Clinical proteomic analysis of scrub typhus infection
Park et al. Clin Proteom
Clinical proteomic analysis of scrub typhus infection
Edmond Changkyun Park 0 2 3
SangY?eop Lee 0 2
Sung Ho Yun 0
ChiW?on Choi 0 4
Hayoung Lee 0 3
Hyun Seok Song 0 2 3
Sangmi Jun 0 2 3
Gun?Hwa Kim 0 3 4
Chang?Seop Lee 1
Seung Il Kim 0 2 3
0 Drug & Disease Target Team, Korea Basic Science Institute (KBSI) , Cheongju 28119 , Republic of Korea
1 Department of Internal Medicine, Chonbuk National University Medical School , Jeonju 54986 , Republic of Korea
2 Center for Convergent Research of Emerging Virus Infection, Korea Research Institute of Chemical Technology (KRICT) , Daejeon 34114 , Republic of Korea
3 Department of Bio?Analytical Science, University of Science and Technology (UST) , Daejeon 34113 , Republic of Korea
4 Division of Life Science, Tunneling Nanotube Research Center, Korea University , Seoul 02841 , Republic of Korea
Background: Scrub typhus is an acute and febrile infectious disease caused by the Gram? negative ?? proteobacterium Orientia tsutsugamushi from the family Rickettsiaceae that is widely distributed in Northern, Southern and Eastern Asia. In the present study, we analysed the serum proteome of scrub typhus patients to investigate specific clinical protein patterns in an attempt to explain pathophysiology and discover potential biomarkers of infection. Methods: Serum samples were collected from three patients (before and after treatment with antibiotics) and three healthy subjects. One? dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis followed by liquid chromatography? tandem mass spectrometry was performed to identify differentially abundant proteins using quantitative proteomic approaches. Bioinformatic analysis was then performed using Ingenuity Pathway Analysis. Results: Proteomic analysis identified 236 serum proteins, of which 32 were differentially expressed in normal subjects, naive scrub typhus patients and patients treated with antibiotics. Comparative bioinformatic analysis of the identified proteins revealed up? regulation of proteins involved in immune responses, especially complement system, following infection with O. tsutsugamushi, and normal expression was largely rescued by antibiotic treatment. Conclusions: This is the first proteomic study of clinical serum samples from scrub typhus patients. Proteomic analysis identified changes in protein expression upon infection with O. tsutsugamushi and following antibiotic treatment. Our results provide valuable information for further investigation of scrub typhus therapy and diagnosis.
Scrub typhus; Clinical proteomics; Orientia tsutsugamushi
Scrub typhus (tsutsugamushi disease) is a
miteborne infectious disease caused by the Gram-negative
?-proteobacterium Orientia tsutsugamushi that is
transmitted through the bite of an infected chigger
(Trombiculidae mite) [
]. After a bite from an infected chigger,
a characteristic necrotic inoculation lesion, termed
eschar, can develop, and the microorganism then spreads
through the lymphatic fluid and blood, causing systemic
manifestations including fever, headache, myalgia,
lymphadenopathy and skin rash. Especially in untreated
cases, patients develop complications with systemic
involvement and disseminated vasculitis, including septic
shock, acute respiratory distress syndrome, acute renal
failure, meningitis, myocarditis, gastrointestinal
bleeding and multiorgan dysfunctions [
]. Scrub typhus
is endemic in the ?tsutsugamsuhi triangle? area, which
extends from Northern Japan and far Eastern Russia in
the North, to Northern Australia in the South, and to
Pakistan and Afghanistan in the East [
]. Over one
billion people are currently living in risk areas, and
approximately one million cases occur annually worldwide .
The average case-fatality rate is usually? ~? 10%, but can
be as high as 35% if antimicrobial treatment is delayed.
In the pre-antibiotic era, case-fatality ratios may have
been as high as 50% [
]. Therefore, early diagnosis of
scrub typhus is essential to prevent complications and to
reduce the mortality rate.
