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. 2024 Nov 2;103(12):104504. doi: 10.1016/j.psj.2024.104504

Avian Pasteurella multocida induces chicken macrophage apoptosis by inhibiting the Zyxin-FAK-AKT-FoxO1/NF-κB axis

Pan Li a,b,1, Guangfu Zhao a,1, Tao Tang a, Fang He a, Xiongli Liu a, Nengzhang Li a, Yuanyi Peng a,⁎
PMCID: PMC11577211  PMID: 39510005

Abstract

Pasteurella multocida (P. multocida) can cause infection in various animals, especially livestock and poultry, which can lead to substantial losses to the breeding industry. However, the pathogenesis of avian P. multocida remains largely unknown. In this study, the mechanisms of avian P. multocida pathogenesis were explored. Chicken macrophage HD11 cells were infected with the avian strain PmQ and the bovine strain PmCQ2. PmQ induced higher cytotoxicity and apoptosis and exerted a stronger anti-phagocytotic effect on HD11 cells than PmCQ2. RNA sequencing analysis revealed that focal adhesion (FA)-related genes were significantly downregulated in PmQ-infected HD11 cells compared with that of PmCQ2. Subsequently, phalloidin staining of the F-actin assembly revealed that PmQ more significantly inhibited the formation of FAs in HD11 than PmCQ2. Western blot analysis revealed that the levels of Zyxin and phosphorylated focal adhesion kinase (FAK) were significantly decreased in PmQ-infected cells, confirming that PmQ inhibited FAs. Consequently, PmQ inhibited the FA downstream factor Akt, which decreased NF-κB and FoxO1 phosphorylation, as evidenced by the decreased expression of downstream anti-apoptotic genes (GADD45B, BCL2L1, BCL2A1, and BIRC2) and increased expression of downstream pro-apoptotic genes (BCL6, PKL2, PKL3, and KLF2). Conversely, pharmaceutically inhibiting FA formation using latrunculin A better enhanced PmCQ2-induced than PmQ-induced apoptosis in HD11 cells. Similarly, the knockdown of Zyxin or FoxO1 by siRNA both boosted the PmCQ2-induced apoptosis rates equal to those of PmQ. These results demonstrated that PmQ inhibited Zyxin-dependent FA formation and disrupted the FAK-AKT-FoxO1/NF-κB pathway to induce apoptosis in chicken macrophages. This study thus offers insights into the pathogenesis of avian P. multocida, which could facilitate the development of new strategies against P. multocida infection.

Keywords: Pasteurella multocida, Zyxin-FAK-AKT-FoxO1/NF-κB pathway, Apoptosis, Macrophage, Pathogenesis

Introduction

Pasteurella multocida is a notorious gram-negative coccobacillus that infects various animals, including mammals and birds (Peng et al., 2019). P. multocida can be classified according to differences in capsular polysaccharides into capsular serotypes A, B, D, E, and F (Harper et al., 2012). Of these, type A strains can be isolated from almost all hosts that P. multocida can infect. It causes fowl cholera in poultry and wild birds; pneumonia in cattle, sheep, rabbits, and pigs; bovine respiratory syndrome in cattle; and atrophic rhinitis in pigs (Wilkie et al., 2012). In particular, fowl cholera is characterized by septicemia and high mortality, resulting in significant economic losses to the poultry industry worldwide (Allen et al., 2024; Blakey et al., 2018). Thus, understanding the pathogenesis of avian P. multocida infections is urgently needed.

Macrophages play a key role in promoting innate immunity, detecting bacterial invasion, initiating the inflammatory response, facilitating host defense, and maintaining tissue homeostasis (Galli et al., 2021). Studies in the 1990s investigated the relationship between macrophages and P. multocida. The P. multocida capsule can mediate adhesion and anti-phagocytosis in macrophages (Harmon et al., 1991; Pruimboom et al., 1996). The less virulent strain was more susceptible to macrophage bactericidal activity (Harmon et al., 1992). Recent studies have shown that different virulent strains with different capsule compositions can induce different levels of cytokine activity in macrophages, thereby demonstrating a distinct pathogenic ability in a mouse model challenge (Fang et al., 2020; He et al., 2021). Conversely, mutant strains with reduced capsular contents could alleviate the inflammatory reaction in macrophages, thereby lowering the mortality rate in mouse models (He et al., 2020; Yang et al., 2021). Transcriptomic analysis of lung tissues collected from chickens infected with the avian strain PmCQ2 and bovine strain PmQ revealed that macrophages were the most enriched immune cell type, and exhibited different responses to the two strains (Li et al., 2020). These studies indicate that macrophages are crucial to P. multocida pathogenesis. However, the underlying mechanism has not been fully elucidated.

