Accepted Manuscript Overexpression of heat shock GroEL stress protein in leptospiral biofilm K. Vinod Kumar, Chandan Lall, R. Vimal Raj, K. Vedhagiri, C. Kartick, P. Surya, K. Natarajaseenivasan, P. Vijayachari PII:
S0882-4010(16)30444-2
DOI:
10.1016/j.micpath.2016.11.010
Reference:
YMPAT 2000
To appear in:
Microbial Pathogenesis
Received Date: 9 August 2016 Revised Date:
24 September 2016
Accepted Date: 14 November 2016
Please cite this article as: Vinod Kumar K, Lall C, Vimal Raj R, Vedhagiri K, Kartick C, Surya P, Natarajaseenivasan K, Vijayachari P, Overexpression of heat shock GroEL stress protein in leptospiral biofilm, Microbial Pathogenesis (2016), doi: 10.1016/j.micpath.2016.11.010. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Overexpression of heat shock GroEL stress protein in leptospiral biofilm
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K. Vinod Kumar1, Chandan Lall1, R. Vimal Raj1, K. Vedhagiri2, C. Kartick1, P Surya1, K.
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Natarajaseenivasan3, P.Vijayachari1. *
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and training in leptospirosis, Port Blair -744101, Andaman and Nicobar Islands, India
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Anna University, Chennai - 600 025, India
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Bharathidasan University, Department of Microbiology, School of Life Sciences Tiruchirappalli – 620 024,
India
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*Address for correspondence
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Dr.PaluruVijayachari
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Regional Medical Research Centre (ICMR),
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Post Bag No.13, Dollygunj, Port Blair 744101,
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Andaman & Nicobar Islands, India
Phone Number
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Fax Number
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E-mail
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Section
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+91 3192 251163
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[email protected]
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Short communication
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Proteomic analysis of Leptospiral biofilms
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National Hub for Healthcare Instrumentation Development, (NHHID), Centre for Biotechnology,
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Regional Medical Research Centre (ICMR), WHO Collaborating Centre for Diagnosis, Reference, research
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ACCEPTED MANUSCRIPT Abstract
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Leptospira is the causative agent of leptospirosis, which is an emerging zoonotic disease. Recent
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studies on Leptospira have demonstrated biofilm formation on abiotic surfaces. The protein
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expressed in the biofilm was investigated by using SDS-PAGE and immunoblotting in combination
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with MALDI-TOF mass spectrometry. The proteins expressed in Leptospira biofilm and planktonic
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cells was analysed and compared. Among these proteins, one (60 kDa) was found to overexpress in
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biofilm as compared to the planktonic cells. MALDI-TOF analysis identified this protein as stress
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and heat shock chaperone GroEL. Our findings demonstrate that GroEL is associated with
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Leptospira biofilm. GroEL is conserved, highly immunogenic and a prominent stress response
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protein in pathogenic Leptospira spp., which may have clinical relevance.
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Keywords: Biofilm, Leptospira, Hsp, Planktonic
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ACCEPTED MANUSCRIPT Introduction
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Leptospirosis is an emerging zoonosis caused by pathogenic spirochetes belonging to the genus
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Leptospira. The disease is transmitted to humans through environmental surface waters
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contaminated by the urine of reservoir host and domestic animals, which are chronically colonized
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with Leptospira. It is also reported that leptospires can survive in nutrient deficient conditions in soil
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and water, for long periods [1]. However, not much is known about the mechanisms by which
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pathogenic leptospires persist in aqueous environments, chronically infected mammals and carrier
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hosts. It is widely accepted that bacteria can exist in two different modes of growth, the first being
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as single planktonic cells and the second as structured, multicellular communities known as biofilms
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[2]. Biofilm formation in Leptospira is a recently studied phenomenon and is demonstrated in
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environment and in vitro [3, 4]. Leptospira biofilm may play a significant role, not only in
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environmental survival but also for successful infection and pathogenesis [5]. Similar to other
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bacterial biofilms Leptospira biofilm is a complex structural arrangement of bacteria that are
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enclosed in an extracellular polymeric substance (EPS)[3]. However the EPS of the leptospiral
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biofilm are yet to be studied.