The main goal of proteomic studies is to elucidate
protein expression and identify changes under the influence
of biological perturbations such as diseases or drug
]. The information obtained from such
analyses provides valuable information on pathophysiology
and potential diagnostic biomarkers of disease . Since
the genome sequence of O. tsutsugamushi was published
], several proteomic studies have focused on
identifying proteins. The first proteomic analysis involved
2D liquid chromatography-tandem mass spectrometry
(LC?MS/MS)-based comparison of protein expression,
and 14 proteins were shown to be differentially expressed
in antibiotic-sensitive and -insensitive O. tsutsugamushi
]. Kishimoto and colleagues also performed shotgun
proteomics analysis of O. tsutsugamushi, which revealed
specific characteristics of this obligate intracellular
bacterial species, and identified potential immunogenic
factors such as type IV secretion system proteins [
recently, comprehensive analysis of global gene and
protein expression in O. tsutsugamushi in fibroblasts and
macrophages showed how the pathogen responds in
different types of host cells [
Although previous proteomic studies on O.
tsutsugamushi provided some evidence about the
pathophysiology of scrub typhus, information about diagnostic
markers is lacking. Currently, complement fixation is one
the earliest tests for diagnosis of scrub typhus [
2, 16, 17
At present, there is no diagnostic test of pathogenic
antigen generated by O. tsutsugamushi. Moreover, proteomic
analysis of O. tsutsugamushi directly isolated from scrub
typhus patients has not been successfully performed,
presumably because O. tsutsugamushi proteins are
present at low concentrations in the blood. In the present
study, as the first step in proteomic analysis of scrub
typhus patients, we investigated the serum proteome to
elucidate physiological changes caused by infection, both
before and after antibiotic treatment.
Patients and clinical samples
The diagnosis of patients with scrub typhus was
confirmed by a positive IgM titre ? 1:160 against O.
tsutsugamushi, or a fourfold or greater rise in the indirect
immunofluorescence assay (IFA, Green Cross Reference
Lab., Yongin, South Korea). Detailed patient
information is listed in Table? 1. Blood was collected from scrub
typhus patients, and peripheral blood mononuclear
cells (PBMCs) were prepared for PCR validation of O.
tsutsugamushi infection. For proteomic analysis, blood
serum samples were prepared from three scrub typhus
WBC white blood cell, AST aspartate aminotransferase, ALT alanine
aminotransferase, PT prothrombin time, INR international normalized ratio, aPTT
activated partial thromboplastin time
patients before and after antibiotic treatment.
Doxycycline 200? mg/day was used to treat for 5 to 7? days. First
blood sample was collected at emergency room before
antibiotics administration and second sample was 5 to
7?days after antibiotics. Sera from three healthy subjects
were used as negative infection controls.
Standard PCR was performed with 20? ng/?l DNA from
PBMCs. For nested PCR, 2??l of a 100-fold diluted
standard PCR mixture was used as template. Primers used for
PCR amplification of a gene encoding a 56? kDa protein
were as follows: standard PCR (862?bp),
5?-CAATGTCTGCGTTGTCGTTGC-3? (forward) and
5?-ACAGATGCACTATTAGGCAA-3? (reverse); nested PCR (509? bp),
5?-CCAGGATTTAGAGCAGAG-3? (forward) and
Preparation of clinical serum samples for proteomic
For proteomic analysis, albumin and IgG were removed
from clinical serum samples using ProteoPrep
Immunoaffinity Albumin and IgG Depletion Kit
(SigmaAldrich, St. Louis, MO, USA). After column equilibration,
100? ?l of serum sample (diluted with 50? ?l of dilution
solution) was loaded on the column and incubated for
10?min at room temperature. The eluate was collected by
centrifugation at 8000?g for 1?min, and reapplied to the
same spin column. The second elute was collected and
used for proteomic analysis.
Sodium dodecyl sulphate?polyacrylamide gel electrophoresis (SDS?PAGE) and tryptic digestion
Prepared clinical serum samples (10? ?g) were
resuspended in sodium dodecyl sulphate?polyacrylamide gel
electrophoresis (SDS-PAGE) sample buffer (1? M TRIS?
HCl pH 6.8, 10% SDS, 1% bromophenol blue, glycerol,
?-mercaptoethanol) and boiled for 10?min. Samples were
separated by 12% SDS-PAGE. The gel was stained with
Coomassie Brilliant Blue R-250 and fractionated
according to molecular weight. Tryptic in-gel digestion was
conducted according to a previous procedure [
peptides were extracted with extraction solution
consisting of 50? mM ammonium bicarbonate, 50% acetonitrile
and 5% trifluoroacetic acid (TFA), and dried. For LC?MS/
MS analysis, samples were dissolved in 0.5% TFA.
