Abstract

Background

   NETs, a unique neutrophil immune mechanism, are vital in defending
   against microbial invasions. Understanding the mechanisms of
   co-infection by Candida albicans and Staphylococcus aureus, which often
   leads to higher mortality and poorer prognosis, is crucial for studying
   infection progression.

Methods

   In our study, we established a mouse model of subcutaneous infection to
   characterize the inflammation induced by co-infection. By purifying and
   extracting NETs to interact with microorganisms, we delve into the
   differences in their interactions with various microbial species.
   Additionally, we investigated the differences in NETs production by
   neutrophils in response to single or mixed microorganisms through the
   interaction between neutrophils and these microorganisms. Furthermore,
   we analyzed the gene expression differences during co-infection using
   transcriptomics.

Results

   In vivo, C. albicans infections tend to aggregate, while S. aureus
   infections are more diffuse. In cases of co-infection, S. aureus
   adheres to and wraps C. albicans. NETs exhibit strong killing
   capability against C. albicans but weaker efficacy against S. aureus.
   When NETs interact with mixed microorganisms, they preferentially
   target and kill the outer layer of S. aureus. In the early stages,
   neutrophils primarily rely on phagocytosis to kill S. aureus, but as
   the bacteria accumulate, they stimulate neutrophils to produce NETs.
   Interestingly, in the presence of neutrophils, S. aureus promotes the
   proliferation and hyphal growth of C. albicans.

Conclusion

   Our research has showed substantial differences in the progression of
   co-infections compared to single-microbial infections, thereby
   providing scientific evidence for NETs as potential therapeutic targets
   in the treatment of co-infections.

   Keywords: Candida albicans, Staphylococcus aureus, subcutaneous
   infection, neutrophil extracellular traps (NETs), co-infection, immune
   response

1. Introduction

   The occurrence of infectious inflammation involves numerous complex
   pathogenic factors, with the disruption of microbial balance and immune
   homeostasis as the primary contributors ([43]1) Polymorphonuclear
   Leukocytes (PMNs), often referred to as neutrophils, constitute the
   first line of defense against pathogen infections ([44]2). Imbalance in
   the interaction between PMNs and microorganisms can lead to disease
   manifestations ([45]3). Upon stimulation by pathogenic microorganisms
   or chemical substances, neutrophils release discrete DNA that combines
   with cytoplasmic granule proteins to form a fibrous network structure
   known as NETs. NETs play a pivotal role in the innate immune response
   mediated by neutrophils ([46]4). Their unique network structure enables
   NETs to capture pathogens and restrict their dissemination, while the
   antimicrobial components they contain exert killing effects on the
   pathogens. The functional role of NETs in the development of diseases
   is a forefront research topic ([47]5, [48]6), and their mechanisms in
   inflammation-related diseases ([49]7–[50]9) have been extensively
   studied. Current research primarily focuses on the interplay between a
   single microbe and NETs, yet coinfection with multiple microorganisms
   is a significant factor that aggravates infections. Co-detection rates
   of C. albicans and S. aureus in infectious diseases are high, and their
   associated mortality rates are similarly elevated ([51]10–[52]14).
   However, the co-interaction mechanisms between these two microorganisms
   with NETs remain unexplored.

   The formation of NETs occurs through two main mechanisms. One involves
   nuclear depolarization, dissolution of the nuclear membrane,
   decondensation of chromatin, and then rupture of the plasma membrane to
   release the NETs. Alternatively, neutrophils can release nuclear
   chromatin and granule proteins while maintaining their non-nucleated
   state, allowing them to continue their phagocytic functions ([53]15).
   Moreover, depending on the type of chemical stimulus or pathogen, NETs
   can be formed through different mechanisms, adding complexity and
   posing a challenge to the study of NETs ([54]16).

   S. aureus induces the production of NETs through the secretion of
   proteins such as Staphylococcal Protein A (SpA) and Leukocidin GH (Luk
   GH) ([55]17–[56]19). To evade the lethal effects of NETs, it employs
   nucleases to degrade the DNA structure of NETs and utilizes
   Extracellular Adherence Protein (Eap) to bind to DNA, inhibiting the
   formation of NETs ([57]20). Interestingly, it has been observed that S.
   aureus can induce excessive formation of NETs, leading to the
   destruction of host tissues, facilitation of bacterial dissemination,
   and maintenance of chronic infections ([58]21).