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Polysaccharides, proteins, nucleic acids, metal ions and other humic materials can be associated
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with the EPS matrix of the biofilms. Embedded cells within this EPS matrix can protect the bacteria
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from environmental stress and may also offer protection from the host immune responses [6]. A
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detailed understanding of the biofilm matrix composition especially proteins are critical for the
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rational understanding of numerous clinical and environmental implication associated with biofilms
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[7].
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The architecture of leptospiral biofilms are rather well studied. However, the molecular mechanism
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of biofilm formation remains to be explored. Proteomic comparison of other bacterial biofilms has
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revealed the presence of several differentially expressed proteins. Heat shock proteins (HSPs) are
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the major and best-studied proteins known for their involvement in bacterial biofilm[8]. These HSPs
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are considered to be important in the pathology of various bacterial and parasitic infections. They
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are reported to protect pathogens against the hostile environment of host phagocytic cell [8]. In this
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study, an attempt was made to analyse the comparitive proteomics of the proteins in planktonic and
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Leptospira biofilm.
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1.
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1.1. Bacterial strains and growth conditions
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Leptospira interrogans strain, Salinem, was originally isolated from human, while Leptospira fainei
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is of animal origin. All strains were obtained from national reference centre for leptospirosis at the
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Regional Medical Research Centre, Port Blair, India. These strains were sub-cultured at 30 °C in
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liquid Ellinghausen-McCullough-Johnson-Harris (EMJH) medium with fatty acid supplements
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(Bovine Serum Albumin + Tween 80) with 1% BSA and were maintained with periodic sub
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culturing every 7 days.
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1.2. Biofilm formation
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The biofilm formation assay was performed for the two strains, L. Interrogans Salinem and L.
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fainei But-6, in EMJH liquid medium, as per the methodology described earlier [9]. Briefly, the
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biofilms were allowed to form in U-bottom 96-well (polyvinyl plate) tissue culture by incubating
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at 30°C for 10 days, without shaking. Every 24 hrs the growth medium was discarded and freshly
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added. Fresh EMJH medium was added to the wells as a control. Each well was washed thrice
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with phosphate-buffered saline (PBS) under aseptic conditions to eliminate unbound bacteria.
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The experiment was repeated at least thrice for the reproducibility. The ability of these strains to
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form biofilm was determined as per the crystal violet staining method, as described by O'Toole [10]
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1.3. Microscopic examination
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Leptospira strains were grown in the EMJH medium to approximately 2 x 108 cells mL-1 (without
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shaking). Sterile glass slides (76 x 26 mm, Hi-Media) were submerged (20 mm) in 50 mL of
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leptospiral cell suspension in EMJH growth medium (initial concentration of 2 x 105 cells mL -1)
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and incubated at 30°C for 7-12 days. After incubation, slides were carefully removed and rinsed
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two twice with PBS (pH- 7.4). One side of the slide was wiped with 70% ethanol and the attached
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Germany)[9].
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1.4. SDS-PAGE and immunoblotting of Biofilm and planktonic cells
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L. interrogans Salinem biofilm was grown in 5ml EMJH liquid medium in a wide mouthed glass
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with stating inoculum size of ~105 cells ml-1 (1:10, vol/vol), incubated at 30°C for 4 to 7 days
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without shaking [11]. Biofilm formation was achieved at air liquid interphase on the walls of the
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tube, the unbounded cells were aspirated and attached cells were gently rinsed with PBS (pH-
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7.4). Biofilm was collected by scraping, using sterile plastic loop. Detachment of the leptospires
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from the biofilm was carried out as described earlier [9]. Further, ethanol insoluble EPS was
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precipitated along with leptospiral cells overnight at 4 °C with 3 volumes of ice-cold ethanol. The
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content was centrifuged at 15000 rpm for 30 min and the pellet was subjected to protein
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estimation and further solubilised in 2X SDS-PAGE sample buffer with 1% 2-mercaptoethanol.
