acute and convalescent phases were
profiled using gene expression microarrays. The clinical
characteristics of the patients are presented in Table 2.
Previously, we showed that typhoid fever is the most
prominent infectious disease among Nigerian children
from our CABSYNC (Community- Acquired Bacteremic
Syndrome in Young Nigerian Children) surveillance data
between September 2008 and November 2009, screening
1,287 children aged 2 months to 5 years [20]. Therefore,
it is critical to elucidate the molecular mechanisms of
host response in African children with typhoid fever, an
under-represented yet extremely important research
cohort. Transcriptional microarray studies require intra-
cellular RNA which can be a challenge since RNA can
be unstable and rapidly degrade within hours after
blood collection. In this pilot study, we showed that it is
feasible to collect whole blood samples from African
children using PAXgene Blood RNA tubes which con-
tain an additive that stabilizes the in vivo gene transcrip-
tion profile by reducing in vitro RNA degradation and
minimizing gene induction. Moreover, PAXgene tubes
can be safely stored or transported at 15-25C for up to
3 days or at 2-8C for up to 5 days, thus suitable for
blood collection in freezer-lacking rural areas in Africa.
Transcriptional profiles of S. typhi patients
To determine whether global transcriptional profiles of
S. Typhi patients were distinct from those of other
Table 1 Specific TaqMan gene expression assays for Q-
PCR and amplicon lengths
Gene TaqMan assay ID Amplicon length (bp)
MYPOP Hs02384801_m1 72
PSAP Hs01551096_m1 74
IL11RA Hs00234415_m1 120
MUC20 Hs00416321_m1 83
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bacteremic infections, namely Acinetobacter, Klebsiella,
and non-typhoidal salmonella, the average signals after
normalization and background correction of each group
during acute phase were compared using the moderate
t-test statistic. Using this exploratory statistical
approach, we focused on up to 100 top up- and down-
regulated genes with P < 0.05 and fold change > 2.5. A
heatmap of top up- and down-regulated genes was gen-
erated, revealing distinctive transcriptional profiles of S.
Typhi patients in comparison with other bacteremic
infections (Figure 1). To determine the host response
transcriptional profiles at acute vs. convalescence phase,
gene expressions during these phases were compared in
the same typhoid fever patients. Our results showed that
the patterns of transcriptional profiles at acute phase
were distinctive from the convalescence phase in S.
Typhi-infected children (Figure 2).
S. Typhi infection-specific genes and pathways
The top 96 differentially-expressed genes (P < 0.05, fold
change > 2.5) between S. Typhi-infected children and
other bacteremia (Figure 1)(Additional file 1) included
genes that are involved in ligand binding and alteration
of cell composition such as lipocalin (LCN2) and prosa-
posin (PSAP). LCN2 was down-regulated, which is an
atypical response for bacterial invasion. In addition,
genes associated with systemic inflammatory response
such as interleukin 4 (IL4) and tumor necrosis factor
(ligand) superfamily, member 9 (TNFSF9) were found
up-regulated, which is another example of an atypical
bacterial invasion response. We used DAVID to assign
annotation groups based on gene ontologies and, as
expected, top ranked clusters included genes related to
extracellular proteins and components as well as sys-
temic defense and inflammatory response pathways
(Additional file 2).
In the human host, detection of pathogens is achieved
through pattern-recognition receptors (PPRs) such as
the membrane-localized Toll-like receptors (TLRs) [21]
and the cytosolic nucleotide-binding and oligomeriza-
tion domain-like receptors [22] of the host innate
immune-surveillance system. PRRs function as bar-code
readers that recognize conserved molecular structures
in microbes known as microbe-associated molecular
patterns [23]. With a focus on the extracellular protein
and component gene clusters (highest enrichment score
in our study), we found down-regulation (-3.6 fold) of
LCN2 (lipocalin-2 or neutrophil gelatinase-associated
lipocalin) in typhoid fever children intriguing. Lipocalins
are extracellular proteins that display different molecu-
lar recognition properties such as ligand and receptor
binding. Hence, they are known to bind with small
hydrophobic molecules, cell surface receptors, as well as
form complexes with other macromolecules. The invar-
iant end of the lipocalin fold may implicate the general
binding to common cell surface receptors, while the
more variable end is adapted to specialized ligand bind-
ing and forming of macromolecular complexes [24].
