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Annexin A2-Mediated Internalization of Staphylococcus aureus into Bovine Mammary Epithelial Cells Requires Its Interaction with Clumping Factor B.

ABSTRACT: Staphylococcus aureus is a leading cause of contagious mastitis in dairy cattle. Internalization of S. aureus by bovine mammary gland epithelial cells is thought to be responsible for persistent and chronic intramammary infection, but the underlying mechanisms are not fully understood. In the present study, we evaluated the role of Annexin A2 (AnxA2), a membrane-binding protein, in S. aureus invasion into bovine mammary epithelial cell line (MAC-T). In vitro binding assays were performed to co-immunoprecipitate the binding proteins of AnxA2 in the lysates of S. aureus. AnxA2 mediated the internalization but not adherence of S. aureus. Engagement of AnxA2 stimulated an integrin-linked protein kinase (ILK)/p38 MAPK cascade to induce S. aureus invasion. One of the AnxA2-precipitated proteins was identified as S. aureus clumping factor B (ClfB) through use of mass spectrometry. Direct binding of ClfB to AnxA2 was further confirmed by using a pull-down assay. Pre-incubation with recombinant ClfB protein enhanced S. aureus internalization, an effect that was specially blocked by anti-AnxA2 antibody. Our results demonstrate that binding of ClfB to AnxA2 has a function in promoting S. aureus internalization. Targeting the interaction of ClfB and AnxA2 may confer protection against S. aureus mastitis.

SUBMITTER: Ying YT

PROVIDER: S-EPMC8538401 | biostudies-literature | 2021 Oct

REPOSITORIES: biostudies-literature

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Annexin A2-Mediated Internalization of <i>Staphylococcus aureus</i> into Bovine Mammary Epithelial Cells Requires Its Interaction with Clumping Factor B.

Ying Yi-Tian YT Ren Wei-Jia WJ Tan Xun X Yang Jing J Liu Rui R Du Ai-Fang AF

Microorganisms 20211003 10

<h4>Background</h4><i>Staphylococcus aureus</i> is a leading cause of contagious mastitis in dairy cattle. Internalization of <i>S. aureus</i> by bovine mammary gland epithelial cells is thought to be responsible for persistent and chronic intramammary infection, but the underlying mechanisms are not fully understood.<h4>Methods</h4>In the present study, we evaluated the role of Annexin A2 (AnxA2), a membrane-binding protein, in <i>S. aureus</i> invasion into bovine mammary epithelial cell line ...[more]

PMID: 34683411

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Project description:Probiotics have been considered as a promising strategy to prevent various diseases in both humans and animals. This approach has gained interest in recent years as a potential means to control bovine mastitis. In a previous study, we found that several L. casei strains, including BL23, were able to inhibit the internalization of S. aureus, a major etiologic agent of mastitis, into bovine mammary epithelial cells (bMEC). This antagonism required a direct contact between L. casei and bMEC or S. aureus, suggesting the inhibition relied on interactions between L. casei cell surface components and bMEC. In this study, we have investigated the impact of some candidates which likely influence bacteria host cell interactions. We have shown that L. casei BL23 fbpA retained its inhibitory potential, indicating that L. casei BL23 antagonism did not rely (solely) on competition between S. aureus and L. casei fibronectin-binding proteins for adhesion to bMEC. We have then investigated the impact of four sortase mutants, srtA1, srtA2, srtC1 and srtC2, and a double mutant (srtA1-srtA2) on L. casei BL23 inhibitory potential. Sortases are responsible for the anchoring on the bacterial cell wall of LPXTG-proteins, which reportedly play an important role in bacteria-host cell interaction. All the srt mutants tested presented a reduced inhibition capacity, the most pronounced effect being observed with the srtA2 mutant. A lower internalization capacity of L. casei srtA2 into bMEC was also observed. This was associated with several changes at the surface of L. casei BL23 srtA2 compared to the wild type (wt) strain, including altered abundance of some LPXTG- and moonlighting proteins, and modifications of cell wall structure. These results strongly support the role of sortase A2 in L. casei BL23 inhibition against S. aureus internalization. Deciphering the contribution of the cell surface components altered in srtA2 strain in the inhibition will require further investigation.

Project description:Bovine mammary epithelial cells (bMECs) contribute to mammary gland defense against invading pathogens, such as Staphylococcus aureus (intracellular facultative), which is recognized by TLR2. In a previous report, we showed that sodium octanoate [NaO, a medium chain fatty acid (C8)] induces (0.25 mM) or inhibits (1 mM) S. aureus internalization into bMECs and differentially regulates the innate immune response (IIR). However, the molecular mechanisms have not been described, which was the aim of this study. The results showed that α5β1 integrin membrane abundance (MA) was increased in 0.25 mM NaO-treated cells, but TLR2 or CD36 MA was not modified. When these receptors were blocked individually, 0.25 mM NaO-increased S. aureus internalization was notably reduced. Interestingly, in this condition, the IIR of the bMECs was impaired because MAPK (p38, JNK, and ERK1/2) phosphorylation and the activation of transcription factors related to these pathways were decreased. In addition, the 1 mM NaO treatment induced TLR2 MA, but neither the integrin nor CD36 MA was modified. The reduction in S. aureus internalization induced by 1 mM NaO was increased further when TLR2 was blocked. In addition, the phosphorylation levels of the MAPKs increased, and 13 transcriptional factors related to the IIR were slightly activated (CBF, CDP, c-Myb, AP-1, Ets-1/Pea-3, FAST-1, GAS/ISRE, AP-2, NFAT-1, OCT-1, RAR/DR-5, RXR/DR-1, and Stat-3). Moreover, the 1 mM NaO treatment up-regulated gene expression of IL-8 and RANTES and secretion of IL-1β. Notably, when 1 mM NaO-treated bMECs were challenged with S. aureus, the gene expression of IL-8 and IL-10 increased, while IL-1β secretion was reduced. In conclusion, our results showed that α5β1 integrin, TLR2 and CD36 are involved in 0.25 mM NaO-increased S. aureus internalization in bMECs. In addition, 1 mM NaO activates bMECs via the TLR2 signaling pathways (p38, JNK, and ERK1/2), which improves IIR before S. aureus invasion. Additionally, NaO (1 mM) might exert anti-inflammatory effects after bacterial internalization.

Dataset Information

Annexin A2-Mediated Internalization of Staphylococcus aureus into Bovine Mammary Epithelial Cells Requires Its Interaction with Clumping Factor B.

Publications

Annexin A2-Mediated Internalization of <i>Staphylococcus aureus</i> into Bovine Mammary Epithelial Cells Requires Its Interaction with Clumping Factor B.

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