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Similarly, in planktonic (unbound cells) leptospiral cells were centrifuged at 12,000 g for 10 min.
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The cell pellet was washed twice in 5 mM MgCl2-phosphate buffered saline (PBS) and protein was
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estimated using BCA method. The protein concentration was optimised to proteins from biofilm
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state and solubilised in 2 X SDS-PAGE sample buffers. After boiling, the content was loaded on a
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10% polyacrylamide gel with pre stained protein marker as a standard (Bio-rad, USA) and
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electrophoresed [12].The resulting lysates of the two phenotypes were utilised as antigens. Pooled
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sera of acute phase leptospiraemic patient’s (Microscopic Agglutination Test Positive and IgM
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ELISA Positive sera) were used as primary antibody for IgM recognition in immunoblotting as per
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the methodology described elsewhere [13, 14].
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1.5. Identification of over expressed proteins by MALDI – TOF
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The target protein band was excised from SDS-PAGE and mass spectrometry analysis was carried
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out. The protein bands were digested following the standard protocol [15]. Peptide mass fingerprint
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was measured on an Opti-TOF 384 well insert (Applied Biosystems/MDS Sciex, Foster City, CA)
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with 0.3 µl of 5 mg ml-1 alpha-cyano-4-hydroxycinnamic acid (Aldrich, St. Louis MO) in 50%
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ACCEPTED MANUSCRIPT CH3CN, 50% 0.1% trifluoroacetic acid. Crystallized samples were washed with cold 0.1%
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trifluoroacetic acid and were analyzed by an Applied Biosystems 4800 MALDI TOF/TOF
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Proteomics Analyzer. An initial MALDI MS spectrum was acquired for each spot (400 laser shots
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per spectrum) and a maximum of 15 peaks, with a signal-to-noise ratio of greater than 20 were
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automatically selected for MS/MS analysis (1000 shots per spectrum) by post-source decay or by
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collisionally-induced dissociation using air at a pressure of 5e-7Torr. Peak lists from the MS/MS
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spectra were submitted for database similarity searching using Protein Pilot (Applied Biosystems)
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Ver. 2.0, Rev. 50861. The involvement of the identified proteins in the molecular function and
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biological process were assigned according to the gene ontology database
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(http://www.geneontology.org) and the Swiss prot/uniprot database (http://beta.uniport.org).
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1.6. RNA extraction and cDNA synthesis
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Total RNA from 1 ml of leptospiral biofilm and planktonic cells from L. interrogans strain Salinem
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was extracted using Trizol reagent (Invitrogen, USA) in accordance with manufacturer’s
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instructions. The air dried RNA pellets were re-suspended in diethyl pyrocarbonate water. RNAase
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free DNAase (Fermentas) was used to remove the genomic DNA content and the concentration was
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estimated at optical density at 260/280 nm. 200 ηg of total RNA was reverse transcribed using
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EuroScript RT kit (Euorgentec, Belgium). After RT, 0.25 U of RNAase (Fermentas) was added and
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the mixture was incubated at 37°C for 20 min to remove any residual RNA from the reaction
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mixture. The cDNA was stored in -20°C until use.
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1.7. Real-time quantitative (qRT- PCR)
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The real time quantitative RT- PCR for the cDNA targeting GroEl gene was performed, using
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16sRNA gene as calibrator. Primers (Table 1) were designed using primer3
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(http://frodo.wi.mit.edu/primer3/). The qRT- PCR was performed for the final volume of 25 µl
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which contained 100 nm each of forward and reverse primer, 1X reaction buffer (2.5 mMdNTPs
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including dUTP, 0.25U Meteor Taq DNA polymerase, 4 mM MgCl2 and SYBR green I). Samples
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in duplicate were kept in 96 well plates and amplified in an automated real time PCR machine
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ACCEPTED MANUSCRIPT (7500, Applied bio-systems). The PCR conditions were followed, viz, 95°C for 5 min, 40 cycles of
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95°C for 15 Sec and 60°C for 1 min. The melt curve was performed at a holding step of 50°C.