During bacterial invasion, TLRs in immune cells stimu-
lates transcription, translation, and secretion of lipocalin
2 protein (Lcn2) to sequester the siderophore entero-
bactin (Ent) and inhibit intracellular microbial growth
by blocking iron intake [25]. Lcn2 may also represent a
novel mechanism of sensing microbial metabolism to
modulate the host response [26]. Lcn2 can be up-regu-
lated by pro-inflammatory cytokines such as IL-17, IL-
22, and IL-1a to provide a robust innate response in a
typical microbial infection [27-30]. However, we found
LCN2 down-regulated in typhoid fever children, an
observation of an atypical antibacterial response. One
explanation is that having the iroA gene cluster in some
bacteria such as Salmonella spp allows bacteria to evade
this effector mechanism of the innate immune system,
rejuvenating their Ent-mediated iron-acquisition path-
way and play a role in their virulence [31]. One can
also debate the potential of the hydrophobic adhesion
protein of S Typhi such as T2544 [32] which binds
directly to Lcn2 or TLRs which bind to Lcn2 and
causes transcriptional disruption and down-regulation
of LCN2. If so, such hydrophobic adhesion proteins can
be a potential immune-modulatory target for typhoid
fever. In general, our findings on differentially-expressed
genes in typhoid fever children can lead us to explore
the genes related with extracellular proteins and/or aty-
pical bacterial inflammatory response for future devel-
opment of typhoid fever-specific diagnostic tools/
biomarker panels and anti-typhoid fever strategies and
therapies.
Table 2 Clinical data of Nigerian children with S. Typhi and other bacteremic infections
Sample Age Sex Bacteria Type Clinical Symptoms
01 6 yrs 4 mo F S. Typhi fever 14 days, diarrhea 4 days
03 3 yrs 4 mo F Acinetobacter spp fever 14 days, cough with difficult breathing 1 day
05 9 mo F non-typhodal Salmonella fever 5 days
08 6 yrs 4 mo M Klebsiella spp fever, vomiting, diarrhea 5 days, cough 7 days
09 5 yrs 1 mo M S. Typhi fever 30 days, diarrhea 14 days
11 12 yrs 1 mo F S. Typhi fever 30 days, diarrhea 14 days
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It has been suggested that host responses are often
typical for groups of pathogens instead of being specific
to individual pathogens, as some pathogens share basic
clinical characteristics such as fever and diarrhea [33].
However, S. Typhi is known to be an atypical bacterium
which does not elicit a typical antibacterial host
response characterized by exudative intestinal inflamma-
tion, but a nonspecific response of interstitial inflamma-
tion resembling viral or parasitic infections [33]. Here,
we showed up-regulation of IL4 and TNFSF9 in S.
Typhi patients. An increased IL4 level is known to be
associated with immune system response in chronic
Figure 1 Distinct transcriptional profiles of S. Typhi patients. A heatmap of differentially-expressed genes showing distinct transcriptional
profiles of S. Typhi patients compared with patients with Acinetobacter, non-typhoidal salmonella or Klebsiella infection. Red indicates over-
expressed transcripts and green represents under-expressed transcripts.
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viral hepatitis [34] while TNFSF9 is reported over-
expressed in primary biliary cirrhosis, an autoimmune
disease of medium and small bile ducts [35]. Both are
cytokines associated with T-cells which have protective
capability for systemic inflammation. We also showed
down-regulation of HRP (haptoglobin-related protein)
and HP (haptoglobin), indicating a systemic response.
HPR is commonly regulated as the innate host immunity
against Trypanosoma, a protozoan parasite [36,37]. Our
results reinforced the opinion that S. Typhi does not eli-
cit classic antibacterial host responses.
Expressed genes and pathways during acute and
convalescence phases
The distinct transcriptional profile of the acute phase
consisted of genes related with hemoglobin
Figure 2 Distinct transcription profiles of acute vs. convalescence phase. A heatmap of differentially-expressed genes showing distinct
profiles between acute (A) and convalescence (C) phase of S. Typhi infection. Red indicates over-expressed transcripts and green represents
under-expressed transcripts.
