FabG moonlights as an extracellular adhesin mediates cytoadhesion of Streptococcus suis via interaction with plasminogen

Abstract

Streptococcus suis serotype 2 (SS2) is an important zoonotic bacterial pathogen that can causes meningitis, septicemia, endocarditis, arthritis, pneumonia, and streptococcal toxic shock-like syndrome in pigs and humans. FabG, an essential reductase in the bacteria type II fatty acid synthesis pathway, is present in the extracellular space and binds to many extracellular matrix components of the host. In this study, we investigated the moonlight function of FabG. First, we found that the transcription of FabG in virulent SS2 strains was significantly higher than in avirulent strains, indicating that FabG may be involved in the pathogenesis of S. suis. After preparation of recombinant proteins, we found that the recombinant FabG could directly bind to host epithelial cells. ELISA, SPR, and Far-Western blot assays showed that rFabG could bind to the host plasminogen. Furthermore, the ability of rFabG to recruit host plasminogen to degrade the host ECM component was visualized by electron microscopy. Moreover, we found that rFabG could bind to complement C3 in serum and enhance the survival ability of S. suis in serum. Collectively, these data imply that FabG is a moonlight adhesin involved in S. suis cytoadhesion and a plasminogen receptor that could help S. suis break through the host barrier or evade immune defenses.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12866-025-04129-7.

Introduction

Streptococcus suis (S. suis), is an important zoonotic bacterial pathogen associated with meningitis, septicemia, and pneumonia [1]. Among the 29 serotypes described, Streptococcus suis serotype 2 (SS2) is one of the most important serotype associated with streptococcosis in pigs and humans [2]. Two deadly human S. suis outbreaks occurred in China in 1998 and 2005 [3, 4]. Although SS2 is most frequently recovered from diseased animals [4], similar to other species of bacteria, different isolates, even in the same serotype of S. suis, can demonstrate different virulence [5], many SS2 isolates can be isolated from healthy pigs.

Plasminogen (Plg) is a 92-kDa single-chain glycoprotein that circulates in plasma as a zymogen. It can be activated and converted to proteolytically active plasmin, which can degrade the extracellular matrix (ECM) and fibrin clots, and block the complement cascade by degrading C3 and C5 [6]. Bacteria can recruit plasminogen via the expression of specific plasminogen receptors to facilitate colonization and evasion from clearance by the immune system [7]. Generally, plasminogen receptors are located on bacterial surfaces, such as the M protein in Group A Streptococci [8]. Recently, several proteins, usually considered to be restricted to the cytoplasm, have been found on the bacterial cell surface or extracellular space, and are involved in interactions with plasminogen, such as glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [9], elongation factor Tu (ef-Tu) [10] and α-enolase [11, 12]. Multiple subcellular localizations of these proteins have been confirmed in multiple species of bacteria [13]. However, these proteins are often metabolically essential for bacterial survival, preventing the construction of isogenic knockout mutants. Thus, research focusing on the moonlight function of these proteins is limited at protein level.

FabG, a bacterial 3-ketoacyl-ACP reductase, catalyzes the essential keto reduction step in the elongation cycle of fatty acid synthase [14, 15]. This enzyme is the only known isozyme of this type in bacteria, and is highly conserved and ubiquitously expressed in bacteria [15]. Thus, FabG has become the focus of numerous attempts to develop antimicrobials as potential drug target [16–18]. In our previous study, we found that FabG of S. suis could be detected in the extracellular space and binds to many extracellular matrix components of the host [19], suggesting a moonlight function of FabG.

In this study, we discuss the moonlight function of FabG and how it contributes to cytoadhesion and exploits host plasminogen/plasmin to assist the pathogenesis of S. suis.

Materials and methods

Bacterial strains and cultures

SS2 strain ZY05719, the representative strain of the Sichuan Outbreak in 2005 in China, was cultured in Todd Hewitt Broth (THB; Becton Dickinson, USA) and used as the model bacteria in this study. E. coli strains DH5α and BL21 (DE3) were cultured in Luria-Bertani broth. Positive recombinant vector transformants were screened using 50 µg/mL ampicillin (Sigma-Aldrich).