   C. albicans, a dimorphic fungus, primarily exhibits its pathogenicity
   in the hyphal phase. The yeast form of C. albicans primarily relies on
   phagocytosis for elimination, whereas the larger hyphal form employs
   the release of NETs for eradication ([59]22, [60]23). Neutrophils
   selectively release NETs based on their perception of the
   microorganism’s size, a process facilitated by Neutrophil Elastase (NE)
   ([61]24). Although C. albicans secretes nucleases, current research
   indicates that DNA degradation alone cannot fully protect the fungus
   from the effects of various antifungal proteins present in NETs
   ([62]25).

   The aforementioned studies have demonstrated that both C. albicans and
   S. aureus possess the ability to induce NETs production as well as
   evade NETs. However, in the context of co-infection, these
   microorganisms engage in interspecies interactions, resulting in the
   formation of mixed biofilms. Research has shown that S. aureus adheres
   to the hyphae of C. albicans through the agglutinin-like sequence 3
   (Als3), and the fibrin formed by S. aureus around C. albicans enables
   the latter to evade the killing effect of immune cells ([63]26,
   [64]27). Moreover, and the virulence factor Eap produced by S. aureus
   inhibits hypha-induced NETs release ([65]23, [66]28). Nevertheless, the
   role of NETs in this process has not been investigated.

   Neutrophils are considered short-lived effector cells that undergo
   apoptosis in damaged tissues during infectious inflammation. In vitro,
   the lifespan of mouse neutrophils is approximately 24 h, but their
   survival is prolonged in inflammatory tissues ([67]29). When
   neutrophils enter peripheral tissues, their phenotype undergoes further
   adjustments. Therefore, during migration and function, although their
   residence time in tissues is limited, they can adapt their
   characteristics to support immune homeostasis in organs, remaining in
   the inflamed tissue for over 24 h ([68]30) and playing a pivotal role.

   Typically, animal infection models related to C. albicans primarily
   involve in situ models of oral mucosa ([69]31, [70]32) and intestinal
   mucosa ([71]33), where colonization often necessitates the creation of
   a susceptible environment with reduced immunity. This is often achieved
   through the administration of immunosuppressive drugs ([72]34) or by
   controlling the age of animal models. Taking into account various
   interfering factors during colonization, such as the impact of
   swallowing or intestinal peristalsis on microbial colonization in the
   oral cavity ([73]35, [74]36) or intestinal mucosa ([75]33), as well as
   the effect of immunosuppressive drugs on neutrophil immunity, we
   ultimately opted for subcutaneous infection to establish a mouse
   co-infection model. The utilization of subcutaneous injection for
   establishing infection models with a single microorganism is a common
   and well-established approach ([76]37, [77]38), analogous to modeling
   cancer cells in situ ([78]39) or ectopically ([79]40, [80]41), which
   involves injecting the microorganisms into a specific location to
   achieve the desired modeling purpose.

   This study found that in the case of co-infection, S. aureus not only
   significantly stimulates the proliferation and hyphal development of C.
   albicans but also envelops it within its distinctive structure. This
   encapsulation effectively impedes the phagocytic capabilities of
   neutrophils and triggers the rapid deployment of NETs. Notably, when
   confronted with this mixed microbiota, NETs primarily target the outer
   layer of S. aureus, exhibiting a comparatively limited killing effect
   on the bacterium. Consequently, this attribute inadvertently aids the
   C. albicans wrapped by S. aureus to evade NETs-mediated killing,
   thereby enhancing its survival rate. This collaborative evasion
   mechanism between S. aureus and C. albicans significantly bolsters
   their co-infection invasiveness, enabling both pathogens to more
   effectively counter the host’s immune defense system.

2. Materials and methods

2.1. Mouse experiments

   The establishment of the animal model was based on a detailed protocol
   outlined in a previous relevant study ([81]42). C. albicans sc5314 and
   S. aureus NCTC-8325-4 were cultured overnight and resuspended in 9%
   sterile saline. The mice(male, C57BL/6 ([82]43), 6-8 weeks old) were
   divided into four groups, with at least six mice per group (Total =
   24): S. aureus group (1 × 10^7 CFUs in total), C. albicans group (1 ×
   10^6 CFUs in total), the co-infection group (S. aureus 1 × 10^7 CFUs +
   C. albicans 1 × 10^6 CFUs in total), and the uninfected control group.
   The injection volume was 2 mL, administered via subcutaneous injection,
   with the concentration of strains adjusted according to the total
   injection volume. Mice were sacrificed at 5, 24, and 48 h
   post-infection (two mice per group per time point, n=2), and tissue
   samples were collected for histopathological and microbial load
   assessments. Uninfected mice served as the control group for
   comparative analysis. The entire experimental procedure adhered to the
   internationally recognized Animal Research: Reporting of In Vivo
   Experiments (ARRIVE) guidelines.