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(hemogloblin-beta, HBB; hemoglobin subunit alpha 1
and 2, HBA 1 and HBA2) and inflammatory (interleukin
11 receptor, alpha, IL11RA; mucin 20, cell surface asso-
ciated, MUC20) that were down-regulated but up-regu-
lated in the convalescence phase (Figure 2)(Additional
file 3). Interestingly, down-regulation of ABC transpor-
ter genes (ATP-binding cassette, subfamily A, member
7, ABCA7; ATP-binding cassette, subfamily C, member
5, ABCC5; ATP-binding cassette, subfamily D, member
4, ABCD4) and enrichment of this pathway in the acute
phase of typhoid fever patients were detected and the
ATPase activity molecular function was clustered as a
top-scoring biological function (Additional file 4).
Genes associated with the distinct transcript profiles
at different phases of illness have been identified in a
cohort of Vietnamese typhoid fever patients (age
unknown) [38]. Here, we presented transcriptional pro-
files and differentially-expressed genes of acute and con-
valescent phases in a small, unique cohort (young
children with African descent) which represents the
most prominent typhoid fever-infected yet under-repre-
sented group in Nigeria. Genes related to hemoglobin
such as HBB, HBA 1 and HBA2 were down-regulated,
reflecting the anemic characteristic of typhoid fever
patients and are consistent with those reported in the
Vietnamese cohort [38]. However, we found down-regu-
lation of inflammatory-related genes such as IL11RA
and MUC20 at the initial acute phase but up-regulated
4 weeks later in the convalescent phase. These results
are different from the Vietnamese cohort where most
immune response genes were over-expressed in acute
phase and down-regulated later in convalescent phase.
While we are uncertain whether the Vietnamese cohort
was sampled before or after initiation of antibiotics
(besides unknown age), our study involved young chil-
dren (average age = 5 years 7 months) that were
sampled before antibiotics prescription in their acute
phase and 2 patients (09 and 11) have prolonged fever
of 30 days. Thus, we can surmise that the innate
response of our patients may not have been fully effec-
tive during the initial acute phase and took a longer
time to recover (hence still maintaining up-regulation of
immunological-related genes after 4 weeks) due to their
young age and not fully developed/weaker immunity. In
addition, it has been reported that a switch from pro-
inflammatory to an anti-inflammatory mode occurred
and inhibited production of cytokines in the acute
phase of typhoid fever patients [39]. It can also be
explained by the stealth tactics of S. Typhi, evading
recognition of TLR4 and TLR5, impairing identification
of its invasion to prevent a typical antibacterial host
response, which results in suppression of neutrophil
recruitment [40] and weak induction of acute phase
responses [41].
From our unique cohort, we also identified down-reg-
ulation of ABC transporter genes (ATP-binding cassette,
subfamily A, member 7, ABCA7; ATP-binding cassette,
subfamily C, member 5, ABCC5; ATP-binding cassette,
subfamily D, member 4, ABCD4) and enrichment of this
pathway in the acute phase of typhoid fever, which have
never been previously reported. ABC proteins transport
a wide variety of substrates ranging from small ions to
macromolecules across the cell membrane. Its export
systems in all living organisms are involved in the extru-
sion of harmful substances and export of extracellular
toxins, which can be S. Typhi bacterium in our case, but
needs further validation. The same explanation can be
given that the ABC transporter genes were down-regu-
lated in the initial acute phase but up-regulated 4 weeks
later due to a slower and less efficient host response
caused by young age and weaker immunity.
Human ATP-binding cassette transporter superfamily
is also known to be involved in drug response pheno-
types. Antimicrobial drug resistance of S. Typhi is well
known [8] and our findings may shed light to the appro-
priate antibiotic treatment to prevent the development
of resistant strains of S. Typhi which is an emerging
problem. While the definite functions of ABCA7 and
ABCD4 are unknown, ABCC5 is a member of the MRP
(multi-resistance protein) subfamily and may contribute
to resistance of certain anti-cancer and anti-HIV drugs
[42-44]. Thus, whether ABCC5 plays a direct or indirect
role in multi-drug resistance to typhoid fever antibiotics
warrants further investigation.