Except ST7 virulent strain ZY05719, two other highly virulent strains HA9801 (the representative strain of the Jiangsu Outbreak in 1998 in China, ST7), P1/7 (the classical European highly virulent strain, ST1), and an avirulent T15 strain (the classical European avirulent strain) were used to determine the RNA abundance; the details of the bacterial strains and plasmid vectors used in this study are listed in Supplementary Table 1.

The genome sequences of 68 S. suis serotype 2 isolates from our previous study were used to determine the distribution of fabG [20].

Expression and purification of recombinant proteins and preparation of polyclonal antiserum

The FabG coding gene (locus tag ZY05719_RS08510, Accession: WP_012027710.1) was amplified by PCR from the genomic DNA of ZY05719 (Accession: NZ_CP007497.1), cloned into pET-32a(+), and verified by Sanger sequencing (Genewiz, China). An Escherichia coli expression system was used to express recombinant protein, which were then purified by Ni-chelating chromatography. The theoretical molecular weight was calculated by using Compute pI/Mw (https://web.expasy.org/compute_pi/). 2 New Zealand white rabbits (purchased from Jiangsu Qinglongshan Biotechnology Co. LTD) were used to prepare polyclonal antiserum. The rabbits were immunized three times (once every two weeks) with 500 µg of rSBP2’ 1:1 emulsified with Montanide ISA 201 adjuvant (Seppic, France).

Western blot analysis

5 µg of protein per lane were used for the western blotting. Protein marker was purchased from Thermo Scientific™ (Cat No. 26620). 12% acrylamide gel was used for SDS-PAGE, and electrophoresis was performed at 80 V for 20 min, and 120 V until the end. After SDS-PAGE, proteins were electrophoretically transferred onto a PVDF membrane by trans-blot turbo transfer system (Bio-Rad). PBST containing 5% BSA was used to block nonspecific binding for 2 h., and the corresponding antibodies at appropriate dilutions were used for detection. The primary antibody was incubated overnight at 4℃, and the secondary antibody was incubated for 1 h at room temperature, the PVDF membrane was washed three times with PBS between each incubation. His-Tag (10E2) mAb (abmart, Cat No. M30111, 1:5000), goat anti-mouse IgG antibody (Boster, Cat No. BA1051, 1:2000), and goat anti-rabbit IgG antibody (Boster, Cat No. BA1055, 1:2000) were used as primary and secondary antibody.

Transcription analysis by qRT-PCR

The RNA sample was collected by using TRIzol-chloroform-isopropanol methods and reverse-transcribed to cDNA. Briefly, 5mL bacteria cultures were cultivated to logarithmic growth phase (OD600 = 0.6–0.8), the volume of bacterial cultures and inoculation ratios were the same. The bacteria samples were collected by centrifugation, resuspended by 1mL TRIzol and added to a lysing matrix tube (MP Biomedicals™, Cat No. 116911050), then grind by using FastPrep-24™ 5G Bead Beating Grinder and Lysis System(MP Biomedicals™). The lysing matrix was removed by centrifuge, and 0.2 mL chloroform was added to extract the RNA, 0.5 mL isopropanol was added to precipitate the RNA, after 2 times washing with ethanol, the RNA sample was collected. HiScript III RT SuperMix for qPCR (+ gDNA wiper) (Vazyme, Cat No. R323-01) was used in reverse transcription, 4 × gDNA wiper mix was added to remove the genome DNA, and 5 × HiScript III qRT SuperMix was added to reverse transcribe the RNA to cDNA. qRT-PCR analysis was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme, Cat No. Q711-02/03). gapdh was used as the control [21]. Primers used are listed in Supplementary Table 1.

Protein binding assay

The protein-binding assay was performed as previously described [22]. Briefly, 1 × 10⁶ STEC (swine tracheal epithelial cells) were collected in a 1.5 mL tube after cultured by DMEM with 10% FBS to form monolayers, 37 °C, 5% CO₂, washed with PBS three times, and incubated with 5 µg rFabG or BSA (negative control) at 37 °C for 2 h in FBS free DMEM, then the cells were centrifuged at 800 × g for 5 min, remove the unbound proteins and washed by PBS three times. The supernatant was removed, the pellet was resuspended in PBS, and SDS-PAGE was performed. The subsequent procedure was the same as that used for the western blotting.

