Table2_Gut microbiota contributes to lignocellulose deconstruction and nitrogen fixation of the larva of Apriona swainsoni.XLSX

Apriona swainsoni is a vital forest pest prevalent in China. The larvae of A. swainsoni live solely in the branches of trees and rely entirely on the xylem for nutrition. However, there is still a lack of in-depth research on the gut microbiota’s use of almost nitrogen-free wood components to provide bio-organic macromolecular components needed for their growth. Thus, in this study, the metagenome, metaproteome, and metabolome of the A. swainsoni larvae in four gut segments (foregut; midgut; anterior hindgut; posterior hindgut) were analyzed by the multi-omics combined technology, to explore the metabolic utilization mechanism of the corresponding gut microbiota of A. swainsoni. Firstly, we found that the metagenome of different gut segments was not significantly different in general, but there were different combinations of dominant bacteria and genes in different gut segments, and the metaproteome and metabolome of four gut segments were significantly different in general. Secondly, the multi-omics results showed that there were significant gradient differences in the contents of cellulose and hemicellulose in different segments of A. swainsoni, and the expression of corresponding metabolic proteins was the highest in the midgut, suggesting the metabolic characteristics of these lignocellulose components in A. swainsoni gut segments. Finally, we found that the C/N ratio of woody food was significantly lower than that of frass, and metagenomic results showed that nitrogen fixation genes mainly existed in the foregut and two hindgut segments. The expression of the key nitrogen fixing gene nifH occurred in two hindgut parts, indicating the feature of nitrogen fixation of A. swainsoni. In conclusion, our results provide direct evidence that the larvae of A. swainsoni can adapt to the relatively harsh niche conditions through the highly organized gut microbiome in four gut segments, and may play a major role in their growth.</p

Table3_Gut microbiota contributes to lignocellulose deconstruction and nitrogen fixation of the larva of Apriona swainsoni.XLSX

Apriona swainsoni is a vital forest pest prevalent in China. The larvae of A. swainsoni live solely in the branches of trees and rely entirely on the xylem for nutrition. However, there is still a lack of in-depth research on the gut microbiota’s use of almost nitrogen-free wood components to provide bio-organic macromolecular components needed for their growth. Thus, in this study, the metagenome, metaproteome, and metabolome of the A. swainsoni larvae in four gut segments (foregut; midgut; anterior hindgut; posterior hindgut) were analyzed by the multi-omics combined technology, to explore the metabolic utilization mechanism of the corresponding gut microbiota of A. swainsoni. Firstly, we found that the metagenome of different gut segments was not significantly different in general, but there were different combinations of dominant bacteria and genes in different gut segments, and the metaproteome and metabolome of four gut segments were significantly different in general. Secondly, the multi-omics results showed that there were significant gradient differences in the contents of cellulose and hemicellulose in different segments of A. swainsoni, and the expression of corresponding metabolic proteins was the highest in the midgut, suggesting the metabolic characteristics of these lignocellulose components in A. swainsoni gut segments. Finally, we found that the C/N ratio of woody food was significantly lower than that of frass, and metagenomic results showed that nitrogen fixation genes mainly existed in the foregut and two hindgut segments. The expression of the key nitrogen fixing gene nifH occurred in two hindgut parts, indicating the feature of nitrogen fixation of A. swainsoni. In conclusion, our results provide direct evidence that the larvae of A. swainsoni can adapt to the relatively harsh niche conditions through the highly organized gut microbiome in four gut segments, and may play a major role in their growth.</p

Table1_Gut microbiota contributes to lignocellulose deconstruction and nitrogen fixation of the larva of Apriona swainsoni.XLSX

Apriona swainsoni is a vital forest pest prevalent in China. The larvae of A. swainsoni live solely in the branches of trees and rely entirely on the xylem for nutrition. However, there is still a lack of in-depth research on the gut microbiota’s use of almost nitrogen-free wood components to provide bio-organic macromolecular components needed for their growth. Thus, in this study, the metagenome, metaproteome, and metabolome of the A. swainsoni larvae in four gut segments (foregut; midgut; anterior hindgut; posterior hindgut) were analyzed by the multi-omics combined technology, to explore the metabolic utilization mechanism of the corresponding gut microbiota of A. swainsoni. Firstly, we found that the metagenome of different gut segments was not significantly different in general, but there were different combinations of dominant bacteria and genes in different gut segments, and the metaproteome and metabolome of four gut segments were significantly different in general. Secondly, the multi-omics results showed that there were significant gradient differences in the contents of cellulose and hemicellulose in different segments of A. swainsoni, and the expression of corresponding metabolic proteins was the highest in the midgut, suggesting the metabolic characteristics of these lignocellulose components in A. swainsoni gut segments. Finally, we found that the C/N ratio of woody food was significantly lower than that of frass, and metagenomic results showed that nitrogen fixation genes mainly existed in the foregut and two hindgut segments. The expression of the key nitrogen fixing gene nifH occurred in two hindgut parts, indicating the feature of nitrogen fixation of A. swainsoni. In conclusion, our results provide direct evidence that the larvae of A. swainsoni can adapt to the relatively harsh niche conditions through the highly organized gut microbiome in four gut segments, and may play a major role in their growth.</p

