Differential alteration in Lactiplantibacillus plantarum subsp. plantarum quorum-sensing systems and reduced Candida albicans yeast survival and virulence gene expression in dual-species interaction

ABSTRACT Candida albicans (C. albicans) and Lactiplantibacillus plantarum subsp. plantarum (L. plantarum) are frequently identified in various niches, but their dual-species interaction, especially with C. albicans in yeast form, remains unclear. This study aimed to investigate the dual-species interaction of L. plantarum and C. albicans, including proliferation, morphology, and transcriptomes examined by selective agar plate counting, microscopy, and polymicrobial RNA-seq, respectively. Maintaining a stable and unchanged growth rate, L. plantarum inhibited C. albicans yeast cell proliferation but not hyphal growth. Combining optical microscopy and atomic force microscopy, cell-to-cell direct contact and co-aggregation with L. plantarum cells surrounding C. albicans yeast cells were observed during dual-species interaction. Reduced C. albicans yeast cell proliferation in mixed culture was partially due to L. plantarum cell-free culture supernatant but not the acidic environment. Upon polymicrobial transcriptomics analysis, interesting changes were identified in both L. plantarum and C. albicans gene expression. First, two L. plantarum quorum-sensing systems showed contrary changes, with the activation of lamBDCA and repression of luxS. Second, the upregulation of stress response-related genes and downregulation of cell cycle, cell survival, and cell integrity-related pathways were identified in C. albicans, possibly connected to the stress posed by L. plantarum and the reduced yeast cell proliferation. Third, a large scale of pathogenesis and virulence factors were downregulated in C. albicans, indicating the potential interruption of pathogenic activities by L. plantarum. Fourth, partial metabolism and transport pathways were changed in L. plantarum and C. albicans. The information in this study might aid in understanding the behavior of L. plantarum and C. albicans in dual-species interaction. IMPORTANCE The anti-Candida albicans activity of Lactiplantibacillus plantarum has been explored in the past decades. However, the importance of C. albicans yeast form and the effect of C. albicans on L. plantarum had also been omitted. In this study, the dual-species interaction of L. plantarum and C. albicans was investigated with a focus on the transcriptomes. Cell-to-cell direct contact and co-aggregation with L. plantarum cells surrounding C. albicans yeast cells were observed. Upon polymicrobial transcriptomics analysis, interesting changes were identified, including contrary changes in two L. plantarum quorum-sensing systems and reduced cell survival-related pathways and pathogenesis determinants in C. albicans.

addition, L. plantarum had been combined with some chemicals to enhance its anti-C.albicans activity.Selenium dioxide-treated L. plantarum or its cell-free spent broth inhibited the growth of C. albicans ATCC 14053 (35).
Collectively, the anti-C.albicans activity of L. plantarum has been explored in the past decades.However, a few aspects had not been considered resulting in the limitation of current studies.First, most studies on the interaction of Lactobacilli and C. albicans eliminated the importance of yeast form.The more invasive hyphal form has been commonly recognized as a major contributor to cause pathogenicity.Thus, it is reasonable that hyphal cells have been paid more attention.But yeast form is an inevitable stage C. albicans cells would experience, especially in environments in which they co-exist with Lactobacilli (low pH).Second, hyphal growth/proliferation was overlooked in most studies.The influence of Lactobacilli on hyphal formation (yeast-tohyphae transition) has been emphasized.Third, the intrinsic genetic interaction between L. plantarum and C. albicans remains unclear.The effect of C. albicans on L. plantarum had also been omitted.Thus, in this study, we took both C. albicans yeast and hyphal form into consideration to explore their interaction with L. plantarum.C. albicans yeast-L.plantarum and C. albicans hyphae-L.plantarum mixed cultures were set up and their proliferation, morphology, as well as transcriptomics-level changes in both species were investigated.