Micro titer plate adhesion assay (MPAA)

MPAA was performed according to a previous study [23], aiming to quantitatively determine the binding of the rFabG protein to host cells. The cell membrane proteins were extracted using the Membrane and Cytosol Protein Extraction Kit (Tiangen Biotech, China), and coated onto a 96-well microtiter plate at 10 µg/mL. After blocking with 5% bovine serum albumin (BSA, Solarbio, Cat No. A8020), the plate was incubated with different concentrations of rFabG. After incubation with mouse anti-His-tag monoclonal antibody and HRP-conjugated goat anti-mouse IgG, the reaction was detected using TMB substrate (Tiangen, Cat No. PA107) for 15 min and stopped with 2M H₂SO₄, absorbance was measured at 450 nm.

Indirect Immunofluorescence assay (IFA)

The IFA was performed as described by Yu et al. [24]. Briefly, the STEC were cultured by DMEM with 10% FBS in glass bottom cell culture dish, 37 °C, 5% CO₂, then fixed with 4% formaldehyde for 10 min when they grew to a monolayer, and washed 3 times with PBS. Then, 10 µg of recombinant protein in PBS solution was added to the culture dish and incubated for 1 h. Subsequently, the anti-rFabG polyserum and the FITC conjugated goat anti-rabbit IgG (H + L) antibody (Boster, Cat No. BA1105, 1:100) were used as primary and secondary antibody to detect the binding proteins. A group in which only cells were not added with bacteria was treated as a blank control. The nuclei were stained with DAPI (Sigma-Aldrich, Cat No. D9542). The fluorescence was observed using a fluorescence microscope (ZEISS, Germany).

Adhesion inhibition assay

An adhesion inhibition assay was performed to evaluate the contribution of FabG to S. suis cytoadhesion. Briefly, STEC cells were cultured by DMEM with 10% FBS in 24-well plates to form monolayers. The number of cells were counted by Countless 3 Automated Cell Counter (Invitrogen). Bacteria grown to log phase were harvest by centrifuge, treated with pre-immune serum or anti-rFabG polyclonal serum before infection for 30 min. After three times washing with PBS, the bacterial numbers were counted and resuspended in DMEM medium, the bacteria number was adjusted according to MOI = 20:1, each 500 µL DMEM was contained 20 times bacteria than cell number of a well. Then the bacteria were added to a 24-well plate, and incubated at 37 °C for 2 h. The cells were then washed with PBS three times, 500 µL ddH₂O was added to the cell well to exposed the adhered bacteria by osmotic pressure. After serially dilute, 20 µL of this solution was spread on the THB plate, the bacteria number was counted after 24 h culture in 37 °C.

Far-western blot of recombinant proteins

Far-western blotting was performed to determine the binding ability of rFabG to the host plasminogen. 5 µg rFabG and rHP07325 were used for western blotting. rHP07325 is a hypothetical protein and predicted located in cytoplasma, it was proved that cannot react with some host extracellular proteins in a previous study [25], which is used as a negative control. After SDS-PAGE, one gel was electrophoretically transferred onto a PVDF membrane and the other was stained with Coomassie blue G-250. The PVDF membrane was further analyzed by western blotting. PBST containing 5% BSA was used to block nonspecific binding, and incubated with human plasminogen (Plg, Sigma-Aldrich, Cat No. SRP6518, 5 µg/mL) overnight at 4 °C, then incubated with rabbit anti-Plg (Sigma-Aldrich, Cat No. HPA021602, 1:2000) and goat anti-rabbit IgG antibody (Boster, Cat No. BA1055, 1:2000) in turn, and ECL reagent (Vazyme) was used to detect the chemiluminescence.

Surface plasmon resonance (SPR) analysis

Surface plasmon resonance (SPR) was used to dynamically determine the interaction between rFabG and plasminogen. The plasminogen was diluted to 10 µg/mL using 10 mM sodium acetate (pH 4.0) and then covalently linked to the carboxylmethylated dextran matrix of a CM5 sensor chip. Binding kinetics were measured by increasing concentrations of rFabG in running buffer (HBS-EP) consisting of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% surfactant P20 (Biacore AB) at a flow rate of 30 µL/min for 180 s over immobilized plasminogen at 20 °C. The resonance unit (RU) value generated by the immobilized soluble protein was 600. The dissociation phase was monitored for 1000 s by allowing the buffer to flow over the chip. Association kinetics were analyzed manually using Biacore X100 Control Software [26].