Focal adhesions (FAs) function as dynamic signaling hubs within the cell and, unsurprisingly, are key targets manipulated by many pathogens (Murphy et al., 2021). The cellular process of FA disassembly is particularly relevant to apoptosis. A study on a chicken fowl cholera model revealed that the expression of the apoptotic effector molecule caspase 3 was significantly elevated in infected chicken liver cells (Tang et al., 2017). Another study demonstrated that liver necrosis is an essential feature in the development of avian cholera, particularly the associated tissue cell inflammation, apoptosis, and necrosis, which involve the activation of the TLR4/NF-κB axis (Cai et al., 2023). Furthermore, serotype A bovine P. multocida can activate the Rassf1-Hippo-Yap pathway to induce pulmonary epithelial apoptosis and disrupt lung tissue integrity to induce pneumonia in mice (Zhao et al., 2024). Thus, the mechanisms of chicken macrophage apoptosis and its role in the pathogenesis of avian P. multocida need to be elucidated.

We previously reported that avian P. multocida infection caused by serotype A PmQ can infect both poultry (e.g., chickens and ducks) and mammals (e.g., mice, rabbits, and pigs), whereas bovine P. multocida infection caused by serotype A PmCQ2 can infect only mammals (Li et al., 2020). We also found that macrophages were the most enriched cell type between chickens infected with PmQ and PmCQ2, suggesting that macrophages play a vital role in the pathogenesis of P. multocida. Therefore, this study explored the pathogenesis of P. multocida by conducting a comprehensive comparison of HD11 chicken macrophages infected with PmCQ2 and PmQ to expand our understanding of the pathogenesis of avian P. multocida and provide insights for the development of strategies against P. multocida infection.

Materials and methods

Bacterial strains and culture condition

Avian strains of capsular serotype A PmQ (accession number: CP033597) sPL665, sPL855, and sPL935 and bovine P. multocida strains of capsular serotype A PmCQ2 (accession number: CP033599), PmCQ1, PmCQ4, and PmCQ5 were isolated by our laboratory and stored at −80 °C. A single colony was selected and cultured in Martin's broth aerobically at 37 °C with shaking at 220 rpm. The cells were counted by plating on Martin's agar plates.

Cell culture and bacterial infection

The immortalized chicken macrophage cell line HD11 (Beug et al., 1979) was kindly provided by Prof. Guoqiang Zhu of Yangzhou University. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM, Gibco, USA) supplemented with 10 % fetal bovine serum (FBS, Gibco), 100  IU/mL penicillin, and 100  μg/mL streptomycin sulfate (Gibco) at 37 °C and 5.0 % CO2 under a humid atmosphere. Before infection, HD11 cells were trypsinized, and live cells were counted via trypan blue exclusion. The cells were seeded at 1 × 106 cells per well, 5 × 105 cells per well, 2 × 105 cells per well, and 1 × 105 cells per well in flat-bottomed 6-well, 12-well, 24-well, and 48-well culture plates (Corning™, Thermo Fisher Scientific, USA). After incubation for 12 h, the cells were rinsed three times with phosphate-buffered saline (PBS), added to DMEM containing 10 % FBS, and infected with a P. multocida strain at a multiplicity of infection (MOI) of 1. The controls included mock-infected cells. For cell morphology analysis, the uninfected/infected cells were fixed with 70 % ethanol for 20  min, stained with 0.5 % crystal violet (Solarbio, Beijing, China) for 20  min, and evaluated under an Olympus microscope.

Quantitation of macrophage associated and phagocytosed P. multocida

As mentioned above, HD11 cells were infected with PmCQ2 and PmQ in 12-well plates at an MOI of 1 for 8 h. Macrophages were washed with cold PBS three times to remove non-associated bacteria and then lysed in PBS containing 0.1 % Triton X-100. The cell lysates were diluted with PBS and grown on Martin's agar plates at 37 °C for 18–24 h, as described previously (He et al., 2020). To count the phagocytosed bacteria, the macrophages were infected with P. multocida as described above and cultured for another 30 min in the presence of 100 μg/mL ciprofloxacin.

Cytotoxicity assays

As mentioned above, HD11 cells incubated in 48-well plates for 12 h were rinsed with PBS three times, added to DMEM containing 1 % FBS, and infected with a P. multocida strain at an MOI of 1. After 2, 4, and 8 h of incubation, the culture media were collected and centrifuged for 5 min at 400 × g. Subsequently, 120 μL of each supernatant was collected to evaluate the release of lactate dehydrogenase (LDH) using an LDH cytotoxicity assay kit (Beyotime, Shanghai, China) according to the manufacturer's instructions. DMEM containing 1 % FBS was used as the blank control, and uninfected cell culture medium was used as the control. The absorbance of the samples was measured at 490 nm using a microplate reader (Multiskan, Thermo Fisher Scientific). The cytotoxicity of the samples was calculated using the following formula:

Cytotoxicity(%)=LDHactivityinthesupernatant/(LDHactivityinthesupernatant+LDHactivityinthecelllysate)×100.

RNA-sequencing and analysis of HD11 Infected with PmCQ2 and PmQ

HD11 cells were seeded at 1 × 106 cells per well in 6-well plates, incubated for 12 h, and then infected with PmCQ2 and PmQ at an MOI of 1. At 8 h post infection, the cells were washed with cold PBS three times and added with 500 μL TRIzol reagent in each well. After the cells were fully lysed on ice, the lysates were collected in 1.5-mL EP tubes, frozen in liquid nitrogen immediately, and sent to the Beijing Genomics Institute (Shenzhen, China) for RNA sequencing and analysis. The data in this study have been deposited with the NCBI Sequence Read Archive (SRA) database under accession number PRJNA786361.