L. plantarum inhibits C. albicans yeast cells' proliferation but not hyphal cells
Upon initial assessment by modified agar overlay assay, clear inhibitory zones were observed around either spotted microcolony or streaked culture line of L. plantarum (Fig. S1), suggesting the inhibition of L. plantarum on C. albicans proliferation.Thus, with single culture as a control, the proliferation of C. albicans and L. plantarum in mixed culture was examined by colony-forming unit (CFU) counting on respective selective agar plates.Considering filamentation is a distinct trait of C. albicans, yeast and hyphal cells were examined separately in the interaction with L. plantarum.
In the mixed culture of L. plantarum and C. albicans yeast cells (Fig. 1A through C), the growth of L. plantarum was stable and consistent with that in the corresponding single culture, indicating L. plantarum cell proliferation was not influenced by C. albicans yeast cells.The growth of C. albicans yeast cells in mixed culture remained stable and uninfluenced within 12 h in all groups.However, in mixed culture, yeast cell proliferation was suspended when L. plantarum entered the stationary phase (16 h in 1:1 and 100:1 groups and 20 h in 1:100 group), resulting in a decreased yeast cell number (80.45%-82.49%less at 24 h) compared with single culture.The significant inhibition appeared at 16 h in the 1:1 and 1:100 groups (Fig. 1A and B) and 20 h in the 100:1 group (Fig. 1C), corresponding with L. plantarum stationary phases.Concerning inhibition rate, in the 1:1 group, 73.66% (7.46 × 10 7 CFU/mL), 80.14% (1.44 × 10 8 CFU/mL), and 82.34% (1.99 × 10 8 CFU/mL) reduction were recorded at 16, 20, and 24 h in C. albicans yeast cell proliferation, respectively.In the 100:1 group, 73.37% (8.80 × 10 6 CFU/mL), 80.31% (1.88 × 10 7 CFU/mL), and 80.45% (2.55 × 10 7 CFU/mL) reduction were recorded at 16, 20, and 24 h in C. albicans yeast cell proliferation, respectively.In the 1:100 group, 69.89% (1.26 × 10 8 CFU/mL) and 82.49% (1.99 × 10 8 CFU/mL) reduction were recorded at 20 and 24 h in C. albicans yeast cell proliferation, respectively.The results indicated that the inhibition of L. plantarum on C. albicans yeast cell proliferation is dependent on the cell density of L. plantarum.
Hyphal cells are more robust and adapt to various environments (36).In the mixed culture of L. plantarum and C. albicans hyphal cells, the proliferation of both strains was not influenced (Fig. 1D).Considering C. albicans stays as yeast cells in the oral cavity, intestinal tract, and genital tract of healthy human bodies, where they co-exist with L. plantarum, it is important to explore their interaction and how they stay (13,14).Thus, the interaction between L. plantarum and C. albicans yeast cells was further explored.

Co-aggregation occurs during dual-species interaction
The morphology of L. plantarum and C. albicans yeast cells was observed under an optical microscope and atomic force microscope (AFM) (Fig. 2).No significant difference in cell shape was identified in optical microscopic images of single and mixed cultures (Fig. 2A through F).Interestingly, in the 24-h mixed culture, more C. albicans yeast cell aggregates surrounded by a few L. plantarum cells were observed (Fig. 2B).Such a phenomenon was also observed in the AFM images of mixed cultures, with single cultures as controls (Fig. 2G through L), demonstrating direct cell-cell contact and interaction between L. plantarum and C. albicans yeast cells.Each C. albicans yeast cell was surrounded by multiple L. plantarum cells (Fig. 2G and H).The surface roughness of L. plantarum and C. albicans yeast cells was calculated based on the 3D images from AFM.The surface roughness of yeast cells was three times higher than that of L. plantarum cells and showed a significant decrease in mixed culture (Fig. 2I).

Role of cell-free culture supernatant in reduced C. albicans yeast cell prolifer ation
With De Man Rogosa Sharpe (MRS) broth and distilled H 2 O as control groups, cell-free culture supernatant (CFCS) collected from 6-, 12-, 24-, and 48-h L. plantarum culture (6 h Lacto S, 12 h Lacto S, 24 h Lacto S, and 48 h Lacto S) was added in C. albicans yeast culture to examine the change in yeast cell proliferation.Different initial concen trations (10 7 and 10 5 CFU/mL) and ratios (1:1, 1:100, and 100:1) of L. plantarum and C. albicans were included in this experiment.Here, the growth of C. albicans yeast cells with the supplement of CFCS collected from L. plantarum culture with an initial concentration of 10 7 CFU/mL is shown as a representative (Fig. 3).The yeast cell proliferation in groups supplemented with 6 h Lacto S and 12 h Lacto S (Fig. 3A) remained unchanged compared to control groups.Significant repression of yeast cell proliferation was observed in groups supplemented with 24 h Lacto S and 48 h Lacto S (Fig. 3A).The culturable yeast cell numbers at 12 and 24 h in groups supplemented with 12 h Lacto S (no inhibition) and 24 h Lacto S (inhibition) were further examined (Fig. 3B).Consistently, no change was observed in the group supplemented with 12 h Lacto S, while significant reduction (59.84% at 12 h and 74.92% at 24 h) was recorded in the group supplemented with 24 h Lacto S. Thus, CFCS from L. plantarum culture played a role in the reduced C. albicans yeast cell proliferation but was maturity dependent.In addition, the initial concentration of C. albicans was positively related to the inhibition rate (Fig. 3C; Fig. S2).
For the groups supplemented with CFCS collected from 6-, 12-, 24-, and 48-h mixed culture [6 h (Lacto + Can) S, 12 h (Lacto + Can) S, 24 h (Lacto + Can) S, and 48 h (Lacto + Can) S], MRS-yeast peptone dextrose (YPD) broth and distilled H 2 O served as control groups.Similarly, significant repression of yeast cell proliferation was observed in groups supplemented with 24 h (Lacto + Can) S and 48 h (Lacto + Can) S (Fig. 3D).Surprisingly, 6 h (Lacto + Can) S and 12 h (Lacto + Can) S also showed significant inhibition on yeast cell proliferation although to a much lower level than 24 h (Lacto + Can) S and 48 h (Lacto + Can) S. The culturable yeast cell numbers at 12 and 24 h in groups supplemen ted with 12 h (Lacto + Can) S and 24 h (Lacto + Can) S were further examined (Fig. 3E).The inhibition rate was 67.32% at 12 h and 67.32% at 24 h for 24 h (Lacto + Can) S group and 46.39% at 12 h and 46.39% at 24 h for 12 h (Lacto + Can) S group.In the groups with different initial concentrations (10 7 and 10 5 CFU/mL) and ratios (1:1, 1:100, and 100:1) of L. plantarum and C. albicans, a similar observation was found (Fig. S3).In comparison with CFCS collected from L. plantarum single culture, corresponding CFCS collected from mixed culture showed stronger inhibition on yeast cell proliferation (Fig. 3F), indicating specific metabolite accumulation during the interaction between L. plantarum and C. albicans yeast cells.

