Exoproteome from Leptospira interrogans and Host Cells during Infection: Leptospiral Virulence Factors and Cellular Proteins Involved in Stress and Inflammation


 Background: Leptospirosis, caused mainly by Leptospira interrogans, is a global zoonotic infectious disease. Macrophages and vascular endothelial cells are the main host cells for L. interrogans during infection, but the proteins released from the pathogen and the two host cells during infection remain mostly unknown.Results: Cellular supernatant proteins (CSPs) from human THP-1 macrophages or umbilical vein endothelial cells (HUVECs) infected with L. interrogans strain Lai were extracted by TCA/FASP methods. The exoproteins in the CSPs were identified by LC-MS/MS. Viability of the leptospires and host cells during infection was confirmed by confocal microscopy and MTT. The results showed that higher co-culture temperature (from 28°C to 37°C) and different biochemical environments cause a large change in the exoproteome of the spirochete. L. interrogans increased levels of leptospiral exoproteins related to stress, signal transduction and virulence factors, while the lipoprotein antigens LipL41, LipL21 and/or Loa22 were not detected. During infection of macrophages and endothelial cells, there was a large increase in host-cell exoproteins involved in stress response, complement pathways (C4/5/7/8), inflammatory cytokines (IL-6, TNF-α, MIF, MCP-1 and GM-CSF), extracellular matrix proteins (FN, LN and COLs), and blood coagulation factors. One-third of the leptospires and infected THP-1 macrophages died during macrophage infection, but nearly all the leptospires and endothelial cells remained viable during endothelial cell infection.Conclusions: Infection causes stress reponses for both leptospires and human macrophages and vascular endothelial cells and release of virulence factors, alteration of surface leptospiral lipoprotein antigens and secretion of complement components and inflammatory cytokines from host cells.

. Therefore, the diffusion from the bloodstream into internal organs and tissues of pathogenic Leptospira species is a key step for the progression and aggravation of leptospirosis.
Infection results from the interaction between microbial pathogens and their hosts. During the process of invasion, surface and released proteins act as the crucial molecular determinants for the pathogens to respond to the microenvironment of infected tissues by change in their expression levels [13]. Previous studies showed that the transcription and expression levels of many genes of pathogenic Leptospirainterrogans during infection of cells are signi cantly changed [14,15]. For example, the expression levels of virulence factors such as adherence factors, invasive enzymes and toxins of L. interrogans are signi cantly up-regulated during infection, but those of surface protein antigens are notably decreased [16][17][18][19][20][21][22][23][24][25]. However, it is believed that only released or surface proteins of microbes can play a direct role in the pathogenic process. Nonetheless, until now, the exoproteome from host cells and leptospires during L. interrogans infection remains poorly understood.
During the process of microbial invasion, the host cells can also respond by changing of their protein expression pro les. Macrophages play an important role in the innate immune response to infection by phagocytosis of the invaded pathogens and the adaptive immunity by presentation of microbial antigens [26]. In addition, macrophages from the bloodstream but not neutrophils have been shown to be the main in ltrating phagocytes in the infectious tissues of leptospirosis patients and L. interrogans-infected animals [27,28]. A previous study reported that the transcription levels of many genes in both human and murine macrophages were altered during infection with L. interrogans [29]. In many leptospirosis patients, leptospires can spread from the bloodstream to internal organs and tissues through small blood vessels by transcytosis [12,30]. Therefore, macrophages and vascular endothelial cells are the two types of host cells suitable for characterization of the exoproteome during leptospiral infection.
Pathogenic Leptospira consists a number of genospecies, in which L. interrogans is the most predominant causative agents of human leptospirosis in the world [31,32]. Although many serogroups and serovars of L. interrogans are prevalent in China, L. interrogans serogroup Icterohaemorrhagiae serovar Lai is responsible for disease in over 60% of leptospirosis patients [4,33]. In addition, the genomic sequence of L. interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai (No. 56601) is known [34]. Therefore, in this study, the proteins released from L. interrogans strain Lai, human macrophages and vascular endothelial cells during infection were characterized. The exoproteome pro les from the pathogen and two types of host cells during infection were examined and the functions of the leptospiral and cellular exoproteins were also analyzed.