Paeonol assists fluconazole and amphotericin B to inhibit virulence factors and pathogenicity of <i>Candida albicans</i>

This study aimed to evaluate the mono- and dual- antifungal activities of paeonol (PAE) and fluconazole (FLZ)/amphotericin B (AmB). To this end, the effects of PAE and FLZ/AmB on cell surface hydrophobicity, hydrolase activity, morphological transition were investigated in vitro and in a Galleria mellonella infection model. The results showed a relatively high minimum inhibitory concentration (MIC) and sessile MIC (SMIC) of PAE alone. However, compared with the single drug, the combined use of PAE and FLZ/AmB had a potent synergistic potential to inhibit the virulence factors for Candida. The concomitant use of two drugs was consistently more effective than either drug alone for increasing survival rate, decreasing the fungal burden, and alleviating the pathological features of G. mellonella infected by the fungus. Taken together, these findings demonstrate the anti-Candida effects of PAE plus FLZ/AmB and their potential to increase the sensitivity of C. albicans to FLZ/AmB of PAE.</p

Table_1_Extraction of Extracellular Matrix in Static and Dynamic Candida Biofilms Using Cation Exchange Resin and Untargeted Analysis of Matrix Metabolites by Ultra-High-Performance Liquid Chromatography-Tandem Quadrupole Time-of-Flight Mass Spectrometry (UPLC-Q-TOF-MS).DOC

Fungal infections caused by Candida albicans poses a great threat to human health. The ability of biofilm formation is believed to be associated with resistance-related Candida infections. Currently, knowledge on extracellular matrix (EM) of C. albicans biofilm is limited. In this study, we introduced ion exchange resin, i.e., cation exchange resin (CER) and anion exchange resin (AER), in EM extraction of C. albicans biofilm as well as several non-albicans Candida (NAC) biofilms under static and dynamic states in combination with vortexing and ultrasonication (VU). The metabolites extracted from the dynamic C. albicans biofilm matrix using the CER-VU and VU were identified with ultra-high-performance liquid chromatography-tandem quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) via untargeted filtration. Compared with other physical and chemical extraction methods, CER-VU was demonstrated to be an ideal approach with high-yield acquisitions of EM constituents including proteins, triglycerides and carbohydrates and low-level damages on fungal cell viability and integrity. The untargeted MS analysis further showed the high efficacy of CER-VU, as a large quantity of metabolites (217 versus 198) was matched comprising a great number of lipids, carbohydrates, amino acids, nucleic acids and their derivatives together with a high involvement of signaling pathways compared with the VU alone. However, combining the results from both the CER-VU and VU methods could generate more metabolites. In summary, the EM analysis of the dynamic C. albicans biofilm expands our understanding upon a comprehensive depiction of matrix components and provides another effective approach for EM extraction.</p

Sodium houttuyfonate enhances the mono-therapy of fluconazole on oropharyngeal candidiasis (OPC) through HIF-1α/IL-17 axis by inhibiting cAMP mediated filamentation in <i>Candida albicans-Candida glabrata</i> dual biofilms

Candida albicans and Candida glabrata are two common opportunistic fungi that can be co-isolated in oropharyngeal candidiasis (OPC). Hypha is a hallmark of the biofilm formation of C. albicans, indispensable for the attachment of C. glabrata, which is seldom in mycelial morphology. Increasing evidence reveals a hypoxic microenvironment in interior fungal biofilms, reminding of a fact that inflammation is usually accompanied by oxygen deprivation. As a result, it is assumed that the disaggregation of hypha-mediated hypoxia of biofilms might be a solution to alleviate OPC. Based on this hypothesis, sodium houttuyfonate (SH), a well-identified traditional herbal compound with antifungal activity, is used in combination with fluconazole (FLU), a well-informed synthesized antimycotics, to investigate their impact on filamentation in C. albicans and C. glabrata dual biofilms and the underlying mechanism of their combined treatment on OPC. The results show that compared with the single therapy, SH plus FLU can inhibit the hyphal growth in the mixed biofilms in vitro, decrease the fungal burden of oral tissues and internal organs, restore mucosal epithelial integrity and function, and reduce hypoxic microenvironment and inflammation in a mice OPC model. The possible mechanism of the combined therapy of SH plus FLU can be attributed to the regulation of HIF-1α/IL-17A axis through direct abrogation of the dual Candida biofilm formation. This study highlights the role of HIF-1α/IL-17A axis and the promising application of SH as a sensitizer of conventional antifungals in the treatment of OPC.</p

Activities of two <i>phz‘</i>-<i>’lacZ</i> transcriptional or translational fusions in strain M18 and its two mutants.

