Therapeutic potential of kaempferol on Streptococcus pneumoniae infection

Abstract

Co-infections with pathogens and secondary bacterial infections play significant roles during the pandemic coronavirus disease 2019 (COVID-19) pathogenetic process, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Notably, co-infections with Streptococcus pneumoniae (S. pneumoniae), as a major Gram-positive pathogen causing pneumonia or meningitis, severely threaten the diagnosis, therapy, and prognosis of COVID-19 worldwide. Accumulating evidences have emerged indicating that S. pneumoniae evolves multiple virulence factors, including pneumolysin (PLY) and sortase A (SrtA), which have been extensively explored as alternative anti-infection targets. In our study, natural flavonoid kaempferol was identified as a potential candidate drug for infection therapeutics via anti-virulence mechanisms. We found that kaempferol could interfere with the pore-forming activity of PLY by engaging with catalytic active sites and consequently inhibit PLY-mediated cytotoxicity. Additionally, exposed to kaempferol significantly reduced the SrtA peptidase activity by occupying the active sites of SrtA. Further, the biofilms formation and bacterial adhesion to the host cells could be significantly thwarted by kaempferol incubation. In vivo infection model by S. pneumoniae highlighted that kaempferol oral administration exhibited notable treatment benefits, as evidenced by decreased bacterial burden, suggesting that kaempferol has tremendous potential to attenuate S. pneumoniae pathogenicity. Scientifically, our study implies that kaempferol is a promising therapeutic option by targeting bacterial virulence factors.

Streptococcus pneumoniae (S. pneumoniae), a typically facultative anaerobic Gram-positive bacterium, triggers a broad range of severe diseases including pneumonia, otitis media, septicemia and meningitis [1]. Overwhelming S. pneumoniae infections contribute to the proliferated mortality and morbidity (an approximated four million illnesses annually in the United States), which are common in young children, elderly persons and immunocompromised patients [2,3]. Alarmingly, S. pneumoniae pathogens were reported as the common bacterial co-infection in coronavirus disease 2019 (COVID-19) patients [4]. The interaction between microbial co-pathogens including S. pneumoniae, SARS-CoV-2 and host seriously threatens the diagnosis, therapy, and prognosis of COVID-19 worldwide [5]. Hence, developing feasible strategies and therapeutic options against S. pneumoniae and identifying the underlying molecular mechanisms would greatly benefit the treatment of SARS-CoV-2 respiratory infections in the current pandemic.

S. pneumoniae secretes multiple virulence factors, including capsule, pneumolysin (PLY), sortase A (SrtA) and hyaluronidase (HylA), play critical roles on bacterial spread and colonization [6]. Notably, the pore-forming toxin pneumolysin (PLY), as a cholesteroldependent cytolysin (CDC) toxin, interferes with the host immune function and forms lytic pores in host cell membranes inducing cell lysis [7]. Additionally, sortase A (SrtA), a membrane-anchored transpeptidase, exhibits the transpeptidation activity at the LPXTG motif to anchor to the cell wall, contributing to the bacterial colonization and invasion of S. pneumoniae [8,9]. Accumulating evidences conclude that feasible approach and strategy targeting PLY and SrtA exhibit great potential for S. pneumoniae infections therapeutics [10].

In the current study, kaempferol, a natural flavonoid present in abundant plant species [11], efficiently reduced sortase A activity and inhibited the pore-forming activity of PLY by occupying the active sites. Inspiringly, kaempferol oral administration could significantly attenuate S. pneumoniae virulence in vivo. Our findings provided an effective therapeutic regimen and a potential candidate for combating S. pneumoniae infections.