Proteomic analysis by LC?MS/MS analysis
Tryptic peptide samples (5? ?l) were separated using an
Ultimate 3000 UPLC system (Dionex, Sunnyvale, CA,
USA) connected to a Q Exactive Plus mass
spectrometer (Thermo Scientific, Waltham, MA, USA) equipped
with a nanoelectrospray ion source (Dionex).
Peptides were eluted from the column and directed onto a
15?cm???75??m i.d. Acclaim PepMap RSLC C18
reversedphase column (Thermo Scientific) at a flow rate of 300?nl/
min. Peptides were eluted by a gradient of 0?65%
acetonitrile in 0.1% formic acid for 180?min. All MS and MS/
MS spectra obtained using the Q Exactive Plus Orbitrap
mass spectrometer were acquired in the data-dependent
top10 mode, with automatic switching between full scan
MS and MS/MS acquisition. Survey full scan MS spectra
(m/z 150-2,000) were acquired in the orbitrap at a
resolution of 70,000 (m/z 200) after accumulation of ions to a
1? ?? 106 target value based on predictive automatic gain
control (AGC) from the previous full scan. MS/MS
spectra were searched with MASCOT v2.4 (Matrix Science,
Inc., Boston, MA, USA) using the UniProt human
database for protein identification. MS/MS search parameters
were set as follows: carbamidomethylation of cysteines,
oxidation of methionines, two missed trypsin cleavages,
mass tolerance for parent ion and fragment ion within
10? ppm, p value? <? 0.01 of the significant threshold. The
exponentially modified protein abundance index (emPAI)
was generated using MASCOT, and mol% was calculated
according to emPAI values [
]. MS/MS analysis was
performed at least three times for each sample.
Statistical analysis and bioinformatics
Analysis was performed only on proteins that were
detected more than two times from triplicate experiments
for each sample. Differentially expressed proteins among
the three groups were identified using Kruskal?Wallis
tests. Non-parametric Mann?Whitney U tests were also
performed to identify differentially expressed proteins
within each group. Proteins were considered significantly
differentially expressed when the p-value was less than
0.05 as calculated using R (http://www.r-project.org).
All identified proteins were subjected to query
canonical pathway analysis using the Ingenuity Pathway
Analysis (IPA) tool (https://www.qiagenbioinformatics.com/
Results and discussion
Evaluation of clinical samples
Sera from three scrub typhus patients were used for
proteomic analysis. We collected blood from patients before
and after antibiotic treatment. To confirm the quality
of clinical blood samples from scrub typhus patients,
PBMCs were isolated from blood, and the presence of
pathogen was evaluated by nested PCR. The results
showed that all three patients were positive for a specific
scrub typhus gene encoding a 56?kDa protein, confirming
infection by O. tsutsugamushi (Additional file?1: Fig. S1).
To identify host proteins affected by scrub typhus
infection, serum samples were prepared from normal subjects
(controls), naive patients (before antibiotic treatment)
and treated patients (after antibiotic treatment), then
analysed by GeLC-MS/MS. From the proteomic
analysis, 174, 155 and 143 human proteins were identified
in the serum of normal subjects, naive scrub typhus
patients and treated patients, respectively (Fig.? 1 and
Additional file? 2: Table S1). Following infection with
O. tsutsugamushi, expression of 70 proteins was
significantly up-regulated, and expression of 94 proteins
Naive patients Treated patients
Fig. 1 Identification of serum proteins in scrub typhus patients. The
Venn diagram shows the number of proteins identified in the serum
of normal subjects, naive scrub typhus patients and patients treated
with the antibiotic doxycycline
was down-regulated, compared with normal subjects
(Table? 2). After antibiotic treatment, 26 proteins were
up-regulated and 36 proteins were down-regulated in
treated patients relative to naive patients (Table?2). These
proteins could be useful for investigating the
pathophysiology of scrub typhus at the molecular level, and for
discovering diagnostic biomarkers of O. tsutsugamushi
infection. The quantitative results also revealed that the
number of proteins expressed at similar levels in normal
subjects and naive patients was 60 (26.8%), while the
number between naive patients and treated patients was
113 (64.6%), indicating that protein expression was more
similar between treated patients and naive patients than
between treated patients and normal subjects (Table? 2).