Transcriptional profiles from the recovery phase were
not available as these samples were not acquired during
this pilot study. One would predict that most recovery
profiles would fail to show distinctive signatures of
acute and convalescent stages. However, patients who
retain the convalescent signature in the recovery phase
may be an indication of carrier state, relapse or suscepti-
ble to reinfection [38]. We are currently planning to
include the recovery phase, beside acute and convales-
cent phases, in a follow-up study with a larger cohort (>
100 African children).
Validation of microarray gene expression with Q-PCR
We confirmed the microarray data by Q-PCR that tar-
geted four differentially-expressed genes, MYPOP, PSAP,
IL11RA, and MUC20. These genes were selected based
on their functions related to extracellular components
or host responses. In our microarray data, MYPOP was
up-regulated in S. typhi-infected patients but down-
regulated in other bacteremic infections. Conversely,
PSAP was down-regulated in S. typhi-infected patients
but up-regulated in other bacteremic infections. Both
IL11RA and MUC20 were down-regulated in acute but
up-regulated in convalescent phase. In addition to the
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original samples used for microarray analysis, three new
samples (two typhoid fever and one enterobacter) were
obtained for Q-PCR validation. We demonstrated that
the Q-PCR results of MYPOP, PSAP, IL11RA, and
MUC20 were consistently correlated with the results
observed in the initial microarray data (Table 3).
Conclusions
It is well known that current diagnostic tests for typhoid
fever are inadequate in many ways and development of
reliable and affordable diagnostic assays is critical to fill
this gap for better disease control and treatment [45]. In
this pilot study, we explored the global transcriptomes
of Nigerian children with typhoid fever. This led to the
characterization of distinct transcriptional profiles for
children specifically with typhoid fever and during acute
vs. convalescent phases. We showed that evaluating
nave patients (before any antibiotic treatment) may
obtain more accurate molecular signatures for diagnostic
and progression of typhoid fever. This unique cohort
(treatment-nave, average age of 5 years 7 months) is
also more suitable for strategizing novel vaccines as
young children are the preferred age for immunization
to induce effective and long-lasting immunity. We con-
firmed that S. Typhi elicits atypical antibacterial host
response. We also identified novel extracellular compo-
nent and ABC transporters as key gene clusters in these
young patients which may lead to a more complete
understanding of typhoid fever. These enriched gene
clusters may represent novel targeted pathways to
improve diagnostic, prognostic, therapeutic and next-
generation vaccine strategies of typhoid fever in Africa.
Additional material
Additional file 1: Most differentially expressed genes for typhoid
fever vs. other bacteremic infections. Log fold change and P value of
top 96 S. Typhi infection-specific genes.
Additional file 2: Top pathway clusters for typhoid fever vs. other
bacteremic infections. Enrichment score and P value of top 10 S. Typhi
infection-specific functional annotation clusters, including extracellular
proteins/components and systemic defense/inflammatory response
pathways.
Additional file 3: Most differentially expressed genes during acute
vs. convalescent phase of typhoid fever. Log fold change and P value
of top 100 up- and down- regulated genes during acute vs. convalescent
phase of typhoid fever.
Additional file 4: Top pathway clusters for acute vs. convalescent
phase of typhoid fever. Enrichment score and P value of top 10
functional annotation clusters during acute vs. convalescent phase of
typhoid fever, with ATPase activity as the highest-ranking molecular
function.
Acknowledgements and Funding
We thanked all children who participated and their parents who gave their
consent for this study. This study is partially funded by the Van Andel
Foundation.
Author details
1
Laboratory of Microarray Technology, Van Andel Research Institute, Grand
Rapids, MI, USA.
2
Laboratory of Cancer Genetics, Van Andel Research
Institute, Grand Rapids, MI, USA.
3
Department of Medicine, University of
Colorado Denver, Aurora, CO, USA.
4
Laboratory of Analytical, Cellular, and
Molecular Microscopy, Van Andel Research Institute, Grand Rapids, MI, USA.
