+ MODEL

Journal of Microbiology, Immunology and Infection xxx (xxxx) xxx

Available online at www.sciencedirect.com

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journal homepage: www.e-jmii.com

Original Article

Plasma proteome profiling reveals

differentially expressed lipopolysaccharide-
binding protein among leptospirosis patients
Cheng-Yee Fish-Low a, Leslie Thian Lung Than a,
King-Hwa Ling b, Qingsong Lin c, Zamberi Sekawi a,*

a
Department of Medical Microbiology and Parasitology, Faculty of Medicine and Health Sciences,
Universiti Putra Malaysia, 43400, Serdang, Selangor, Malaysia
b
Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universiti Putra
Malaysia, 43400, Serdang, Selangor, Malaysia
c
Department of Biological Sciences, National University of Singapore, 117543, Singapore

Received 18 October 2018; accepted 22 December 2018

Available online - - -

KEYWORDS Abstract Background: Human leptospirosis, or commonly known as “rat urine disease” is a
Leptospirosis; zoonotic disease that is caused by the bacteria called Leptospira sp. The incidence rate of
Lipopolysaccharide- leptospirosis has been under-reported due to its unspecific clinical symptoms and the limita-
binding protein; tions of current laboratory diagnostic methods. Leptospirosis can be effectively treated with
Plasma proteome antibiotics in the early stage, and it is a curable disease but the accuracy to diagnose the infec-
tion is rarely achieved.
Methods: The present pilot study investigated plasma protein profiles of leptospirosis patients
and compared them against two control groups which consisted of dengue patients and healthy
individuals. The plasma protein digests were analyzed using shotgun approach by liquid
chromatography-tandem mass spectrometry (LC-MS/MS). Protein abundances were estimated
from the exponentially modified protein abundance index (emPAI) values. Plasma proteins in
leptospirosis patients with at least two-fold differential expression compared to dengue and
healthy control groups (p < 0.05, ANOVA) were identified.
Results: Lipopolysaccharide (LPS)-binding protein (LBP) was found to be the only protein that
has significant different expression between leptospirosis and the two control groups. The
expression levels of leucine-rich alpha-2-glycoprotein (LRG1) and alpha-1-antichymotrypsin
(ACT) were different significantly between leptospirosis and healthy group but not to the
dengue control group.
Conclusion: This is the first plasma proteome-based study on leptospirosis that reports the dif-
ferential expression of LBP compared to both dengue and healthy controls, which has not been
previously reported in the context of leptospirosis.

* Corresponding author. Fax: þ603 8947 2585.

E-mail address: zamberi@upm.edu.my (Z. Sekawi).