2.2. Neutrophil isolation

   The isolation of neutrophils was carried out following the protocol
   established by Yansen Xiao ([83]44), with several modifications to
   optimize the process. To isolate neutrophil from bone marrow, bone
   marrow cells from 6 to 8-weeks-old C57BL/6 mice were harvested in
   sterile Hank’s buffered salt solution (HBSS) without Ca^2+/Mg^2+
   (Solarbio, China). The cell suspension was gently layered on top of a
   density gradient consisting of Ficoll Plus 1.077 (Solarbio, China) and
   11191#RNBK6705 (Sigma, Germany), followed by centrifugation at 1,000 g
   for 30 min at 25°C. Unless noted otherwise, neutrophils were cultured
   in RPMI 1640 medium containing 10% FBS.

2.3. Purification of NETs

   We isolated NETs from primary neutrophils of bone marrow using a
   previously described method with slight modifications ([84]45, [85]46).
   Neutrophils were treated with 500 nM PMA (APExBIO, USA) ([86]47) for 4
   h. After removal of the supernatant, NETs adhered at the bottom were
   washed down by pipetting 2 mL of cold PBS and were centrifuged at 1,000
   g at 4°C for 10 min. The cell-free supernatant containing NETs
   (DNA–protein complex) was collected. The DNA concentration of NETs was
   measured by spectrophotometry and the NETs were used for further
   experiments.

2.4. In vitro NET analysis

   To assess NETs formation, neutrophils (1×10^5 cells ([87]48)) were
   seeded on coverslips coated with poly-L-lysine (Sigma, Germany) in
   24-well plates for 30 min before adding S. aureus (MOI=30, MOI=100), C.
   albican (MOI=10 ([88]49)) with S. aureus (MOI=100). After 4 h ([89]50)
   at 37°C, neutrophils were fixed with 4% paraformaldehyde (PFA,
   Biosharp, China) for 10 min, washed three with PBS and permeabilized in
   0.1% Triton X-100 for 5 min. Cells were blocked in PBS containing 5%
   BSA for 30 min, then incubated with anti-histone H3 ([90]51) (1:200,
   17168-1-AP, Proteintech, USA) and anti-MPO (1:200, 66177-1-Ig,
   Proteintech, USA) in blocking buffer overnight at 4°C. After three
   washes in PBS, cells were incubated with fluorochrome-conjugated
   secondary antibodies (1:500, ab97014, ab150077, Abcam, UK) for 1 h, and
   then counterstained with DAPI (Solarbio, China) before mounting
   Observation and photographing were performed with the confocal
   microscopy, and image processing and analysis. The procedures and
   methods were referenced from prior research ([91]44).

2.5. Scanning Electron Microscopy

   The fabrication process for SEM specimens was derived from preceding
   research ([92]52). Primary neutrophils were also incubated in 24-well
   plates with C. albicans, S. aureus or mixed-species at specified MOIs.
   After gentle rinsing, electron microscope fixative was added and left
   for 2-16 h at 4°C. After fixation, the samples were dehydrated with a
   graded ethanol series (25–100%) and washed with 100% ethanol before
   critical point drying. Finally, they were observed using scanning
   electron microscopy.

2.6. Non-contact induction of neutrophils

   Referring to previous studies ([93]53), using a Transwell cell culture
   plate (0.4 µm) as a barrier, a system was established for non-contact
   induction of neutrophils by S. aureus, where the cells were placed in
   the upper chamber and the microorganism in the lower chamber. In each
   chamber, 1×10^5 cells were inoculated, and S. aureus was added at a
   multiplicity of infection (MOI) of 100. After 4 h of co-culture, the
   upper chamber cells were collected and mounted on a confocal dish. The
   immunofluorescence staining method was consistent with the above, and
   the samples were observed under a confocal microscope.

2.7. Antimicrobial analysis

   C. albicans and S. aureus were collected as described above. NETs were
   pre-incubated at 37°C, and diluted with sterile PBS. Gradient-diluted
   NETs were dispensed into 96-well plates, and C. albicans (1×10³ CFUs
   ([94]54, [95]55)) or S. aureus (1×10^6 CFUs ([96]56)) were added,
   separately or combined. After incubation at 37°C for 2, 4, and 8 h,
   samples were collected, diluted, plated, and CFU counts were performed.
   C. albicans cultures were further incubated at 37°C for 24 h, while S.
   aureus cultures were incubated at 35°C for 24 h, before CFU
   enumeration.