Direct binding assay

Direct binding assay were performed as previous study [27]. A 96-well micro titers plate was coated with 100 µL rFabG or plasminogen at a concentration of 5 µg/mL overnight at 4 °C. After blocking with 5% BSA for 2 h, rFabG or plasminogen was incubated with immobilized plasminogen or rFabG at increasing concentrations. An equal concentration of BSA was used as a negative control. The corresponding antibody was used to determine binding activity, and the absorbance of the plate was measured at 450 nm.

Degradation of ECM by the combination of rFabG and plasminogen

rFabG or BSA was conjugated to 1.1 μm polystyrene beads (Sigma-Aldrich, Cat No. LB11). Briefly, 20 µL beads (1% v/v) were incubated with 1 mL 1.5 mg/mL rFabG or BSA overnight at 4 °C, and then blocked with 5% BSA. Next, 10 µg/mL plasminogen was added and incubated at 37 °C for 3 h, followed by 200 ng/mL tissue-specific plasminogen activator (tPA, Sigma-Aldrich, Cat No. T0831). After washing, the beads were resuspended in 1 mL of PBS.

Matrigel (Corning, USA), a commercial extracellular matrix mixture, was used to evaluate the degradation of rFabG-bound plasminogen. The Matrigel was diluted with prechilled PBS (1:3) and layered on the upper chamber of a 3 μm Transwell insert (Corning, USA) at 37 °C overnight to let it gelled. Before starting the experiment, the Matrigel was rehydrated in 70 µL PBS at 37 °C for 1 h. 70 µL beads were added to the upper chamber, whereas the lower compartment contained 700 µL PBS. The chambers were incubated at 37 °C for 40 h. The obtained Transwell membrane were then gently washed with PBS, fixed with 2.5% glutaraldehyde, gradient dehydration with ethanol, then coated in metallic media, and examined under a Zeiss EVO-LS10 scanning electron microscope (Zeiss, Germany).

Binding activity of rFabG to C3 from serum and serum survival assay

A 96-well micro titers plate was coated with 100 µL rFabG at a concentration of 5 µg/mL overnight at 4 °C, an equal concentration of BSA was used as a negative control. After blocking with 5% BSA for 2 h, 200µL of normal swine serum was added to each well and serially diluted. Rat anti-C3 antibody (abcam, Cat No. ab11862) and Goat anti-Rat IgG antibody (abcam, Cat No. ab97057) were used to determine the binding ability of rFabG to C3.

Furthermore, a serum survival assay was performed. Briefly, the SS2 strain ZY05719 was cultured in THB until the mid-exponential growth phase. Then, 5 × 10⁶ cfu of bacteria were resuspended in 200 µL normal swine serum (absin, Cat No. abs934), then incubated at 37 °C for 1 h, with or without rFabG. After the incubation, the serum contains resident bacteria was serially diluted to the appropriate concentration, 20 µL of this solution was spread on the THB plate, the bacteria number was counted after 24 h culture in 37 °C.

Statistical analysis

An unpaired two-tailed Student’s t-test was used to compare the difference between the means of two groups of data, and one-way ANOVA was used to perform the multiple comparisons test with Dunnett’s as correct. The data were visualized using GraphPad Prism 7 software.

Results

Determination of differential transcription of FabG in SS2 strains of different virulence

qRT-PCR was performed to determine the transcription level of fabG in SS2 strains of different virulence levels. As shown in Fig. 1, the transcript levels of fabG are significantly higher in the three virulent SS2 strains (HA9801, P1/7, ZY05719) than in the avirulent SS2 strain (T15).

Fig. 1.