1. Material and methods

1.1. Reagents

Kaempferol (purity >98%) were obtained from Chengdu Herbpurify Co., Ltd. (China) and dissolved in dimethylsulfoxide (DMSO, Sigma–Aldrich). Fluorescent synthetic peptide substrate Dabcyl-QALPETGEE-Edans used in Sortase A activity inhibition assay was purchased from GL Biochem (Shanghai, China). A Cytotoxicity Detection Kit (Roche) and LIVE/DEAD™ Viability/Cytotoxicity Kit (Invitrogen) were applied to evaluate cell death caused by pneumolysin (PLY).

1.2. Bacterial strain and cell cultures

S.pneumoniae strain D39 (NCTC 7466) was cultured overnight in ToddHewitt broth containing 2% yeast extract (THY) in the condition of stilling culture at 37 °C. The human lung A549 epithelial cells and mouse macrophage-like cell line J774A.1 were grown in Dulbecco's modified Eagle's medium (DMEM, Gibco) with 10% fetal bovine serum at 37 °C for the cytotoxicity and cytoprotection analysis.

1.3. Construction, expression and purification of SrtA and PLY

The specific sequence of SrtA and PLY was amplified from S. pneumoniae D39 genomic DNA using the primers listed in Supplementary Table 1 and the recombinant proteins were respectively expressed using pGEX-6P-1- and pET28a-based expression system as described in previous study [12,13].

1.4. SrtA activity inhibition assay

SrtA protein in the diluting buffer (50 mM Tris–HCl, 150 mM NaCl, pH 8.0) was cultured in the presence or absence of kaempferol (0, 2, 8, 32 μg/mL) for 30 min at 37 °C. Subsequently, the cultures were mixed with the fluorescent peptide substrate Dabcyl-QALPETGEE-Edans (10 μM) for 60 min in the dark. And protease K (Merck) culture was served as a positive control. SrtA activity was determined by measuring with excitation wavelength at 350 nm and emission wavelength at 520 nm.

1.5. Growth curve

S. pneumoniae D39 supplemented with different concentrations of kaempferol (0, 2, 4, 8, 16, 32 μg/mL) were cultured in a motionless condition at 37 °C for continuously 5 h. The absorbance values at OD_{600 nm} were monitored by a UV spectrophotometer at 1-h intervals to evaluate the effect of kaempferol on bacterial growth.

1.6. In vitro anti-biofilm activity determination

For the bacterial biofilm formation, the logarithmic growth of S. pneumoniae D39 (2 × 10⁶ CFU/mL) with fresh THY broth in the presence or absence of kaempferol (0, 4, 8, 16, 32 μg/mL) were cultured in a 24-well plate at 37 °C for 10 h or 20 h, respectively. Additionally, the biofilms were pre-formed for 10 h and then cultured with the indicated concentrations of kaempferol for an additional 10 h, according to the above procedures. Then, the supernatants were discarded and biofilms were washed twice with PBS and stained with 0.1% crystal violet (CV) for 1 h. The CV-staining biofilms were dissolved with 30% acetic acid and measured at OD_{570 nm} using a microplate reader.

1.7. Hemolysis inhibition assay

PLY exposed to different concentrations of kaempferol (0, 2, 4, 8, 16, 32 μg/mL) was incubated at 37 °C for 30 min and mixed with 2.5% (v/v) fresh rabbit red blood cells (RBCs) for the hemolytic activity determination. Additionally, RBCs were cultured with the presence or absence of kaempferol (2–128 μg/mL) for 1 h at 37 °C for cytotoxicity analysis. Samples in the absence of PLY and treated with ddH₂O was respectively used as a vehicle control and positive control. Finally, the hemolytic activity was determined as following formula: Hemoglobin release rate (%) = (Samples_{OD570 nm} − Vehicle_{OD570 nm})/(100% lysis_{OD570 nm} − Vehicle_{OD570 nm}) × 100%.