Results
Overview of CSPs and exoproteins from L. interrogans and host cells before infection.
The ow cytometric examination showed that 95.7% of THP-1 monocytes were differentiated into CD68 + macrophages after PMA treatment ( Figure 1A). The SDS-PAGE examination showed the pro les of CSPs from L. interrogans strain Lai cultured in EMJH medium at 28°C or in 2.5% FCS RPMI-1640 medium at 37°C for 24 h and from THP-1 macrophages and HUVECs cultured in 2.5% FCS RPMI-1640 medium at 37°C for 24 h ( Figure 1B). The LC-MS/MS identi ed 57 or 61 leptospiral exoproteins from EMJH (28°C) or RPMI-1640 (37°C) medium but 62.3% (38/61) of the exoproteins were different (Table 1 and 2). In addition, the LC-MS/MS identi ed 27 or 28 exoproteins in the CSPs from THP-1 macrophages or HUVECs during incubation and 75.0% (21/28) of the exoproteins from different cells were identical (Table 3 and   4). The category analysis indicated that the main exoproteins of the spirochetes from EMJH-medium at 28ºC were outer membrane/surface proteins and lipoproteins but the toxic proteins from the spirochetes were notably increased during incubation in RPMI-1640 medium at 37ºC ( Figure 1C). The data suggested that L. interrogans largely changed its released proteins in response to higher environmental temperature and different biochemical environments.
Increased secretion of exoproteins from L. interrogans during infection of cells.
The SDS-PAGE showed the pro les of total CSPs from co-cultures of L. interrogans strain Lai with THP-1 macrophages or HUVECs (Figure 2A). The LC-MS/MS identi ed 115 or 98 leptospiral exoproteins in the CSPs during infection of THP-1 macrophages or HUVECs, of which 45 or 40 exoproteins were also present in the leptospiral CSPs from EMJH and/or RPMI-1640 media and 25 exoproteins were identical during infection of THP-1 macrophages and HUVECs but 45 or 33 exoproteins were different during infection of THP-1 macrophages or HUVECs (Table 5 and 6). The heatmaps of leptospiral exoproteins during infection of cells were shown in Figure 2B. The outer membrane/surface proteins (33.87%) and toxic proteins (20.62%) were the main exoproteins of L. interrogans during infection ( Figure 2C). Compared to the exoproteins from L. interrogans before infection, oxidoreductases, virulence factors and two-component signaling proteins were found as the main increased leptospiral exoproteins during infection (Table 5 and 6). In particular, outer membrane lipoprotein 32 (LipL32) was present in all the CSPs before and during infection of cells but OmpA family lipoprotein 22 (Loa22) and LipL41 disappeared during infection of THP-1 macrophages and LipL21 and LipL41 were absent during infection of HUVECs (Table 1, 2, 5 and 6). The data suggested that L. interrogans changes its metabolism during infection of human macrophages and vascular endothelial cells by increasing secretion of exoproteins involved in virulence, oxidative stress and signal transduction but decreasing secretion of some lipoprotein antigens.
Increased secretion of exoproteins from host cells during infection with L. interrogans.
The LC-MS/MS identi ed 95 or 163 cellular exoproteins in the total CSPs from co-cultures of L. interrogans strain Lai with THP-1 macrophages or HUVECs, of which 26 or 27 exoproteins were also present in the CSPs from THP-1 macrophages and/or HUVECs incubated in RPMI-1640 medium and 40 exoproteins were identical in the CSPs from THP-1 macrophages and HUVECs during infection but 24 or 90 exoproteins were different from THP-1 macrophages or HUVECs during infection (Table 7 and 8). The heatmaps of cellular exoproteins during infection were shown in Figure 3A and 3B. In the exoproteins, stress/immune response proteins (38.61% for THP-1 macrophages and 39.14% for HUVECs), such as multiple heat shock proteins (HSPs), oxidoreductases and complement components, and adhesion proteins (21.67% for THP-1 macrophages and 20.81% for HUVECs), such as bronectin (FN), laminin (LN) and many collagens (COLs) in extracellular matrix (ECM), were as the main exoproteins ( Figure 3C, Table   7 and 8). In addition, the THP-1 macrophages released more antibacterial response proteins (17.97%) and fewer inhibitor activity proteins (1.92%) than HUVECs (6.14% and 13.16%) during infection ( Figure 3C). In particular, THP-1 macrophages or HUVECs during infection released different in ammatory cytokines (IL-6 and TNF-α from THP-1 macrophages, MCP-1 and GM-CSF from HUVECs) ( Table 7 and 8). Interestingly, both types of host cells released multiple blood coagulation factors during infection, such as coagulation factor X, thrombospondin-1/4 and prothrombin. The data suggested that L. interrogans also causes a signi cant metabolic change in human macrophages and vascular endothelial cells by increasing secretion of exoproteins involved in heat/oxidative stress, in ammation and immune response.
Functional classes of leptospiral exoproteins before and during infection.
The main exoproteins of L. interrogans strain Lai incubated in EMJH medium at 28°C play roles in twocomponent signaling system and agellar assembly, but secretion of leptospiral ABC transporters was increased during incubation in RPMI-1640 medium at 37°C ( Figure 4A and 4B). When the spirochetes were incubated with THP-1 macrophages and HUVECs, there was a signi cant increase in leptospiral exoproteins involved in oxidoreduction and metabolism for the stress response ( Figure 4C and 4D). The data suggested that L. interrogans expresses more exoproteins to response to the infection of human macrophages and vascular endothelial cells.
Functional classes ofcellular exoproteins before and during infection.
The main exoproteins of THP-1 macrophages and HUVECs incubated in RPMI-1640 medium play roles in protein binding, extracellular exosome and signal transduction ( Figure 5A and 5B). When the cells were infected with L. interrogans strain Lai, the cellular exoproteins with functions in complement and coagulation cascades, ECM-receptor interaction and cytoskeleton rearrangement signal transduction were signi cantly increased ( Figure 5C and 5D). The data suggested that human macrophages and vascular endothelial cells also express more exoproteins in response to the infection with L. interrogans.

Viability of L. interrogans and host cells during infection
The confocal microscopic examination showed that 19.2%-34.6% of the leptospires during infection of THP-1 macrophages had died, but nearly all of the leptospires during infection of HUVECs were still alive ( Figure 6A and 6B). On the other hand, the MTT test showed that the THP-1 macrophages showed 65.6% viability percentage at 24 h during infection with L. interrogans strain Lai but nearly all of the HUVECs persistently maintained their viability during the whole infection process ( Figure 6C). The data suggested that L. interrogans and human macrophages are mutually damaged during infection, but both leptospires and host cells remained viable during infection of vascular endothelial cells.

Discussion
Infection is an interactive process that takes place between pathogens and hosts [23,35]. During this interaction, hosts raise body temperature and mount as in ammatory reaction in order to eliminate the pathogens, but conversely, prokaryotic pathogens also change their metabolism in order to respond to the adverse environment for survival in the hosts. Host and pathogen proteins, including enzymes, play a major role in host defense against the pathogen and metabolic changes in the pathogen for environmental adaptation. Therefore, a characterization of exoproteins released from L. interrogans and host cells during infection can enable us to further understand the interaction between the pathogen and host.
The Leptospira genus includes a large group of helical, didermal prokaryotic microbes that can be classi ed into pathogenic and non-pathogenic saprophytic Leptospira genospecies [32]. The optimal growth temperature of Leptospira in medium in vitro is 28°C. At this culture temperature, L. interrogans strain Lai in EMJH medium released the OmpA family lipoprotein 22 (Loa22) and outer membrane lipoproteins 21, 32 and 41 (LipL21, LipL32 and LipL41). When the spirochetes were incubated in cellular RPMI-1640 medium, LipL21 and LipL41 were absent. The two lipoproteins have been described as the major surface antigens of pathogenic Leptospira species [36][37][38]. In addition, most of Clp family proteins, which is composed of chaperones/protease complexes responsible for degradation of abnormal proteins, also disappeared in the CSPs from L. interrogans strain Lai during incubation in cellular RPMI-1640 medium at 37°C while co-chaperonin GroEL and chaperone DnaK, also called heat shock protein 60 cofactor (Co-HSP60) and HSP70, and VagC, a toxic protein in the VagCB toxin-antitoxin module, were present in the supernatants [34,39,40]. The data indicated that higher cell-culture temperature and different chemical environments stimulate metabolic change of L. interrogans by decreasing expression of surface lipoprotein antigens and increasing expression of heat stress and virulence proteins.
interrogans can rapidly invade the bloodstream to cause septicaemia and diffuse from the bloodstream into internal organs and tissues, such as lungs, liver, kidneys and cerebrospinal uid [12,30,32]. On the other hand, macrophages but not neutrophils have been con rmed as the main in ltrating phagocytes involved in the immune response during leptospirosis [28]. Therefore, PAM-differentiated human THP-1 macrophages and HUVECs were used as host cells in this study to generate cell infection models of L. interrogans.
When L. interrogans strain Lai was incubated with THP-1 macrophages or HUVECs, the spirochete quickly increased secretion of exoproteins related to outer membrane/surface protein antigens, virulence, oxidative stress and two-component signaling systems. Among the exoproteins, von Willebrand factor type A (vWA) domain-containing proteins (LB_054/055), ColA collagenase (LA_0872), Sph2 and TlyA hemolysins (LA_1029/0327) have been con rmed as pulmonary hemorrhage inducers, invasive enzyme and in ammatory stimulators of L. interrogans, respectively [20,21,25]. Importantly, LipL21 and LipL41 disappeared from the supernatants during infection of THP-1 macrophages while Loa22 and LipL41 were absent during infection of HUVECs, but LipL32 was persistently present before or after infection of the two types of host cells. The data indicated that L. interrogans can adapt to each speci c host cell and respond to the environments by increasing expression of virulence factors and decreasing surface lipoprotein antigens.
Macrophages, which are also the main leptospiral phagocytes in leptospirosis [28], play an important role in innate and adaptive anti-infection immunity due to its ability to phagocytose pathogens and present their antigen. Vascular endothelial cells also participate in anti-infection immunity through in ammatory reaction by release of multiple cytokines [41]. When THP-1 macrophages and HUVECs were infected with L. interrogans in vitro, the two types of host cells released many HSPs, oxidoreductases, complement components and in ammatory cytokines (IL-6 and TNF-α from THP-1 macrophages, MCP-1, MIF and GM-CSF from HUVECs). In particular, the mitochondrial HSP10, HSP60 and HSP70 imply a role for mitochondrial stress in the two types of host cells during infection [42]. TLR4 is often considered as an important pattern recognition receptor on phagocytes in response to bacterial infection [20,43]. In recent years, many components in the ECM of cells, such as FN, LN and COL1/3/4, have been shown to be receptors for many bacteria including L. interrogans [44]. In the present study, THP-1 macrophages during infection with L. interrogans expressed TLR4, FN and COL3, while the infected HUVECs presented FN, LN and COL3. The data suggested that human macrophages and vascular endothelial cells respond to infection of L. interrogans through rapid generation of a heat/oxidative stress response an in ammatory reaction, and increased ability to recognize pathogens.
Pulmonary hemorrhage is a typical histopathological change observed in leptospirosis patients [10][11][12]. von Willebrand factor (vWF) plays a crucial role in blood coagulation by inducing platelet aggregation and activating blood coagulation factors [45]. Our previous study revealed that the proteins containing vWF A-region domains (vWA) from L. interrogans cause pulmonary hemorrhage by competitive inhibition of vWF-mediated platelet aggregation [25]. In this study, THP-1 macrophages and HUVECs were shown to produce coagulation factor X during incubation in medium. To our surprise, during the infection process with L. interrogans, both THP-1 macrophages and HUVECs are predicted to prevent hemorrhage by secretion of many blood coagulation factors, such as prothrombin, brinogen, coagulation factor V, and thrombospondin-1/4. The infected THP-1 macrophages or HUVECs also released coagulation factor IX or vWF. A recent study revealed that coagulation factors such as VII/IX/X factors act as effective antibacterial agents [46]. On the other hand, approximately one-third of the leptospires and infected THP-1 macrophages died during infection of macrophages, but almost all the leptospires and infected HUVECs survived during infection of the endothelial cells. These results are consistent with previous reports describing the interaction between the L. interrogans and phagocytes (macrophages) or nonphagocytes (HUVECs) [30,36,37].
Taken together, our results show that infection of human macrophages or vascular endothelial cells by L. interrogans can lead to heat and oxidative stress responses for both the pathogen and host cells, but release of virulence factors from L. interrogans and in ammatory cytokines from both host cells. Moreover, the change in leptospiral surface lipoprotein antigens released during infection could be helpful in selection of suitable protein immunogens and development of speci c vaccines against leptospirosis.