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Quantification was carried out using the comparative cycle threshold (CT) and demonstrated relative
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transcription or the (2-∆∆ Ct) n-fold difference relative to calibrator gene.
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2. Results and discussion
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The biofilms of L. Interrogans Salinem and L. fainei But-6 were examined by using DFM. Briefly,
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leptospiral cells were allowed to adhere on glass slides, glass tubes and wells of PVC plates. These
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were observed by DFM at different time intervals (Fig. 1). Leptospira formed a dense layer on glass
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slides, which was observed to increase in a time-dependent manner, reaching a stationary phase after
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4-10 days. In addition, surface attached biomass (Biofilm) of Leptospira was quantified after 7 days
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by crystal violet staining. L. interrogans Salinem and L. fainei But-6 yielded surface attached
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biomass at OD550 nm =1.08 ± 0.06 SD and OD550 nm =1.60 ± 0.17 SD respectively (Fig. 1, IV). The
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formation of biofilm at the air liquid interphase by L. interrogans Salinem was described earlier [3]
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as observed in many other leptospiral strains. However, in this study, L. fainei But-6 was used for
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first time which had comparatively higher intensity of attachment than the L. interrogans Salinem.
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Salinem showed low affinity to bind to abiotic surface and there was a delay in adherence (after 6
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days) when compared to the strain, But 6.
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Protein extracted from biofilm and planktonic cells were visualized by SDS – PAGE. Although
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several bands were observed in the biofilm state, protein band with molecular weight, 60 kDa from
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the biofilm cells (Fig. 2, A) was overexpressed, as compared those in planktonic cells.
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Immunoblotting revealed that this overexpressed protein reacted with the pooled sera of the patient,
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and was immunoreactive with a molecular mass of 60 kDa (Fig. 2, B). The protein identities
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including its theoretical and experimental molecular weights, pIs, sequence coverage and MASCOT
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score was noted. The sequence coverage is the fraction of the complete protein sequence identified
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and MASCOT score is given as S= -10 x log (P), where P is the probability that the observed match
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would be a random event. MASCOT score values higher than 80 are considered to be significant (P
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ACCEPTED MANUSCRIPT < 0.05). The analysis revealed that 60 kDa protein band corresponded to leptospiral chaperon
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protein GroEL (PI=5.30) with a protein homology similar to L. interrogans serovar Lai str. IPAV.
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A recent study by Iraola, Spangenberg [5] using RNAseq of Leptospira biflexa biofilm also showed
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several chaperon proteins to be differentially expressed, i.e; dnaJ and dnaK, grpE (HSP),
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LEPBI_I1822, LEPBI_I3214, LEPBI_II0248, LEPBI_I0452 and LEPBI_I0885. However, GroEL
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was not observed in their differentially expressed gene data. This may be due to the conserved
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sequence of GroEL proteins among L. interrogans.
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The genomospecies L. biflexa is a free living saprophytic bacteria, even though it shared majority of
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genes with its pathogenic counterpart (L. interrogans) and are involved in metabolic function and
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environmental survival. L. biflexa is genetically distinct and lack the genes involved in pathogenesis
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and host adaptations [16]. Therefore, it was worthwhile to study the proteomics involved in the
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biofilm of pathogenic and intermediated strains such as L. interrogans Salinem and L. fainei But-6.
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Moreover, these strains showed comparatively higher biofilm formation in our study[9].
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GroEL belongs to the chaperonin family of molecular chaperones. It is present in several bacterial
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fractions, including the cytosol, cell membrane, and extracellular material [17]. It is essential for
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biofilm formation in the both Gram negative and Gram positive bacteria [18]. GroEL are stress-
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response proteins that have important roles inducing generation or alteration of proteins involved in
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adhesion and maintenance of biofilm [19] and also promotes refolding of misfolded polypeptides,
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especially under stressful conditions and high cell density [20]. GroEL is reported to perform a
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regulatory function that is not required in planktonic growth but is needed for biofilm maturation
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[21].