This may suggest that treated patients are in the process
of recovery, but not fully recovered.
Comparative analysis of canonical pathways
Discovery of canonical pathways enriched in different
conditions and investigation of their differences upon
infection with O. tsutsugamushi could provide potential
information on the pathophysiology of scrub typhus. To
this end, the identified proteins were analysed using the
IPA bioinformatics tool. Bioinformatic analysis revealed
that the identified proteins are mainly involved in
immune responses. In all groups, acute phage response
signalling and complement system were ranked in the
top five in the canonical pathway (Additional file?1: Fig.
S2). In addition, proteins related to LXR/RXR
activation, FXR/RXR activation and coagulation system were
enriched in all samples (Additional file?1: Fig. S2).
To better understand the differences in expression of
proteins and changes in the canonical pathways following
infection with O. tsutsugamushi and subsequent
antibiotic treatment, we compared the identified proteins and
their enriched canonical pathways. The results revealed
dynamic changes in the expression of proteins involved
in immune responses (Fig.? 2). In particular, expression
ilitrcsssseuahppeeonegnagnA ii/ttcvLaaonXXRRR ii/ttcvaanoFXXRRR lttsyspoeeneCmmm littsysagounoaeCm iliiilittt-rycsssdeadenahodneognangCm liiiltrrcssssheoeongangA iliiiIt-rsc12gnaLgannduopdonn rcsaoaphegm iiiiittItttrrrsccvyophonbnanoapaahnwm iiiiitfttrrrcxccvnoonudodoeandeaeP iirysxsccsengeponeapoaghem ii/ttcvLaaonXXRRR ilitrcssssuepahepeoneangngA ii/ttcvaaonFXXRRR lttysspoeeneCmmm iliiilittt-rcysssedaednahodenognangCm liiiltrrcssssheeooanggnA iiliiIt-rsc12angLagnnduopdonn rcsaophaegm littyssgoauonaeCm iiiiitfttrrrccxcvoonnudoodeandeaeP iirysxcscsnegpeoneapoaghem iiiiitIttttrrrcscvyohpobnanaopnhaanwm
Fig. 2 Top 10 canonical pathways most significantly altered in naive scrub typhus patients compared with normal subjects (a) and in patients
treated with antibiotics compared with naive patients (b). The stacked bar chart displays the percentage of proteins up?regulated (red) and down?
regulated (green), and of proteins not overlapping with the dataset (white), in each canonical pathway. The numerical value at the top of each
bar represents the total number of genes in the canonical pathway. The secondary y?axis (right) shows the ?log of the p value calculated by the
of proteins associated with complement system was
upregulated by infection with O. tsutsugamushi (Fig.? 2a)
and down-regulated by antibiotic treatment (Fig.? 2b).
Investigation of signalling pathways also confirmed that
complement system was significantly activated in scrub
typhus patients, and activity was down-regulated by
antibiotic treatment (Fig.?3).
Differentially expressed proteins
Next, we identified proteins displaying statistically
significant differential expression between the three groups,
and 32 differentially expressed proteins were identified
(Table? 3), of which 27 were up-regulated by infection
with O. tsutsugamushi and down-regulated by
antibiotic treatment. Representative proteins up-regulated in
naive patients include serum amyloid proteins,
complement component proteins, protein S100-A8 and
C-reactive protein, which are mainly related to immune
responses (Table?3). Up-regulation of immune responses
is expected since scrub typhus infection induces a
combination of non-specific symptoms that overlap with
other infections [
]. Interestingly, platelet factor 4 was
exclusively expressed in scrub typhus patients (Table? 3).
This small cytokine is secreted from platelets during
platelet aggregation, and promotes blood coagulation
]. Recent studies reported that blood coagulation
is activated by infection with O. tsutsugamushi [
Five proteins, serotransferrin, tetranectin, ficolin-3,
selenoprotein P and adiponectin, were down-regulated in
the serum of scrub typhus patients and up-regulated in
patients treated with antibiotics (Table? 3). Proteins
displaying differential expression among normal subjects,
naive patients and treated patients could be potential
biomarkers for diagnosis and/or prediction of therapy
responses during scrub typhus infection.