5
Department of Pediatrics and Human Development, College of Human
Medicine, Michigan State University, East Lansing, MI, USA.
Authors contributions
SKK contributed to the microarray experiment study design, performed the
RNA extraction and microarray procedures, and wrote the manuscript. DP
carried out the real-time PCR assays and analyses. MP and ACT provided
expertise in gene expression microarray analysis. JHR participated in its
design and supervised the study. SKO conceived the study, participated in
its design and coordination, and contributed to writing the manuscript. All
authors read and approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 4 May 2011 Accepted: 13 September 2011
Published: 13 September 2011
References
1. Bhutta ZA, Threlfall J: Addressing the global disease burden of typhoid
fever. JAMA 2009, 302(8):898-899.
2. WHO: Background document: the diagnosis, prevention and treatment
of typhoid fever. WHO 2003, 19-23.
3. Feasey NA, Archer BN, Heyderman RS, Sooka A, Dennis B, Gordon MA,
Keddy KH: Emerg Infect Dis 16(9):1448-1451.
4. Bhutta ZA: Impact of age and drug resistance on mortality in typhoid
fever. Arch Dis Child 1996, 75(3):214-217.
5. Parry CM, Hien TT, Dougan G, White NJ, Farrar JJ: Typhoid fever. N Engl J
Med 2002, 347(22):1770-1782.
6. Dutta S, Sur D, Manna B, Sen B, Deb AK, Deen JL, Wain J, Von Seidlein L,
Ochiai L, Clemens JD, et al: Evaluation of new-generation serologic tests
for the diagnosis of typhoid fever: data from a community-based
surveillance in Calcutta, India. Diagn Microbiol Infect Dis 2006,
56(4):359-365.
7. Mweu E, English M: Typhoid fever in children in Africa. Trop Med Int
Health 2008, 13(4):532-540.
8. Cooke FJ, Wain J: The emergence of antibiotic resistance in typhoid
fever. Travel Med Infect Dis 2004, 2(2):67-74.
9. Klugman KP, Koornhof HJ, Robbins JB, Le Cam NN: Immunogenicity,
efficacy and serological correlate of protection of Salmonella typhi Vi
capsular polysaccharide vaccine three years after immunization. Vaccine
1996, 14(5):435-438.
10. Whitaker JA, Franco-Paredes C, del Rio C, Edupuganti S: Rethinking typhoid
fever vaccines: implications for travelers and people living in highly
endemic areas. J Travel Med 2009, 16(1):46-52.
11. Loke P, Hammond SN, Leung JM, Kim CC, Batra S, Rocha C, Balmaseda A,
Harris E: Gene expression patterns of dengue virus-infected children
from nicaragua reveal a distinct signature of increased metabolism. PLoS
Negl Trop Dis 4(6):e710.
Table 3 Validation of gene expression using Q-PCR
Average fold change using 2-C
T
method
MYPOP PSAP IL11RA MUC20
S. Typhi infection 2.9 1.6 - -
Other bacteremic infection 1.7 2.7 - -
S. Typhi acute phase - - 1.8 1.9
S. Typhi convalescent phase - - 3.2 4.1
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12. Ardura MI, Banchereau R, Mejias A, Di Pucchio T, Glaser C, Allantaz F,
Pascual V, Banchereau J, Chaussabel D, Ramilo O: Enhanced monocyte
response and decreased central memory T cells in children with
invasive Staphylococcus aureus infections. PLoS One 2009, 4(5):e5446.
13. Andersson H, Hartmanova B, Back E, Eliasson H, Landfors M, Naslund L,
Ryden P, Sjostedt A: Transcriptional profiling of the peripheral blood
response during tularemia. Genes Immun 2006, 7(6):503-513.
14. Kim HS, Choi EH, Khan J, Roilides E, Francesconi A, Kasai M, Sein T,
Schaufele RL, Sakurai K, Son CG, et al: Expression of genes encoding
innate host defense molecules in normal human monocytes in response
to Candida albicans. Infect Immun 2005, 73(6):3714-3724.
15. Griffiths MJ, Shafi MJ, Popper SJ, Hemingway CA, Kortok MM, Wathen A,
Rockett KA, Mott R, Levin M, Newton CR, et al: Genomewide analysis of
the host response to malaria in Kenyan children. J Infect Dis 2005,
191(10):1599-1611.