1.8. Western blotting analysis

The S. pneumoniae D39 cells cultured with kaempferol (0, 8, 16, 32 μg/mL) were collected and obtained an OD_{600 nm} of 1.0. The samples were mixed with loading buffer and boiled at 100 °C for 10 min for immunoblotting analysis. Briefly, samples were separated by 12% SDS-PAGE and transferred onto the polyvinylidene fluoride (PVDF) membrane. After blocking, the membranes were incubated with anti-pneumolysin antibody (1:3000; Abcam) and HRP-conjugated goat anti-rabbit secondary antibodies (1:3000; Proteintech). Then, the membranes were visualized using an enhanced chemiluminescence substrate.

1.9. Oligomerization formation assay

Purified PLY was incubated with kaempferol (0, 4, 8, 16, 32 μg/mL) at 37 °C for 1 h. Then, the samples were mixed with 0.5% RBCs at 4 °C for 10 min and treated with loading buffer at 55 °C for an additional 10-min incubation. In vitro oligomerization of PLY was determined by western blotting using an antibody against His-tag (Immunoway), as previously described [14]. And the densitometry of PLY oligomers and monomers was quantified with ImageJ software.

1.10. Cytotoxicity and cytoprotection test

A549 cell line and J774A.1 macrophages were cultured in 96-well plates at a density of 3 × 10⁴ cells per well and respectively incubated with the indicated concentrations of kaempferol at 37 °C for 6 h for cytotoxicity analysis. Additionally, J774A.1 cells were treated with or without kaempferol in the presence of PLY (5 μg/mL), or incubated with different concentrations of PLY. After incubation for 6 h, the LDH in the cultured supernatants were detected and cell viability was determined using a Cytotoxicity Detection Kit. And the cell treated with 0.2% Triton X-100 was employed as a positive control.

Visually, a LIVE/DEAD™ Viability/Cytotoxicity Kit was further employed to observe the cell viability under the treatment of kaempferol. Briefly, J774A.1 cells in the cytoprotection test were stained with Calcein-AM (live cells) and Ethidium homodimer-1 (dead cells) for 30 min and observed using an inverted fluorescence microscope (Olympus). And the relative fluorescence density was quantified using ImageJ software.

1.11. Bacterial adhesion assay

A549 cells were cultured in a 24-well plate at a density of 2 × 10⁵ cells per well and infected with S. pneumoniae D39 (MOI = 20) in the presence or absence of kaempferol (0, 4, 8 μg/mL). At 3 h or 6 h post-infection, bacteria adhered to the host cells were transplanted onto the tryptic soy broth (TSB) agar plates. Finally, the bacterial colonies were counted after incubation for 12 h.

1.12. Molecular modelling

The initial structures of SrtA and PLY were obtained from protein crystal models (PDB: 4O8L and 4QQA). The kaempferol/SrtA complex and kaempferol/PLY complex were docked and analyzed using Sail Vina v1.0 and protein-ligand interaction profler (PLIP) website (https://plip-tool.biotec.tu-dresden.de/plip-web/plip/index) [15].

1.13. Animal study

BALB/c female mice (6–8 weeks, 20–23 g) used in our study were obtained from Changsheng Biotechnology Co. Ltd. (Liaoning, China). All animal study strictly followed the guidelines of the Jilin University Institutional Animal Care Committee and approved by this the committee. The mice were housed in a specific pathogen-free (SPF) and comfortable environment (23 ± 2 °C, 55 ± 10% humidity).

After acclimating for 1 week, the mice were lightly anesthetized and infected with S. pneumoniae D39 (1 × 10⁸ CFU/per mouse) through the nose. The infected mice were randomly divided into 3 groups (the vehicle control group (0.5% sodium carboxymethylcellulose (0.5% CMC)), 40 mg/kg/day kaempferol (resuspended in 0.5% CMC), 80 mg/kg/day kaempferol), and the drugs or solvent were treated by oral administration at 12-h intervals, respectively.

At 48 h post-infection, the mice were euthanized for bacterial burden analysis. The lung were homogenized in sterile PBS and plated for bacterial counting. Furthermore, lungs in each groups were fixed with 10% formalin for pathological observation by H&E staining.