The relative transcription level of fabG between virulent SS2 strains and the avirulent strain. T15 was set as 1.0. The relative change in gene expression ratios of selected genes was normalized to the expression of the housekeeping gene, gapdh. The change trends in three highly virulent strains were calculated using the 2^−ΔΔCT method. Results are expressed as means ± SD of three experiments with triplicate samples (**** p < 0.0001). Error bars represent the variation within groups

Expression and purification of rFabG

The FabG coding gene was cloned from S. suis strain ZY05719 and cloned into the pET-32a(+) expression vector and the recombinant proteins was expressed in the E. coli strain BL21(DE3) induced by IPTG. The expression and purification of rFabG were verified using SDS-PAGE (Fig. 2A) and western blotting using an Anti-His-tag antibody (Fig. 2B). The theoretical molecular weight of rFabG was calculated as 44.8 kDa, and the band just lower than the 55 kDa marker, indicating the recombinant protein was successfully obtained. The polyclonal antiserum against rFabG was prepared and verified by western blotting (Fig. 2C). Pre-immune serum has been confirmed does not recognize rFabG (Supplementary Figure 1A).

Fig. 2.

Expression and purification of rFabG. A SDS-PAGE analysis; B Reaction with anti-His-tag antibody; C Reaction with anti-rFabG polyclonal antibody. The marker was loaded in the first lane and the rFabG protein was loaded in the second lane. His-Tag (10E2) mAb and rabbit anti-rFabG polyclonal antibody were used as primary antibody, goat anti-mouse IgG antibody and goat anti-rabbit IgG antibody were used as secondary antibody. The arrow pointed the recombinant protein and the specific reaction band. The exposure time is 2s

rFabG could bind to host epithelial cells

The binding ability of rFabG to host epithelial cells was evaluated in swine tracheal epithelial cells (STEC). Cell samples were washed 3 times with PBS after interaction with rFabG and BSA in 1.5 mL tubes and loaded onto gels for western blotting, and rFabG could be detected, suggesting that rFabG could directly bind to STEC (Fig. 3A). The anti-rFabG antiserum has been confirmed does not bind to STEC cells themselves (Supplementary Figure 1B). To further quantify the binding, a microtiter plate adhesion assay (MPAA) was performed, and the binding of rFabG to the membrane proteins of STEC cells was determined in a dose-dependent manner from 1 µg/mL to 100 µg/mL (Fig. 3B). Moreover, the binding of rFabG to STEC cells was visualized using fluorescence microscopy, and green fluorescence was be observed on the surface of STEC cells after treatment with rFabG (Fig. 4).

Fig. 3.

rFabG could directly bind to host epithelial cells. A Protein binding test, the STEC cells are digested and collected to a tube and 5 µg rFabG was added to test the binding ability, the exposure time is 5 s; B Microtiter plate adhesion assay (MPAA) for the binding of the rFabG protein to cell membrane proteins. The X axis represented the concentration of rFabG added to the well of 96-well plate. The experiment has been repeated three times independently. Error bars represent the variation within groups

Fig. 4.

Cytoadhesion of the rFabG protein detected by indirect immunofluorescence assay. The experiment has been repeated three times independently

Anti-rFabG polyclonal antibody could inhibit the adhesion of Streptococcus suis to host cell

To further evaluate the role of FabG in Streptococcus suis cytoadhesion, an adhesion inhibition assay was performed. The bacteria was pre-incubated with anti-rFabG antiserum, then added to the 24 well plate to interact with STEC cells, the result show that the anti-rFabG antiserum treated group was almost a 50% reduction in adherence relative to cells treated with pre-immune serum (Fig. 5), indicating that FabG is involved in the cytoadhesion of S. suis.

Fig. 5.

Adhesion inhibition assay of anti-rFabG antibody. The bacterial adhesion in the pre-immune serum treatment group was set as the baseline (100%). Results are expressed as means ± SD of three experiments with triplicate samples (**p < 0.01). Error bars represent the variation within groups

FabG is a host plasminogen receptor of Streptococcus suis

The binding ability of rFabG to the host plasminogen was first determined by the Far-Western blotting. Specific binding was detected at the site corresponding to the purified rFabG band (Fig. 6A). Anti-plasminogen antibody has been confirmed does not recognize rFabG (Supplementary Figure 1C). Surface plasmon resonance (SPR) was conducted to further investigate the kinetics of the interaction, and the affinity and high affinity between rFabG and the host plasminogen were observed, with a dissociation equilibrium constant (KD) of 1.724 × 10⁻⁹ M (Fig. 6B). ELISA was used to quantify the binding of rFabG to host plasminogen, and dose-dependent binding of soluble plasminogen to immobilized rFabG was observed; the reverse experiment also showed similar results (Fig. 6C).