16. Wittwer M, Grandgirard D, Rohrbach J, Leib SL: Tracking the transcriptional
host response from the acute to the regenerative phase of experimental
pneumococcal meningitis. BMC Infect Dis 10:176.
17. Huang da W, Sherman BT, Lempicki RA: Systematic and integrative
analysis of large gene lists using DAVID bioinformatics resources. Nat
Protoc 2009, 4(1):44-57.
18. Huang da W, Sherman BT, Lempicki RA: Bioinformatics enrichment tools:
paths toward the comprehensive functional analysis of large gene lists.
Nucleic Acids Res 2009, 37(1):1-13.
19. Livak KJ, Schmittgen TD: Analysis of relative gene expression data using
real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods
2001, 25(4):402-408.
20. Obaro S, Lawson L, Essen U, Ibrahim K, Brooks K, Otuneye A, Shetima D,
Ahmed P, Ajose T, Olugbile M, et al: Community acquired bacteremia in
young children from central Nigeria- a pilot study. BMC infectious diseases
2011, 11:137.
21. Iwasaki A, Medzhitov R: Toll-like receptor control of the adaptive immune
responses. Nat Immunol 2004, 5(10):987-995.
22. Franchi L, Park JH, Shaw MH, Marina-Garcia N, Chen G, Kim YG, Nunez G:
Intracellular NOD-like receptors in innate immunity, infection and
disease. Cell Microbiol 2008, 10(1):1-8.
23. Hoebe K, Janssen E, Beutler B: The interface between innate and adaptive
immunity. Nat Immunol 2004, 5(10):971-974.
24. Flower DR: Multiple molecular recognition properties of the lipocalin
protein family. J Mol Recognit 1995, 8(3):185-195.
25. Flo TH, Smith KD, Sato S, Rodriguez DJ, Holmes MA, Strong RK, Akira S,
Aderem A: Lipocalin 2 mediates an innate immune response to bacterial
infection by sequestrating iron. Nature 2004, 432(7019):917-921.
26. Bachman MA, Miller VL, Weiser JN: Mucosal lipocalin 2 has pro-
inflammatory and iron-sequestering effects in response to bacterial
enterobactin. PLoS Pathog 2009, 5(10):e1000622.
27. Aujla SJ, Chan YR, Zheng M, Fei M, Askew DJ, Pociask DA, Reinhart TA,
McAllister F, Edeal J, Gaus K, et al: IL-22 mediates mucosal host defense
against Gram-negative bacterial pneumonia. Nat Med 2008, 14(3):275-281.
28. Hiroshima Y, Bando M, Kataoka M, Inagaki Y, Herzberg MC, Ross KF, Hosoi K,
Nagata T, Kido JI: Regulation of antimicrobial peptide expression in
human gingival keratinocytes by interleukin-1alpha. Arch Oral Biol .
29. Conti HR, Shen F, Nayyar N, Stocum E, Sun JN, Lindemann MJ, Ho AW,
Hai JH, Yu JJ, Jung JW, et al: Th17 cells and IL-17 receptor signaling are
essential for mucosal host defense against oral candidiasis. J Exp Med
2009, 206(2):299-311.
30. Raffatellu M, George MD, Akiyama Y, Hornsby MJ, Nuccio SP, Paixao TA,
Butler BP, Chu H, Santos RL, Berger T, et al: Lipocalin-2 resistance confers
an advantage to Salmonella enterica serotype Typhimurium for growth
and survival in the inflamed intestine. Cell Host Microbe 2009,
5(5):476-486.
31. Fischbach MA, Lin H, Zhou L, Yu Y, Abergel RJ, Liu DR, Raymond KN,
Wanner BL, Strong RK, Walsh CT, et al: The pathogen-associated iroA gene
cluster mediates bacterial evasion of lipocalin 2. Proc Natl Acad Sci USA
2006, 103(44):16502-16507.
32. Ghosh S, Chakraborty K, Nagaraja T, Basak S, Koley H, Dutta S, Mitra U,
Das S: An adhesion protein of Salmonella enterica serovar Typhi is
required for pathogenesis and potential target for vaccine development.