https://doi.org/10.1016/j.jmii.2018.12.015
1684-1182/Copyright ª 2019, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an open access article under the CC
BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article as: Fish-Low C-Y et al., Plasma proteome profiling reveals differentially expressed lipopolysaccharide-binding
protein among leptospirosis patients, Journal of Microbiology, Immunology and Infection, https://doi.org/10.1016/j.jmii.2018.12.015
 + MODEL
2 C.-Y. Fish-Low et al.
Copyright ª 2019, Taiwan Society of Microbiology. Published by Elsevier Taiwan LLC. This is an
open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
Introduction
Leptospirosis is a disease with significant health impact in
many regions of the world, particularly in the Americas and
Asia. Clinical presentation of leptospirosis is often non-
specific and known to mimic clinical profile of other preva-
lent febrile illnesses, such as dengue, influenza and malaria.
Clinical manifestations of leptospirosis range from mild
course, such as fever, headache, myalgia, conjunctival suf-
fusion, and abdominal pain, to severe complications char-
acterized by organ failure and bleeding which may lead to
death.1 Different clinical courses experienced by different
patients complicate the diagnosis of the disease. Misdiag-
nosis and delayed diagnosis of leptospirosis are common as
the accuracy to diagnose the infection is rarely achieved due
to a broad spectrum of signs and symptoms and the limita-
tions of available tests, which may result in inaccurate
treatment and poor outcomes.2 Currently, diagnosis of
leptospirosis is performed through the detection of IgM an-
tibodies against the pathogen by microscopic agglutination
test (MAT), which is time consuming and insensitive in the
early acute-phase specimens due to late sero-conversion.
It is hypothesized that the perturbations in the host
proteome profile can differentiate not only between in-
dividuals with leptospirosis and other clinically resem-
blance diseases, but also between leptospirosis patients
with different disease severity. Srivastava et al.3 reported
the serum proteome of leptospirosis patients showed
differentially expressed proteins compared to malaria pa-
tients, among those were a-1-antitrypsin, vitronectin,
ceruloplasmin, G-protein signaling regulator, and apolipo-
protein A-IV. A more recent serum proteome-based study
demonstrated that apolipoprotein A-I, serum amyloid A,
transferrin, haptoglobin, and transthyretin have significant
differential expression between mild versus severe lepto-
spirosis patients.4
Considering that leptospirosis may mimic other febrile
illnesses, it is necessary to include other closely resem-
blance febrile patients in the study controls to differentiate
leptospirosis from not only healthy individuals but also
other febrile patients. Misdiagnosis of leptospirosis has
been associated with dengue fever, H1N1 influenza, and
malaria.2,5,6 In this pilot study, we compared the plasma
proteome profiles of leptospirosis and dengue patients in
view of their overlapping geographic distributions and
indistinguishable clinical presentations.
Methods
Informed consent
The study protocol was approved by the Institutional Re-
view Board of Faculty of Medicine and Health Sciences,
Please cite this article as: Fish-Low C-Y et al., Plasma proteome profiling reveals differentially expressed lipopolysaccharide-binding
protein among leptospirosis patients, Journal of Microbiology, Immunology and Infection, https://doi.org/10.1016/j.jmii.2018.12.015
Universiti Putra Malaysia, and the Medical Research Ethics
Committee (MREC), Ministry of Health Malaysia (NMRR-15-
2148-27536). Written informed consent was obtained from
all included subjects prior to the sample and data
collection.
Enrolment and sample collection
Enrolment of subjects included all clinically suspected
leptospirosis and dengue patients who were admitted in
Hospital Serdang, Malaysia from June to December 2016.
Patients in the paediatric age group (below 18 years old)
and patients with past history of autoimmune diseases or