2.8. cDNA library preparation and sequencing

   The MOI of interaction between microorganisms and neutrophils was
   selected according to the above experimental results. C. albicans MOI
   was 10 and S. aureus MOI was 100. Each chamber had 1×10^5 cells. After
   a co-incubation period of 4 h, the supernatant was discarded, and the
   remaining samples were promptly frozen in liquid nitrogen. Total RNAs
   was extracted for transcriptome analysis. RNA quality was determined by
   examining A260/A280 with Nanodrop™ OneCspectrophotometer (Thermo Fisher
   Scientific Inc). An amount of 2 μg of total RNAs was used for stranded
   RNA sequencing library preparation using KC-Digital™ Stranded mRNA
   Library Prep Kit for Illumina^® (Catalog NO. DR08502, Wuhan Seqhealth
   technology Co., Ltd., Wuhan, China). The kit eliminates the duplication
   bias during PCR and sequencing steps by using a UMI of 8 random bases
   to label the pre-amplified cDNA molecules. The kit eliminates
   duplication bias in PCR and sequencing steps, by using unique molecular
   identifier (UMI) of 8 random bases to label the pre-amplified cDNA
   molecules ([97]57–[98]59).

2.9. RNA-Seq data analysis

   Raw sequencing data was first filtered by Trimmomatic (version 0.36),
   low-quality reads were discarded and the reads contaminated with
   adaptor sequences were trimmed. Deduplicated Reads were mapped to the
   reference genome of C. albicans (ASM18296v3) and S. aureus (ASM1342v1)
   using STRA software (version 2.5.3a) with default parameters. Reads
   mapped to the exon regions of each gene were counted by featureCounts
   (Subread-1.5.1; Bioconductor) and then RPKM was calculated. RPKM (Reads
   per Kilobase per Million Reads) was used as A measure of gene
   expression ([99]60).

2.10. Statistical analysis

   Experimental data are expressed as mean ± SD. When the experimental
   data were normally distributed, t test or one-way analysis of variance
   was been used. Otherwise, Kruskall-Wallis analysis was been used.
   Differences were considered statistically significant when P < 0.05.
   The statistical analysis of the experimental data obtained was
   performed using GraphPad Prism 7.

3. Results

3.1. Co-infection with C. albicans and S. aureus aggravated the infection

   To compare the characteristics of single-infections and co-infections
   in vivo, we have established a mouse subcutaneous infection model
   ([100] Figure 1 ). Our findings showed ([101] Figure 1A ) that when C.
   albicans infects alone, localized aggregations of infection appear
   within 5 h, developing into spherical or ovoid, yellowish, purulent
   lesions that progressively enlarge a period of 24 to 48 h. In contrast,
   S. aureus infection manifests as diffuse subcutaneous thickening
   accompanied with a yellowish hue. In the co-infection group, comprising
   C. albicans and S. aureus, localized subcutaneous thickening centered
   around a yellowish aggregate is observed at 24 hours. After 48 h, the
   color fades while the thickness intensifies.

Figure 1.

   [102]Figure 1
   [103]Open in a new tab

   Co-infection of C. albicans and S. aureus leads to increased infection.
   (A) Formation of infected masses in mice 5, 24, and 48 h after
   subcutaneous injection. (B) Microbial colony counts of subcutaneous
   infected masses. *** P < 0.001, **** P < 0.0001, ns: no significance.
   (C) Representative microphotographs of pathological changes at
   indicated times in subcutaneous infected mass. Scale bars, 750 μm (D)
   PAS staining of C. albicans infected mass at 24 h, 48 h. Scale bars,
   200 μm. S. a: S. aureus; C. a: C albicans; Red arrow: infected area;
   Yellow arrow: C. albicans hyphae.

   To illuminate the effects of co-infection on the proliferative
   potential of these microorganisms, we conducted microbial load counting
   on the infected tissues ([104] Figure 1B ). Our results indicate that
   the tissue load of C. albicans is higher in the co-infection group
   compared to the single-microbial infection at 24 h. Similarly, the
   tissue load of S. aureus is also elevated in the co-infection group, as
   compared to the single-microbial infection, at 5, 24, and 48 h.

   HE staining ([105] Figure 1C ) showed that C. albicans infection
   exhibits a progressively expanding aggregation pattern over time,
   whereas S. aureus infection demonstrates a diffuse pattern in the
   infected area specifically at 48 h. Intriguingly, in the co-infection
   group, an infection band is noticeable at 5 h, which intensifies over
   time, ultimately culminating in a two-layered structure at 48 h: an
   outer diffuse layer, surrounding an inner aggregated layer.

   To further investigate the characteristics of C. albicans to resist
   neutrophil-mediated immunity In vivo, we performed Periodic Acid-Schiff
   (PAS) staining on C. albicans-infected tissues at 24 and 48 h ([106]
   Figure 1D ). The staining showed the presence of hyphae of C. albicans
   at the edges of the infected sites.

