Bacterial signal C10-HSL stimulates spore germination of Galactomyces geotrichum by transboundary interaction
Xin Lu , Yue Wang , Zhixuan Feng , Liang Fu , Dandan Zhou
Galactomyces geotrichum ubiquitously inhabits in a wide range of environments, such as air, water, soil, plant, silage, and milk, dominating 22.58% of yeast-like fungi [1-3]. Massive numbers of fungal spores are exposed to fungal dissemination and survival, especially for G. geotrichum. Spore germination occurs due to stress and regeneration of cell walls under favorable environmental conditions [4, 5]. The spore density also has an influence in terms of equilibrating the populations of germinated spores [6]. However, the social behaviors of G. geotrichum in a microbial community have not been well understood.
The polymicrobial interactions play an important modulatory role in the succession of a microbial community [7, 8]. Bacterial community behavior was recognized as a population-coordinated mechanism regulated by acyl-homoserine lactone (AHL) molecules, for example; this was called quorum sensing (QS) [9, 10]. AHL signals can be used by Gram-negative bacteria to monitor and sense their cell density. They are composed of a lactonized homoserine moiety with a varying-length acyl chain between 4 and 18 carbons by increments of 2 carbon units (C4, C6, C8, and so on) [11, 12]. AHL signals bind to the receptor protein (LuxR) to form the activated AHL–LuxR protein complex, which activates the transcription of target genes by homodimerizing and binding adjacent to QS promoters [11]. Interestingly, AHLs are identified as "transboundary languages" that can be monitored and signaled by eukaryotes [13-16]. Further, 3-oxo-C12-HSL prevents filamentation by inhibiting the expression of the cyclic adenosine monophosphate (cAMP)/PKA pathway in Candida [17-20]. Moreover, C12-HSL can suppress the filamentation of G. geotrichum to inhibit fungal bulking in an activated sludge microecosystem [21].
Various AHL signals widely exist in a microecological environment such as plant rhizosphere, biological wastewater treatment systems, animals, human body, and food industry [22-24]. In multiple natural ecosystems, C10-HSL, C12-HSL, and 3-oxo-C12-HSL have been reported as the dominant AHLs [11]. C12-HSL and 3-oxo-C12-HSL both contain a 12-carbon backbone. They are similar to farnesol generated by C. albicans [17]. These two signals can mimic the actions of farnesol by binding to the inhibitory site in the cAMP/PKA pathway, thus disturbing fungal morphogenesis [17]. They have the potential to share their signals and communicate with each other due to the long-term coevolution and coexistence between bacteria and fungi [25]. However, C10-HSL has a shorter acyl chain and can improve exopolysaccharide metabolism and enhance bacterial cell activity [26, 27]. It has effects opposite to those of C12-HSL and 3-oxo-C12-HSL. Therefore, we hypothesized that bacterial C10-HSL could play a distinguished role in yeast, which was not verified yet.
Considering the population of G. geotrichum in aquatic ecosystems, it is meaningful to understand the social interactions between G. geotrichum and coexisting bacterial community. In this study, 5 µmol/L C10-HSL was added to pure G. geotrichum to explore the mechanism of transboundary communication between fungi and bacteria. Higher spore germination rates and longer germ tube length were stimulated by bacterial C10-HSL, accompanied by more carbohydrate production. C10-HSL mediated an upregulated expression of genes related to the cell wall biosynthesis pathway due to its contribution to carbohydrate metabolism. Also, an interaction network of the metabolic pathways in spore germination was established in C10-HSL selection. The results provided new sights into the coexistence and coevolution between yeast-like fungi and bacteria.
The pure G. geotrichum was purchased from China General Microbiological Culture Collection Center (CGMCC 2.4043). The fungal strains were activated by inoculating in potato dextrose agar medium at 28 ℃ overnight. The spore suspension was obtained by filtering strains harvested from plates with four layers of a nonwoven fabric. The initial optical density at 600 nm was 0.03 for each experiment, and the suspension was finally transferred into a liquid medium (1% glucose, 1% peptone, and 0.5% yeast extract) [21]. Further, 500 nmol/L commercial C10-HSL standard was added in an experimental group; strains without the treatment of C10-HSL served as a control.
The germinated spores were collected after 2 h, centrifuged at 10,000 g for 10 min, and fixed with 2.5% glutaraldehyde at 4 ℃ overnight. The prepared samples were washed three times with 20 mmol/L PBS (pH 7.0), and dehydrated using an ethanol gradient of 10%, 30%, 50%, 70%, 90%, and 100% (v/v) for 30 min [21]. The micro-morphologies were observed under a scanning electron microscope (SEM) (XL-30 ESEM FEG, Thermo, USA).
