This study was conducted from May 2024 to January 2025 at Chinese Academy of Medical Sciences and Peking Union Medical College. M. catarrhalis strain 73-OR, derived from prior studies, was a macrolide-sensitive clone belonging to clonal complex CC446 (lacking the A2330T mutation), which exhibits robust invasive capacity in human alveolar type II epithelial cells (A549) (16). The wild-type (WT) strain M. catarrhalis ATCC 25238 was purchased from the American Type Culture Collection (ATCC).

Actin-related protein 2/3 complex (Arp2/3) inhibitor CK-636 (CK-0944636, Cat. No. IC3650, Beijing Solarbio Science &Technology Co., Ltd), CDC42 GTPase inhibitor ML 141 (ab145603, Cat. No. 10155-1-AP, Proteintech Group, Inc. Chicago, USA), actin polymerization inhibitor: Latrunculin A (Cat. No. HY-16929, MedChem Express LLC), Rho GTPase inhibitor: Simvastatin (Cat. No. IS0170, Beijing Solarbio Science &Technology Co., Ltd.), Rac1/CDC42 activator: CN02-B (Cat. No. CN02-B, Cytoskeleton, Inc.).

The human alveolar type II epithelial cell line A549 was purchased from the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences (1101HUM-PUMC000002). CRISPR-Cas9 knockout of CDC42^-/- A549 cells, Rac1^-/- A549 cells, ArpC2^-/- A549 cells and ArpC4^-/- A549 cells were achieved via electroporation using dual-sgRNA pairs designed to excise critical genomic regions, ensuring frameshifts and complete loss-of-function. For all resulting monoclonal lines (CDC42^-/^-, Rac1^-/^-, ArpC2^-/^-, ArpC4^-/^-), successful knockout was rigorously confirmed by Sanger sequencing, which unambiguously identified the intended large-fragment deletions or indels. This direct genomic validation circumvents potential ambiguities of protein-based methods, such as persistent protein detection due to long half-life or antibody specificity issues in Western blotting. The morphology of the cells after recovery revealed no discernible impact of genetic knockout on cellular architecture. Detailed results are presented in Figure 1.

All five cell lines were cultured in F-12K medium supplemented with 10% fetal bovine serum (FBS), 100 μg/mL penicillin and streptomycin, and incubated at 37 °C in a humidified 5% CO₂ incubator. Culture medium was replaced every 1–2 days. Upon reaching 95%–100% confluence, cells were detached using 0.25% trypsin-EDTA, and experiments were conducted with cells at passages 3–5.

Wild-type A549 cells, CDC42^-/- A549 cells, Rac1^-/- A549 cells, ArpC2^-/- A549 cells and ArpC4^-/-A549 cells were inoculated in 6-well culture plates respectively, with a density of 2.5×10⁵ cells/well. Cells were infected with M. catarrhalis at an appropriate multiplicity of infection (MOI) of 10:1 (2.5×10⁶ bacteria/well) for 4 hours. Unattached bacteria were removed by washing with phosphate-buffered saline (PBS; pH 7.2–7.4). To eliminate extracellular bacteria, cells were treated with 300 μg/mL gentamicin for 1 hour, followed by PBS washes to remove residual antibiotic. Cells were detached with 800 μL 0.25% trypsin (Sigma) for 10 minutes, and then lysed with 1200 μL 0.1% saponin (Sigma) for 10 minutes. The lysates were transferred to 1.5 mL Eppendorf tubes and homogenized by repeated inversion. Serial 10-fold dilutions of lysates were prepared. Both undiluted and diluted lysates (100 μL) were plated onto 5% sheep blood agar plates for the number of internalized bacteria. Each dilution was plated in quadruplicate technical replicates, and experiments were repeated in three independent biological replicates.

Wild-type A549 cells, CDC42^-/- A549 cells, Rac1^-/- A549 cells, ArpC2^-/- A549 cells and ArpC4^-/-A549 cells were seeded into 6-well cell culture plates at a density of 2.5×10⁵ cells/well. The plates were incubated at 37 °C in a humidified 5% CO₂ incubator for 24 hours. Each cell line was assigned to three experimental groups: blank control, 73-OR strain infection, and ATCC-25238 strain infection. M. catarrhalis suspension (2.5×10⁶ CFU/well, MOI 10:1) was added to the designated wells. After 4 hours of co-cultivation, cells were washed twice with phosphate-buffered saline (PBS) and detached using 0.25% trypsin at 37 °C until mild detachment occurred. The enzymatic reaction was terminated by adding complete culture medium. The detached cells were transferred to 1.5 mL Eppendorf tubes and centrifuged. After discarding the supernatant, the cells were fixed with 2.5% glutaraldehyde for 48 hours. Fixed samples were processed for TEM. The intracellular and extracellular distribution of M. catarrhalis in A549 cells was analyzed. Additionally, the macropinocytosis-mediated engulfment of bacteria and the volume of resultant macropinosomes were compared across different experimental groups.