Fig. 6.

Binding activity of the rFabG protein to host plasminogen. A Far-Western blot analyses the binding ability between rFabG to host plasminogen. rFabG and a negative control rHP07325 were loaded on gel to perform SDS-PAGE, After SDS-PAGE, (a) one gel stained by Coomassie blue G-250 and, (b) another gel was electrophoretically transferred onto PVDF membrane to perform Far-Western blot, the exposure time is 5 s; B Sensorgrams depicting the binding of immobilized plasminogen to rFabG. Increasing concentrations of FabG (18.75, 37.5, 75, 150, and 300 µg/mL) were injected at a flow rate of 30 µL/min for 180 s over immobilized plasminogen. RU = resonance units; C Detect the dose-dependent binding between rFabG and plasminogen by ELISA. The experiment has been repeated three times independently

FabG mediates host ECM degradation by hijacking host plasminogen

One of the most crucial physical defenses against bacterial infections for host epithelial cells is the extracellular matrix (ECM). Bacteria may produce plasminogen receptors to hijack host plasminogen and breakthrough the ECM component that helps in bacterial invasion of host cells, given that plasminogen is responsible for the dissolution of host ECM components in host homeostasis. To find out if rFabG is capable of this, we conducted an ECM degradation experiment in this study.

The upper chamber of the 3 μm filter Transwell cell inserts was loaded with matrigel. After being treated with plasminogen and tPA, a plasminogen activator, polystyrene beads coated with rFabG were introduced to the chamber. Electron microscopy was used to observe the restored basement membrane’s breakdown following a 40-hour incubation period. The reconstituted basement membrane treated with rFabG-coated beads showed clearly damage, and the filter membrane’s perforations were visible where the beads might fall (Fig. 7). This result suggests that rFabG can recruit host plasminogen and promote the invasion of S. suis by exploiting its ability to degrade host ECM.

Fig. 7.

Electron microscopic visualization of the degradation of Matrigel reconstituted basement membrane. The rFabG-harboring polystyrene beads (A, B) or the BSA-harboring beads (C, D) were incubated with plasminogen and tPA and then added on the 3-µm filters in Transwell cell culture chamber inserts previously coated with Matrigel reconstituted basement membrane. After an incubation of 40 h, the filters were fixed with 2.5% glutaraldehyde, and examined in a scanning electron microscope. B and D are the enlarged view of a part of A and C, respectively. The experiment has been repeated three times independently

Binding activity of rFabG to C3 from serum and interferes with complement activity

C3 is a central protein of the host complement system. All three complement activation pathways generate C3 convertases that cleave C3 to perform their functions. Bacterial pathogens can generate proteins that competitively inhibit C3 activity. Moreover, considering that the coagulation and complement cascades of the human organism are tightly connected, and as the key enzyme of the coagulation system, the C3 cleavage ability of plasminogen has been proven. Here, we analyzed the binding ability of rFabG to C3 and discussed its potential role in S. suis immune evasion.

A 96-well microtiter plates coated with rFabG were incubated with serially diluted normal swine serum. After reaction with the corresponding antibody, the results showed that rFabG could recruited C3 in the serum (Fig. 8A). To further determine whether this kind of recruitment affects S. suis escape from complement killing, a serum survival assay was performed. We found that the number of surviving bacteria of S. suis is significantly increased after adding rFabG (Fig. 8B).

Fig. 8.

rFabG contributes to S. suis resistance host complement killing. A Binding ability of the rFabG protein to host complement C3 from normal swine serum, the X axis represented the concentration of rFabG and BSA added to the well of 96-well plate; B The survival ability of S. suis in normal swine serum was significantly increased after rFabG was added. The experiment has been repeated three times independently. Error bars represent the variation within groups