<p>The <i>phz1‘-’lacZ</i> and <i>phz2‘-’lacZ</i> transcriptional or translational fusion in plasmids pME6522 (A) or pME6015 (B). Their transcriptional (C) or translational activities (D) in wild type strain <i>Pseudomonas</i> sp. M18 (square), <i>phzA1-G1</i> inactivated mutant M18ΔP1 (triangle) and <i>phzA2-G2</i> inactivated mutant M18ΔP2 (circle). Symbols: open, <i>phz1‘-’lacZ</i> fusion; solid, <i>phz2‘-’lacZ</i> fusion. All experiments were performed in triplicate, and each value is presented as the average ± standard deviation.</p

Secondary structures in the 5′-UTRs of two <i>phz</i> gene clusters were predicted by RNA fold.

<p>The predicted secondary structures in 5′-UTR of <i>phzA1-G1</i> (1–337 nt) and <i>phzA2-G2</i> (1–198 nt) gene clusters (A). The conserved RNA secondary structures were predicted from the alignment of <i>Pseudomonas</i> sp. M18 and <i>P. aeruginosa</i> PA7. The base pairing probabilities were annotated with colors. Three suboptimal secondary structures were also predicted for a portion of the 5′-UTR of the <i>phzA1-G1</i> gene cluster in <i>Pseudomonas</i> sp. M18 (B). Gibbs free energies (ΔG) of the three suboptimal local structures from left to right are −39.3 kcal/mol, −38.1 kcal/mol and −36.7 kcal/mol, respectively.</p

Integrative relationship of the Gac/Rsm signal transduction pathway and the expressions of two <i>phz</i> clusters.

<p>Diagrammatic representation of the integrative relationship of the Gac/Rsm signal transduction pathway and the expressions of two <i>phz</i> clusters in <i>Pseudomonas</i> sp. M18. Less efficiency of expression of <i>phzA2-G2</i> gene cluster produce a small amount of PCA signal molecule to auto-induce transcriptional activity itself and the expression of <i>phzA1-G1</i> gene cluster (red fine lines). The high efficiency expression of <i>phzA1-G1</i> at transcriptional level were blocked by its 5′-UTR region and could be relieved partial in post-transcriptional level by PCA or some unknown factor(s) (red thick lines). The interactions of induction and self-induced among two <i>phz</i> clusters resulted in a large amount of PCA production as antibiotics for bio-control. The <i>phzA2-G2</i> expression was negatively controlled mainly at the post-transcriptional level by regulator GacA in respond to environmental signals at overall level. Symbols: solid circle, inhibition; solid arrow, activation; diamond, environmental signals; X, unknown factor(s).</p

The physical map, growth and PCA production of strain M18 and its two <i>phz</i> mutants.

<p>The two inactivated <i>phz</i> gene clusters with their flanking genes in <i>Pseudomonas</i> sp. M18 (A) and the growth curves (B) and PCA production (C) in wild type strain and the two mutants M18ΔP1 and M18ΔP2 with or without exogenous PCA molecules. Symbols in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019413#pone-0019413-g001" target="_blank">figure 1A</a>: red solid arrows denote the two <i>phz</i> core gene clusters; empty arrows, the <i>phz</i> flanking genes; fine line, Gm, genamycin resistence gene; +1, indicates the transcriptional start site (TSS) and the numbers indicate the relative length from TSS; black lines, intergenic regions between <i>phz</i> gene clusters and flanking genes; black solid lines denote the 88 bp sequence homologous to that in <i>P. aeruginosa</i> PAO1; grey solid line denotes additional 56 bp sequence in strain M18. Symbols in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0019413#pone-0019413-g001" target="_blank">figure 1B and 1C</a>: square, wild type strain M18; triangle, mutant M18ΔP1; circle, mutant M18ΔP2; red solid and blue empty denote presence or absence of exogenous PCA in culture, respectively. All experiments were performed in triplicate, and each value is presented as the average ± standard deviation.</p