CFCS mimicking acidified medium is not sufficient to cause reduced C. albicans yeast cell proliferation
Acid production is a typical trait of L. plantarum, which may elicit an adverse environ ment for C. albicans growth (37,38).In order to test the role of acidic environment in reduced C. albicans yeast cell proliferation in mixed culture, the pH value of CFCS was determined.The pH values of MRS broth, MRS-YPD, 12 h Lacto S, 24 h Lacto S, 12 h (Lacto + Can) S, and 24 h (Lacto + Can) S were 5.64, 4.17, 3.88, 5.80, 4.44, and 3.82, respectively.The pH of CFCS collected from L. plantarum single culture showed no significant difference from that collected from the mixed culture.However, CFCS from mixed culture showed stronger inhibition on C. albicans yeast cell proliferation.Thus, pH might not play a key role.For verification, CFCS mimicking acidified medium was prepared by adjusting the pH of MRS and MRS-YPD broth to the same as CFCS and was used as a growth medium for C. albicans yeast cells.Undoubtedly, CFCS mimicking acidified medium was not sufficient to cause reduced C. albicans yeast cell proliferation (Fig. 3G and H). C. albicans yeast cells were able to adapt to acidic environment yield by L. plantarum.

Transcriptomes of L. plantarum and C. albicans yeast
To explore the intrinsic change in L. plantarum and C. albicans yeast cells, single and mixed cultures were collected at 12 and 24 h in biological triplicates and designated as Lh_12 h (12-h L. plantarum single culture), Lh_24 h (24-h L. plantarum single culture), Ca_12 h (12-h C. albicans single culture), Ca_24 h (24-h C. albicans single culture), LhCa_12 h (12-h mixed culture), and LhCa_24 h (24-h mixed culture).To represent the status before and after reduced C. albicans yeast cell proliferation, 12-and 24-h samples were selected.The samples were adapted to RNA-sequencing (RNA-seq) and down stream bioinformatics analysis.The reads from Lh_12 h and Lh_24 h were mapped to the reference genome of L. plantarum WCFS1, and the reads from Ca_12 h and Ca_24 h were mapped to the reference genome of C. albicans SC5314.Especially, reads from LhCa_12 h and LhCa_24 h were mapped to both reference genomes and annotated separately.Fragments per kilobase of transcript per million mapped reads (FPKM) value was used to determine the relative gene expression level of each sample.To identify differentially expressed genes (DEGs), eight comparative groups were investigated, including four groups comparing L. plantarum genes (group 1: Lh_12 h vs LhCa_12 h, group 2: Lh_24 h vs LhCa_24 h, group 3: Lh_12 h vs Lh_24 h, and group 4: LhCa_12 h vs LhCa_24 h) and four groups comparing C. albicans genes (group 1: Ca_12 h vs LhCa_12 h, group 2: Ca_24 h vs LhCa_24 h, group 3: Ca_12 h vs Ca_24 h, and group 4: LhCa_12 h vs LhCa_24 h).Relatively large amounts of DEGs were identified in each group (Fig. 4A through H; Tables S1 and S2).Considering the growth deficiency of C. albicans yeast in mixed culture appeared at 24 h but not at 12 h, overlapped and unique DEGs between comparative groups (L.plantarum group 1 vs 2, 3 vs 4 and C. albicans group 1 vs 2, 3 vs 4) were determined to specifically focus on genes potentially inducing such difference (Fig. 4I through L; Table S3).A total of 435 and 412 unique DEGs were identified in L. plantarum group 2 (Fig. 4I) and C. albicans group 2 (Fig. 4K), respectively, representing the key genes changed in 24-h mixed culture.Regarding potential genes causing reduced C. albicans yeast cell proliferation, 150 and 99 DEGs were uniquely changed in L. plantarum group 4 (Fig. 4J) and C. albicans group 4 (Fig. 4L), respectively.The DEGs in each group were subsequently adapted to annotation against the non-redundant (Nr) database, the Gene Ontology (GO) database, and the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database, as well as enrichment analysis.