Discussion

The crucial stage in the infection of many bacteria is establishing colonization of epithelial cells. The interaction of bacterial surface or extracellular proteins with host plasminogen, extracellular matrix, or cell membrane components typically results in the multi-factor synergistic colonization process for Streptococcus suis [2]. Bacterial infection is a consequence of multiple factors, a single phenotype can result from multiple proteins, and a single protein can involve a number of different processes. In S. suis, a lot of proteins were identified as host-binding proteins, and they can be separated into two groups, specialized cell surface receptors and cytoplasmic and glycolytic pathway proteins. In the past, most of the identified host-binding proteins are cell surface proteins, such as SsPepO [28] and muramidase-released protein (MRP) [29], which have been reported in S.suis. However, in recent years, a number of proteins that are typically thought to be limited to the cytoplasm and involved in metabolism have been discovered on the extracellular space or cell wall of S. suis and have been linked to interactions with host components. These include α-enolase (Eno) [30], elongation factor Tu (EF-Tu) [19], and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) [9]. The crucial keto reduction step in the fatty acid synthase elongation cycle is often catalyzed by the bacterial 3-ketoacyl-ACP reductase FabG. Our earlier investigation reported that FabG could be detected in the cell wall proteins of S. suis, and it could interact with host’s extracellular matrix [19], and the results of this study also suggesting the cell surface distribution of FabG, these finding indicating that the FabG have a potential to be a moonlight protein. The cell surface localization mechanism of this kind of proteins are not defined, however, some of these proteins such as GAPDH [31] and PGK [32], have been confirmed localized in the cell surface in multiple species of bacteria.

Unlike traditional microbial surface components that recognize adhesive matrix molecules (MSCRAMMs), these kinds of proteins are usually not directly involved in the adhesion of bacteria to host cells but play their roles in competing with the host immune-associated molecules or hijacking host components to facilitate bacterial pathogens break through the barrier [33]. In this study, we discussed the second function of FabG and found that it can bind to host epithelial cells and mediate ECM degradation by commandeering the host plasminogen, as a part of the cell surface proteins of S. suis, the binding between FabG and host plasminogen could meditate the interact between bacterial cell and host plasminogen, considering the plasminogen responsible to the degradation of extracellular matrix and complement factor. These findings suggest that FabG is a novel plasminogen receptor in S. suis and could be involved in the first step of colonization of S. suis, helping it to break through the host barrier through recruit the host plasminogen to the cell surface.

Through screening the prevalence of FabG in 68 S. suis serotype 2 isolates, we found that FabG exists in all S. suis serotype 2 isolates and show 100% similarity within virulent isolates (Supplementary Figure 2A), so we speculate that FabG shows similar moonlighting functions in virulent S. suis serotype 2. Further, through detecting FabG in all available genome sequences from public database GenBank, we found that FabG is conserved as a member of the core-genome of S. suis with more than 90% similarity. Moreover, FabG is widely distributed in other Streptococci (Supplementary Figure 2B), and the role of FabG in these Streptococci is also interesting. Meanwhile, there are also some publications about FabG in other bacteria, such as Escherichia coli [34] and Pseudomonas aeruginosa [35], but most of them focus on the role of FabG in metabolism. We also analyzed the difference of FabG sequence between the virulent and the avirulent isolates of S. suis type 2, only 2–3 amino acid difference were found. Probably some of these changes involve or are close to important places such as active sites. Combined with the different transcript levels, we speculate that these differences may account for the different functions of FabG in virulent and avirulent isolates of S. suis type 2. However, it is important to note that higher fabG transcription levels in highly virulent strains may not necessarily correlate with higher protein expression levels, numerous post-translational modifications exist in bacteria.