1.14. Statistical analysis

All the data analysis were performed as the mean ± SEM (n ≥ 3) and statistical significance was analysed with GraphPad Prism 8.0.1 using Student's t-test. The significance levels (∗P < 0.05 and ∗∗P < 0.01) were indicated in the figures and figure legends.

2. Results

2.1. Kaempferol incubation attenuates the biological activity of SrtA and inhibits biofilms formation

The inhibition effect of kaempferol on the SrtA activity was determined using the a fluorescence resonance energy transfer (FRET) assay. Kaempferol (Fig. 1 A), a natural flavonoid, was identified to dramatically inhibit the activity of SrtA (Fig. 1C) in nonbacteriostatic concentrations (Fig. 1B). Previous reports have emerged indicating that SrtA-mediated modification of surface proteins plays a crucial role in biofilm formation [16]. Consistently, as shown in Fig. 1D and 1E, exposed to kaempferol could significantly suppress the bacterial biofilms formation at 10 h and 20 h post-inoculation. Additionally, the inhibitory effect of kaempferol on the development of biofilms were also observed (Fig. 1F–G). Collectively, these results indicated that kaempferol incubation could inhibit the S. pneumoniae biofilms formation and maturation.

Fig. 1.

Kaempferol inhibits the transpeptidation activity of SrtA and suppresses the bacterial biofilms formation. (A) Chemical structure of kaempferol. (B) Growth curve of S. pneumoniae supplemented with different concentrations of kaempferol (0, 2, 4, 8, 16, 32 μg/mL). (C) Inhibition of SrtA activity exposed to kaempferol (0, 2, 8, 32 μg/mL). (D–E) Quantification and macroscopic images of bacterial biofilms formation by crystal violet (CV) staining at 10 h or 20 h post-infection. (F–G) Destruction of bacterial biofilms with the presence or absence of kaempferol was determined by crystal violet (CV) staining. All the data were analysed as means ± SEM (n ≥ 3). ∗∗P < 0.01, ∗P < 0.05.

2.2. Kaempferol inhibits the pore formation activity of PLY

S. pneumoniae evolved numerous virulence factors, including pneumolysin (PLY), which possesses the potent pore formation activity via the oligomerization of soluble monomers. Significantly, kaempferol incubation reduced the PLY-mediated hemolytic activity in a concentration-dependent manner (Fig. 2A–B), by contrast, kaempferol exhibited no effect on pneumolysin expression (Fig. 2 C). Further mechanism studies indicated that the formation of PLY oligomers was obviously inhibited by the addition of kaempferol (Fig. 2D–E). Taken together, our findings showed that kaempferol treatment significantly inhibited the hemolytic activity of PLY, attributing to the interference with the formation of high-molecular-weight complexes.

Fig. 2.

Kaempferol interferes with the pore-forming potency of PLY. (A–B) Inhibition effects of kaempferol on the hemolysis. Samples in the absence of PLY and treated with ddH₂O was respectively served as a negative control and positive control and hemoglobin release was measured at A570 nm. (C) Effects of kaempferol on the PLY production in the precipitates of S. pneumoniae culture. (D–E) Oligomerization of PLY with the presence of indicated concentrations of kaempferol (0, 4, 8, 16, 32 μg/mL) was determined using western blotting and quantified with ImageJ software. All the data were analysed as means ± SEM (n ≥ 3). ∗∗P < 0.01.

2.3. Identification of the binding mode of kaempferol with SrtA and PLY

The molecular docking of SrtA/kaempferol complex and PLY/kaempferol complex were performed for further mechanisms exploration of the inhibition effects of kaempferol on the transpeptidation activity of SrtA and pore-formation ability of PLY. As shown in Fig. 3 A, ASP209, ALA211, HIS141, VAL205, ALA139 and LEU113, as active residues of SrtA, showed potent interactions with kaempferol. Additionally, the molecular docking results indicated that the binding sites of PLY with kaempferol were ASN346, SER167, PHE334, THR57 and ILE60 (Fig. 3B). Our results confirmed that kaempferol displayed anti-virulence potency by combined interactions formed by kaempferol interaction on PLY and SrtA active residues.

