Potential therapeutic application of bacteriophages and phage-derived endolysins as alternative treatment of bovine mastitis

The increase in bacterial drug resistance causes major difficulties in the clinical treatment of a growing number of bacterial infections worldwide. Consequently, there is an urgent need to develop novel anti-bacterial agents to control these resistant pathogens and to complement the currently used antibiotics. Mastitis is the most prevalent disease impacting dairy cattle, and therefore one of the costliest diseases in the global dairy industry. The excessive use of curative as well as preventive antibiotics in this sector entails a real risk for the emergence of antimicrobial resistance. Moreover, these traditional antimicrobial agents are often ineffective and lead to residues in the milk, which can affect dairy product consumers. As an alternative therapeutic approach, bacteriophages and phage-encoded endolysins have been proposed and are currently (re)investigated as potential antibacterial agents against mastitis

Characterization of the bacteriophage-derived endolysins PlySs2 and PlySs9 with in vitro lytic activity against bovine mastitis streptococcus uberis

Bovine mastitis, an infection of the cow’s mammary gland, is frequently caused by Streptococcus uberis and causes major economic losses in the dairy industry. The intramammary administration of antibiotics currently remains the predominant preventive and therapeutic measure. These antimicrobial compounds, ofwhich some are consideredcritical in human health care, are frequently applied as dry therapy resulting in their consistent overuse. Therefore, the use of antibiotics in the dairy sector is being questioned. We here identified two endolysins, i.e., PlySs2 and PlySs9, respectively derived from Streptococcus suis serotype-2 and -9 prophages, with lytic activity against S. uberis in an in vitro setting. Both endolysins gave clear lysis zones in spot-on-plate assays and caused a reduction of the optical density in a turbidity reduction assay. In depth characterization identified PlySs9 as the more potent endolysin over PlySs2 with a lower MIC value and about one additional log of killing. PlySs2 and PlySs9 were challenged to a panel of subclinical and clinical S. uberis milk isolates and were both able to lyse all strains tested. Molecular dissection of these endolysins in catalytic and cell wall binding subdomains resulted in major loss of killing and binding activity, respectively. Taken together, we here propose PlySs2 and PlySs9 as candidate compounds to the current antimicrobial arsenal known against bovine mastitis-causing S. uberis as future add-on or replacement strategy to the currently used intramammary antibiotics

Enterobactin Deficiency in a Coliform Mastitis Isolate Decreases Its Fitness in a Murine Model:A Preliminary Host–Pathogen Interaction Study

Iron is an essential nutrient for bacterial growth. Therefore, bacteria have evolved chelation mechanisms to acquire iron for their survival. Enterobactin, a chelator with high affinity for ferric iron, is secreted by Escherichia coli and contributes to its improved bacterial fitness. In this preliminary study, we evaluated enterobactin deficiency both in vitro and in vivo in the context of E. coli mastitis. Firstly, we showed that expression of lipocalin 2, a protein produced by the host that is able to both bind and deplete enterobactin, is increased upon E. coli infection in the cow’s mastitic mammary gland. Secondly, we demonstrated in vitro that enterobactin deficiency does not alter interleukin (IL)-8 expression in bovine mammary epithelial cells and its associated neutrophil recruitment. However, a significantly increased reactive oxygen species production of these neutrophils was observed. Thirdly, we showed there was no significant difference in bacterial in vitro growth between the enterobactin-deficient mutant and its wild-type counterpart. However, when further explored in a murine model for bovine mastitis, the enterobactin-deficient mutant vs. the wild-type strain revealed a significant reduction of the bacterial load and, consequently, a decrease in pro-inflammatory cytokines (IL-1a,−1b,−4,−6, and−8). A reduced neutrophilic influx was also observed immunohistochemically. These findings therefore identify interference of the enterobactin iron-scavenging mechanism as a potential measure to decrease the fitness of E. coli in the mastitic mammary gland

OMO-1 reduces progression and enhances cisplatin efficacy in a 4T1-based non-c-MET addicted intraductal mouse model for triple-negative breast cancer

c-MET is considered a driver of cancer progression, impacting tumor growth and tumor-supporting stroma. Here, we investigated the therapeutic efficacy of OMO-1, a potent and selective c-MET inhibitor, in an immunocompetent intraductal mouse model for triple-negative breast cancer (TNBC). OMO-1 reduced non-c-MET addicted 4T1 tumor progression dose dependently as monotherapeutic and provided additional disease reduction in combination with cisplatin. At the stromal level, OMO-1 significantly reduced neutrophil infiltration in 4T1 tumors, promoted immune activation, and enhanced cisplatin-mediated reduction of tumor-associated macrophages. OMO-1 treatment also reduced 4T1 tumor hypoxia and increased expression of pericyte markers, indicative for vascular maturation. Corroborating this finding, cisplatin delivery to the 4T1 primary tumor was enhanced upon OMO-1 treatment, increasing cisplatin DNA-adduct levels and tumor cell death. Although verification in additional cell lines is warranted, our findings provide initial evidence that TNBC patients may benefit from OMO-1 treatment, even in cases of non-c-MET addicted tumors

Comparative profiling of metastatic 4T1- vs. non-metastatic Py230-based mammary tumors in an intraductal model for triple-negative breast cancer

The transition of ductal carcinoma in situ (DCIS) to invasive carcinoma (IC) in breast cancer can be faithfully reproduced by the intraductal mouse model. Envisaging to use this model for therapeutic testing, we aimed to in-depth characterize the tumor immunity associated with the differential progression of two types of intraductal tumors. More specifically, we focused on triple-negative breast cancer (TNBC) and intraductally inoculated luciferase-expressing metastatic 4T1 and locally invasive Py230 cells in lactating mammary glands of syngeneic BALB/c and C57BL/6 female mice, respectively. Although the aggressive 4T1 cells rapidly formed solid tumors, Py230 tumors eventually grew to a similar size through enhanced proliferation. Yet, ductal tumor cell breakthrough and metastasis occurred earlier in the 4T1- compared to the Py230-based intraductal model and was associated with high expression of matrix metalloproteinase (MMP)-9, vascular endothelial growth factor (VEGF), chitinase 3-like 1 (CHI3L1) and lipocalin 2 (LCN2) as well as an increased influx of immune cells (mainly macrophages, neutrophils and T-cells). Moreover, activated cytotoxic T-cells, B-cells and programmed death-1 (PD-1)-positive cells were more prominent in the 4T1-based intraductal model in line with enhanced pro-inflammatory cytokine and gene expression profiles. Py230-based tumors showed a more immunosuppressed anti-inflammatory profile with a high amount of regulatory T-cells, which may account for the decreased T-cell activation but increased proliferation compared to the 4T1-based tumors. Taken together, our results highlight the differential immunological aspects of aggressive metastatic and non-aggressive intraductal progression of 4T1- vs. Py230-based tumors, providing a base for future studies to explore therapy using these intraductal TNBC models

The PlySs9 endolysin contains unique catalytic domains and is a potential therapeutic against Streptococcus suis

Increasing resistance to antibiotics amongst livestock has forced the discovery of alternative techniques to continue treating bacterial infections successfully. Bacteriophage-encoded peptidoglycan hydrolases, also referred to as endolysins, are able to lyse the bacterial cell wall and offer possible applications in food safety, human health and veterinary science. The treatment of Streptococcus suis infections in pigs specifically involves the latter three. Its zoonotic nature is a potential human health threat and the economic loss of $100 million per year is devastating on the swine industry. Preventing on-farm disease outbreaks is extremely difficult and current approaches to eradicate S. suis from herds are often ineffective. Therefore, a pressing need to identify and evaluate S. suis-specific endolysins arises. A bioinformatic approach was conducted to identify proteins in bacteriophage genomes with similar homology to known endolysin catalytic domains. We chose five candidates for synthesis, expression, purification and characterization upon discovery of lytic activity assayed by turbidity reduction. Binding capacity was evaluated by fluorescent microscopy. PlySs9 represents our lead candidate and is predicted to contain an N-terminal amidase catalytic domain, a central LysM-based cell wall binding domain, and a C-terminal CHAP catalytic domain. We have determined the optimal conditions for the lytic activity of PlySs9, characterized its broad activity spectrum, and investigated its ability to disrupt biofilms. Active-site residues were detected through site-directed mutagenesis. We also assessed the contribution of each individual domain to activity or binding. Lastly, a triple-acting enzyme of PlySs9 was engineered using three unique, potentially synergistic lytic domains to reduce the risk of resistance development. These results indicate that the broad lytic spectrum of PlySs9 and its derivatives have the potential to be used as therapeutic agents against S. suis infections

One cisplatin dose suffices to maintain anti-tumor immunity and enhances anti-PD-1 efficacy in an intraductal model for triple-negative breast cancer

Cytotoxic cisplatin chemotherapy has a direct killing effect on tumor cells and significantly modulates the host immune system. This cisplatin-mediated immunomodulation is attractive in combination with immune checkpoint blockers, as it may enhance their efficacy e.g. in immunotherapy-resistant triple-negative breast cancer (TNBC). To better understand the combination therapy-induced immune response, an immunocompetent 4T1-based intraductal model was used that recapitulates the complete human TNBC process. Treatments with cisplatin and programmed death (PD)-1 blocking antibody were started once primary tumors met the invasive carcinoma stage and continued until deadly metastases developed. The model showed resistance to single weekly anti-PD-1 therapy, but became responsive upon combination therapy with cisplatin. Interestingly, repetitive cisplatin dosing (every 5 days) provided similar disease reduction as one cisplatin dose at start of treatment, indicating that early cisplatin-mediated immunomodulation suffices to stimulate and maintain anti-tumor immunity in support of PD-1 blockade. In line with this observation, cytotoxic T-cells (CTLs) were increased in lymphoid tissues (i.e. spleen and lymph nodes) while immunosuppressive myeloid cells were reduced in both the primary tumor as well as lymphoid tissues at 2 days after the first cisplatin dose. Both significant cisplatin-mediated changes remained for 2 weeks and the combination with anti-PD-1 further increased the number of CTLs, providing an add-on disease reduction. Collectively, our results highlight that single-dosed cisplatin is a promising immunotherapeutic ally, holding potential for reducing toxicity without treatment efficacy loss in TNBC