any known comorbidities were excluded. Healthy volun-
teers were recruited as the control group. Blood sample
was collected in ethylenediaminetetraacetic acid (EDTA)
tube and serum-separating tube. The similar collection,
processing and storage conditions were maintained for all
diseased and control samples in order to minimize any pre-
analytical variations.
Case definition and patient selection
Both the leptospirosis and dengue tests were performed by
the microbiology laboratory of Hospital Serdang and our
own laboratory. Briefly, leptospirosis was confirmed by MAT
according to the standard protocol7 using a 20-serovar
panel, which consisted of Australis, Autumnalis, Bataviae,
Canicola, Celledoni, Djasiman, Grippotyphosa, Hardjopra-
jitno, Icterohaemorrhagiae, Javanica, Patoc, Pomona,
Pyrogenes, Tarassovi, IMR LEP 1, IMR LEP 115, IMR LEP 175,
IMR LEP 803/11, IMR LEP 27, and IMR LEP 22. A confirmed
leptospirosis case is defined as single serum titre of 1:400 or
at least a four-fold rise in titre of paired sera.8 Dengue was
confirmed by positive detection of non-structural protein 1
(NS1) antigen and/or IgM. Healthy subjects were volunteers
who were disease-free for two months and with no known
medical illnesses when having blood drawn. Out of 33
clinically suspected cases, three of leptospirosis and three
of dengue confirmed cases, together with two age- and
gender-matched healthy control subjects were selected for
proteome profiling.
Plasma sample preparation for mass spectrometry
Plasma protein was estimated by colorimetric Bradford
method using Bio-Rad Protein Assay (Bio-Rad, USA). For
each sample, about 150e200 mg of protein in 0.5 M trie-
thylammonium bicarbonate (TEAB) buffer with 0.1% sodium
dodecylsulfate (SDS) was reduced with 2 mM tris(2-
carboxyethyl)phosphine (TCEP; SigmaeAldrich, USA) at
56  C for 60 min and alkylated with 20 mM methyl meth-
anethiosulfonate (MMTS; SigmaeAldrich, USA) at room
 + MODEL
Differentially expressed LBP in leptospirosis
temperature for 30 min. Sequencing grade modified trypsin
(Promega, USA) was added to the sample at a trypsin:pro-
tein ratio of 1:100 and incubated at 37  C for 16 h. Com-
plete digestion was confirmed by comparing an aliquot of
sample before and after the tryptic digestion on a one-
dimensional SDS-PAGE. The digested peptides were cleaned
up using strong cation-exchange (SCX) cartridge system (AB
SCIEX, USA) and the SCX eluent was desalted using Pierce
C18 tips (Thermo Scientific, USA).
Liquid chromatography tandem mass spectrometry
The protein identification was performed using shotgun
approach by liquid chromatography-tandem mass spec-
trometry (LC-MS/MS) analysis. In brief, the desalted eluent
was adjusted to about 1.0 mg/mL before loading to the
cHiPLC-nanoflex system (Eksigent, USA) coupled to a Tri-
pleTOF 5600 system (AB SCIEX, USA). The tandem mass
spectra of peptides were extracted from raw files and
searched against Swiss-Prot protein database with the
taxonomy restricted to Homo sapiens (released on 16
February 2017, 20,172 sequences) using MASCOT Server
2.4.0 (Matrix Science, UK). The cut-off threshold for protein
identification was set at 1% false discovery rate (FDR). The
protein abundance was estimated based on exponentially
modified protein abundance index (emPAI) score.9
Statistical analysis
Proteins that detected in leptospirosis samples and with at
least two-fold differential expression compared to the
dengue or healthy control groups were determined. The
difference of protein abundance was compared statistically
by analysis of variance (ANOVA) multiple comparison test
followed by Tukey post hoc test using SPSS Statistics v25
(IBM, USA) and those with statistical significance (p < 0.05)
were identified.
Results
Patient clinical characteristics
Three leptospirosis patients (denoted as LEPTO-1, LEPTO-2,
LEPTO-3; age range 18e37; mean 25.7; standard deviation
8.2) and three dengue patients (age range 22e37; mean
27.7; standard deviation 6.6), together with two healthy
individuals (age range 21e35; mean 28; standard deviation
7.0) were selected for proteomics profiling and comparison.