Proteins related to immune responses
For more detailed investigation of proteins related to
immune responses, protein network analysis was
performed. The results clearly showed that expression levels
a Gene ontology is classified according to UniProt
b Not detected
GO Biological functiona
Antigen processing and
Innate immune response
O? glycan processing
Adherens junction organi?
Iron ion homeostasis/Platelet
Response to oxidative stress/
of proteins involved in acute phase response signalling
and complement system were up-regulated by
infection with O. tsutsugamushi (Fig.?4a) and down-regulated
by treatment with the antibiotic doxycycline (Fig.? 4b).
Acute phase response signalling is a rapid
inflammatory response that provides protection against pathogens
using non-specific defence mechanisms [
]. We also
found that proteins involved in inflammation, such as
serum amyloid proteins, protein S100-A8 and C-reactive
protein, were up-regulated in naive patients (Table?3).
The complement system is part of the innate immune
system that enhances the ability of antibodies and
phagocytic cells to clear microbes and damaged cells from
the host organism, promotes inflammation and attacks
the plasma membrane of the pathogen [
]. Our results
showed that, among the 32 differentially expressed
proteins, six of those up-regulated were complement
component proteins (Table? 3). Proteomic analysis also
revealed that the polymeric immunoglobulin receptor
was exclusively expressed following infection with O.
tsutsugamushi (Table?3). The polymeric immunoglobulin
receptor facilitates the secretion of IgA and IgM [
]. It is
well known that both IgM and IgG initiate complement
system activation [
]. Therefore, infection by O.
tsutsugamushi induces IgM production, and this may lead to
the expression of complement component proteins and
activation of complement system, resulting in activation
of the innate immune system, including inflammation.
In this study, we performed proteomic analysis of blood
serum from scrub typhus patients to investigate the
pathophysiological mechanisms and discover
potential diagnostic biomarkers for scrub typhus infection.
Comparative analysis revealed that proteins involved
in immune responses and blood coagulation were
dynamically regulated following infection with O.
tsutsugamushi and subsequent antibiotic treatment. In
particular, complement system was activated in scrub typhus
patients. We also discovered proteins that are
differentially expressed among normal subjects, naive patients
and patients treated with antibiotics. To our knowledge,
this is the first analytical study of clinical samples from
scrub typhus patients. Our results provide valuable
information for further investigation of scrub typhus therapy
Additional file 1: Figure 1. Nested PCR of 56 kDa gene confirmed the
infection of O. tsutsugamushi in scrub typhus patients. Figure 2. Func?
tional annotation of the proteins identified in each group.
Additional file 2: Table 1. Total list of identified proteins.
ALT: alanine aminotransferase; aPTT: activated partial thromboplastin time;
AST: aspartate aminotransferase; PBMC: peripheral blood mononuclear cell;
FXR: frarnesoid X receptor; GeLC?MS/MS: one ? dimensional sodium dodecyl
sulfate?polyacrylamide gel electrophoresis followed by liquid chromatogra?
phy?tandem mass spectrometry; INR: international normalized ratio; LXR: liver
X receptor; PT: prothrombin time; RXR: retinoid X receptor; WBC: white blood
ECP, SYL, and SIK designed and wrote manuscript. CWC and SHY conducted
proteomic analysis. HL conducted data analysis. HSS, SJ, and GHK helped in
data analysis. CSL collected clinical samples and helped in manuscript writing.
All authors read and approved the final manuscript.
We would like to thank Dr. Yeong?Seon Lee at Korea National Institute of
Health for providing O. tsutsugamushi genomic DNA and her advice on this
The authors declare that they have no competing interests.
Availability of data and materials
All data generated during this study are included in this published article. Total
list of identified proteins has been uploaded as additional files.
Consent for publication
Ethics approval and consent to participate
This study protocol was approved by Institutional Review Board of Chonbuk
National University Hospital (IRB Number; CUH 2016?04?007) and all patients
provided informed consent.
This work was supported by Korea Health Technology R&D Project
(HI16C0312) through the Korea Health Industry Development Institute (KHIDI)
funded by the Ministry of Health & Welfare, the National Research Council
of Science & Technology (NST) grant by the Korea government (MSIP) (No.
CRC?16?01?KRICT), and the Korea Basic Science Institute research program
Springer Nature remains neutral with regard to jurisdictional claims in pub?
lished maps and institutional affiliations.
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