Quantitative real-time-PCR (qRT‒PCR)

The total cellular RNA was extracted using a Cell Total RNA Isolation Kit (Foregene, Chengdu, China) with a DNA-cleaning column. cDNAs were synthesized using a FastKing gDNA Dispelling RT SuperMix Kit (TIANGEN, Beijing, China). Quantitative reverse transcription polymerase chain reaction (qRT‒PCR) was performed using a SsoFast EvaGreen Supermix Kit (Bio-Rad, CA, USA) according to the manufacturer's instructions and a CFX96 system (Bio-Rad). The relative expression levels of genes were normalized to that of glyceraldehyde phosphate dehydrogenase (GAPDH) using the 2−(△△Ct) method (Livak et al., 2001). All primers used in this study are listed in Supplementary Table 1.

Immunofluorescence assays

HD11 cells were seeded at 2 × 105 cells/well in 12-well plates, placed on a 20-mm-diameter cell slide, and incubated for 12 h. After incubation, the cells were rinsed three times with PBS and infected with PmCQ2 and PmQ at an MOI of 1. At 8 h post infection, the cells were washed with cold PBS three times and fixed in 4 % paraformaldehyde for 30  min at room temperature. The cells were rinsed three times in cold PBS and incubated with PBS containing 0.5 % Triton X-100 for 10  min at room temperature. The cells were then rinsed again with cold PBS and incubated with YF®594-Phalloidin (US Everbright Inc., Suzhou, China) for 30 min at room temperature in the dark. The cell slides were rinsed with PBS three times and covered with a glass slide containing a drop of fluorescent mounting medium with 4′, 6-diamidino-2-phenylindole (DAPI) (ZSGB-BIO, Beijing, China). Finally, the cells were marked, and images were acquired using a confocal microscope (Leica, Germany).

Apoptosis determination

HD11 cells were seeded at 2 × 105 cells/well in 12-well plates and incubated for 12 h. The cells were rinsed three times with PBS and infected with PmCQ2 and PmQ at 1 MOI for 8 h. Both the culture medium and trypsin-digested HD11 were collected by centrifugation at 1000 × g for 5 min. The cells were then resuspended in annexin V–fluorescein isothiocyanate (FITC) binding buffer and stained with FITC-conjugated annexin V and propidium iodide (PI) using an Annexin V-FITC Apoptosis Detection Kit (Beyotime, Shanghai, China) according to the manufacturer's recommendation. The samples were subjected to flow cytometry (Agilent, NovoCyte 1000, USA), and the data were analyzed via NovoExpress® software (1.3.1, ACEA Biosciences, Inc., San Diego, CA, USA).

Measurement of mitochondrial membrane potential

The mitochondrial membrane potential (MMP) assay was performed via JC-1 (Beyotime, Shanghai, China). HD11 cells were rinsed three times with PBS, placed in 12-well plates, and infected with PmCQ2 and PmQ at an MOI of 1 for 8 h. The cells were then washed twice with PBS, digested with trypsin, and collected by centrifugation at 600 × g for 4 min at 4 °C. The cells were subsequently resuspended in DMEM containing 10 % FBS and stained with JC-1 according to the manufacturer's protocols. The samples were analyzed by flow cytometry (Agilent, NovoCyte 1000, USA), and the data were analyzed via NovoExpress.

For fluorescence microscopy analysis, HD11 cells were placed in 12-well plates and infected with PmCQ2 and PmQ at an MOI of 1 for 8 h. The positive cells were treated with 10 µM carbonyl cyanide 3-chlorophenylhydrazone (CCCP) for 20 min before testing. All the samples were washed with PBS once, added with 1 mL JC-1 staining working solution, and incubated for 20 min at 37 °C. Then, the cells were washed twice with JC-1 staining buffer and added with DMEM containing 10 % FBS. Finally, images of the samples were captured under a fluorescence microscope (Olympus IX51, Japan).

Western blotting

The antibodies used in this study were mouse anti-GAPDH monoclonal antibody (Solarbio, Beijing, China); rabbit anti-phospho-FAK (Tyr397; Affinity, Jiangsu, China); cleaved caspase-3 (Asp175; 5A1E; rabbit mAb; CST, USA); phospho-AKT (Ser473; D9E rabbit mAb; CST); rabbit anti-phospho-FoxO1 antibody (Ser256; CST, US); zyxin rabbit mAb (ABclonal, Wuhan, China); phospho-NF-kB p65 (Ser529; rabbit pAb and phospho-IKB alpha [Ser32/Ser36] rabbit pAb; Zen-bioscience, Chengdu, China); goat anti-mouse IgG secondary antibody (horseradish peroxidase, HRP; Invitrogen, USA); and goat anti-rabbit IgG secondary antibody (HRP; Invitrogen). HD11 cells cultured in 6-well plates were infected or not infected with PmCQ2 or PmQ for 8 h (n = 3), rinsed with cold PBS, digested with trypsin, and collected. Total protein extracts from the cells were prepared via radioimmunoprecipitation assay buffer supplemented with 1 mM phenylmethylsulfonyl fluoride (Beyotime, Shanghai, China). Protein concentrations were determined via a modified bicinchoninic acid protein assay kit (Sangon Biotech, Shanghai, China). The protein samples were then placed on sodium dodecyl sulfate–polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Bio-Rad, USA). Western blotting was performed using the appropriate antibody. Antibody/antigen complexes were detected via an enhanced chemiluminescence reagent (Bio-Rad), and images were acquired using a chemifluorescence detection system (Tanon, Shanghai, China) at room temperature. The signal intensities of target proteins were quantified and normalized to GAPDH abundance using Image J software (v1.8.0.112).