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GroEL protein plays a significant role in the adherence and subsequent biofilm formation in many
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bacterium, viz, Mycobacteriumsmegmatis [21], Clostridium difficile [22], Haemophilus influenza
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[18], Streptococcus mutans [23] and Campylobacter jejuni [2] ect. However, the role of GroEL in
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adherence mechanism is yet to be explored.
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In leptospires, Brihuega, et al (2012) observed cell aggregation of L. interrogans serovar Pomona, in
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the placental tissue of pregnant guinea pigs[24]. We hypothesize that the cell adherence and biofilm
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ACCEPTED MANUSCRIPT formation could be occurring in kidney tubular cells and might be a major factor in chronic
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leptospirosis of animals as well as in humans. Role of GroEL (HSP60) in Leptospira during these
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situations need to be studied.
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The expression study by real time PCR showed that during the attachment of leptospiral cell at air-
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liquid interface, an up-regulation of GroEl mRNA synthesis (Fig 2, C) occurs. Up-regulation of
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GroEl protein during biofilm formation suggests the importance of GroEl in the process. HSPs are
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expressed at basal level during normal growth conditions but are over expressed during stress. In
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Leptospira, this condition can occur due to abnormal temperatures, poor nutrient conditions, adverse
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host factors, oxidative stress etc. [1, 3, 25]. Remarkably, at higher temperatures, L. interrrogans has
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shown to upregulate the synthesis of stress response and HSP’s, including the chaperones, DnaK,
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GrpE, and GroEL, as well as the HSP100/Clp chaperone ClpB and proved essential for the virulence
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and resistance to various environmental stress [26]. In the host, HSPs are considered to be the major
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antigens among a number of bacterial pathogens and several micro-organisms, which express
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immunogenic GroEL homologues, giving rise to an immunogenic host response [27]. Bacterial
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infections may cause up-regulation of bacterial HSPs, in response to various stress conditions.
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This study only focused on single HSP, However there could be other important proteins in co-
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expression network which may have significant role in leptospiral biofilm. Especially proteins
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involved in chemotaxis, synthesis of EPS material, Outer membrane proteins (OMPs), Metabolism
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of sugars and lipids etc.[5]
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In the immunoblot assay using pooled patient sera, it was found that the GroEL protein (60 kDa)
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produced by the biofilm of both the strains was overexpressed and immunogenic. This is indicative
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of the interaction of leptospiral cells with host during infection. In our earlier study, GroEL was
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identified as one of the common immunoreactive protein and was suggested for use in the diagnosis
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of leptospirosis[28]. GroEL (HSP60) proteins has been identified as an immunogenic protein and
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predicted as a potential vaccine candidate against bacterial infections in animal models, viz.
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Salmonella typhi [29], Streptococcus pneumonia [30], Bacillus anthracis[31].
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ACCEPTED MANUSCRIPT Similarly in our previous study, GroEL (HSP60) was identified to be one of the few proteins
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eliciting long lasting immune response up to 4 years (data no shown). GroEL in some bacteria can
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induce high antibody titers, promote lymphocyte proliferation, and induce both humoral and cell-
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mediated immune [32]. Furthermore, investigations are needed to evaluate the capacity of GroEL to
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trigger a protective immune response against Leptospira.
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Bacterial growth in a biofilm provides many advantages for them, including, enhanced resistance to
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environmental stress, such as desiccation and antimicrobials, as well as an increased resistance to
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host defence mechanisms. For the first time, we demonstrated the proteomic analysis of Leptospira
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biofilms for the pathogenic strains. Proteomic comparison of biofilm-grown and planktonic cells
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demonstrated the difference in protein expression between the two states of growth, particularly in
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the expression of one of the proteins involved during stress response. In conclusion, we provide
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evidence in this communication that GroEL of Leptospira may have involvement in bacterium
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adhesion as in other bacteria. Further investigations are needed to determine the mechanism of
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GroEL in leptospiral biofilm formation.
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Acknowledgement
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The authors are thankful to the Department Of Science & Technology (DST), Government of India,
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Ministry of Science and Technology for providing the extramural grant under Science and
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Engineering Research Board (SERB), DST/SERB no. SR/SO/HS-0114/2012 for the study. The
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authors are thankful to Dr. I. P. Sunish for his help in preparation of this manuscript.
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Ethical clearance – This study was approved by Institutional Ethics Committee
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Conflict of Interest: no conflict of interest declared
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[28] Natarajaseenivasan K, Artiushin S, Velineni S, Vedhagiri K, Vijayachari P, Timoney J. Surface-
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associated Hsp60 chaperonin of Leptospira interrogans serovar Autumnalis N2 strain as an immunoreactive
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protein. Euro J Clin Microbiol Infect Dis. 2011;30:1383-9.
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[29] Panchanathan V, Naidu BR, Devi S, Di Pasquale A, Mason T, Pang T. Immunogenic epitopes of
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Salmonella typhi GroEL heat shock protein reactive with both monoclonal antibody and patients sera.
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Immunology letters. 1998;62:105-9.
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[30] Khan MN, Shukla D, Bansal A, Mustoori S, Ilavazhagan G. Immunogenicity and protective efficacy of
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GroEL (hsp60) of Streptococcus pneumoniae against lethal infection in mice. FEMS immunology and medical
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microbiology. 2009;56:56-62.
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[31] Sinha K, Bhatnagar R. GroEL provides protection against Bacillus anthracis infection in BALB/c mice.
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Molecular immunology. 2010;48:264-71.
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[32] Yi L, Wang Y, Ma Z, Lin HX, Xu B, Grenier D, et al. Identification and characterization of a
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Streptococcus equi ssp. zooepidemicus immunogenic GroEL protein involved in biofilm formation. Veterinary
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research. 2016;47:50.
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ACCEPTED MANUSCRIPT Figure legends
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Figure 1.
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A. Biofilm formation by Leptospira, L. fainei But-6; (i) planktonic cells and (ii) biofilm allowed toform on the
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glass slide, observed under Dark Ground Microscope (DFM) under 20X magnification. B. Demonstration of
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biofilm formation at the air liquid in a 10 ml glass tubes, C. Biofilm formation at the air liquid interface in the
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wells of 96 well Polyvinyl chloride U bottom plate and D. Crystal violet staining of the biofilm formed by
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Leptospiral strain in comparison to planktonic cells. PL-1: Planktonic cells of L. interrogans Selinem, PL-2:
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Planktonic cells of L. fainei But-6; BA-1: Biofilm of L. interrogans Selinem; BA-2: Biofilm of L. fainei But-6.
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Figure 2. A: SDS-PAGE and B: immunoblots assay. P: Planktonic Leptospira whole cell lysate and BF;
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biofilm whole cell lysate.
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C) 16S rRNA and GroEL gene expression in culture in EMJH medium pathogenic Leptospira. RT-PCR was
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performed to demonstrate leptospiral 16S rRNA (calibrator) and GroEL (test) gene expression. Comparative
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cycle threshold (CT) and demonstrated relative transcription or the (2-∆∆ Ct) n-fold difference relative to
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calibrator gene (the error bar is SE).
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Table 1. List of primers used Sl no.
Genes
Primers sequence (5’->3’)
Product length
Forward 5’- AAATTCGGAGCACCTACCAT -3’ 1
GroELint
125 bp
Reverse 5’- ACGTCGTTCGTCTTAGTGGA-3’ Forward 5’- GTCGGAATCGCTAGTAATCG -3’
185 bp
16s rRNA Reverse 5’- CCGATACGGCTACCTTGTTA -3’
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Highlights •
Biofilm formation in Leptospires is a new area of study and its molecular mechanism has not been fully explored yet. This study demonstrate the over expression of a stress protein associated with the Leptospira biofilm formation.
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GroEL is conserved, known to be highly immunogenic and a prominent stress response protein
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among Leptospira spp., which may have the clinical relevance
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This study will help in understanding the mechanisms of biofilm formation in Leptospira.
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