Spore germination rates and germ tube lengths were used for testing spore germination efficiency [6]. The spore germination rates were calculated using the proportion of germinated spores to total spore numbers in 2 h. The lengths of germ tubes were measured every 2 h using a microscope (BX53, Olympus, Japan). The data of 100 samples were evaluated as the average value.
The extracellular polymeric substance was extracted by the heating method [27]. The fungal solution was resuspended in 0.9% NaCl solution after discarding the nutrient solution. The fungal solution was heated at 60 ℃ for 30 min and centrifuged at 4 ℃ and 10,000 rpm for 10 min. The samples were filtered through a glass fiber membrane filter (GF/B 47 mm, Whatman, UK) and used for measuring the carbohydrate content by the phenol-sulfuric acid method.
The transcriptomic technology was applied to analyze the pathway of cell growth regulated by C10-HSL. The cell samples were collected by centrifuging at 10,000 g for 10 min, immediately frozen in liquid nitrogen, and stored at –80 ℃ in an ultra-low temperature freezer (PLATILAB 340, Angelantoni). The total extraction process and RNA-seq library construction were performed by Novogene Co., Ltd. (Beijing, China) following the manufacturer's protocol [21]. The high-quality data were finally acquired through removing reads containing adapter, poly-N, and poor-quality reads from raw data. The subsequent assembly data were analyzed by the no-reference-genome method with default parameters. The upregulated and downregulated expression of differentially expressed genes (DEGs) was defined by the log2-fold change, logFC > 1 and logFC < –1, respectively, using the DEGSeq (1.12.0). The gene function annotation was referred to the National Center for Biotechnology Information nonredundant database. Further, the Gene Ontology (GO) terms of proteins were used to determine the functional classification of each gene based on the GO database.
The differences between samples with and without C10-HSL were calculated based on means and standard errors of replicates, which were evaluated by a significance threshold of P < 0.05 using the Student t-test in SPSS software (Version 23). The relationship between the carbohydrate content and the germ tube lengths was analyzed by linear regression fitting. An interaction network among pathways was constructed using GlueGO, a plug-in of Cytoscape (Version 3.8.0), with differential gene numbers enriched in the biological process of the GO database.
During spore germination, C10-HSL significantly increased the rates of spore germination by 22% (Fig. 1a). Also, the germ tubes were much longer (9%−35%) in the AHL-treated group than in the control group (Fig. 1b). SEM images (Figs. 1c and d) also directly revealed more germinated spores and longer hyphal lengths under treatment with C10-HSL. These results indicated that C10-HSL had positive effects on the density of germinated spores of G. geotrichum.
C12-HSL plays a negative role in prokaryote–eukaryote communication by inhibiting the sludge bulking in G. geotrichum [21]. However, C10-HSL had a contrast ecological function in G. geotrichum [17]. In a bacterial community, C10-HSL signal sensing was beneficial to cell growth by binding to receptor proteins, which mediated intracellular biochemical reactions [7, 11]. Nevertheless, G. geotrichum could coordinate similar social behaviors with the bacterial community under the stimulation of C10-HSL [25]. It seemed that bacterial C10-HSL used "transboundary languages" across the prokaryote–eukaryote boundary, also monitored and signaled the G. geotrichum population to promote spore germination.
Fig. 2 shows the extracellular carbohydrate production from G. geotrichum induced by C10-HSL during spore germination. C10-HSL could stimulate higher carbohydrate production, gradually improving the extracellular concentration of carbohydrate substrates by 25%−62% (Fig. 2a). A positive linear regression model (r = 0.97, P < 0.05) was established between the carbohydrate concentration and the spore germination rates (Fig. 2b), indicating that the morphological transition could accelerate the excretion of carbohydrate production. Carbohydrate is an essential constituent for cell wall assembly, necessarily restricting spore germination and the extension of the hyphal tip [28]. Hence, bacterial C10-HSL could regulate the morphology of germinated spores by increasing the carbohydrate metabolite production in fungal submerged cultures. These results demonstrated that C10-HSL was an effective stimulator for improving carbohydrate biosynthesis.