Key DEGs alteration in L. plantarum in 12-h interactome
A total of 1,194 genes (234 upregulated and 960 downregulated genes) in L. plantarum showed significant expression change in group 1, representing 12-h interactome with C. albicans (Fig. S1).Based on their fold change and encoding protein, the unique DEGs in L. plantarum group 1 were compared to group 2 and analyzed (Table S3).Quorum sensing (QS) has been documented as a cell-cell communication system that coordinates interactions both within one species and between different species (39).Signal transduction is also a key trait in cell-cell communication.Thus, we paid more attention to the genes involved in quorum-sensing system and signal transduction.In addition, genes that showed no expression in one sample but highly expressed in the other sample within the same group were more likely to contribute to the microbial interaction.For unique DEGs in group 1, 69 genes showed upregulation, including three signaling factors (citF: 5.47-fold, citE: 4.92-fold, and mae: 2.40-fold), and 449 genes showed downregulation, including 23 genes that stopped expression in sample LhCa_12 h.
Next, the unique DEGs were adapted to GO term enrichment analysis.Up and downregulated DEGs were analyzed separately.Uniquely upregulated DEGs in group 1 (Fig. 5A) were significantly enriched in "carbohydrate binding, " "carbohydrate transmem brane transporter activity, " and "carbohydrate transmembrane transporter activity" in molecular function (MF) and "D-ribose metabolic process" and "pentose metabolic process" in biological process (BP).Uniquely downregulated DEGs in group 1 (Fig. 5B) were significantly enriched in "DNA polymerase complex" and "transferase complex" in cellular component (CC), "ligase activity, forming carbon-oxygen bonds, " "ligase activity, forming aminoacyl-tRNA and related compounds, " and "nucleotidyltransferase activity" in MF, "cellular component organization or biogenesis, " "single-organism cellular process, " and "tRNA metabolic process" in BP.

Key DEGs alteration in L. plantarum in 24-h interactome
A total of 1,135 genes (477 upregulated and 658 downregulated genes) in L. plantarum showed significant expression change in group 2, representing 24-h interactome with C. albicans (Fig. S1).Based on their fold change and encoding protein, the unique DEGs in L. plantarum group 2 were compared to group 1 and analyzed (Table S3).Among the unique DEGs in group 2, 312 genes were upregulated, including nine signaling factors (lamB: 21.71-fold, lamC: 6.87-fold, lamA: 16.56-fold, sip1: 3.56-fold, hpk3: 2.64-fold, sip3: 2.23-fold, zmp1: 3.73-fold, ica1: 3.27-fold, lytN: 20.25-fold, and isaA: 564.18-fold), and 147 genes were downregulated.Genes (lamA, lamB, lamC, and lamD) involved in the twocomponent regulatory quorum-sensing system Lam were uniquely upregulated in group   2, indicating C. albicans yeast cells induced the activation of L. plantarum QS system Lam in mixed culture at 24 h but not at 12 h.The expression of downstream genes regulated by the Lam system also changed correspondingly, including membrane proteinencoding genes (ydaS: 0.06-fold, folT: 4.79-fold, and ywzA: 0.05-fold) and stress response genes (asp1: 0.18-fold, asp2: 0.01-fold, and kat: 0.43-fold) (Fig. 7A).In the previous report, deletion of lamA gene showed no difference in growth rate and cell or colony morphol ogy but acquired reduced adherence to glass surfaces (40).Thus, the lamA gene was closely related to cell adherence ability, and its upregulation might contribute to the adherence of L. plantarum cells to C. albicans yeast cells in mixed culture observed under microscopy.

