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Cathelicidins Mitigate Staphylococcus aureus Mastitis and Reduce Bacterial Invasion in Murine Mammary Epithelium.

ABSTRACT: Staphylococcus aureus, an important cause of mastitis in mammals, is becoming increasingly problematic due to the development of resistance to conventional antibiotics. The ability of S. aureus to invade host cells is key to its propensity to evade immune defense and antibiotics. This study focuses on the functions of cathelicidins, small cationic peptides secreted by epithelial cells and leukocytes, in the pathogenesis of S. aureus mastitis in mice. We determined that endogenous murine cathelicidin (CRAMP; Camp) was important in controlling S. aureus infection, as cathelicidin knockout mice (Camp^-/- ) intramammarily challenged with S. aureus had higher bacterial burdens and more severe mastitis than did wild-type mice. The exogenous administration of both a synthetic human cathelicidin (LL-37) and a synthetic murine cathelicidin (CRAMP) (8??M) reduced the invasion of S. aureus into the murine mammary epithelium. Additionally, this exogenous LL-37 was internalized into cultured mammary epithelial cells and impaired S. aureus growth in vitro We conclude that cathelicidins may be potential therapeutic agents against mastitis; both endogenous and exogenous cathelicidins conferred protection against S. aureus infection by reducing bacterial internalization and potentially by directly killing this pathogen.

SUBMITTER: Cavalcante PA

PROVIDER: S-EPMC7309602 | biostudies-literature | 2020 Jun

REPOSITORIES: biostudies-literature

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Cathelicidins Mitigate Staphylococcus aureus Mastitis and Reduce Bacterial Invasion in Murine Mammary Epithelium.

Cavalcante Paloma Araujo PA Knight Cameron G CG Tan Yi-Lin YL Monteiro Ana Paula Alves APA Barkema Herman W HW Cobo Eduardo R ER

Infection and immunity 20200622 7

Staphylococcus aureus, an important cause of mastitis in mammals, is becoming increasingly problematic due to the development of resistance to conventional antibiotics. The ability of S. aureus to invade host cells is key to its propensity to evade immune defense and antibiotics. This study focuses on the functions of cathelicidins, small cationic peptides secreted by epithelial cells and leukocytes, in the pathogenesis of S. aureus mastitis in mice. We determined that endog ...[more]

PMID: 32341117

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Project description:BackgroundThe most important disease of dairy cattle is mastitis, caused by the infection of the mammary gland by various micro-organisms. Although the transcriptional response of bovine mammary gland cells to in vitro infection has been studied, the interplay and consequences of these responses in the in vivo environment of the mammary gland are less clear. Previously mammary gland quarters were considered to be unaffected by events occurring in neighbouring quarters. More recently infection of individual quarters with mastitis causing pathogens, especially Escherichia coli, has been shown to influence the physiology of neighbouring uninfected quarters. Therefore, the transcriptional responses of uninfected mammary gland quarters adjacent to quarters infected with two major mastitis causing pathogens, E. coli and Staphylococcus aureus, were compared.ResultsThe bacteriologically sterile, within-animal control quarters exhibited a transcriptional response to the infection of neighbouring quarters. The greatest response was associated with E. coli infection, while a weaker, yet significant, response occurred during S. aureus infection. The transcriptional responses of these uninfected quarters included the enhanced expression of many genes previously associated with mammary gland infections. Comparison of the transcriptional response of uninfected quarters to S. aureus and E. coli infection identified 187 differentially expressed genes, which were particularly associated with cellular responses, e.g. response to stress. The most affected network identified by Ingenuity Pathway analysis has the immunosuppressor transforming growth factor beta 1 (TGFB1) at its hub and largely consists of genes more highly expressed in control quarters from S. aureus infected cows.ConclusionsUninfected mammary gland quarters reacted to the infection of neighbouring quarters and the responses were dependent on pathogen type. Therefore, bovine udder quarters exhibit interdependence and should not be considered as separate functional entities. This suggests that mastitis pathogens not only interact directly with host mammary cells, but also influence discrete sites some distance away, which will affect their response to the subsequent spread of the infection. Understanding the underlying mechanisms may provide further clues for ways to control mammary gland infections. These results also have implications for the design of experimental studies investigating immune regulatory mechanisms in the bovine mammary gland.

Project description:Outcome and resolution of an udder inflammation (mastitis) depends on the type of the invading pathogen. Gram-negative pathogens, such as Escherichia coli mostly trigger strong inflammation and full local activation of the immune defense eventually resulting in pathogen elimination. Staphylococcus aureus and other Gram-positive pathogens elicit only weak immune reactions possibly allowing for pathogen persistence. Little is known about the very early molecular determinants of this pathogen-species specific immune response of the udder. We therefore infected healthy mid-lactating heifers with a high dose of defined E. coli1303 or S. aureus1027 pathogens, sampled udder tissues at 1, 2, and 3 h post infection and globally profiled the transcriptome of samples from the gland cistern. We expectedly found that the E. coli infection elicited a fast and strong immune response. Bioinformatics analyses suggested that pathogen recognizing receptors triggered the activation of the IkB/NF-kB signaling cascade inducing the expression of a wealth of inflammation related genes, but also many immune dampeners. S. aureus infection failed to elicit inflammation and activate IkB/NF-kB signaling. Instead, it triggered immune suppressive mechanisms which were, however not outweighed by strong induction of inflammatory genes. Moreover, it activated the wnt/b-catenin signaling cascade resulting in active suppression of NF-kB signaling and rearrangement of the actin-cytoskeleton through modulating expression of the Rho GTPases. These alterations facilitate invasion of pathogens into host cells. We validated experimentally that pathogenic S. aureus, but not E. coli may invade mammary epithelial cells (MEC). Microscopy revealed an altered structure of the actin-cytoskeleton of primary MEC having internalized live GFP-tagged S. aureus pathogens. Our study reveals for the first time the earliest host responses in the udder differentiating E. coli from S. aureus mastitis. The latter is characterized by eliciting immune suppression rather than inflammation and invasion of S. aureus into the epithelial cells of the host.

Project description:Staphylococcus aureus intramammary infection is one of the most common causes of chronic mastitis in dairy cows, whose development may be associated with epigenetic changes in the expression of important host defense genes. This study aimed to construct a genome-wide DNA methylation profile of the mammary gland of Chinese Holstein cows (n = 3) following experimentally induced S. aureus mastitis, and to explore the potential gene regulatory mechanisms affected by DNA methylation during S. aureus mastitis. DNA was extracted from S. aureus-positive (n = 3) and S. aureus-negative (n = 3) mammary gland quarters and subjected to methylation-dependent restriction-site associated DNA sequencing (Methyl-RAD Seq). Results showed that CmCGG/CmCWGG DNA methylation sites were unevenly distributed and concentrated on chromosomes 5, 11, and 19, and within intergenic regions and intron regions of genes. Compared with healthy control quarters, 9,181 significantly differentially methylated (DM) CmCGG sites and 1,790 DM CmCWGG sites were found in the S. aureus-positive quarters (P < 0.05, |log2FC| > 1). Furthermore, 363 CmCGG differently methylated genes (DMGs) and 301 CmCWGG DMGs (adjusted P < 0.05, |log2FC| > 1) were identified. Gene ontology and KEGG enrichment analysis indicated that CmCGG DMGs are involved in immune response pathways, while the CmCWGG DMGs were mainly enriched in gene ontology terms related to metabolism. The mRNAs of 526 differentially methylated CmCGG genes and 124 differentially methylated CmCWGG genes were also significantly differentially expressed (RNA-Seq data) in the same samples, herein denoted differentially methylated and expressed genes (DMEGs) (P < 0.05). Functional enrichment analysis of DMEGs revealed roles related to biological processes, especially the regulation of immune response to diseases. CmCGG DMEGs like IL6R, TNF, BTK, IL1R2, and TNFSF8 enriched in several immune-related GO terms and pathways indicated their important roles in host immune response and their potential as candidate genes for S. aureus mastitis. These results suggest potential regulatory roles for DNA methylation in bovine mammary gland processes during S. aureus mastitis and serves as a reference for future epigenetic regulation and mechanistic studies.

Dataset Information

Cathelicidins Mitigate Staphylococcus aureus Mastitis and Reduce Bacterial Invasion in Murine Mammary Epithelium.

Publications

Cathelicidins Mitigate Staphylococcus aureus Mastitis and Reduce Bacterial Invasion in Murine Mammary Epithelium.

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