The transcriptome technique was applied to analyze the differential genes in cell samples between the AHL-treated and control groups to reveal the mechanism of spore germination in G. geotrichum regulated by C10-HSL. The expression of 45 genes was significantly upregulated (P < 0.05) in the AHL-treated group compared with that in the control group (Fig. 3). These DEGs were responsible for polysaccharide synthesis and cell wall assembly. During germination, spores developed a polar tube that relied on the surface expansion of outer spores, hatching out the remodeling cell wall [4-6]. The cell wall extension was promoted due to the 1.27-fold upregulation of the target gene Smi1 controlled by C10-HSL. The genes Utr2 and Chs2 were associated with the biosynthesis of chitosan, which is a major component in the cell wall, and also had upregulated expression by 1.02-fold and 1.36-fold, respectively. Further, a majority of the upregulated genes belonged to intracellular carbohydrate metabolism, ultimately linking with the integration of biochemical pathways, including cell cycle, endoplasmic reticulum processing, Golgi transportation, and so forth [28]. These data suggested that C10-HSL facilitated spore germination in G. geotrichum by upregulating the cell wall biogenesis pathway.
Fig. 4 shows an interaction network of metabolic pathways, which was established based on the upregulated genes (more than one fold) regulated by bacterial C10-HSL. The pivotal hub gene Rho1 in the interaction network had an upregulated expression (2.05-fold) in C10-HSL signal transduction, which was the central linkage of cell cycle, carbohydrate metabolism, and cell wall biogenesis process. The receptor gene Rho1 was critical for cell polarity and cell wall synthesis, which signaling pathway were triggered and resulted in the up-regulation of Rho1 and its downstream to factors promote the hypha-to-yeast transitions [29-32]. The genes associated with the cell cycle were upregulated by 1- to 2.5-fold, and were involved in cell division, cytokinesis, and chromatid cohesion. It indicated that the cell cycle was positively regulated by C10-HSL, leading to the motivation of cell growth and division at the genetic level. Moreover, the carbohydrates metabolic pathway had close linkages with cell cycle and cell wall biogenesis, confirming that carbohydrate played a central role in physiological activities with the stimulation of C10-HSL. The metabolic network revealed that the hub gene Rho1 was responsible for bacterial C10-HSL regulation, up-regulating the cascade reactions to induce filamentation in downstream.
Theoretically, we deduced the transboundary interaction between G. geotrichum and bacteria. The C10-HSL that freely diffused into fungal cells was sensed by the intracellular AHL receptor Rho1. Subsequently, the growth and division of G. geotrichum responded to C10-HSL by upregulating the cell cycle. The downstream carbohydrate metabolic pathway was activated to produce polysaccharide-likes substances, which were used for cell wall assembly. Ultimately, this process affected fungal morphological changes and induced spore germination and extension. The transboundary information is channeled to benefit fungi, G. geotrichum, maintain a synergistic relationship with C10-HSL secreted by QS bacteria. The cooperation between fungi and bacteria protects the diversity of the ecological system.
Bacterial C10-HSL was capable of communicating with G. geotrichum and promoting spore germination by remodeling cell wall biogenesis. The receptor Rho1 was a communication bridge between fungi and bacteria, it recognized signals and coordinated interactions by modifying the gene expression. These findings verified the regulatory mechanism of bacterial C10-HSL to G. geotrichum, which was similar to the pathways acting on the bacterial community. Deeply understanding the interaction between eukaryotic and prokaryotic species and enhancing the spore germination of yeast are essential.
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
The authors thank the National Natural Science Foundation of China (Nos. 52070036, U20A20322), and the Fundamental Research Funds for the Central Universities (No. 2412018ZD042) for their financial support.
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Figure 1 Spore germination rates and germ tube length of G. geotrichum in the control (a) and the AHL-treated groups (b). The micro-morphology of germinated spores in the control (c) and the AHL-treated groups (d). The scale bar is 10 µm. Statistics for the data were calculated based on 100 images. ** represents P < 0.01, and *** represents P < 0.001.
Figure 2 Effects of C10-HSL on the carbohydrate content of G. geotrichum during (a) and its relationship with germ tube extension (b). All error bars indicate the standard deviation of the means. * represents P < 0.05 and ** represents P < 0.01.
Figure 3 Regulatory model of genes associated with the spore germination in G. geotrichum in a comparison dataset: C10-HSL vs. control. Boxes represent differentially expressed genes (> onefold change, P < 0.05). The colored bar in the bottom right corner represents the expression of upregulated differentially expressed genes. ER, endoplasmic reticulum; G, Golgi body; V, vesicles.
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