Upon admission, the three leptospirosis and the three
dengue patients presented with some common clinical
manifestations, including fever, gastrointestinal symptoms,
and myalgia. None of the leptospirosis patients were tested
positive in dengue detection. The LEPTO-1 patient had MAT
titer 1:400 as early as in day-3 since the disease symptom
onset. Despite the highest level of C-reactive protein and
creatine kinase as well as high creatinine recorded in
LEPTO-1 among the three leptospirosis patients, his clinical
symptoms quickly resolved after treatment. Similarly,
LEPTO-2 patient gradually recovered throughout hospitali-
zation. However, his diagnosis for leptospirosis was not
Please cite this article as: Fish-Low C-Y et al., Plasma proteome profiling reveals differentially expressed lipopolysaccharide-binding
protein among leptospirosis patients, Journal of Microbiology, Immunology and Infection, https://doi.org/10.1016/j.jmii.2018.12.015
3
confirmed until his paired sera were tested. In contrast,
LEPTO-3 patient was admitted to the hospital due to
persistent fever and diarrhea for two weeks. The patient
presented with mild jaundice, shortness of breath, mel-
aena, hepatorenal syndrome, and severe metabolic
acidosis. His chest x-ray showed minimal bilateral pleural
effusions. Detection of IgM against Leptospira in his blood
upon admission was confirmed by MAT. He was cared for in
the intensive care unit and eventually discharged after
recovery. As for the dengue patients, their paired sera were
tested negative for MAT.
Differentially expressed proteins
Lipopolysaccharide (LPS)-binding protein (LBP) was the only
plasma protein that was detected in the leptospirosis pa-
tients with more than two-fold differential expression
compared to both dengue and healthy control groups. On
the other hand, the protein abundances of leucine-rich
alpha-2-glycoprotein (LRG1) and alpha-1-antichymotrypsin
(ACT) in leptospirosis were more than two-fold compared to
the healthy control group but lower than two-fold differ-
ence compared to the dengue control group (Table 1).
Discussion
Infection with pathogenic Leptospira species causes
leptospirosis. Leptospira is a genus of Gram-negative
spirochaete bacteria with more than 250 Leptospira sero-
vars have been reported based on the heterogeneity in
carbohydrate component of LPS.1 The serovar identity is
attributed to the differences in O-antigen polysaccharide of
LPS,10 which is determined by MAT that uses a panel of live
leptospires representing different serogroups as antigens
for detection of agglutinating antibodies. Besides the role
in serological classification, leptospiral LPS serves as the
principal antigen that is recognized by the human immune
system during leptospiral infections. Administration of
monoclonal antibodies directed against LPS determinants in
an animal model confers protection against the lethality of
leptospirosis.11
LBP, a soluble positive acute phase protein plays a
central role in the early step of host defense to Gram-
negative bacterial infections or to LPS. The level of LBP in
normal serum is about 5e10 mg/mL, and may elevate to
above 200 mg/mL in acute-phase serum.12 Lamping et al.13
proposed a concentration-dependent dual role of LBP in the
pathogenesis of Gram-negative bacterial sepsis. Low con-
centrations of LBP was reported to enhance LPS-induced
tumor necrosis factor-alpha (TNF-a) synthesis whereas
acute phase concentrations of LBP inhibit this effect. High
concentration of LBP has been shown to reduce LPS activity
and to neutralize LPS.14,15 In addition, it also results in
better disease outcome.16 Collectively, high LBP concen-
tration in host during sepsis has been described as a pro-
tective function from uncontrolled inflammatory responses.
Interactions of CD14 with LBP is necessary to activate
toll-like receptors 2 and 4 (TLR2 and TLR4) signaling
cascade in response to LPS.17 TLR4 is known as the pre-
dominant receptor of the innate immune system that rec-
ognizes Gram-negative LPS,18 which is in contrast to TLR2
 + MODEL
4 C.-Y. Fish-Low et al.