3.2. Killing ability of NETs against C. albicans and S. aureus

   After conducting initial in vivo experiments aimed at examining the
   co-infection characteristics of two microorganisms, we intended to
   further investigate whether these identified characteristics are
   associated with NETs. To accomplish this, we conducted a purified
   extraction of NETs in vitro and exposed them to both single-species and
   mixed-species cultures of the microorganisms. This approach was
   designed to characterize the direct effects of NETs on the
   microorganisms. Under resource constraints, we utilized varying
   concentration gradients to evaluate the antibacterial efficacy of NETs
   against C. albicans and S. aureus ([107] Figures 2A, B ). At the peak
   concentration of 20 ng/μL ([108] Figure 2A ), both microbial species
   experienced pronounced inhibition of colony growth, with C. albicans
   exhibiting notably higher sensitivity compared to S. aureus at the 4 h
   interval. In a further exploration of NETs’ ability to inhibit the
   growth of C. albicans ([109] Figure 2B ), we observed no substantial
   changes in the number of C. albicans cells after 2 h of NETs exposure.
   However, after 4 h of treatment, a distinct difference in the number of
   C. albicans cells compared to the control group was observed.

Figure 2.

   [110]Figure 2
   [111]Open in a new tab

   C. albicans growth inhibited by NETs and protected by S. aureus during
   co-infection. (A) Colony counts of C. albicans and S. aureus assessed
   at 2, 4, and 8 h post-treatment with NETs at 20, 10, and 5 ng/μL. (B)
   Colony counts of C. albicans interacting with NETs evaluated at 2.5, 5,
   10, and 20 ng/μL after 2 and 4 h incubation. * P < 0.05. (C) SEM
   visualization of C. albicans and S. aureus individually or combined
   with NETs (20 ng/μL) for 4 h. Scale bars, 20 μm.

   Following the quantification of NETs’ differential killing effects on
   the two microorganisms, we further examined the morphological changes
   in the microorganisms after NETs treatment using SEM. SEM analysis
   showed ([112] Figure 2C ) that when NETs were applied solely to C.
   albicans, fungal hyphae were still observed to elongate notably, with
   NETs adhering to the surface of the hyphae. However, in the case of
   co-infections, there was a pronounced reduction in the number of S.
   aureus cells on the outer layer, while the hyphae of C. albicans did
   not exhibit elongation compared to the blank control group.

3.3. C. albicans and S. aureus stimulate neutrophils to generate NETs

   In our experiments investigating the interaction between NETs and
   microorganisms, we observed significant differences in the killing
   effect of NETs against two distinct microorganisms. Specifically, the
   killing ability of NETs against S. aureus caught our attention, leading
   us to hypothesize that neutrophils exhibit substantial differences in
   their specific immune responses towards these two distinct microbial
   entities. To further explore the immune mechanisms between neutrophils
   and these morphologically distinct microorganisms, we isolated primary
   neutrophils and examined their interactions with the microorganisms.

   In the co-culture group of S. aureus and neutrophils ([113] Figure 3A
   ), neutrophils in the low MOI group exhibited a significant number of
   phagocytosed S. aureus. However, in the high MOI group, a substantial
   production of NETs was detected. To investigate the role of
   phagocytosis in the formation of NETs induced by S. aureus, we utilized
   a transwell assay to mimic the interaction between neutrophils and S.
   aureus while preventing direct physical contact between them. Through
   this approach, we observed that neutrophils were still capable of
   inducing NETs generation even without phagocytosing S. aureus.

Figure 3.

   [114]Figure 3
   [115]Open in a new tab

   C. albicans and S. aureus stimulate neutrophils to generate NETs. (A)
   Visualization of the interaction between S. aureus and neutrophils
   using confocal microscopy after incubation for 4 h under different
   infection doses (MOI: 30, 100), and isolation of S. aureus (MOI: 100)
   with simultaneous visualization of NET production. Scale bars, 50 μm.
   (B) SEM visualization of C. albicans (MOI: 10) and S. aureus (MOI: 100)
   individually or in combination with neutrophils. Scale bars, 20 µm. (C)
   Confocal microscopy images showing co-localization of Histone-H3 and
   MPO. Neutrophils were induced for 4 h by co-stimulation with C.
   albicans (MOI: 10) and S. aureus (MOI: 100). Scale bars, 40 μm. The
   NETs were stained with Histone-H3 (green), MPO (red) and DAPI (blue).