Key DEGs during maturation of L. plantarum from 12 to 24 h
Representing the key genes contributing to the reduced proliferation rate of C. albicans yeast cells in mixed culture, 2 up-and 98 downregulated (seven had no expression in sample LhCa_24 h) DEGs were identified uniquely in C. albicans group 4. Regarding GO terms, downregulated DEGs (Fig. 5O) were significantly enriched in "ribonucleoprotein complex, " "polymer complex, " "ribosome, " and "cytoplasm" in CC, "structural molecular activity" in MF, and "gene expression, " "citric acid metabolism, " and "tricarboxylic acid metabolism" in BP.A total of five pathways were significantly enriched in unique DEGs in group 4 (Fig. 6I), including "ribosomes, " "TCA cycle, " "oxidative phosphorylation, " "carbon metabolism, " and "glyoxylic acid and dicarboxylic acid metabolism." In addition, the enriched pathways were compared among the four groups (Fig. 6J).

DISCUSSION
Studies on human genital and gastrointestinal tract microbiota have revealed Lactobacil lus spp.as a frequently identified, even dominant bacterial genus (29,(41)(42)(43), mostly associated with health benefits, and C. albicans as an opportunistic fungal pathogen inducing candidiasis (12).L. plantarum strains from diverse niches, including the human oral cavity, vagina, feces, and fermented foods, had been tested to acquire anti-C.albicans activity and were considered a promising probiotic treatment for VVC (30)(31)(32)(33)(34).However, the anti-C.albicans activity differs among Lactobacillus species and strains and is influenced by various factors (19)(20)(21)(22).In addition, most studies have focused on the anti-hyphal formation effect, eliminating the importance of C. albicans yeast form, the proliferation of hyphae cells, and the effect of C. albicans posed on L. plantarum.In this study, a mixed culture model of L. plantarum BM-LP14723 and C. albicans SC5314 yeast and hyphae cells, respectively, was setup, and the changes in proliferation, morphology, and transcriptomes of both strains were monitored.
In the mixed culture of L. plantarum and C. albicans yeast cells, yeast cell proliferation was suspended when L. plantarum entered the stationary phase, while the growth of L. plantarum was stable and uninfluenced.In addition, the inhibition of L. plantarum on C. albicans yeast cell proliferation depended on L. plantarum cellular density.The inhibi tion effect was somewhat consistent with previous studies showing that L. plantarum strains acquired anti-C.albicans activity (30)(31)(32)(33)(34).However, no change was observed in proliferation level in the mixed culture of L. plantarum and C. albicans hyphal cells.As a polymorphic fungus, specific environmental cues can trigger C. albicans to undergo morphological transition from yeast to hyphae (25).Hyphal cells are known to be more robust and acquire stronger resistance to stress, which might contribute to their unchanged proliferation level in the mixed culture with L. plantarum.Considering C. albicans stays as yeast cells in the oral cavity, intestinal tract, and genital tract of healthy human bodies where they co-exist with L. plantarum, it is important to explore their interaction and how they stay (13,14).In a subsequent investigation, we emphasized the interaction between L. plantarum and C. albicans yeast cells.
Short-term co-aggregation through physical interaction had previously been revealed in the early stage of L. plantarum and C. albicans interaction (44).To examine the cell-to-cell interaction between L. plantarum and C. albicans yeast cells, optical micro scopy and AFM were applied to observe the morphological changes.Interestingly, co-aggregation with L. plantarum cells surrounding C. albicans yeast cells was recorded, demonstrating direct cell-cell contact and interaction.The co-aggregation rate between L. plantarum 319 and C. albicans cells had been reported to be the highest among five strains of intestinal Lactobacilli and seven clinical isolates of Candida (44).Among the five strains, Lacticaseibacillus rhamnosus IMC 501, Lacticaseibacillus paracasei IMC 502, and SYNBIO were able to produce H 2 O 2 to co-aggregate and exert antimicrobial activity against pathogenic Candida strains (44).Biosurfactants and bacteriocins secreted by Lactobacilli have also been suggested to contribute to its increased adhesion (45,46).Besides biosurfactants and bacteriocins, H 2 O 2 , lactic acid, exopolysaccharides, and fatty acids produced by Lactobacilli have been key factors contributing to the inhibition of C. albicans growth (9,18,19).In the current study, we also tested the role of L. plantarum CFCS in reduced C. albicans yeast cell proliferation.CFCS from mature L. plantarum culture (>24 h) and CFCS from L. plantarum-C.albicans mixed culture to a higher level contributed to the reduced C. albicans yeast cell proliferation.However, the inhibition rates were lower than L. plantarum culture, suggesting the important role of L. plantarum cells in the interaction.After co-existence for 24 h, the pH of Lactobacilli-C.albicans mixed culture (3.7-4.2) had been identified to be significantly lower than C. albicans single culture (5.3-5.8)(47).Thus, the decreased C. albicans cell number in mixed culture has been suggested due to the lactic acid produced during Lactobacilli metabolism (47).However, the lactic acid produced by Lacticaseibacillus rhamnosus GG has been shown to have no effect on C. albicans growth (19).In this study, the pH values of CFCS from L. plantarum single culture and L. plantarum-C.albicans culture were determined, and pH-adjusted media were used to examine the influence of low pH on C. albicans proliferation.The results showed that CFCS mimicking acidified medium was not sufficient to cause reduced C. albicans yeast cell proliferation.The acidic environment created by L. plantarum could not inhibit C. albicans growth.
Phenotypically, in L. plantarum-C.albicans mixed culture, the proliferation of L. plantarum remained unchanged and that of C. albicans yeast cell was inhibited after 12 h.But it did not mean no change was made by L. plantarum cells.Intrinsic changes should have happened in both L. plantarum and C. albicans cells, leading to the reduced C. albicans yeast cell proliferation, co-aggregation, and higher-level influence of CFCS from mixed culture on C. albicans yeast cells.To explore such changes, regular RNAseq was performed on C. albicans single culture and L. plantarum single culture, and polymicrobial RNA-seq was performed on L. plantarum-C.albicans mixed culture.Two time points were selected, with 12 h as unchanged cell proliferation on both cells and 24 h as reduced C. albicans cell proliferation.Downstream key DEGs' identification and GO term and KEGG pathway enrichment were performed to acquire the gene regulation and pathway changes.In previous studies, genetic changes in C. albicans, especially the expression level of selected virulence factors, had been studied by reverse transcrip tion-polymerase chain reaction (RT-PCR).Considering the limitation of RT-PCR, which could identify single gene expression and complication of genetic polymorphism, global transcriptomics analyses by RNA-seq and bioinformatics were conducted in this study.Importantly, the global changes in both L. plantarum and C. albicans were analyzed, with a few interesting points elucidated.