Conclusions
Leptospirosis is a zoonotic infectious disease of global importance. Leptospira interrogans, a major pathogenic genospecies of this disease, can invade the bloodstream to cause septicaemia and then diffuse from the bloodstream into many internal organs and tissues. Monocyte-differentiated macrophages but not neutrophils are the main in ltrating phagocytes in the immune response against the pathogen in vitro and in vivo. Therefore, the interaction between L. interrogans and macrophages and vascular endothelial cells, and the neighboring tissues via their secreted exorpoteins, are expected to play a major role in the pathogenesis of leptospirosis. In this study, we found that both leptospires and host cells secreted many exoproteins during infection. The main exoproteins from the spirochete during infection were involved in stress responses, virulence and signal transduction while those from the infected cells were host stress proteins, complement proteins, in ammatory cytokines, extracellular matrix proteins and blood coagulation factors. Analysis of the secretome indicates that infection induces stress response in both pathogen and host cell, while leptospiral virulence factors and in ammatory cytokines from the host may alter the physiology of microenvironment and the host immune response.

Extraction of supernatant proteins from co-cultures
Supernatants of the leptospire-cell co-cultures were centrifuged at 12,000×g for 15 min (4°C) and then passed through 0.45 and 0.22 µm lters (Millipore, USA) in turn. Trichloroacetic acid (TCA, Sigma) was added into the ltered supernatants at a nal concentration 10% (W/V) TCA to precipitate the co-culture supernatant proteins (CSPs) overnight (4°C). After centrifugation at 17,200×g for 15 min (4°C), the CSP pellets were washed twice with ice-cold acetone and methanol to remove TCA [20,24]. In addition, the CSPs from L. interrogans strain Lai in EMJH medium at 28°C and THP-1 macrophages or HUVECs in 2.5% FCS antibiotic-free RPMI-1640 medium for a 6-, 12-or 24-h incubation at 37°C were also extracted as above. All the CSP extracts were dissolved in ultrapure double-distilled water (ddH 2 O, Millipore) for further analysis.

Examination of CSP extracts by SDS-PAGE
The CSP extracts were quanti ed using a BCA Protein Assay Kit (Thermo Scienti c, USA) and then examined under a Gel Image Analyzer (Bio-Rad, USA) after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Pretreatment of CSP extracts
The CSP extracts were puri ed using a ProteoPrep Blue Albumin and IgG Depletion Kit (Sigma) to remove FCS proteins and then treated by lter aided sample preparation (FASP) method as previously described [38][39][40]. Brie y, after determination of protein concentration as above, the CSP extracts were administrated with an ultra ltration lter (10 kDa units, Millipore) at 12,000×g. The lter was rinsed with 200 µL urea buffer (6 M urea-10 mM Tris-HCl, pH 6.8) and then incubated with 10 mM 1,4-dithiothreitol (DTT, Sigma) for 1 h at 37°C. After the centrifuge, the samples were alkylated in 40 mM iodoacetamide (IAA, Sigma) for 30 min at room temperature in dark. After washing with urea buffer, then washed twice with 50 mM ammonium bicarbonate, the harvested CSPs were hydrolyzed with trypsin (Sigma) at a 1:50 (enzyme: protein) mass ratio at 37 °C for 12 h. After the centrifuge, the hydrolytic peptides were collected and dissolved with 0.1% tri uoroacetic acid (TFA). The samples were desalted with Ziptip according to the instruction before freeze-dried preservation.