Numerous proteins that bacteria code for have the ability to attach to host molecules and take control of them in order to bypass immune system defenses or penetrate the host barrier. The case of factor H binding proteins is one notable example. Pathogenic immune escape techniques specifically target the complement system, which distinguishes between innate and adaptive immunity [36]. In the alternative complement pathway, which shields host cells from complement-mediated harm, factor H is a crucial soluble inhibitor of complement activation [37]. Three methods are frequently used by bacterial pathogens to avoid host complement killing: recruiting host complement regulatory factors, generating complement inhibitory factors, and expressing proteases to directly cleave complement factors [38]. Numerous factor H binding proteins are expressed by bacteria in order to recruit host factor H to their cell surface. This allows them to inactivate or accelerate the intrinsic decay of C3 or C5 in order to avoid complement killing [33]. Using a C3-/-mouse model, earlier research has shown that the complement system plays a critical role in avoiding the morbidity and mortality brought on by S. suis infection, suggesting that S. suis infection may require evading complement activation [39]. A number of S. suis complement escape factors have been discovered, including factor H binding proteins [40], C5a peptidase SspB [41], and IgM protease Ide_Ssuis [42]. Plasminogen interaction has grown in importance in host-pathogen interactions in recent years. Human activators, such as urokinase-type (uPA) and tissue-type plasminogen activators, can transform plasminogen, one of the essential coagulation system enzymes, into the active serine protease plasmin. Plasmin can mediate fibrinolysis, ECM degradation, and complement inhibition [43]. We discovered in this investigation that FabG binds to host C3, increasing S. suis’s survival. With the recruitment of plasmin(ogen) to sites of bacterial infection, bacteria could utilize this function of plasmin to suppress the host immune response, then implement the immune evasion. According to our findings, FabG could bind to both host plasminogen and complement factor C3, after plasminogen was activated by activators such as tPA/uPA to plasmin, the plasmin will degrade those complement factors, and finally assists S. suis in avoiding complement death. Recruitment of plasminogen to the bacterial cell surface is mediated directly by either specialized cell surface receptors or cytoplasmic proteins localized to the bacterial cell surface, or indirectly via interactions with host plasma proteins such as IgG, fibrinogen, complement factors [44]. Thus, despite our finding suggests that FabG could bind to both host plasminogen and C3, it still need further discuss about the binding mechanism, is the binding direct or indirect or both, because there is possible that their binding could lead by a pre-binding by other two molecules.

It is worth noting that FabG is part of the fatty acid metabolism system that is metabolically essential for bacterial survival, a property that prevents the construction of isogenic knockout mutants. Thus, although we tried all the existing deletion mutant construction methods for S. suis, the FabG deletion mutant could not be constructed. Therefore, our study focused on the moonlight function of FabG, using the recombinant protein to simulate the role of the native protein. This may have caused bias during the experiments. Further studies are required to explore the complete function of this type of protein. Meanwhile, according to previous reports, there are many reasons that affect the definition of S. suis virulence, such as the use of different animal models, different in vitro methods, and even different routes of infection [45]. The different models used in the study may lead to some biased results. In this study, both human and pig models were used to evaluate the role of FabG in S. suis pathogenesis, but we need to note that although both pigs and humans are hosts of S. suis, there may still be some bias in the results. Although many factors have been evaluated, such the similarity of plasminogen from different sources, it is not straightforward to speculate that the results obtained in one model can be directly transferred to another.

In summary, we report that S. suis FabG is a multifunctional protein that is not only responsible for lipogenesis but also acts as an extracellular adhesin involved in the cytoadhesion of S. suis. It can bind host plasminogen and ECM components to mediate ECM degradation, and scramble for host complement C3 to enhance the survival of S. suis in vivo. These findings suggest that FabG plays an important role in the first step colonization of S. suis.

Authors’ contributions

GGL, ZY and ZW designed and planned this study; GGL, LP and LQ performed the acquisition, analysis of data; YYF Validated the study; GGL and ZW drafted the manuscript; All authors reviewed the manuscript.

Funding

This study was supported by the Programs of National Natural Science Foundation of China (Grant No. 32402898), Shandong Academy of Agricultural Sciences, Agricultural Science and Technology Innovation Project (CXGC2025C18), Guizhou Province Science and Technology Plan Project (Grant No. QKH[2023]008), National Natural Science Foundation of China (Grant No. 32172860), and Jiangsu Association for Science and Technology Youth Talent Support Program (Grant No. JSTJ-2023-XH031).

Data availability

The datasets generated and/or analysed during the current study are available in the GenBank repository, accession of ZY05719 is NZ_CP007497.1, accession of FabG is WP_012027710.1.

Declarations

Ethics approval and consent to participate

The animals were anaesthetized and unconscious, pentobarbital sodium was used as anaesthetic, injection dosage is 100 mg/kg (over 3 times of anesthesia dose), anaesthetized by constant speed intravenous injection, the animals were observed, Animals should be observed to lose consciousness within one minute with respiratory arrest. All animal studies were approved by the Experimental Animal Welfare and Ethics Committee of Nanjing Agricultural University (Approval ID: SYXK(SU)2021-0086) and were performed in accordance with the Animal Welfare Agency Guidelines.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.