First, two L. plantarum QS systems (lamBDCA and luxS) showed significant changes in mixed culture.Two types of QS systems, including two-component signaling system and luxS/AI-2 signaling system, had been identified in L. plantarum.Concerning two-compo nent signaling system, lamBDCA and lamKR systems have been found to be cooperative in L. plantarum WCFS1, homologous to the agrBDCA system in Staphylococcus aureus and the fsrABC system in Enterococcus faecalis (40,48).The transcriptional regulation of the lam QS system relies on growth status.In the single culture of L. plantarum, the genes in the lam QS system were found to start expression at 5 h and maintain continuous high expression during post-logarithmic phase and stationary phase (48).In this study, the genes in the lamBDCA system showed significant upregulation in L. plantarum-C.albicans mixed culture, indicating the existence of C. albicans yeast cells induces the activation of the lamBDCA system.The mutation of the lamA gene in the lamBDCA system had been linked to reduced adhesion activity (48), suggesting the possible contribution of the upregulated lamBDCA system to the co-aggregation of L. plantarum and C. albicans cells.Similarly, in S. aureus-C.albicans mixed culture, the lamBDCA homologous agrBDCA system in S. aureus had been revealed to be induced by C. albicans (49,50).Such "coincidence" may indicate that there is a possible link in such interactions.Another QS system, luxS/AI-2, is a common system in many Gram-negative and Gram-positive bacteria, contributing to interspecific communication (51).In Lactobacilli, the luxS gene had been shown to be significantly upregulated in the interaction with Escherichia coli O157:H7, Listeria monocytogenes, and S. aureus (52,53).However, in the current study, the L. plantarum luxS gene was significantly downregulated in mixed culture with C. albicans.Genes involved in the AI-2 production pathway, including metE, metH, and metK (54), were also downregulated in mixed culture, indicating the inactivation of the luxS/ AI-2 system in L. plantarum by C. albicans.A large scale of genes previously identified to be upregulated by AI-2 in Lactiplantibacillus paraplantarum L-ZS9 (54) were also mostly downregulated in this study.The discrepancy elucidated the difference between L. plantarum and C. albicans in communication with other bacteria and suggested the diversity in bacteria-bacteria communication and bacteria-fungi communication.The difference was potentially because the QS systems carried by other bacteria are unique and not identified in fungi (55).Especially the acyl homoserine lactone QS system carried in Gram-negative strains might play a role in inducing the activation of the luxS/AI-2 system in L. plantarum.Besides the signaling factors involved in QS systems, some other signaling factors also showed significant change, mostly upregulation, which are potential stimulating factors for C. albicans.
Second, in mixed culture, the upregulation of stress response-related genes and downregulation of cell cycle, cell survival, and cell integrity-related pathways were identified in C. albicans, possibly connected to the stress posed by L. plantarum and the reduced yeast cell proliferation.The upregulated stress response-related genes were involved in response to oxidative stress, external chemical stimuli, acidic pH, nutritional environment, organic substances, and metal ions.Simultaneously, C. albicans downregu lated ribosomal neogenesis (Fig. 8A), spliceosomes, RNA polymerase (Fig. 8B), matrix transcription factors, cell cycle, meiosis, and MAPK signaling pathway (Fig. 8C) in the early stage, and downregulated ribosomes, ribosome neogenesis, RNA polymerase, and MAPK signaling pathways in the later stage of mixed culture.Ribosome neogenesis includes the synthesis and precise assembly of four rRNAs and approximately 80 ribosomal proteins (56).The downregulation of the ribosome neogenesis pathway might lead to the termination of microbial growth.The cell cycle is a process of repeated DNA replication and mitosis and its intermediate stages (57).The downregulation of the cell cycle pathway indicated that C. albicans slowed down the progress of the cell cycle in mixed culture, potentially resulting in slower proliferation.The downregulation of RNA polymerase and matrix transcription factor pathways indicated a decrease in C. albicans RNA synthesis.The downregulation of the mRNA monitoring pathway, which is a quality control mechanism for detecting and degrading abnormal mRNA, reduced the ability of C. albicans to monitor abnormal mRNA, which might have a greater impact on the growth and metabolism due to the presence of abnormal mRNA.The transition between growth and meiosis in yeast is regulated by nutrient signals, and the downregulation of meiotic pathways indicates that external nutrient signals regulate the conversion of its focus of gravity to growth (58).Therefore, it is possible that C. albicans downregulated cell cycle, RNA synthesis, abnormal mRNA monitoring, and protein synthesis in the early stage and RNA and protein synthesis in the later stage of mixed culture, resulting in a decreased proliferation rate.The partial MAPK signaling pathway [especially the high-osmolarity glycerol pathway contributing to adaption to stress, morphogenesis, and cell wall formation (59)] (Fig. 8C) was also downregulated in the mixed culture, potentially contributing to the reduced proliferation rate of C. albicans.
Third, a large scale of pathogenesis and virulence factors were downregulated in C. albicans (Fig. 5C), including adhesion/invasion (als1, mnt1, and mnt2), phenotypic switch (cph1, efg1, and tec1), proteases (sap1, sap2, sap5, sap6, sap8, sap9, and sap10), nutrient acquisition (pra1), environmental adaption and biofilm formation (bcr1, tec1, and efg1), pH sensing (phr1, rim101, dfg16, and rim21), thigmotropism (mid1), drug resistance (cdr1, cdr2, upc2, erg3, erg11, erg6, and fcy2), and immune evasion (pra1, ace2, xog1, and ece1)associated genes.The adhesion and invasion ability enable C. albicans to adhere and penetrate into host cells (60).The downregulation of adhesion/invasion-related genes in mixed culture indicated the potential interruption of L. plantarum to C. albicans adherence/invasion ability.However, the expression of als3 and hwp1 (61, 62), which are major adhesin and invasion in C. albicans, remained stable in mixed culture, indicating the limited influence of L. plantarum on C. albicans adhesion/invasion.The downregula tion of als3 and hwp1, as well as transcriptional regulator bcr1 and cph1, had been previously identified in C. albicans co-cultured with Lacticaseibacillus rhamnosus (63).In C. albicans pathogenesis, adhesion to host cells is followed by hyphae formation, which is accompanied by the expression of hypha-associated proteins with known damage and immune activation capabilities (64)(65)(66).Cph1 and efg1 deletion mutants had been well documented to lose the ability to form hyphae.In the mixed culture of L. plantarum and C. albicans, hyphae formation-associated genes, cph1, efg1, and tec1, were significantly downregulated, indicating the reduced yeast-hyphae transition ability of C. albicans in the presence of L. plantarum.Secretion of proteases facilitates active penetration of hyphae into epithelia contributing to extracellular nutrient acquisition (67).The downre gulation of some secreted aspartic proteinases (Saps) and nutrient acquisition proteinencoding genes in mixed culture revealed the compromised penetration activity of C.  and C. albicans (E).Red: both upregulated in groups 1 and 2; green: both downregulated in groups 1 and 2; blue: downregulated in groups 1; purple: downregulated in group 2; and yellow: upregulated in group 2.
albicans.Importantly, the ece1 gene, which encodes a polypeptide generating candidaly sin, showed significant downregulation (32-fold) in mixed culture.Candidalysin is a newly discovered toxin contributing to the pathogenesis of C. albicans in multiple infection models (68)(69)(70).The repression of the ece1 gene by L. plantarum indicated that it significantly impacts the pathogenesis of C. albicans.
Fourth, partial metabolism and transport pathways were changed in L. plantarum and C. albicans in mixed culture.In L. plantarum, significantly changed pathways included upregulation of pyruvate and carbohydrate metabolism and downregulation of amino acid (histidine, glycine, serine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, and methionine) metabolism pathways in early stage and upregulation of pyruvate metabolism pathway and downregulation of amino acid (phenylalanine, tyrosine, and tryptophan) metabolism pathways in the later stage.During the whole process, multiple ABC transporters were consistently up/downregulated in L. plantarum (Fig. 8D).In C. albicans, the downregulation of the glycerophospholipid metabolism pathway in the early stage and purine metabolism, taurine and hypotaurine metabolism, pyrimidine metabolism, and cysteine and methionine metabolism pathways in the later stage was identified.