Fig. 3.

The predicted binding modes of SrtA/kaempferol (A) and PLY/kaempferol (B). The binding sites of kaempferol with SrtA (ASP209, ALA211, HIS141, VAL205, ALA139, LEU113) and PLY (ASN346, SER167, PHE334, THR57, ILE60) were identified by molecular docking.

2.4. Kaempferol decreases PLY-induced cytotoxicity and reduces bacterial adhesion on the host cells

Safety evaluation is of important significance for drug development and we further evaluated the safety of kaempferol on either fresh rabbit red blood cells (RBCs) or two cell lines (A549 and J774A.1 cell lines). As expected, kaempferol exhibited no haemolysis activity at high levels (2–128 μg/mL) (Fig. 4 A). As depicted in Fig. 4B, the effective concentrations of kaempferol in the current study (0–16 μg/mL) showed no obvious cytotoxicity in A549 cells and J774A.1 macrophages.

Fig. 4.

Kaempferol prevents PLY-induced cell damage and inhibits bacterial adhesion to the host cells. (A and B) The cytotoxicity evaluation of kaempferol on the RBCs, J774A.1 and A549 cell lines. (C and D) LDH released in the cocultured supernatants of J774A.1 cells in the presence of PLY with or without indicated concentrations of kaempferol were quantified and the cultured cells were stained using an LIVE/DEAD™ Viability/Cytotoxicity Kit (E). (F) And live/dead cells (green/red fluorescence) present in each samples were quantified using ImageJ software. (G) The bacterial adhesion to A549 cells in the presence or absence of kaempferol at 3 h or 6 h post-infection were assessed by microbiological plating. All the data were analysed as means ± SEM (n ≥ 3). ns, P > 0.05, ∗P < 0.05.

PLY, as a pore-forming toxin, could perforate host cells and cause cell death. Consistently, PLY-induced cytotoxicity was observably boosted with the increased concentrations of PLY (Fig. 4C). And J774A.1 cells exposed to kaempferol showed an effective cytoprotective activity against PLY-mediated cell death, as evidenced by markedly decreased LDH release (Fig. 4D) and reduced red fluorescence (Fig. 4E–F) in kaempferol-treated cells.

Additionally, bacterial adhesion is a pivotal step for tissue colonization and ingrowth and we investigated the effects of kaempferol on bacterial adhesion to A549 lung epithelial cells. Encouragingly, kaempferol treatment could observably inhibit bacterial adherence to the host cells in the late stages of S. pneumoniae infection, as evidenced by the decrease in CFUs at 6 h post-infection (Fig. 4G), suggesting that kaempferol has a promising anti-infection efficacy on the adherence of S. pneumoniae.

2.5. Kaempferol attenuates S. pneumoniae virulence in vivo

S. pneumoniae, as an important pathogen, has impacted bacterial lung injury in different ways with rapid dissemination of co-infections with pathogens or virus. Hence, clarification of the potency of kaempferol shows great significance for S. pneumoniae infection therapeutics. The experimental model of pulmonary infection with S. pneumoniae was established as described in Fig. 5 A. As expected, the bacterial burden in lungs were significantly decreased by kaempferol (80 mg/kg) treatment, compared with the S. pneumoniae-infected control (Fig. 5B). Congruent with our results, the macroscopic observation and pathological analysis showed that S. pneumoniae infection observably led to pathological injury in the lungs, as evidenced by dark redness of macroscopic characteristic, hyperemia, edema and accumulation of inflammatory cells in lung tissues, whereas, which were markedly alleviated by kaempferol treatment (Fig. 5C). Collectively, kaempferol therapy provided potent protection against S. pneumoniae infection in vivo and showed great potential for combating co-infections and secondary bacterial infections.