   Comparing the mechanisms of NETs formation induced by C. albicans and
   S. aureus, scanning electron microscopy showed ([116] Figure 3B ) that
   in the C. albicans group, significant fungal hyphal growth was
   observed, and neutrophils released abundant fibrous NETs that entangled
   and wrapped the C. albicans. In contrast, in the S. aureus group,
   neutrophils released cytoplasmic NETs that wrapped S. aureus. In
   comparing the interaction mechanisms between neutrophils and single
   versus mixed microorganisms, SEM showed that upon the introduction of
   neutrophils, the number of S. aureus that surrounding C. albicans was
   reduced. Additionally, in the mixed infection group, neutrophils
   released cytoplasmic NETs, with a notable increase in the volume of
   some neutrophils.

   To confirm the release of NETs during co-infection, we utilized
   co-localization fluorescence labeling of NETs’ signature proteins
   ([117] Figure 3C ). The experiment showed co-localized fluorescence
   expression around the microorganisms and neutrophils, with the
   fluorescence primarily concentrated around the outer layer of S.
   aureus, while exhibiting reduced expression around the hyphae of C.
   albicans in the interior.

3.4. S. aureus promotes the proliferation and hyphal growth of C. albicans

   To further investigate the interaction between S. aureus and C.
   albicans during co-infections, we employed an RNA-Seq analysis. Given
   the opportunistic nature of C. albicans as an infectious fungus, with
   its hyphal form being the primary pathogenic factor, we focused our
   attention on how this fungus behaves in the presence of S. aureus,
   particularly on its ability to resist neutrophils. To this end, we
   designed three experimental groups to monitor the gene expression
   patterns of C. albicans in detail. Before and after the introduction of
   S. aureus, we observed a significant increase in the number of
   differentially expressed genes in C. albicans ([118] Figure 4A ).Among
   these, the expression levels of hypha-related genes such as HWP1, UME6,
   CPH1, EFG1, and IHD1, as well as biofilm-related genes NDT80 and BRG1,
   were upregulated. While adhesin genes ALS1 and IHD1 showed
   downregulation, HYR1 and HWP1 exhibited upregulation. Furthermore,
   genes related to extracellular matrix formation (IFD6), virulence
   factors associated with core microcolony formation (HWP1, HYR1, SAP5,
   PLB1), and drug resistance (ERG11 and MDR11) were all upregulated
   ([119] Figure 4B ).

Figure 4.

   [120]Figure 4
   [121]Open in a new tab

   Effect of S. aureus on the global gene expression profile of C.
   albicans. (A) Volcano plot illustrating differential gene expression
   (DEG) patterns pre- and post-inoculation with S. aureus. (B) Selected
   genes differentially expressed and associated with specific mechanisms
   are shown in the heat map. (C) GO enrichment analysis of differentially
   expressed genes. Red or blue dots represent upregulated or
   downregulated genes in response to S. aureus, respectively. The inner
   cycle bars represent the statistical significance. Log[2](FC),
   Log[2](SCP group/CP group), denotes the ratio of expression levels
   between two samples (groups). (D) Enrichment analysis of KEGG pathways.
   Color scale represents the ratio of differentially expressed genes
   under the metabolic pathway to all genes annotated to the pathway, and
   the size of circles indicates gene numbers. X-axis, P Value. Y-axis, GO
   term.

   To gain a deeper understanding of these changes, we performed GO (Gene
   Ontology) functional enrichment analysis ([122] Figure 4C ) and KEGG
   (Kyoto Encyclopedia of Genes and Genomes) pathway enrichment analysis.
   GO analysis showed an upregulation of genes related to RNA metabolic
   processes, nucleobase-containing compound metabolic processes, and gene
   expression regulation, while genes involved in drug metabolic processes
   were downregulated. KEGG pathway analysis ([123] Figure 4D ) indicated
   that the introduction of S. aureus primarily upregulated genes related
   to meiosis and cell cycle in C. albicans, suggesting a potential
   stimulatory effect on proliferation. Conversely, genes involved in
   energy metabolism were significantly downregulated.

4. Discussion

   Over the course of the study, male mice were utilized for animal
   modeling. Previous studies have indicated that estrogen can impact the
   function and recruitment of neutrophils ([124]43, [125]44). Therefore,
   selecting male mice with stable hormone levels for immune modeling can
   enhance the generalizability of our experiment. The escalating
   invasiveness of co-infections ([126] Figure 1A ) highlights the growing
   dominance of S. aureus in interspecies competition ([127] Figure 1B ),
   suggesting that the immune system’s response may be influenced by
   microbial interactions. Closely tied to their colonization and
   dissemination abilities is the infective morphology of microorganisms.
   S. aureus, owing to its small, ovoid shape, exhibits a reduced tendency
   for colonization, facilitating the ease of infection spread ([128]61).
   This is reflected in the diffuse infection patterns observed in models
   involving S. aureus ([129] Figure 1C ), which aligns with findings from
   other studies utilizing animal models injected with the bacterium
   ([130]38). Conversely, C. albicans relies on its intricate hyphal
   structure for dissemination and colonization ([131]62), leading to
   localized, aggregated infections.

   When these two microorganisms co-infect, they exhibit a distinctive
   infection pattern: internal aggregation and external dissemination
   ([132] Figure 1A, C ). This pattern not only ensures the pathogens’
   colonization capabilities but also promotes their further dissemination
   and proliferation. The encapsulation phenomenon observed in vivo may
   show a synergistic mechanism for pathogens to evade the immune system.
   Our in vitro experiments ([133] Figures 2C ; [134]3B ) further validate
   the existence of this encapsulation phenomenon, strengthening our
   hypothesis. The primary mode of invasion by C. albicans in vivo occurs
   through outward hyphal infection ([135] Figure 1D ), consistent with
   neutrophils’ rapid response to hyphae ([136]63), which are the primary
   targets of immune cells in vivo.

   Within the context of co-infections, we have delved into the functional
   mechanisms of neutrophil-released NETs. When evaluating the
   bactericidal effect of NETs against two different microorganisms, we
   observed contrasting results ([137] Figure 2A ). NETs exhibited
   relatively weaker killing capability against the smaller S. aureus. In
   contrast, NETs displayed robust bactericidal activity against C.
   albicans, and our experiments further analyzed the killing effect of
   NETs on C. albicans, showed significant effectiveness within 4 h ([138]
   Figure 2B ).

   Intriguingly, when NETs acted solely on C. albicans ([139] Figure 2C ),
   they significantly induced the elongation of its hyphae. However, when
   NETs were exposed to a mixed population of S. aureus and C. albicans,
   no significant changes in the hyphal morphology of C. albicans were
   observed. This finding suggests that S. aureus may somehow counteract
   the potential effect of NETs on the hyphal growth of C. albicans.

   Concurrently, we observed that during extraction, resuspension,
   isolation, or the process of cell death and lysis, NETs might lose some
   of their inherent properties, including cellular control and functional
   activity. This loss significantly impacted their morphological scaffold
   structure, resulting in the absence of a fibrous backbone ([140]64,
   [141]65).

   The methods of NETs production vary among different microbial species,
   resulting in distinct morphological variations ([142]66). Our research
   showed that while both C. albicans and S. aureus can induce NETs
   formation, the resulting NETs exhibit significant morphological
   differences ([143] Figure 3B ). Specifically, C. albicans-induced NETs
   are fibrous and filamentous, whereas S. aureus-induced NETs primarily
   adopt a cytoplasmic morphology. This underscores the distinct pathways
   employed by the two microorganisms in triggering NETs production. When
   confronted with neutrophils, S. aureus is initially phagocytosed by
   neutrophils in large numbers, followed by the release of NETs for
   eradication.

   Notably, during C. albicans’ defense against neutrophils, we did not
   observe phagocytosis. While numerous studies indicate that yeast-form
   C. albicans can be phagocytosed by neutrophils, in actual infection
   environments, the rapid release of NETs promptly stimulates the hyphal
   formation of C. albicans ([144]22, [145]23). This process not only
   inhibits the phagocytosis of neutrophils towards the morphologically
   enlarged C. albicans, but the abundant hyphal growth also accelerates
   the recognition of the pathogen by neutrophils, thus promoting the
   continuous release of NETs.

   In the process of S. aureus inducing NETs, it is generally believed
   that this occurs through a non-lethal pathway, enabling neutrophils to
   maintain their phagocytic capabilities after releasing NETs ([146]66,
   [147]67). However, previous studies have not provided a clear
   understanding of the specific temporal relationship between these two
   mechanisms. To gain further insights, we conducted comparative
   experiments using high and low MOIs (multiplicity of infection) to
   observe the functional mechanisms of neutrophils at the same time
   points ([148] Figure 3A ). Our research findings indicate that
   neutrophils prioritize phagocytosis to clear S. aureus pathogens,
   followed by releasing NETs to target bacteria that escape phagocytosis.
   Notably, the direct phagocytic capacity of neutrophils against S.
   aureus is quantitatively limited. Through further analysis using a
   transwell system to separate S. aureus from neutrophils, we discovered
   that the production of NETs induced by S. aureus does not solely rely
   on phagocytosis as a prerequisite. Instead, certain extracellular
   substances released by microorganisms can trigger the release of NETs.
   This finding provides a novel perspective on understanding the complex
   interactions between neutrophils and S. aureus.

   In our in vitro experiments, we observed that when two microbial
   species co-infect ([149] Figure 3B ), S. aureus tends to adhere to the
   hyphae of C. albicans, forming a unique biofilm structure ([150]68,
   [151]69). This specific configuration effectively segregates C.
   albicans from direct contact with neutrophils, prompting the
   neutrophils to primarily target S. aureus. However, this adhesion
   phenomenon poses a challenge to the phagocytic function of neutrophils,
   rendering them less effective in clearing S. aureus.

   Furthermore, due to the adherence of S. aureus, which leads to an
   increase in the size of the pathogen complex, NE within the neutrophils
   is rapidly activated ([152]24), accelerating the release of NETs.
   Nevertheless, as C. albicans is partially shielded by the S. aureus
   coating, it is not fully exposed to the NETs, resulting in a reduced
   direct killing effect of NETs on C. albicans. Notably, the morphology
   of NETs induced by co-infection resembles that triggered solely by S.
   aureus. This observation aligns with our hypothesis that the primary
   target of NETs shifts from C. albicans to the S. aureus.

   In a transcriptome analysis ([153] Figure 4 ), it was discovered that
   S. aureus not only stimulated an upregulation of hypha-related genes
   ([154]70, [155]71) in C. albicans but also significantly upregulated
   genes involved in pathways representing proliferation rates. S. aureus
   stimulated key genes related to core microcolonies formation ([156]72)
   in C. albicans hyphae and certain biofilm-related genes ([157]70),
   facilitating rapid colonization and increasing drug resistance
   ([158]73). This discovery is mutually corroborated by our findings of
   an increased invasive capability of C. albicans in vivo and the
   production of hyphae observed in vitro. Moreover, during the
   colonization of epithelial cells by C. albicans hyphae, the adhesive
   proteins Als3 and HWP1 play pivotal roles ([159]74, [160]75). Notably,
   in the transcriptome, we found that while Als3 expression remained
   largely unchanged, the expression of HWP1 was upregulated, further
   confirming the importance of Als3 in C. albicans for maintaining the
   stability of epithelial cell-associated adhesion complexes. The
   variation in virulence factors of S. aureus is closely associated with
   its infection stages. Remarkably, the matrix secreted by S. aureus
   while wrapping C. albicans may be a critical factor contributing to
   increased drug resistance ([161]76). This enhanced resistance has the
   potential to adversely affect the bactericidal effect of NETs.
   Specifically, by stimulating the formation of hyphae in C. albicans, S.
   aureus not only impedes the direct phagocytosis of C. albicans by
   neutrophils but also accelerates the release of NETs, potentially
   promoting the progression of infection.

   Based on our results, there appears to be a connection between the
   progression of co-infection and the effects of NETs release. We
   speculate that the release and killing efficiency of NETs under
   co-infection conditions may be influenced by various factors that are
   less significant in mono-infection. Therefore, further research is
   required to delve deeper into these differential mechanisms,
   particularly identifying which factors impact NETs release and function
   compared to mono-infection.

5. Conclusions

   This study conducted a preliminary investigation into the progression
   of co-infection between C. albicans and S. aureus, as well as that of
   single-microbe infection, by constructing a co-infection model. Our
   findings showed significant differences in NETosis during co-infection
   compared to single-microbe infection, offering novel insights for
   controlling the progression of co-infection.

Data availability statement

   The data presented in the study are deposited in the NCBI Sequence
   Readrchive (SRA) repository, accession number: PRJNA1123423.

Ethics statement

   The animal study was approved by the policy of Sichuan University and
   West China School of Stomatology, and the protocol was approved by the
   Ethical Committee of West China School of Stomatology, Sichuan
   University (Chengdu, China) (Project identification code:
   WCHSIRB-D-2020-326, approval date: 10 September 2020). The study was
   conducted in accordance with the local legislation and institutional
   requirements.

Author contributions

   QJ: Data curation, Formal analysis, Funding acquisition, Methodology,
   Writing – original draft. RL: Data curation, Methodology, Validation,
   Writing – original draft. QsJ: Data curation, Methodology, Validation,
   Writing – original draft. YL: Data curation, Methodology, Validation,
   Writing – original draft. JH: Data curation, Formal analysis,
   Methodology, Writing – review & editing. XZ: Data curation, Formal
   analysis, Methodology, Writing – review & editing. OY: Data curation,
   Formal analysis, Methodology, Supervision, Writing – review & editing.
   C-HC: Data curation, Formal analysis, Methodology, Supervision, Writing
   – review & editing. LC: Investigation, Supervision, Validation, Writing
   – review & editing. BR: Investigation, Supervision, Validation, Writing
   – review & editing. ML: Conceptualization, Data curation, Formal
   analysis, Funding acquisition, Investigation, Methodology, Supervision,
   Writing – review & editing.

Acknowledgments