Conclusion
In this study, taking both yeast and hyphal forms of C. albicans into consideration, we set up a dual-species interaction model of L. plantarum and C. albicans and investigated their changes in proliferation, morphology, and transcriptomes (Fig. 8E).Maintaining stable and unchanged growth rate, L. plantarum inhibited C. albicans yeast cell prolifer ation but failed to induce reduced hyphal growth.The inhibition of L. plantarum on C. albicans yeast cell proliferation was dependent on L. plantarum cell density.Combin ing optical microscopy and AFM, cell-to-cell direct contact and co-aggregation with L. plantarum cells surrounding C. albicans yeast cells were observed during dual-species interaction.Reduced C. albicans yeast cell proliferation in mixed culture was partially due to L. plantarum CFCS but not acidic environment.Upon polymicrobial transcriptom ics analysis, interesting changes were identified in both L. plantarum and C. albicans gene expression.First, two L. plantarum QS systems showed contrary changes, with the activation of lamBDCA and repression of luxS.Second, the upregulation of stress response-related genes and downregulation of cell cycle, cell survival, and cell integrityrelated pathways were identified in C. albicans, possibly connected to the stress posed by L. plantarum and the reduced yeast cell proliferation.Third, a large scale of pathogen esis and virulence factors were downregulated in C. albicans, indicating the potential interruption of pathogenic activities by L. plantarum.Fourth, partial metabolism and transport pathways were changed in L. plantarum and C. albicans.The information yield in this study might aid in understanding the behavior of L. plantarum and C. albicans in dual-species interaction.