Wild-type A549 cells, CDC42^-/- A549 cells, Rac1^-/- A549 cells, ArpC2^-/- A549 cells and ArpC4^-/-A549 cells were respectively seeded into 96-well plates at a density of 2×10⁴ cells/well. Subsequently, M. catarrhalis suspension was added to infect the cells at a multiplicity of infection (MOI) of 10:1 for 4 hours. After infection, cells were fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 20 minutes. The solution was aspirated, and cells were washed 2–3 times with PBS. Permeabilization was performed using 0.1% Triton X-100 in PBS under dark conditions for 30 minutes, followed by three PBS washes. Then, cells were incubated with 50 μL/well of staining solution containing phalloidin (for F-actin labeling) and Hoechst 33342 (for nuclear staining) in PBS at room temperature for 1 hour in the dark. After removing the staining solution, cells were washed twice with PBS, and 200 μL of PBS was added to each well for imaging.

Fluorescence imaging was performed using a Perkin Elmer high-content imaging system (Operetta CLS; PerkinElmer, Hamburg, Germany). The DAPI channel (excitation/emission: 360/460 nm) was utilized to detect Hoechst 33342-labeled nuclei, while the FITC channel (excitation/emission: 488/520 nm) captured phalloidin-stained F-actin. Quantitative analysis of nuclear morphology (count, area) and F-actin content (fluorescence intensity, area) was conducted using dedicated analysis software (Harmony 4.9; PerkinElmer, Hamburg, Germany).

The F-Actin/G-Actin In Vivo Assay Kit (Catalog #BK037; Cytoskeleton, Inc., Denver, CO, USA) was employed to quantitatively assess the ratio of globular actin (G-actin) to filamentous actin (F-actin). Cultured cells were divided into two groups: (1) control group treated with culture medium, and (2) experimental group infected with M. catarrhalis strains 73-OR and ATCC-25238 at MOI of 10:1. Cells were lysed using a F-actin-stabilizing lysis buffer to preserve the structural integrity of F-actin while solubilizing G-actin. The lysate was subjected to sequential centrifugation. First centrifugation at 1,000×g for 5 minutes at 25 °C to remove cellular debris; Second centrifugation for the supernatant was further centrifuged at 100,000×g to separate insoluble F-actin (pellet) from soluble G-actin (supernatant).

Proteins were resolved via 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes for Western blotting. Anti-actin monoclonal antibody (Clone 7A8.2.1, Catalog #AAN02; Cytoskeleton, Inc.) was used for immunodetection, ensuring specificity for both G-actin and F-actin isoforms. Actin bands were quantified using densitometric scanning. The F-actin/G-actin ratio was calculated based on the integrated optical density (IOD) values of the corresponding bands. Intensities of protein bands in Western blots were determined with Image J (National Institutes of Health) (17).

Statistical analysis was conducted using GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). Statistical comparisons between two groups were performed using the unpaired Student’s t-test for normally distributed data or the Mann-Whitney U test for non-normally distributed data. Western blot results were analyzed using ImageJ 1.53i (National Institutes of Health, Bethesda, MD, USA). A statistically significant difference was defined as P<0.05.

No human subjects were involved in this study. The study was approved by the Human Research Ethics Committee of the Peking Union Medical College (No. I-23PJ2153).

This study investigated the involvement of key factors in the Rho GTPase signaling pathway regulating actin polymerization/depolymerization in A549 cells. Among them, A549 cells were pretreated with Arp2/3 complex inhibitor CK-636, CDC42 GTPase inhibitor ML 141, actin polymerization inhibitor Latrunculin A, Rho GTPase inhibitor Simvastatin, and Rac1 and CDC42 activator CN02-B. The untreated A549 cells served as the control group. Then, all groups were infected with the hypervirulent M. catarrhalis strain 73-OR. By invasion assay, the Rho GTPase pathway was confirmed to mediate M. catarrhalis internalization into A549 cells (Figure 2A). TEM revealed that CN02-B induced larger endosomal compartments after M.catarrhalis invaded A549 cells, suggesting enhanced actin-driven membrane remodeling. Conversely, the endosome volumes in the cells pretreated with Simvastatin and ML 141 were smaller. Simvastatin and ML 141 resulted in smaller endosomes, indicative of suppressed actin polymerization and impaired vesicle expansion (Figure 2B).

In this study, we investigated whether CDC42, a key factor in the Rho GTPase signaling pathway, regulated actin polymerization/depolymerization dynamics and thereby modulated invasive efficiency of M. catarrhalis. The invasion assay showed the bacterial counts of 73-OR and ATCC 25238 strains invading CDC42^-/- A549 cells were significantly reduced compared to wild-type A549 cells (Figure 3A). For the 73-OR strain, invasion counts were dramatically reduced in CDC42^-/- A549 cells (Mean:3,510 ± 1,790 CFU/well) compared to wild-type A549 cells (Mean: 37,145 ± 15,501 CFU/well). This represents a mean difference of 33,635 ± 15,604 CFU/well. While the P-value was 0.161, the analysis yielded a very large effect size (Cohen’s d = 1.760), strongly indicating a highly potent biological dependence of 73-OR invasion on CDC42. Similarly, for the ATCC 25238 strain, the invasion reduction was even more profound (Mean Difference: 104,712 ± 46,045 CFU/well), with counts dropping from 110,433 ± 46,016 CFU/well in wild-type A549 cells to only 5,722 ± 1,639 CFU/well in CDC42^-/- A549 cells. Although the calculated P-value was 0.151, the effect size analysis again demonstrated a very large magnitude (Cohen’s d = 1.857), confirming that the invasive efficiency of both M. catarrhalis strains is highly dependent on CDC42 activity. Furthermore, TEM results directly show that CDC42^-/- A549 cells exhibited fewer internalized bacteria and smaller macropinosomes compared to wild-type A549 cells post-infection with both M. catarrhalis strains (Figures 3B, C). In addition, that CDC42^-/- A549 cells showed a significantly increased F-actin/G-actin ratio following bacterial infection compared to wild-type A549, indicating impaired actin depolymerization (Figure 3D). At the same time, by analyzing the fluorescence expression levels of microfilaments after infection of A549 cells by M. catarrhalis, the results also proved that compared with wild-type A549 cells infected by Moraxella catarrhalis, the expression level of microfilaments in CDC42^-/- A549 cells increased (Figures 3E, F). Thereby, CDC 42 modulates M. catarrhalis invasion by regulating actin cytoskeletal remodeling, as evidenced by reduced bacterial uptake, smaller macropinosomes, and elevated microfilaments in CDC42-deficient cells.

In this research section, we investigated whether Rac1, a pivotal component of the Rho GTPase signaling pathway, regulates actin polymerization/depolymerization dynamics and thereby modulates invasive efficiency of M. catarrhalis. Among them, the invasion assay results showed that compared with wild-type A549 cells, the number of bacteria invading Rac1^-/- A549 cells by 73-OR and ATCC 25238 decreased significantly (Figure 4A); For the 73-OR strain, invasion counts were substantially lower in Rac1^-/- A549 cells (Mean:9,833 ± 3,436 CFU/well) compared to WT A549 cells (Mean: 76, 562 ± 53, 978 CFU/well). While the P-value was 0.342, failing to reach conventional statistical significance, the analysis of the mean difference (66,728 ± 54, 087 CFU/well) revealed a large effect size (Cohen’s d=1.007), suggesting a high biological requirement for Rac1 in 73-OR internalization. Similarly, for the ATCC 25238 strain, invasion counts dropped from 203,750 ± 135,232 CFU/well in WT A549 cells to 71,173 ± 60,217 CFU/well in Rac1^-/- A549 cells (Mean Difference:132,577 ± 148,033 CFU/well). The difference was not statistically significant (P = 0.421),but the associated effect size (Cohen’s d = 0.731) indicates a moderate to large biological effect of Rac1 depletion on the invasive process. Also, the transmission electron microscopy results could directly show that the number of bacteria invading Rac1^-/- A549 cells by 73-OR and ATCC 25238 was significantly reduced, and the volume of macropinosomes formed was smaller (Figures 4B, C). Moreover, the results of detecting the expression levels of F-actin/G-actin indicated that compared with wild-type A549 cells infected by M. catarrhalis, the expression ratio of F-actin/G-actin in Rac1^-/- A549 cells was increased (Figure 4D). Meanwhile, by analyzing the fluorescence expression levels of microfilaments after infection of A549 cells by M. catarrhalis, the results also proved that compared with wild-type A549 cells infected by M. catarrhalis, the expression level of microfilaments in Rac1^-/- A549 cells was increased (Figures 4E, F). Therefore, Rac1 modulates M. catarrhalis invasion by governing actin cytoskeletal dynamics, as evidenced by reduced bacterial uptake, diminished macropinosome formation, and increased microfilaments in Rac1^-/- A549 cells.

Through this study, we investigated whether ArpC2, a critical component of the Rho GTPase signaling pathway, modulates invasive efficiency of M. catarrhalis by regulating actin polymerization/depolymerization dynamics. The invasion assay suggested bacterial counts of M. catarrhalis strains 73-OR and ATCC 25238 invading ArpC2^-/- A549 cells showed no significant difference compared to wild-type A549 cells (Figure 5A). The invasion assay suggested that bacterial counts for both M. catarrhalis strains invading ArpC2^-/-A549 cells showed no significant difference compared to wild-type A549 cells. For the 73-OR strain, invasion counts were 25,700 ± 4,197 CFU/well in the WT group and 21,653 ± 13,043 CFU/well in the knockout group (Mean Difference: 4,047 ± 13,701 CFU/well). This minor reduction was highly non-significant (P = 0.782), with a negligible effect size (Cohen’s d = 0.241), suggesting ArpC2 is not a primary factor regulating this process. Similarly, for the ATCC 25238 strain, the results were even more pronounced, with invasion remaining almost identical between the WT group (122,533 ± 37,828 CFU/well) and the ArpC2^-/- group (121,350 ± 34,178 CFU/well). The difference was statistically negligible (P = 0.983), corresponding to a trivial effect size (Cohen’s d = 0.019). TEM results could directly show that the number of bacteria invading ArpC2^-/- A549 cells by 73-OR and ATCC 25238 was not reduced, and the volume of the macropinosomes formed was larger (Figures 5B, C). Additionally, F-Actin/G-Actin in vivo analysis indicated no significant alteration in the F-actin/G-actin ratio was detected between infected wild-type A549 and ArpC2^-/- A549 cells (Figure 5D). Consistent with the above results, F-actin expression levels in ArpC2^-/- cells remained significantly unchanged compared to wild-type A549 controls following bacterial infection (Figures 5E, F). Thus, ArpC2 did not participate in regulating the polymerization/dissociation process of actin and subsequently modulating the number of M. catarrhalis invading A549 cells.

We investigated whether ArpC4, an essential factor of the Rho GTPase signaling pathway regulates actin polymerization/depolymerization dynamics and thereby modulates invasive efficiency of M. catarrhalis. Both M. catarrhalis strains 73-OR and ATCC 25238 invading ArpC4^-/- A549 cells demonstrated no significant difference compared to wild-type A549 cells. The invasion assay suggested that bacterial counts for both M. catarrhalis strains invading ArpC4^-/-A549 cells demonstrated no significant difference compared to wild-type A549cells. However, detailed analysis showed distinct trends. For the 73-OR strain, invasion counts were notably reduced in ArpC4^-/- A549 cells (Mean: 19,990 ± 10,832 CFU/well) compared to WT cells (Mean: 78,212 ± 53,344 CFU/well). While this difference did not reach statistical significance(P = 0.345), the analysis revealed a large effect size (Cohen’s d = 0.873) suggesting a strong biological tendency for ArpC4 to regulate 73-OR internalization. In contrast, for the ATCC 25238 strain, the invasion counts remained nearly identical between WT cells (237,300 ± 127,533 CFU/well) and ArpC4^-/-cells (192,750 ± 126,182 CFU/well). The difference was statistically negligible (P = 0.816), corresponding to a trivial effect size (Cohen’s d=0.203). (Figure 6A). Electron microscopy showed that the number of 73-OR and ATCC 25238 invading ArpC4^-/- A549 cells was reduced, and the size of the macropinosomes formed was large (Figures 6B, C). While ArpC4^-/- A549 cells showed altered F-actin/G-actin ratios under basal conditions, no infection-dependent modulation was detected (Figure 6D). The analysis of the fluorescence expression of microfilaments in A549 cells infected with M. catarrhalis also proved that ArpC4 may affect the expression of microfilaments, but it is not related to M. catarrhalis infection (Figures 6E, F). The above results indicate that ArpC4 participates in the regulation of actin cytoskeletal dynamics but does not contribute to M. catarrhalis invasion into A549 cells, as evidenced by unaltered bacterial uptake and infection-independent actin stabilization.