Table 1 Differentially expressed proteins of leptospirosis compared to the control groups.

UniProtKB Protein Name LEPTOa vs. DEN-CTRLb LEPTOa vs. HLTY-CTRLc
Fold Change *p-value Fold Change *p-value
P18428 Lipopolysaccharide-binding protein 5.03 0.000022 15.67 0.000016
P02750 Leucine-rich alpha-2-glycoprotein 0.37 0.458,692 3.63 0.045,576
P01011 Alpha-1-antichymotrypsin 1.01 0.008408 2.56 0.002862
a
Average protein abundance of leptospirosis patients.
b
Average protein abundance of dengue patients as a control group.
c
Average protein abundance of healthy individuals as a control group.
*p-value was calculated using ANOVA multiple comparison test followed by Tukey post hoc test.

that recognizes Gram-positive bacteria19 and other non-LPS
products.20 Surprisingly, leptospiral LPS is able to activate
both TLR2 and TLR4 in murine cells21 but activates only
TLR2 pathway in human cells.22 It is possible that the effi-
cient detection of LPS by both the TLR2 and TLR4 signaling
cascades has rendered the mice resistant to leptospirosis.
Though the whole Leptospira or their purified immunogenic
proteins may trigger pro-inflammatory responses via TLR-
dependent signaling,21,22 Chassin et al.23 have demon-
strated that TLR signaling plays a protection role instead of
inducing inflammation in susceptible mice.
As the name implies, LBP is previously thought to bind
and respond only to Gram-negative LPS when it was first
characterized.24 However, many studies revealed that the
LBP involvement in immune responses does not limit to
against LPS in Gram-negative infections. It has been proven
to interact with other components, including lipoteichoic
acid (LTA) and peptidoglycans of Gram-positive bacteria,25
LTA-like lipoglycans of spirochaetes,26 glycolipids,27 and
lipoproteins.28 Several clinical studies have reported
elevated LBP levels in patients with Gram-negative bacte-
ria, Gram-positive bacteria, fungal infections as well as
those with other medical conditions.14,16 Levels of LBP
during dengue infection are inconsistent. Decrease of LBP is
reported among dengue patients with severe plasma
leakage,29 which contrasts with increase of LBP that
observed in severe dengue patients as reported by Yong
et al.30 Even though the presence of LBP itself may not
indicate a bacterial infection, a statistically significant
differential expression of LBP may aid to differentiate a
bacterial and viral or non-bacterial infection,31e33 which
supports the findings of this study that aimed to differen-
tiate leptospirosis from dengue fever.
Interestingly, differential levels of LBP among leptospi-
rosis patients have not been previously reported. Compared
to the other proteome-based studies on human leptospi-
rosis, the present study is the first to report LBP as one of
the significant differentially expressed proteins, which is
not noted in the studies of Srivastava et al.3 as well as Ting
et al.4 One explanation could be that different study de-
signs and control groups were used in making the compar-
ison of protein profiles. Srivastava et al.3 compared the
serum proteome profile of leptospirosis against both ma-
laria patients and healthy volunteers whereas Ting et al.4
compared that of between mild versus severe leptospi-
rosis against healthy individuals. In considering that addi-
tional sample pretreatments prior to the mass
spectrometry analysis, including albumin/IgG depletion and
protein separation by two-dimensional gel electrophoresis
(2-DE), loss of LBP during the laboratory procedures or
masking of LBP by other highly abundant proteins on the 2-
DE gels cannot be ruled out.
LRG1 is found in the serum of healthy individuals at
about 50 mg/mL.34 It is also an acute phase protein, which
the level elevated in patients with bacterial and viral in-
fections,34,35 as well as in patients with different types of
cancers.36,37 LPS has been demonstrated to induce a dose-
dependent expression of LRG1 in a murine study.38 Eleva-
tion of another acute phase protein, ACT has been associ-
ated with cognitive decline and Alzheimer’s disease.39 As
reported in this study, it is not unusual to find the levels of
both LRG1 and ACT elevated significantly among leptospi-
rosis patients compared to healthy individuals but not to
the dengue patients.
Serum is routinely collected in clinical setting to di-
agnose leptospirosis thus making it a conveniently obtain-
able sample for leptospirosis-related studies. However,
according to the recommendations of Human Proteome
Organization Plasma Proteome Project (HUPO-PPP), plasma
is preferable over serum in a proteomic biomarker study
due to several reasons:40
i. Many uncontrollable factors, such as temperature and
time taken for clot formation during clotting may
alter the serum content;
ii. Proteins of interest may bind to the clot and may be
removed from serum during separation; and
iii. Detection of endogenous peptides may be impeded
by highly concentrated and intense peptide signals
that present in serum.
Nevertheless, serum is still widely used in leptospirosis
studies due to its availability and may be preferable when
coagulation factors are to be depleted prior to the study.
Consistent with previous reports, LBP may aid to
differentiate a bacterial and viral or non-bacterial infec-
tion. Together with other previously reported biomarkers,
it can assist in improving the diagnostic accuracy of the
disease. The study can be further improved by testing on
larger cohort of patients with different spectrum of the
disease and also on other febrile illnesses besides dengue.
Considering the nature of shotgun mass spectrometry with
limited dynamic range coverage, coupled with under-
sampling associated with the method should be acknowl-
edged in the current study. The results obtained from this
small number of patients may not represent the clinical

Please cite this article as: Fish-Low C-Y et al., Plasma proteome profiling reveals differentially expressed lipopolysaccharide-binding
protein among leptospirosis patients, Journal of Microbiology, Immunology and Infection, https://doi.org/10.1016/j.jmii.2018.12.015
 + MODEL
Differentially expressed LBP in leptospirosis
course of leptospirosis in general. In addition to the need of
larger cohort size with different degree of clinical mani-
festations, inclusion of other febrile diseases as control
groups for further validation may be required. Instead of
using LBP as a single diagnostic biomarker, a biomarker
panel comprises of one or more of host- and pathogen-
derived proteins should be developed.
Conflicts of interest
All authors declare no conflicts of interest.
Acknowledgements
This work was funded by the Ministry of Higher Education,
Malaysia Long Term Research Grant Scheme (LRGS) [UPM/
700-2/1/LRGS/5526400]. We would like to thank the Di-
rector General of Health Malaysia for his permission to
publish this article. The authors are indebted to all the
clinicians and supporting staff from Hospital Serdang,
Malaysia. The authors also express their appreciation to all
the members of Malaysia Leptospirosis Research Network
(MyLepto) and to all the study participants.
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