Strains and growth conditions
C. albicans strain SC5314 and L. plantarum strain BM-LP14723 were maintained as glycerol stock stored at −80°C.A small amount of glycerol stock was spread onto appropriate agar and incubated at optimum conditions (37°C for 48 h for C. albicans and 30°C for 24 h for L. plantarum) to obtain single colonies.A single colony was transferred to 2 mL of appropriate broth and incubated at optimum temperature with shaking at 200 rpm overnight prior to further experiments.MRS medium was used for the optimal growth of L. plantarum.For C. albicans, YPD and Roswell Park Memorial Institute-1640 medium were used for the growth of yeast and hyphal cells, respectively.culture mixed in the ratio of 1:1 with MRS-YPD broth (1:1 mixture of MRS and YPD broth) or distilled H 2 O was used as control.All experimental groups were incubated at 37°C with shaking at 200 rpm.Optical density value at 600 nm (OD 600 ) was determined every 2 h using an InfiniteM200 Pro multimode plate reader (Tecan, Switzerland), and CFU counting on selective YPD agar was performed at 12 and 24 h. to identify significantly enriched GO terms.A P value < 0.05 was considered statistically significant.

FIG 3
FIG 3 Role of CFCS in reduced C. albicans yeast cell proliferation.(A-C) Yeast cell proliferation in groups supplemented with CFCS from L. plantarum single culture examined by OD 600 (A) and CFU (B), with different initial concentrations (C).(D-F) Yeast cell proliferation in groups supplemented with CFCS from mixed culture examined by OD 600 (D) and CFU (E), with different initial concentrations (F).(G and H) C. albicans yeast cell proliferation in CFCS from L. plantarum single culture (G) and mixed culture (H) mimicking acidified media.All experiments were conducted in biological triplicates.*P < 0.05; **P < 0.01; and ***P < 0.001.

FIG 5
FIG 5 Significantly enriched GO terms for unique DEGs in L. plantarum groups 1 (A and B), 2 (C and D), and 4 (E and F), respectively, and overlapped DEGs in L. plantarum group 1 vs 2 (G and H), as well as in C. albicans groups 1 (I and J), 2 (K and L), and 4 (M and N), respectively, and overlapped DEGs in C. albicans group 1 vs 2 (O).

FIG 8
FIG8 Gene expression changes in ribosome neogenesis (A), RNA polymerase (B), MAPK signaling pathway (C), pathways from C. albicans, and ABC transporter pathway (D) from L. plantarum, as well as the dual-species interaction between L. plantarum and C. albicans (E).Red: both upregulated in groups 1 and 2; green: