Human lung epithelial (A549) cells were obtained from the American Type Culture Collection (ATCC, CCL-185). Cells were cultured in Kaighn’s modification of Ham’s F-12 medium (F-12K medium), supplemented with 10% fetal bovine serum (FBS; Gibco, Thermofisher, US) and 1% Antimycotic Antibiotics 100X (Gibco, Thermofisher, US). Cultures were maintained at pH 7.42 at 37°C, and 5% CO₂ in a humidified incubator. Cells were grown in ventilated tissue culture flasks and maintained in growth medium until use.

Bacterial species. Both Group A streptococcus (Streptococcus pyogenes Rosenbach, ATCC – 19615) and S. pneumoniae (Streptococcus pneumonia (Klein) Chester, ATCC – 49619) were obtained from ATCC. Both bacteria strains were inoculated on blood agar plates and incubated at 37°C and 5% CO₂ humidity. Bacterial growth was monitored microscopically to ensure viability and purity.

Human Enterovirus D68 (HEV-D68) strain US/IL/14-18952 (VR-1824™) with taxonomy ID: 167331, was obtained from the ATCC (Manassas, VA). The virus was propagated in A549 cells at 33°C, following protocols described by Elrick et al. (2021) (Elrick et al., 2021). The viral growth medium consisted of DMEM/F-12 medium supplemented with 10% FBS. Cytopathic effect (CPE), characterized by rounded and refractile cells forming clusters across cell layers, were observed four days past infection (DPI). Viral titers were determined, and samples were aliquoted and stored at –80°C. An overview of the cell lines, bacterial strains, and viral propagation conditions used in this study is provided in Table 1.

To determine the infectious dose of HEV.D68, a TCID₅₀ assay was performed. Serial dilutions of the virus ranging from 10⁰ to10^-8, were prepared, and 200 μl from each dilution was added to wells in 96-well plates containing A549 cells. Plates were incubated under optimal conditions until the CPE was observed. After two days, the medium was removed, and the wells were washed, fixed with methanol, and stained with 0.1% crystal violet. Absorbance was measured at 570 nm using an ELISA plate reader. TCID₅₀ value were calculated using the Reed and Muench method (REED and MUENCH, 1938), based on the amount of cell lysis across dilutions. The TCID₅₀ was determined to be 10^-3.

To ensure consistency across experimental conditions, cell viability was monitored 0-, 6-, 12- and 24-hours post-infection. The infectious pressure was evident from the beginning of the experiment and peak around the 6-hour time point. After sample collection at 6 hours (T6), the medium was refreshed to reduce the infectious load. However, infection continued due to pathogens already attached to the epithelial cells at the T6. Figure 2 illustrates the progression of infectious pressure over time, based on morphological changes observed under the microscope. These changes, indicative of CPE, were used to assess the impact of infection. A549 lung epithelial cells typically display a spindle-shaped morphology, forming interconnected networks. Cells were infected at approximately 70% confluence to ensure optimal growth and responsiveness.

HEV-D68 infection resulted in visible CPE, with cells becoming rounded and detaching from the culture surface. At 70-80% confluence, A549 cells are in exponential growth phase, which coincides with active viral replication. By adding the virus at this confluence level, continuous cell splitting occurs parallel to increased CPE as the virus spread through the culture. Continuous monitoring of cell morphology and confluence provided valuable insight into the progression of viral infection and its impact on cell viability.

Due to the increased CPE and lytic effects caused by both HEV-D68 and S. pneumoniae, the number of viable cells available for RNA isolation decreased significantly after 24 hours. Therefore, the experimental endpoint was set at 24 hours. To further assess cell viability, an MTT assay was performed after 24 hours (1 DPI) in A549 cells infected with S. pneumoniae and GAS. This assay confirmed the viability of cells and the ability of both bacterial strains to grow on A549 cells as showed in Figure 3.

Throughout the experiment, an increase in CPE was observed between 12 and 24 hours in both virus-only and co-infected conditions. All samples were visually inspected under an optical microscope prior to harvesting to confirm cell viability. Quality control was also performed before the experiment began. After 36 hours, pronounced differences in CPE were observed, reinforcing the decision to set the experimental cutoff at 24 hours. For future experiment requiring longer durations, the initial infection pressure would need to be reduced to maintain cell viability over extended periods.

Mucin genes play an important role in protecting epithelial cells from external threats (Mettelman et al., 2022). Mucins are classified into membrane-associated and secreted types. Secreted mucins, such as MUC2 and MUC5AC, are highly glycosylated proteins that form a physical barrier to protect cells (Hollingsworth and Swanson, 2004). Investigating these genes provides insight into pathogenesis and host defense mechanisms. TNFα is a key component of the primary immune response to viral infections, promoting cytokine production and contributing to inflammation (Newton et al., 2016). The MAP kinase family, including p38, is involved in cellular processes such as growth, differentiation, and apoptosis (Hollingsworth and Swanson, 2004; Ono and Han, 2000). p38 is activated by extracellular stimuli, making it a relevant marker for studying cellular stress responses (Banerji and Saroj, 2021).

In mono-viral infections, gene expression levels were consistently higher, indicating a stronger cellular response. In contrast, co-infections with GAS or S. pneumoniae showed more distinct gene changes, as showed in Figure 4. This suggest that bacterial co-infection may modulate or suppress the host’s response to viral infection.

The heatmap in Figure 4, reveals that both co-infection conditions follow a similar expression pattern, while the viral-only condition shows distinct upregulation. This supports that viral infection alone elicits a more dynamic and pronounced cellular response. Notably, MUC5C displayed a delayed but stronger upregulation compared to MUC2, indicating differential regulation of mucin genes during infection.

During the first 6 hours, when infectious pressure was highest, MUC2 was upregulated in viral infections. After the medium was refreshed at T6, expression declined by 12 hours, suggesting reduced mucin production as the immediate viral threat subsided. By 24 hours, MUC2 expression increased slightly, possibly reflecting a secondary response to ongoing infection. Both MUC2 and MUC5AC are secreted, gel-forming mucins (Sheng and Hasnain, 2022), and their expression is closely tied to pathogen presence in the environment. The initial upregulation corresponds to active pathogen interaction, while later changes reflect the host’s adaptive response as showed in Figure 5.

TNFα expression peaked between 6- and 12-hours post-infection, coinciding with active viral replication and high infectious pressure as shown in Figure 6. After the medium was refreshed at T6, expression declined, indicating a reduced immune response as extracellular pathogen levels decreased. However, as intracellular replication continued, expression began to rise again toward 24 hours. In co-infections, TNFα expression was less pronounced, suggesting that bacterial presence may dampen the immune response triggered by the virus. This supports the observation that mono-viral infections elicit a stronger cellular reaction compared to co-infections.

The interaction between viruses and sialic acid receptors is believed to trigger the sialylation biosynthesis (Kim, 2020). One of the most common forms of sialic acid in humans is N-acetylneuraminic acid (Neu5Ac), which contributes to mucus formation in the respiratory epithelium by capping the ends of glycan chains in both N-glycosylation and O-glycosylation processes (Haines-Menges et al., 2015; Brockhausen et al., 2009; Traving and Schauer, 1998). O-glycosylation of mucins is a critical post-translational modification (PTM) required to ensure proper protein function (Tarp and Clausen, 2008).

Key enzymes involved in sialic acid biosynthesis include CMAS, GNE and NANS which represent different steps in the sialylation pathway (Lee et al., 2021). Additionally, Siae modifies sialic acid on the cell surface through O-acetylation, making it available for viral binding (Ide et al., 2024).

Despite the high initial infection pressure, only a small proportion of viruses are taken up by the cells in the early stages. The biosynthesis of sialic acid is triggered as a cellular response to infection, particularly when the availability of sialic acid receptors is depleted. After the initial exposure, viruses attach to or are internalized by cells begin replicating, releasing new virions that go on to infect neighboring cells. This cascade increases the demand for sialic acid biosynthesis.

It is evident that CMAS and NANS are more dynamically regulated than GNE, particularly in mono-viral infections where both genes as upregulated, as shown in heatmap (Figure 7). In contract, all co-infection conditions show consistent downregulation across all four biomarkers, suggesting that bacterial presence may supress the sialylation response.

Figure 8 presents the relative gene expression of NANS that shows a downregulation for all the infections. At 6 hours (T6), viral attachment begins, and a small number of viruses are internalized. Between 6 and12 hours, viral uptake increase, initiating biosynthesis. By 24 hours, the expression of NANS rises further in mono-viral infections, reflecting the growing demand for sialic acid synthesis. However, in co-infections with GAS and S. pneumoniae, NANS expression is significantly downregulated, despite the presence of infectious pressure. This suggests that bacterial co-infection may interfere with og supress the host’s biosynthetic response.

Siae is the enzyme responsible for modifying sialic acid on the cell surface, making it accessible for viral binding. Interestingly, while viruses directly utilize sialic acid as a receptor, bacteria such as S. pneumoniae and GAS cleave the molecule but do not consume the receptor in the same way. In co-infection with S. pneumoniae, Siae expression is significantly downregulated after 6 hours (T6) when the infectious pressure is high. Still the gene expression of Siae in the co-infection is significantly downregulated after 24 hours. These findings highlight the distinct impact of bacterial co-infection on host cell biosynthetic pathways compared to viral infection alone.

Samples collected after 36 hours were excluded due to the high infection pressure, which rendered them unrepresentative. At this time point, approximately 50% of the cells has died. The remaining cells at 36 hours (T36) would consist of a mix of infected and non-infected cells, including both dead infected cells and viable non-infected cells. Since we measure the total gene expression based on an equal number of viable cells, the cell population at 36 hours would not accurately reflect the expression profile of pathogen-affected cells.

To extend the experiment duration, we could have reduced the infection pressure from the beginning. This would allow us to isolate RNA from a consistent proportion of viable cells across all samples. Theoretically, based on the viral replication cycle illustrated in Figure 2, the ratio of cells to virus at 36 hours would resemble that observed at 12 hours. A prolonged experiment with reduced infection pressure would likely result in a delayed infection process, with the time points such as 36 and 48 hours reflecting similar gene expression patterns to those seen at 12 and 24 hours.

The immune response is a coordinated process involving various components of the immune system working together to protect cells from external threats. It is evident that the cellular response is entirely different during co-infection, indicating that distinct processes are occurring in the cellular environment. When epithelial cells encounter extracellular pathogens, they initiate immune responses. Mucins, which are expressed in epithelial cells, play a crucial role in promoting cell survival and protecting against external stimuli. Mucus serves a dual purpose: it acts as a vital defense mechanism in the respiratory tract by trapping and clearing pathogens, while also being exploited by pathogens (Zanin et al., 2016).

Epithelial cells are known to produce mucus in response to viral infections such as influenza virus, RSV, and SARS-CoV-2 (Mettelman et al., 2022). This is also observed with HEV-D68, as shown in Figure 3. Mucins are a part of the cellular defense mechanism that prevents pathogens from anchoring to the cells. The upregulation of MUC2 during viral infection is expected, as cells attempt to block pathogens attachment (Rose et al., 2001). The slight upregulation of MUC2 may indicate a secondary response or a return to baseline levels as the cells adapt to the new environment. The trends for all the distinct infections show differences in gene expression.

HEV-D68 infection triggers a dynamic cellular response, as illustrated in Figure 5(MUC2). During co-infections, however, changes in the surrounding environment alter the cellular response. The virus appears to affect cells differently when competing with bacteria, resulting in gene expression patterns that diverge from those seen in mono-viral infections. Bacterial virulence factors, such as pneumolysin from S. pneumoniae disrupt epithelial integrity and suppress antiviral responses by modulating host signaling pathways (Rai et al., 2016). This suppression may explain the reduced expression of mucin and cytokine genes during co-infections. Bacterial factors interfere with host defenses, leading to a more complex and potentially attenuated immune response compared to mono-viral infections (Fieber and Kovarik, 2014; Melvin and Bomberger, 2016).

This downregulation may indicate that the immune system is navigating a more complex regulatory environment. GAS and S. pneumoniae produce virulence factors that facilitate immune evasion, contributing to the downregulation of TNFα (Rahman and McFadden, 2006). The protein kinase p38 MAPK is activated by various stimuli, including molecules like TNFα. p38 is a key enzyme in intracellular signaling pathways involved in stress-induced production of IL-1β and TNFα (Bachstetter et al., 2011). Activation of stress-induced pathways such as p38 MAPK may lead to barrier disruption and enhanced bacterial invasion.

The genes GNE, NANS, CMAS and Siae are integral components of the sialic acid biosynthesis pathway, which plays a crucial role in cell-cell interactions and immune responses. These biomarkers are part of the same pathway and are considered interdependent. Upregulation or downregulation of on gene can lead to compensatory changes in the expression of others to maintain balance in the sialic acid production (Awasthi et al., 2021). During viral infection, all enzymes in this pathway are expected to be upregulated in response to the need to restrain viral spread and maintain cellular functions. The upregulation was confirmed, as shown in Figure 7. GNE, the first enzyme in the biosynthesis pathway, is considered the “master regulator”. Its upregulation during viral infection is expected, as cells must ensure the availability of ManNAc-6- for subsequent steps in the pathway (Bhide and Colley, 2017). The stronger upregulation of NANS may reflect increased demand for Neu5Ac during viral infection. The difference in upregulation between GNE and NANS can be explained by their respective roles in maintaining the flow and output of the pathway (Bhide and Colley, 2017). CMAS upregulation is also triggered by the increased demand for sialic acid. CMAS activates free sialic acids by transferring cytidine monophosphate (CMP) from cytosine triphosphate (CTP) to the hydroxyl group at C2 - a process that initiates sialylation, where sialic acid is added to glycolipids and glycoproteins (Bhide and Colley, 2017).

The major differences in infections lie in how efficiently pathogens bind and anchor to host-cell receptor. In co-infection with GAS, all genes show slight upregulation to meet the increased cellular demand for more sialic acid. However, despite the presence of virus - which typically triggers downstream gene expression - the expected changes do not follow the infection’s progression. For both co-infections, gene expression does not align with the anticipated development of infection. Interestingly, in co-infection with S. pneumoniae, there is no early response or change in gene expression, implying that S. pneumoniae exerts an immediate effect on host cells. Both GAS and S. pneumonia have evolved strategies to interfere with host cell signaling (Fieber and Kovarik, 2014; Melvin and Bomberger, 2016). The presence of bacterial pathogens may alter the host’s gene expression profile, suppressing the demand for sialic acid production and impacting the progression of the viral infection (Melvin and Bomberger, 2016). The bacterial impact in co-infection appears to be more pronounced that the typical demand triggered by mono-viral infection. While mono-viral infection stimulates cells to produce more sialic acid, co-infections do not. One possible explanation is that pathogens compete for receptors on the cell surface, which are not consumed, reducing the need for turnover and production of new sialic acid molecules – resulting in reduced gene expression, as shown in Figure 9. Bacteria compete with viruses for species-specific resources, nutrients, receptors and cell viability.

The variability observed between mono-viral infection and bacterial co-infection can be attributed to difference in pathogen replication rates. S. pneumoniae has a generation time of approximately 24–36 under optimal conditions, whereas HEV-D68 has a replication cycle of about 8–12 hours. This cycle includes viral entry, replication, assembly of new particles, and release (Wang et al., 2017). These differences highlight the faster reproduction rate of viruses compared to bacteria. Viral replication is influenced by host cell conditions, while bacterial growth depends on nutrient availability and the microenvironment shaped by viral infection. Virus-induced cell lysis releases nutrients, which can enhance bacterial proliferation. Additionally, changes in pH and oxygen levels caused by viral infection may affect bacterial growth. To facilitate the interpretation, the genes are categorized into two groups: cellular response and sialic acid biosynthesis.

The primary goal of this experiment was to investigate the lung epithelial cellular response to infection. Our findings reveal that the cellular response differs between mono-viral infections and bacterial co-infections. This variation is likely linked to the virus’s mechanism of binding to and consuming sialic acid receptors, which in turn triggers the sialylation biosynthesis pathway. While our study provides valuable preliminary insights, further research is necessary to confirm the specific impact of HEV-D68 and bacterial co-infection on epithelial cells. HEV-D68 may interact with host cells in ways that differ from other respiratory viruses, and these unique interactions warrant deeper investigation. The use of A549 cells offers a practical model for studying respiratory epithelial responses; however, it comes with limitations. A549 cells may not fully replicate the receptor expression profiles or immune responses of primary lung epithelial cells. This could influence the interpretation of our results, particularly regarding receptor-mediated interactions and immune signaling. Future studies should incorporate primary lung and airway epithelial cells to validate these findings and better assess the microenvironment created during infection.

Another important consideration is the influence of the infection on the growth environment for both virus and bacteria. Viral replication and bacterial proliferation are shaped by the microenvironment, including nutrient availability, pH, oxygen levels, and cell viability. A more detailed understanding of how these factors interact and evolve during co-infection could provide critical insights into pathogen dynamics and host responses.

To advance this research, future studies should focus on validating findings in primary epithelial cell models to ensure physiological relevance and investigate the competitive interactions between viruses and bacteria for cellular receptors and nutrients. By targeting key points in interaction – such as receptor binding, immune evasion, and nutrient acquisition – novel therapeutic strategies could be developed. These may include blocking viral attachment and entry, inhibiting bacterial nutrient uptake, or modulating host cell signaling pathways to enhance immune defense. Such approaches could significantly reduce the severity of respiratory infections and improve treatment outcomes.