A metabolite from commensal Candida albicans enhances the bactericidal activity of macrophages and protects against sepsis

Abstract

The gut microbiome is recognized as a key modulator of sepsis development. However, the contribution of the gut mycobiome to sepsis development is still not fully understood. Here, we demonstrated that the level of Candida albicans was markedly decreased in patients with bacterial sepsis, and the supernatant of Candida albicans culture significantly decreased the bacterial load and improved sepsis symptoms in both cecum ligation and puncture (CLP)-challenged mice and Escherichia coli-challenged pigs. Integrative metabolomics and the genetic engineering of fungi revealed that Candida albicans-derived phenylpyruvate (PPA) enhanced the bactericidal activity of macrophages and reduced organ damage during sepsis. Mechanistically, PPA directly binds to sirtuin 2 (SIRT2) and increases reactive oxygen species (ROS) production for eventual bacterial clearance. Importantly, PPA enhanced the bacterial clearance capacity of macrophages in sepsis patients and was inversely correlated with the severity of sepsis in patients. Our findings highlight the crucial contribution of commensal fungi to bacterial disease modulation and expand our understanding of the host-mycobiome interaction during sepsis development.

Keywords: Bacterial clearance; Candida albicans; Macrophage; Phenylpyruvate; Sepsis.

Fig. 1

Effects of gut commensal C. albicans on bacterial sepsis. A C. albicans burden in bacterial sepsis patients (n = 30) and healthy subjects (HS, n = 14) was determined using a C. albicans SYBR qPCR Kit. B Experimental design of the CLP model and pretreatments. C, D Mice received an oral gavage of 300 μL blank medium (Med), supernatant of C. albicans ATCC 10231 [Sup (ATCC 10231)] or SC 5314 [Sup (SC 5314)] 3 h before CLP. The survival rate (n = 10–15) was determined up to 72 h after CLP using the Kaplan‒Meier method with the log-rank test. E, F Serum ALT and AST levels (n = 4–12). G, H HE staining and histological score of the lungs (n = 4–8). Scale bar, 50 μm. I–K Blood, peritoneal lavage fluid (PLF), and liver samples were collected from the mice at 12 h after CLP. After the samples were serially diluted, 100 μL of each dilution was plated on Columbia blood agar plates and incubated at 37 °C for 14–16 h under aerobic or anaerobic conditions. Representative images and quantified results of bacterial load in blood (I), PLF (J) and liver (K) samples (n = 6–13). L Representative fluorescence images of medium- or supernatant (ATCC 10231)-pretreated BMDMs with pHrodo red E. coli bioparticles and blue nuclear staining (DAPI) over the course of 45 min. Scale bar, 10 μm. M, N Phagocytosis of E. coli or S. aureus by BMDMs after 3 h of pretreatment with medium or supernatant (n = 6–12). O, P Bacterial killing by BMDMs based on the gentamicin protection assay at 60 min post E. coli or S. aureus infection after 3 h of pretreatment with medium or supernatant (n = 6–12). Data are presented as the mean ± SEM. *p < 0.05 by two-tailed Student’s t test

Fig. 2

The C. albicans metabolite PPA protected against polymicrobial sepsis. A, B PCA scatter plots and volcano plots of metabolomics data based on blank medium and supernatant from C. albicans culture (n = 4–8). C Molecular structure of PPA. D Relative abundance of PPA detected by nontargeted metabolomics. E PPA levels in the medium and supernatant detected by GC/MS (n = 5). F–K Mice were treated with saline or 40 mg/kg PPA 3 h before CLP. F Survival rates of saline- or PPA-pretreated CLP mice were analyzed using the Kaplan‒Meier method with the log-rank test (n = 38). G HE staining and histological scores (n = 3–5). H Serum ALT and AST levels (n = 4–10) and bacterial load in the I blood, J PLF and K liver (n = 14–20) samples. Scale bars, 50 μm. Data are presented as the mean ± SEM. *p < 0.05 by two-tailed Student’s t test. ns not significant

Fig. 3

Decreased PPA production by C. albicans impaired protection against sepsis. A Schematic diagram illustrating the workflow for aromatic-amino-acid:2-oxoglutarate transaminase-overexpressing C. albicans and the metabolic process of C. albicans. PPA is mainly produced by the conversion of phenylalanine (Phe) by aromatic-amino-acid:2-oxoglutarate transaminase. B PPA levels in the supernatant of wild-type and ARO9-overexpressing C. albicans detected by GC/MS (n = 4). C PPA levels in Sup^phe+ and Sup^phe− (supernatant of C. albicans cultured in SLD medium containing 300 or 0 μg/mL Phe, respectively) detected by GC/MS (n = 4–7). D Survival rate of CLP mice (n = 17) treated with Sup^phe+ or Sup^phe− 3 h before CLP. HE staining and histological scores (E, n = 8), serum ALT and AST levels (F, n = 8), and bacterial load in blood (G), PLF (H), and liver (I, n = 9) samples. Scale bars, 50 μm. Phagocytosis and bacterial killing by BMDMs with Sup^phe+ or Sup^phe- pretreatment prior to E. coli (J) or S. aureus infection (K, n = 11–12). L Survival rate of CLP mice treated with wild-type or ARO9-overexpressing C. albicans 3 h before CLP (n = 15). HE staining and histological scores (M, n = 5), serum ALT and AST levels (N, n = 10), and bacterial load in blood (O, n = 6), PLF (P, n = 6), and liver (Q, n = 6) samples. Scale bars, 50 μm. Data are presented as the mean ± SEM. *p < 0.05 by two-tailed Student’s t test. The survival rates of septic mice were analyzed using the Kaplan‒Meier method with the log-rank test

Fig. 4

Macrophages were required for the PPA-mediated beneficial effects in CLP-induced sepsis. A Confocal micrographs showing the phagocytosis of pHrodo red E. coli bioparticles by BMDMs with or without pretreatment with PPA (100 μM). Scale bars, 10 μm. B, C Phagocytosis by BMDMs with or without pretreatment with PPA (100 μM) for 18 h prior to E. coli or S. aureus infection (MOI, 20) for 45 min (n = 6). Separate samples were incubated for another hour to assess the bacterial killing activity (n = 6). D, E Neutrophils were pretreated with or without PPA for 18 h and then infected with E. coli or S. aureus (MOI, 100) for 45 min. Representative images and quantification results of phagocytosis and bacterial killing are presented (n = 6). Serum ALT and AST levels (F, n = 5-6), HE staining and histological scores (G, n = 5-6) and bacterial load (H) in blood, PLF, and liver (H, n = 5–6) samples from macrophage-depleted mice with or without PPA treatment. Scale bars, 50 μm. Serum ALT and AST levels (I, n = 6), HE staining and histological scores (J, n = 6) and bacterial load (H) in blood, PLF, and liver (K, n = 9) samples from neutrophil-depleted mice with or without PPA treatment. Scale bars, 50 μm. Data are presented as the mean ± SEM. *p < 0.05 by two-tailed Student’s t test. ns not significant

Fig. 5

PPA inhibited SIRT2 activity and increased ROS production in BMDMs. A Schematic diagram showing target identification of PPA in macrophage lysates. Immunoblot analysis of SIRT2 in pronase-digested (B) BMDMs and (C) Raw 264.7 cell lysates (n = 3). D Surface-plasmon resonance (SPR) analysis of PPA binding to SIRT2. E Docking analysis of the interactions between PPA and SIRT2. The SIRT2 residues that are likely to participate in the interactions with PPA are labeled. BMDMs were treated with or without PPA (100 μM) for 18 h. G6PD activity (F), NADPH levels (G), and ROS levels (H) in E. coli- or S. aureus-infected BMDM lysates (n = 3–6). I–L Phagocytosis and bacterial killing by BMDMs with or without N-acetylcysteine (NAC) or mitoTEMPOL (MiT) pretreatment (n = 4). M, N Phagocytosis and bacterial killing by BMDMs transfected with SIRT2 siRNA or negative control siRNA for 48 h in the presence or absence of PPA (n = 6). O G6PD activity, P NADPH levels, and Q ROS levels in E. coli- or S. aureus-infected BMDM lysates with the indicated treatments (n = 3–6). R, S Phagocytosis and bacterial killing by BMDMs with SIRT2 overexpression or empty plasmid transfection for 48 h in the presence or absence of PPA (n = 5–6). T G6PD activity, U NADPH levels, and V ROS levels in E. coli- or S. aureus-infected BMDM lysates with the indicated treatments (n = 3–6). Data are presented as the mean ± SEM. *p < 0.05 by two-tailed Student’s t test. ns not significant

Fig. 6

C. albicans culture supernatant and PPA protect against E. coli-induced sepsis in pigs. A Basic scheme of the experimental protocol. Serum ALT and AST before (B) Sup or (C) PPA pretreatment and after euthanasia of the pigs (n = 3). D Representative HE staining image of the lungs of septic pigs with or without Sup treatment. Scale bar, 50 μm. E Representative HE staining image of the lungs of septic pigs with or without PPA treatment. Scale bar, 50 μm. Effect of Sup (F–H) or PPA (I–K) on bacterial load in the lungs, liver, and kidneys of septic pigs. Bacterial colonies were cultured on plates, and CFUs were counted (n = 3). Data are presented as the mean ± SEM. *p < 0.05 by two-tailed Student’s t test. ns not significant

Fig. 7

PPA modulated bacterial killing by human phagocytes and was associated with the disease outcome of sepsis patients. A, B Representative images and quantitative results of phagocytosis and killing of E. coli and S. aureus by human blood monocyte-derived macrophages (MDMs) from sepsis patients in the dimethyl sulfoxide (DMSO) and PPA groups (n = 5). Sepsis patients were divided into two groups based on APACHE II scores as follows: high group (>median) and low group (<median) (C, n = 63). PPA levels were compared between the two groups (D, n = 31). E, F Sepsis patients were divided into two groups based on the circulating levels of PPA as follows: a high group (>median) and a low group (<median) (E, n = 63). The APACHE II score was compared between the two groups (F, n = 31). G Correlation analysis of plasma PPA levels with APACHE II scores (n = 63). H Schematic diagram depicting enhanced bacterial clearance by C. albicans-derived PPA via regulation of SIRT2-dependent ROS production in macrophages. Data are presented as the mean ± SEM. *p < 0.05 by two-tailed Student’s t test. ns not significant