Label free LC-MS/MS detection
The trypsin-digested proteins from the hydrolyzed CSPs were identi ed by high performance liquid chromatography plus tandem mass spectrometry (LC-MS/MS) as previously described [23,53]. Brie y, 1 ug of the trypsin-digested protein preparation was dissolved in 5 μL of 0.1% formic acid (FA, Sigma) solution. The peptide solution was loaded onto an in-house packed C18 trap column (100 μm ID × 2 cm, 5 μm, Reprosil-Pur C18 AQ, Dr. Maisch, Germany) for separation in an in-house packed C18 analytical column (75 μm ID × 20 cm, 3 μm, Reprosil-Pur C18 AQ, Dr. Maisch) using mobile phase A solution (0.1% FA in ultrapure ddH 2 O) and 5-95% gradient mobile phase B solutions (0.1% FA in acetonitrile) for a 78-min separation with a 280 NL/min ow rate in a LC system (EASY-nLC-1000, Thermo Scienti c). The separated peptides were then identi ed in a MS/MS system (LTQ-Orbitrap-Elite, Thermo Scienti c) using positive ion mode. The full-scan fragmentation MS/MS spectrum (300-1600 m/z) of each of the peptides carrying 2-5 positive charges were obtained from the Orbitrap analyzer at a high resolution of 70,000 (m/z 200) with an automatic gain control and a maximum ll time of 60 ms. All the obtained fragmentation MS/MS spectral data were further analyzed using Xcalibur v2.2 software.

Determination of the leptospiral or cellular exoproteins
The LC-MS/MS-identi ed peptides were searched for their corresponding exoproteins from L. interrogans strain Lai, THP-1 macrophages or HUVECs based on match of amino acid sequences. The exoproteins from the spirochete were determined using both NCBI and UniProt/Swiss Prot databases while the exoproteins from THP-1 macrophages and HUVECs were determined using UniProt/Swiss Prot database.

Bioinformatic analysis
The categories of exoproteins from L. interrogans strain Lai, THP-1 macrophages and HUVECs were classi ed into clusters of orthologous groups (COGs) using MicroScope and GenoScope softwares [54].
The heatmaps and clusters of the leptospiral and cellular exoproteins were generated using Complex-Heatmap package and R software [55]. GO and KEGG pathway analysis including cellular component, molecular function and biological process in gene ontology were analyzed as previously described [23,56-58].

Detection of viabilities of L. interrogans and cells during infection
THP-1 macrophages or HUVECs were infected with L. interrogans strain Lai as described above. After trypsinization, the co-cultures were centrifuged at 500×g for 10 min (4°C) to precipitate the extracellular leptospire-free cells and the supernatants were centrifuged at 10,000×g for 30 min (4°C) to precipitate the extracellular leptospires. The cells and leptospires were washed twince with PBS and then centrifuged as above. The viability of infected cells was detected by methyl thiazolyl tetrazolium (MTT) test using a Cell Proliferation Kit (Sigma) while the viability of collected leptospires was detected by confocal microscopy (Zeiss, Germany) (485/630 or 485/530 nm excitation/emission wavelengths for SYTO 9 or PI detection) using a LIVE/DEAD Bacterial Viability Kit (Invitrogen, USA) and the fold changes of green uorescence intensity (FI) were analyzed for semi-quanti cation of the leptospiral viability [23,31]. In the tests, L. interrogans strain Lai, THP-1 macrophages and HUVECs without infection were used as the controls.

Statistical analysis
Data from a minimum of three independent experiments for detection of viability of L. interrogans, macrophages and HUVECs during infection were averaged and then presented as mean ± standard deviation (SD). Signi cant differences were determined by t and χ 2 tests. Statistical signi cance was de ned as p <0.05. Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Availability of data and materials
The data that support the ndings of this study are available from the corresponding author upon reasonable request.

Competing interests
The authors declare that they have no competing interests.