Latrunculin A treatment

HD11 cells were seeded in 6-well or 12-well culture plates, incubated for 12 h, infected with PmCQ2 and PmQ at an MOI of 1, and treated with 500 nM latrunculin A (Lat. A). At 8 h post infection, the cells were analyzed through immunofluorescence assay, apoptosis test, and western blotting.

siRNA interference manipulations

The mRNA sequences of chicken zyxin and FoxO1 were obtained from the NCBI database and imported into an online website (http://sidirect2.rnai.jp/) (Ui-Tei et al., 2004) to gain the candidate siRNA sequences. Two-pair sequences of each gene were selected and synthesized by Sangon Biotech. The sequences are listed in Supplementary Table 2. HD11 cells were seeded at 2 × 105 or 5 × 105 cells/well in 6-well or 12-well culture plates and incubated for 12 h. The cells were transfected with Lipofectamine™ 3000 reagent (Invitrogen, USA) following the manufacturer's instructions. Scrambled siRNA was transfected as the negative control. After transfection for 24 h, the expression levels of target genes were verified by qRT‒PCR. After the same treatment, the cells were infected with PmCQ2 and PmQ at an MOI of 1 for 8 h and subjected to apoptosis tests and western blotting.

Statistical analysis

All data are expressed as mean ± standard error of the mean, while the remainder of the data expressed as mean ± standard deviation (SD). The data were analyzed using unpaired, two-tailed Student's t-test performed with GraphPad Prism. Significant differences were considered at P < 0.05 (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).

Results

PmQ induced higher cytotoxicity and exhibited stronger anti-phagocytic ability than PmCQ2 in chicken macrophages

To clarify the underlying pathogenic mechanism of avian P. multocida, chicken macrophage HD11 were infected with PmCQ2 and PmQ at an MOI of 1. PmCQ2-infected cells differentiated into a spindle shape, whereas PmQ-infected cells became round (Fig. 1A). The LDH content in the cell culture supernatant was measured at 2, 4, and 8 h post infection. The amount of LDH in the PmQ-infected HD11 supernatant was significantly higher than PmCQ2 at 4 and 8 h, indicating that PmQ induced higher cytotoxicity (Fig. 1B). Plate colony-counting revealed that more PmCQ2 cells were associated with and phagocytosed by HD11 cells than PmQ (Fig. 1C), demonstrating that PmQ exhibited higher cytotoxicity and stronger anti-phagocytic ability than PmCQ2 in chicken macrophages.

Fig. 1.

Fig. 1

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HD11 infected with PmCQ2 and PmQ. (A) Images of HD11 morphology infected with PmCQ2 and PmQ at 8 h via crystal violet staining (bar = 50 μm). (B) Cytotoxicity of PmCQ2- and PmQ-infected HD11 at 2, 4, and 8 h. (C) Statistics of associated and phagocytic bacteria in HD11 at 8 h post infection (ns represents no significance; *** represents p < 0.001).

PmQ induced a higher apoptosis rate in chicken macrophages compared with PmCQ2

To further assess the cytotoxicity of PmQ, the apoptosis rate of HD11 cells infected with PmCQ2 and PmQ was quantified by JC-1 and annexin V-FITC apoptosis staining. PmQ markedly disrupted the MMP in HD11 cells, indicating that more HD11 cells underwent apoptosis (Fig. 2A and B). Similarly, flow cytometry analysis of cell apoptosis by annexin V-FITC revealed a significantly higher percentage of PmQ-infected HD11 cells than PmCQ2-infected ones (Fig. 2C and D). Moreover, fluorescence microscopy revealed that PmQ more severely reduced MMP in HD11 cells (Fig. 2E). Altogether, these results indicate that PmQ induced a higher apoptosis rate than PmCQ2 in HD11 cells.

Fig. 2.

Fig. 2

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Apoptosis test of HD11 infected with PmCQ2 and PmQ. Fowl cytometry measurement of HD11 infected with PmCQ2 and PmQ at 8 h stained with JC-1 (A) and Annexin V/PI (B). Fowl cytometry statistical results of JC-1 (C) and Annexin V/PI (D) (n = 3, data represent the means ± SD). (E) Representative immunofluorescence images of HD11 infected with PmCQ2 and PmQ at 8 h stained with JC-1 (Positive, CCCP treated HD11 cells; red, normal cells; green, apoptotic cells with decreased mitochondrial membrane potential, scale bar = 100 μm).

RNA-seq analysis of HD11 infected with PmQ and PmCQ2

To understand how PmQ induced chicken macrophage apoptosis, the transcriptomes of HD11 cells infected with PmCQ2 and PmQ were obtained. The raw data and mapped reads (more than 99 % clean reads and 88 % unique mapping ratios; Supplementary Table 3) indicate the good quality and reliability of the data. Pearson correlation analysis of the expression patterns in each sample revealed that the intra-groups were highly correlated, whereas the inter-groups were clearly separated (Fig. 3A). Differentially expressed gene (DEG) analysis of the three groups identified 3111 DEGs (1871 upregulated and 1240 downregulated) in PmCQ2-infected HD11 cells (HD11_PmCQ2) compare with the control group (HD11_control); 2700 DEGs (1,502 upregulated and 1,198 downregulated) in PmQ-infected cells (HD11_PmQ) compare with the HD11_control; and 900 DEGs (323 upregulated and 577 downregulated) in HD11_PmCQ2 versus HD11_PmQ (Fig. 3B, Supplementary Table 4). Hierarchical clustering analysis of the DEG profiles also revealed that each of the three biological triplicates clustered together and that significant differences were observed between HD11_PmCQ2 and HD11_PmQ (Fig. 3C). Venn analysis of DEGs in the three group comparisons identified 159 DEGs shared between HD11_PmCQ2 and HD11_PmQ; 294 DEGs shared between HD11_Control and HD11_PmCQ2, and 173 DEGs shared between HD11_Control and HD11_PmQ (Fig. 3D). The full list of Venn analysis results are presented in Supplementary Table 5. The results of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analyses are presented in Supplementary Tables 6 and 7, respectively.

Fig. 3.

Fig. 3

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Transcriptomic analysis and clustering results of HD11 infected with PmCQ2 and PmQ. (A) Pearson correlation between each sample. (B) Statistical results of DEGs. (C) Heatmap of DEGs clustering. (D) Venn diagram of three comparison groups.

Avian PmQ inhibited HD11 FA formation

Because the morphology of HD11 cells was altered by infections with PmCQ2 and PmQ, we screened the DEGs associated with FAs. Clustering analysis revealed that genes in the PmCQ2 infection group were expressed significantly higher than those in the PmQ infection group (Fig. 4A). Among these DEGs, a common reference gene, beta actin (ACTB), was noted, and qRT‒PCR verification confirmed that the Ct value of ACTB qRT‒PCR in HD11_Control, HD11_PmCQ2, and HD11_PmQ were significantly different (Fig. 4B). Compared with another reference gene GAPDH, ACTB expression increased in PmCQ2 infected HD11 but decreased in PmQ infection (Fig. 4C). Zyxin played a crucial role in FA, was present in the DEGs and significantly reduced in PmQ infected HD11 compare to PmCQ2 (Fig. 4D–F). Another FA hallmark, the phosphorylation of focal adhesion kinase (p-FAK), was significantly decreased in PmQ-infected cells, while that in PmCQ2-infected cells increased (Fig. 4E, G). Further verification by phalloidin staining analysis also revealed that F-actin enrichment significantly decreased in PmQ infected HD11 cells but increased in PmCQ2-infected HD11 cells (Fig. 4H). Furthermore, in cells infected with the bovine P. multocida strains PmCQ1, PmCQ4, and PmCQ5, F-actin formation increased, whereas in cells infected with the avian strains sPL665, sPL855, and sPL935, F-actin assembly was suppressed (Supplementary Fig. 1). These results demonstrate that avian P. multocida strains can more significantly inhibit FA formation in chicken macrophages than the bovine strains.

Fig. 4.

Fig. 4

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FAs is dramatically inhibited in PmQ-infected HD11 cells. (A) Heatmap of FA-related gene expression clustering. (B) ACTB qPCR amplification curves. (C) Relative expression levels of ACTB normalized to GAPDH (n = 4). (D) qRT‒PCR validation of Zyxin (n = 4). (E) Western blot images of Zyxin and p-FAK (n = 3). (F) and (G) Densitometric analysis of Zyxin and p-FAK by ImageJ (n = 3, data represent the means ± SD). (H) Confocal fluorescence images of HD11 infected with PmCQ2 and PmQ at 8 h (Red color represents phalloidin staining; blue color represents DAPI staining, scale bar = 25 μm).