Fig. 5.

Kaempferol oral administration potently alleviates S. pneumoniae virulence in vivo. (A) Scheme of the experimental procedures of mice pulmonary infection by S. pneumoniae. (B) The bacterial burden in the lungs of mice (n = 6 per group) were determined by microbiological plating. (C) And pathological observation of lungs in different groups were evaluated by H&E staining. Scale bar, 100 μm and 200 μm ∗P < 0.05.

3. Discussion

Targeting bacterial virulence as a feasible alternative strategy has been extensively explored and showed great potential for infection therapeutics, involving in multiple defence mechanisms by destruction of bacterial adhesion, invasion, and subversion of host immune defense [17]. Of particular concern is the pore-forming toxin pneumolysin (PLY), the toxic effects of which chiefly attribute to the binding and polymerization in cholesterol-structure of cell membranes, to form the transmembrane β-barrel pores that induce hemolysis [18,19]. In our study, natural flavonoid kaempferol was identified as a specific PLY inhibitor, as evidenced that significantly suppressed the hemolytic activity of PLY by the interference with the oligomerization of PLY and directly engaged with catalytic active sites. Accumulating evidences have emerged indicating that PLY disrupts the host immune defenses against pathogens infection and leads to cell death [20,21]. In our work, PLY as a pore-forming cytotoxin exhibited a potent cytotoxic activity in J774A.1 macrophages, conversely, exposed to kaempferol significantly ameliorated the cytotoxic effects of PLY, which suggesting that kaempferol may contribute to the attenuated pathogenicity of S. pneumoniae.

Sortase A (SrtA), a membrane-anchored transpeptidase, has attracted great attention as a potential antimicrobial target [22]. The current evidences availably support the concept that SrtA could anchor multiple surface protein virulence factors onto the cell wall, contributing to the bacterial adhesion/colonization and biofilms formation [8,23]. Remarkably, inhibition of SrtA activity by kaempferol observably decreased biofilms formation and bacterial adhesion to the host cells. Congruent with in vitro efficacies, kaempferol confers potent protection against S. pneumoniae infection in vivo, as evidenced by the decreased bacterial burden and alleviative pathological lesions.

Notably, with the emergence and dissemination of bacterial co-infections in COVID-19 pneumonia, co-infections with S. pneumoniae seriously impair the host immune defense, followed by severe secondary bacterial pneumonia, especially in elderly persons and immunocompromised patients [24]. Encouragingly, as illustrated in Fig. 6 , our study proposed a feasible therapeutic strategy to attenuate S. pneumoniae virulence by simultaneously inhibiting the PLY-mediated pore-forming activity and the peptidase activity of SrtA. Moreover, plant natural products possess abundant pharmacological activities, with no poisonous effect, which have been extensively explored for the pathogens infections therapeutics. In the current study, the natural flavonoid kaempferol, as a potential anti-virulence candidate drug, highlights great promise for combating co-infections with S. pneumoniae.

Fig. 6.

Scheme summarizing the potency of kaempferol against S. pneumoniae infection. Natural flavonoid kaempferol, as a potent antagonist of SrtA and PLY, significantly attenuates S. pneumoniae virulence in vivo/in vitro by interference with the pore-forming activity of PLY and inhibition of the SrtA peptidase activity, which contribute to the diminished cell death and reduced biofilms formation.

Author contributions

LX, HHF and JFW conceived and designed this project. LX, JF and JWX performed the experiments. LX, DYO, XMD and GFC analyzed the data. LX, HHF and JFW drafted this manuscript. All authors have read, reviewed and approved the final manuscript.

Data availability

The data are available from the corresponding author on reasonable request.

Declaration of competing interest

All authors declare no conflicts of interest.

Appendix A. Supplementary data

The following is the Supplementary data to this article: