Biology subjects

da Silva, R. A. G.

Publications and source records attributed to da Silva, R. A. G..

3 recordsLinked to original sources

bioRxiv2023

Bosutinib stimulates macrophage survival, phagocytosis and intracellular killing of bacteria

Host-acting compounds are emerging as potential alternatives to combat antibiotic resistance. Here, we show that bosutinib, an FDA-approved chemotherapeutic for treating chronic myelogenous leukemia, does not possess any antibiotic activity but enhances macrophage responses to bacterial infection. In vitro, bosutinib stimulates murine and human macrophages to kill bacteria more effectively. In a murine wound infection with vancomycin-resistant Enterococcus faecalis, a single intraperitoneal bosutinib injection or multiple topical applications on the wound reduces bacterial load by approximately 10-fold, which is abolished by macrophage depletion. Mechanistically, bosutinib stimulates macrophage phagocytosis of bacteria by upregulating surface expression of bacterial uptake markers Dectin-1 and CD14 and promoting actin remodelling. Bosutinib also stimulates bacterial killing by elevating the intracellular levels of reactive oxygen species. Moreover, bosutinib drives NF-{kappa}B activation which protects infected macrophages from dying. Other Src kinase inhibitors such as DMAT and Tirbanibulin also upregulate expression of bacterial uptake markers in macrophages and enhance intracellular bacterial killing. Finally, co-treatment with bosutinib and mitoxantrone, another chemotherapeutic in clinical use, results in an additive effect on bacterial clearance in vitro and in vivo. These results show that bosutinib stimulates macrophage clearance of bacterial infection through multiple mechanisms and could be used to boost host innate immunity to combat drug-resistant bacterial infections. SignificanceThis study shows that bosutinib, an FDA-approved chemotherapeutic, stimulates macrophage responses to antibiotic-resistant bacterial infection by enhancing phagocytosis and intracellular killing of bacteria and promoting survival of infected macrophages. These findings suggest that bosutinib could serve as an adjuvant therapy to combat drug resistant bacterial infections and opens the possibility to target Src kinases to boost innate immunity in general.

microbiology↗

bioRxiv2022

Mitoxantrone Targets Both Host and Bacteria to Overcome Vancomycin Resistance in Enterococcus faecalis

Among Enterococci, intrinsic and acquired resistance to antibiotics such as {beta}-lactams and vancomycin critically limit treatment options for infection with these opportunistic pathogens. Antimicrobials that enhance the host immune response are emerging as alternative approaches, with the potential to overcome bacterial resistance. Here, we investigate the antibiotic and immunological activity of the anticancer agent mitoxantrone (MTX) in vitro and in vivo against vancomycin resistant Enterococcus faecalis (VRE). We show that, in vitro, MTX is a potent antibiotic against Gram-positive bacteria with a minimal inhibitory concentration (MIC) of ~1 g/ml through induction of reactive oxygen species and DNA damage. MTX synergises with vancomycin and lowers the vancomycin concentration required to kill VRE by over 140-fold. This synergy is specific to vancomycin-resistant, but not susceptible strains because vancomycin rendered the resistant strains more permeable to MTX and thus MTX-mediated DNA damage. In a murine wound infection model, MTX treatment effectively reduced VRE bacterial numbers by 120-fold and with further reduction when combined with vancomycin. Wounds treated with MTX had significantly higher numbers of macrophages and higher pro-inflammatory cytokines compared to untreated wounds. In addition, MTX augmented intracellular bacterial killing by both murine and human macrophages by upregulating the expression of lysosomal hydrolases cathepsins D and H, and {beta}-Hexosaminidase. These results show that MTX is a potent antibiotic against Gram-positive bacteria, synergizes with vancomycin, enhances macrophage recruitment and intracellular bactericidal activity, and represents a promising dual bacterium- and host-targeted therapeutic for overcoming vancomycin resistance. One sentence summaryMitoxantrone synergizes with vancomycin against vancomycin resistant bacterial strains via direct antibiotic activity and by augmenting both host macrophage recruitment to the site of infection and macrophage bactericidal activity.

microbiology↗

bioRxiv2021

Enterococcus faecalis persists and replicates within epithelial cells in vitro and in vivo during wound infection

Enterococcus faecalis is a frequent opportunistic pathogen of wounds, whose infections are associated with biofilm formation, persistence, and recalcitrance toward treatment. We have previously shown that E. faecalis wound infection persists for at least 7 days. Here we report that viable E. faecalis are present within both immune and non-immune cells at the wound site up to 5 days after infection, raising the prospect that intracellular persistence contributes to chronic E. faecalis infection. Using an in vitro keratinocyte infection model, we show that a subpopulation of E. faecalis becomes internalized via macropinocytosis into single membrane-bound compartments, where they can survive and replicate. These intracellular E. faecalis can persist in late endosomes up to 72 hours after infection in the absence of colocalization with the lysosomal protease cathepsin D or apparent fusion with the lysosome, suggesting that E. faecalis blocks endosomal maturation. Indeed, intracellular E. faecalis infection results in a marked reduction in Rab7 expression, a small GTPase required for endosome-lysosome fusion. Finally, we demonstrate that intracellular E. faecalis derived from infected keratinocytes are significantly more efficient in reinfecting new keratinocytes. Together, these data suggest that intracellular proliferation of E. faecalis may contribute to its persistence in the face of a robust immune response, providing a primed reservoir of bacteria for subsequent reinfection. Author SummaryEnterococcus faecalis is often isolated from chronic wounds. Prior to this study, E. faecalis has been observed within different cell types, suggesting that it can successfully colonize intracellular spaces. However, to date, little is known about the mechanisms E. faecalis use to survive intracellularly. Here, we describe key features of the intracellular lifestyle of E. faecalis. We show that E. faecalis exists in an intracellular state within immune cells and non-immune cells during mammalian wound infection. We show that E. faecalis can survive and replicate inside keratinocytes, and intracellularly replicating E. faecalis are primed to more efficiently cause reinfection, potentially contributing to chronic or persistent infections. In order to establish this intracellular lifestyle, E. faecalis is taken up by keratinocytes via macropinocytosis, whereupon it manipulates the endosomal pathway and expression of trafficking molecules required for endo-lysosomal fusion, enabling E. faecalis to avoid lysosomal degradation and consequent death. These results advance our understanding of E. faecalis pathogenesis, demonstrating mechanistically how this classic extracellular pathogen can co-opt host cells for intracellular persistence, and highlight the heterogeneity of mechanisms bacteria can use to avoid host-mediated killing in order to cause disease.

microbiology↗