PmQ inhibited AKT activation and NF-κB and FoxO1 phosphorylation to mediate apoptosis in chicken macrophages

To elucidate the relationship between FAs and HD11 apoptosis, the activity of AKT (serine-threonine kinase) downstream of FAK, which is vital for cell survival and proliferation, was investigated. Western blot analysis of p-AKT revealed that it was significantly downregulated in PmQ-infected HD11 cells compared with PmCQ2-infected HD11 cells (Fig. 5A and B). Furthermore, the key executor effects protein involved in cell apoptosis, cleaved caspase-3, in HD11_PmQ was significantly increased than HD11_PmCQ2 and HD11_Control (Fig. 5A and C). Furthermore, analysis of downstream AKT revealed that the phosphorylation of FoxO1 (p-FoxO1) and IKBα/NF-κB (p-IKBα/p-P65) was significantly downregulated in HD11 cells infected with PmQ compared with those infected with PmCQ2 (Fig. 6A–D). The phosphorylation of FoxO1 can induce its exonuclear transfer, thereby reducing its transcriptional activity. Consistent with these findings, the expression of downstream genes regulated by FoxO1 (BCL6, PKL2, PKL3, and KLF2), which are pro-apoptotic genes, increased in PmQ-infected HD11 cells (Fig. 6E). However, the downregulation of NF-κB phosphorylation significantly decreased the expression of its downstream genes (GADD45B, BCL2L1, BCL2A1, and BIRC2), which are apoptosis suppressor genes, in PmQ-infected HD11 cells (Fig. 6F). Taken together, in PmQ-infected HD11 cells, the activation of the AKT-NF-κB/FoxO1 pathway was inhibited, thereby inducing chicken macrophage apoptosis.

Fig. 5.

Fig. 5

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PmQ infected HD11 inhibits AKT activation and induces Caspase-3 mediated apoptosis. (A) Western blot images of p-AKT and cleaved caspase-3 of HD11 infected with PmCQ2 and PmQ at 8 h. (B) & (C) Densitometric analysis of p-AKT and cleaved caspase-3 (n = 3, data represent the means ± SD).

Fig. 6.

Fig. 6

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PmQ infected HD11 hinders NF-κB and FoxO1 activation to mediate apoptosis. (A) Expression levels of p-FoxO1, p-IKBα and p-P65 of HD11 infected with PmCQ2 and PmQ at 8 h. (B), (C) and (D) Densitometric analysis of p-FoxO1, p-IKBα and p-P65 (n = 3, data represent the means ± SD); Downstream gene expression qRT‒PCR verification of FoxO1 (E) and NF-κB (F) (n = 4).

Inhibition of FAs promoted PmCQ2-induced apoptosis in chicken macrophage

To further confirm the occurrence of the apoptosis in chicken macrophages infected with P. multocida, the HD11 cells were incubated with latrunculin (Lat.A) to inhibit FAs before infection. After FA inhibition, there was no significant difference on the expression of p-FAK and cleaved caspase-3 in HD11 cells infected with PmCQ2 and PmQ (Fig. 7A–C). Additionally, the difference of apoptotic cell proportion in PmCQ2- and PmQ-infected HD11 was disappeared (Fig. 7D, E). Studies have shown Zyxin could promote actin polymerization involving in FA formation (Hirata et al., 2008). siRNA treatment reduced Zyxin expression by approximately 60 % (Fig. 7F), consequently leading to the expression of cleaved caspase-3 (Fig. 7G, H) and apoptosis rates in PmCQ2- and PmQ-infected HD11 cells reached similar levels (Fig. 7I, J).

Fig. 7.

Fig. 7

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Inhibition of FAs formation promotes HD11 apoptosis infected with PmCQ2. (A) Western blot images of p-AKT and cleaved caspase-3 of HD11 infected with PmCQ2 and PmQ after FAs inhibition. (B) and (C) Densitometric analysis of p-FAK and cleaved caspase-3. (D) and (E) Apoptosis test and statistical analysis of Lat.A inhibition of HD11 infected with PmCQ2 and PmQ at 8 h (n = 3, data represent the means ± SD). (F) qRT‒PCR analysisi of Zyxin expression levels after siRNA treatment (n = 4). (G) and (H) Western blot results and densitometric analysis of cleaved caspase-3 of HD11 infected with PmCQ2 and PmQ after Zyxin siRNA (n = 3, data represent the means ± SD). (I) and (J) Apoptosis test and statistical analysis of HD11 infected with PmCQ2 and PmQ after Zyxin siRNA (n = 3, data represent the means ± SD).

Similarly, siRNA knockdown decreased the expression of FoxO1 has lowered by 50 %, as confirmed by qRT‒PCR (Fig. 8A). Following, western blot analysis of the key apoptosis execute protein cleaved caspase-3 indicated there was no significant difference in PmCQ2- and PmQ-infected HD11 cells (Fig. 8B, C). Moreover, flow cytometry analysis revealed that infection of HD11 cells with PmCQ2 induced a proportion of cell apoptosis similar to that induced by PmQ infection (Fig. 8D, E). These results demonstrated that inhibition of FAs could promote the avirulent PmCQ2 to achieve a similar behavior to that of PmQ infection, which implied that FAs might be a target to prevent P. multocida infection.

Fig. 8.

Fig. 8

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FoxO1 knockdown stimulates PmCQ2-induced apoptosis in HD11 cells. (A) qRT‒PCR analysis of FoxO1 expression level after siRNA treatment (n = 4). (B) and (C) Western blot results and densitometric analysis of cleaved caspase-3 of HD11 cells infected with PmCQ2 and PmQ after FoxO1 siRNA (n = 3, data represent the means ± SD). (D) and (E) Apoptosis test and statistical analysis of HD11 infected with PmCQ2 and PmQ after FoxO1 siRNA knockdown (n = 3, data represent the means ± SD).

Discussion

Macrophages are an important component of the host immune system. They are the first responders to local damage and against infection (Mu et al., 2021). Our previous study on the lung transcriptome of mouse infected with PmCQ2 revealed that overwhelming infectious inflammation is an important cause of host death (Wu et al., 2017). Moreover, He et al. reported that L-serine treatment in mice decreased macrophage-induced inflammation and increased survival rates. (He et al., 2019). Furthermore, L-ascorbic acid (Zhao et al., 2021) and melatonin (He et al., 2022) also reduce the macrophage inflammatory response and mortality in mice. A transcriptomic study of chickens infected with PmCQ2 and PmQ also confirmed that macrophages play a vital role in the diverse outcomes of chicken infections (Li et al., 2020). Moreover, the transcriptomes of mouse macrophages infected with PmCQ2 and PmQ presented very few DEGs with no significantly enriched pathways, which is consistent with the fact that both strains are fatal to mice (data not shown). To deepen current understanding on the role of macrophage regulation in the pathogenesis of P. multocida infection, HD11 chicken macrophages were infected with bovine PmCQ2 and avian PmQ.

Although the bovine PmCQ2 is avirulent to poultry, it exhibited very mild cytotoxicity to chicken macrophages at the cellular level, which was dramatically lower than that of avian PmQ. In addition to the higher cytotoxicity and stronger anti-phagocytotic activity of PmQ than those of PmCQ2, the altered cell phenotypes of infected HD11 cells differed significantly. This morphological change facilitated the investigation of FA formation in HD11 cells. As we suspected, PmCQ2 promoted FA formation, whereas PmQ suppressed it. This finding was also confirmed by transcriptomic analysis. Because FAs were the binding points between the cytoskeleton and matrix adhesion, they play a crucial role in many biological processes, including cell movement, cell proliferation, cell differentiation, the regulation of gene expression, and cell survival (Carragher et al., 2004; Petit et al., 2000). Thus, FAs have become key targets for pathogens to manipulate the host cells (Murphy et al., 2021). In the FA complex, Zyxin is localized and enriched along actin filaments to promote the stabilization or repair of protein fibers (Wang et al., 2019). During human papillomavirus infection, the E6 protein in attenuated strains can induce Zyxin accumulation in the cell nucleus, whereas the virulent strains cannot (Degenhardt et al., 2001). F-actin assembly and retrograde flow are indispensable for integrin-based FA assembly in protruding lamellipodia-mediated cell migration (Thievessen et al., 2013). In some pathogens, such as Shigella dysenteriae, the IpaA could tightly bind with vinculin to form a vinculin–IpaA complex, which affects the interaction of vinculin with F-actin and subsequent depolymerization of actin filaments to promote pathogen invasion (Bourdet-Sicard et al., 1999). Therefore, FA may be critical factors for the ability of macrophages to clear nonvirulent bacteria. Conversely, the highly pathogenic PmQ can deactivate FA to prevent being phagocytosed.

FAK is one of two major tyrosine kinases in FAs (Kleinschmidt et al., 2017). It has a phosphorylation site on Tyr397 that is frequently in communication with other pathways (Eide et al., 1995; Kim et al., 2015). During Chlamydia caviae infection, the LD domain of its TarP interacts with FAK to mediate Cdc42- and Arp2/3-dependent actin assembly, which promotes pathogen invasion (Thwaites et al., 2014). Enteropathogenic Escherichia coli-derived EspC can directly cleave FA proteins, such as FAK, triggering FAK dephosphorylation and degradation and eventually leading to cell rounding and cell detachment, thereby causing cell death (Navarro-Garcia et al., 2014). A thymidine kinase encoded by Kaposi's sarcoma-associated herpesvirus (KSHV) Orf21 gene exhibits tyrosine kinase activity and can translocate FAK back into the cell, leading to membrane blebbing and cell detachment (Gill et al., 2015). Thus, FAK and its downstream pathways play vital roles in infectious disease and are important potential targets for the treatment of various infections (Zhao et al., 2011). The opposite regulation of FAK in HD11 cells infected with PmCQ2 and PmQ implies that FAK could be one reason for the difference in pathogenicity between PmCQ2 and PmQ.

The AKT signaling pathway downstream of FAK-Src-PI3K is vital to cell survival in various apoptotic paradigms (Brazil et al., 2001; Franke et al., 2003; Thamilselvan et al., 2007). In bacterial pathogenesis, many pathogens develop the ability to induce cell apoptosis in immune cells, such as macrophages and neutrophils, to escape phagocytosis (Cailhier et al., 2005; Kobayashi et al., 2003). Staphylococcus aureus and Listeria monocytogenes secrete virulence factors to activate the host cell apoptosis regulation mechanism (Lamkanfi et al., 2010), whereas Shigella flexneri can directly induce cell apoptosis in macrophages (Zychlinsky et al., 1992), and secrete versatile effectors, such as VirA (Bergounioux et al., 2012), via the type III secretion system to prevent epithelial cell death, thereby maintaining its foothold for replication (Ashida et al., 2021). These studies have shown that S. flexneri can perfectly regulate cell death in the host to promote its survival and proliferation. Studies on the chicken fowl cholera model have demonstrated that numerous liver and lung cells are undergoing caspase 3-mediated apoptosis (Tang et al., 2017). In our study, the more virulent PmQ infection inhibited FAK-AKT activation to induce caspase 3-mediated cell apoptosis in HD11 cells, which is likely to be a successful approach for the pathogenesis of avian P. multocida. Conversely, the non-pathogenic bovine PmCQ2 activated AKT phosphorylation and inhibited apoptosis in chicken macrophages, which seems to be an improper strategy for the development of infection in chickens.

Conclusions

In conclusion, this study revealed that the avian PmQ strain inhibited FAK phosphorylation and Zyxin-dependent FAs, resulting in the downregulation of AKT activation, consequently impeding the phosphorylation of NF-κB and FoxO1 and inducing apoptosis in chicken macrophages. The pathogenic mechanism of apoptosis caused by PmQ provides insights into the pathogenesis of avian P. multocida and potential therapeutic targets for pasteurellosis prevention.

Funding

This work is supported by the earmarked fund of the China Agriculture Research System (Beef/Yak Cattle, CARS-37), the National Natural Science Foundation of China (32302876), the Taiyuan Institute of Technology Scientific Research Initial Funding (2022KJ015), the Fundamental Research Program of Shanxi Province (202303021212276), and the Chongqing Municipal Postdoctoral Science Special Foundation (2023CQBSHTB2030). The funding organizations had no role in the study design, data collection or analysis, decision to publish or preparation.

CRediT authorship contribution statement

Pan Li: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Software, Validation, Visualization, Writing – original draft, Writing – review & editing. Guangfu Zhao: Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Methodology, Software, Visualization, Writing – original draft, Writing – review & editing. Tao Tang: Data curation, Formal analysis, Investigation, Methodology, Software, Validation, Writing – original draft. Fang He: Formal analysis, Data curation, Methodology, Software, Validation, Visualization, Writing – review & editing. Xiongli Liu: Data curation, Methodology, Software, Validation, Visualization. Nengzhang Li: Conceptualization, Supervision, Writing – review & editing. Yuanyi Peng: Conceptualization, Project administration, Resources, Funding acquisition, Writing – review & editing.

Disclosures

The authors declare no conflicts of interest.

Acknowledgments

We are grateful to Prof. Zhu (Yangzhou University) for his gifted HD11 cell line.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.psj.2024.104504.

Appendix. Supplementary materials

mmc1.docx (4MB, docx)
mmc2.docx (21.6KB, docx)
mmc3.docx (19.6KB, docx)
mmc4.docx (22.2KB, docx)
mmc5.xlsx (1.8MB, xlsx)
mmc6.xlsx (2.1MB, xlsx)
mmc7.xlsx (1,000.3KB, xlsx)

Supplementary Fig. 1. Confocal fluorescence images of HD11 infected with different P. multocida strains at 8 h (PmCQ1, PmCQ4, and PmCQ5 are bovine strains; sPL665, sPL855, and sPL935 are avian strains). Red color represents phalloidin staining; blue color represents DAPI staining, scale bar = 25 μm.

Supplementary Table 1. Primer sequences used for qRT‒PCR validation.

Supplementary Table 2. Primer sequences used for siRNA interference.

Supplementary Table 3. Statistical results of the transcriptomic sequencing data.

Supplementary Table 4. Full lists of DEGs.

Supplementary Table 5. Full lists of Venn diagrams.

Supplementary Table 6. Full GO analysis lists.

Supplementary Table 7. Full KEGG analysis lists.

mmc8.xlsx (39.3KB, xlsx)

Data availability

All the data in this study have been deposited to NCBI Sequence Read Archive (SRA) database and the accession number is PRJNA786361.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

mmc1.docx (4MB, docx)
mmc2.docx (21.6KB, docx)
mmc3.docx (19.6KB, docx)
mmc4.docx (22.2KB, docx)
mmc5.xlsx (1.8MB, xlsx)
mmc6.xlsx (2.1MB, xlsx)
mmc7.xlsx (1,000.3KB, xlsx)

Supplementary Fig. 1. Confocal fluorescence images of HD11 infected with different P. multocida strains at 8 h (PmCQ1, PmCQ4, and PmCQ5 are bovine strains; sPL665, sPL855, and sPL935 are avian strains). Red color represents phalloidin staining; blue color represents DAPI staining, scale bar = 25 μm.

Supplementary Table 1. Primer sequences used for qRT‒PCR validation.

Supplementary Table 2. Primer sequences used for siRNA interference.

Supplementary Table 3. Statistical results of the transcriptomic sequencing data.

Supplementary Table 4. Full lists of DEGs.

Supplementary Table 5. Full lists of Venn diagrams.

Supplementary Table 6. Full GO analysis lists.

Supplementary Table 7. Full KEGG analysis lists.

mmc8.xlsx (39.3KB, xlsx)

Data Availability Statement

All the data in this study have been deposited to NCBI Sequence Read Archive (SRA) database and the accession number is PRJNA786361.

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