Proc Natl Acad Sci USA 108(8):3348-3353.
33. Tsolis RM, Young GM, Solnick JV, Baumler AJ: From bench to bedside:
stealth of enteroinvasive pathogens. Nat Rev Microbiol 2008,
6(12):883-892.
34. Goncharova IA, Beloborodova EV, Freidin MB, Beloborodova EI, pyzurev VP:
[Association of immune system gene polymorphisms with quantitative
features which are pathogenetically important in chronic viral hepatitis].
Mol Biol (Mosk) 2008, 42(2):242-246.
35. Xia R, Tang Y, Huang Y, Yao D, Zhou Y, Chen B, Chen Y, Chen S, Deng A,
Zhong R: TNFSF9 expression in primary biliary cirrhosis and its clinical
significance. Cytokine 50(3):311-316.
36. Vanhollebeke B, De Muylder G, Nielsen MJ, Pays A, Tebabi P, Dieu M,
Raes M, Moestrup SK, Pays E: A haptoglobin-hemoglobin receptor
conveys innate immunity to Trypanosoma brucei in humans. Science
2008, 320(5876):677-681.
37. Thomson R, Samanovic M, Raper J: Activity of trypanosome lytic factor: a
novel component of innate immunity. Future Microbiol 2009, 4(7):789-796.
38. Thompson LJ, Dunstan SJ, Dolecek C, Perkins T, House D, Dougan G,
Nguyen TH, Tran TP, Doan CD, Le TP, et al: Transcriptional response in the
peripheral blood of patients infected with Salmonella enterica serovar
Typhi. Proc Natl Acad Sci USA 2009, 106(52):22433-22438.
39. Keuter M, Dharmana E, Gasem MH, van der Ven-Jongekrijg J,
Djokomoeljanto R, Dolmans WM, Demacker P, Sauerwein R, Gallati H, van
der Meer JW: Patterns of proinflammatory cytokines and inhibitors
during typhoid fever. J Infect Dis 1994, 169(6):1306-1311.
40. Raffatellu M, Santos RL, Chessa D, Wilson RP, Winter SE, Rossetti CA,
Lawhon SD, Chu H, Lau T, Bevins CL, et al: The capsule encoding the viaB
locus reduces interleukin-17 expression and mucosal innate responses
in the bovine intestinal mucosa during infection with Salmonella
enterica serotype Typhi. Infect Immun 2007, 75(9):4342-4350.
41. Wilson RP, Raffatellu M, Chessa D, Winter SE, Tukel C, Baumler AJ: The Vi-
capsule prevents Toll-like receptor 4 recognition of Salmonella. Cell
Microbiol 2008, 10(4):876-890.
42. Wijnholds J, Mol CA, van Deemter L, de Haas M, Scheffer GL, Baas F,
Beijnen JH, Scheper RJ, Hatse S, De Clercq E, et al: Multidrug-resistance
protein 5 is a multispecific organic anion transporter able to transport
nucleotide analogs. Proc Natl Acad Sci USA 2000, 97(13):7476-7481.
43. Oguri T, Isobe T, Suzuki T, Nishio K, Fujiwara Y, Katoh O, Yamakido M:
Increased expression of the MRP5 gene is associated with exposure to
platinum drugs in lung cancer. Int J Cancer 2000, 86(1):95-100.
44. Ravna AW, Sager G: Molecular modeling studies of ABC transporters
involved in multidrug resistance. Mini Rev Med Chem 2009, 9(2):186-193.
45. Baker S, Favorov M, Dougan G: Searching for the elusive typhoid
diagnostic. BMC Infect Dis 10:45.
Pre-publication history
The pre-publication history for this paper can be accessed here:
http://www.biomedcentral.com/1471-2334/11/241/prepub
doi:10.1186/1471-2334-11-241
Cite this article as: Khoo et al.: Host response transcriptional profiling
reveals extracellular components and ABC (ATP-binding cassette)
transporters gene enrichment in typhoid fever-infected Nigerian
children. BMC Infectious Diseases 2011 11:241.
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