Biology subjects

Antypas, H.

Publications and source records attributed to Antypas, H..

6 recordsLinked to original sources

bioRxiv2026

Enterococcus faecalis biofilm rewires neutrophil metabolism to suppress antimicrobial functions

Enterococcus faecalis is an opportunistic pathogen that persists in biofilm-associated infections despite robust neutrophil recruitment. How E. faecalis evades neutrophils remains poorly understood. Here, we investigated how E. faecalis biofilms alter effector functions in human neutrophils and a murine wound infection model. We found that E. faecalis biofilm suppresses neutrophil extracellular trap (NET) formation, bacterial engulfment, and neutrophil-mediated control of biofilm growth. This suppression is partly driven by production of lactic acid through the lactate dehydrogenases (LDH) of E. faecalis, which acidifies the extracellular environment, lowers neutrophil intracellular pH, and disrupts glycolysis, the tricarboxylic acid cycle, and the oxidative pentose phosphate pathway. In a murine wound infection model, loss of LDH increased neutrophil recruitment and NETosis. We also found that high bacterial density, a defining feature of biofilms, suppresses NETosis through a lactic acid-independent mechanism. Collectively, these findings identify biofilm immune evasion strategies mediated by bacterial metabolism and the high-density structural organization of biofilm-associated bacteria. TeaserEnterococcus faecalis biofilms suppress neutrophil defenses through lactic acid and high bacterial density.

microbiology↗

bioRxiv2025

Gelatinase regulates the egress of intracellular replicating populations during Enterococcus faecalis infection

Enterococcus faecalis is a common opportunistic pathogen, frequently isolated from chronic wounds, yet the mechanisms underlying its virulence and persistence in this niche remain incompletely understood. We previously showed that a subpopulation of E. faecalis can survive intracellularly for several days during murine wound infection and can replicate within macrophages, revealing an unexpected intracellular phase for this traditionally extracellular bacterium. Here, we identify the secreted metalloprotease gelatinase (GelE) and its regulator, the Fsr quorum sensing system, as key modulators of E. faecalis intracellular survival and replication. Mechanistically, Fsr quorum sensing is induced during intracellular replication, promoting GelE-dependent host cell lysis and bacterial egress. In the absence of active GelE, E. faecalis accumulates as large intracellular clusters, a phenotype observed consistently across GelE deficient wound isolates. In a mouse wound model, GelE deficient E. faecalis similarly exhibited higher intracellular numbers within wound infection-associated host cells. Together, our study uncovers GelE as a central effector that orchestrates the transition between intracellular and extracellular lifestyles of E. faecalis, providing a possible explanation for its persistence in chronic wound infection. Author summaryPathogenic bacteria are traditionally classified as either "intracellular" or "extracellular", but growing evidence suggests that many extracellular bacteria also adopt transient intracellular lifestyles that promote persistent and recurrent infection. Enterococcus faecalis, a leading cause of chronic wound infection, exemplifies this duality. We discovered that E. faecalis strains lacking the secreted protease gelatinase accumulate to high numbers inside host cells such as macrophages. Our data indicate that gelatinase facilitates bacterial escape following intracellular replication, regulating the transition between an intracellular and extracellular lifestyle. During infection, gelatinase-deficient bacteria remain hidden within various wound cell types, potentially evading immune clearance and antibiotic treatment. This work reveals a previously unrecognized role for gelatinase in controlling E. faecalis intracellular dynamics, highlighting a mechanism that may underline chronic and persistent infection.

microbiology↗

bioRxiv2025

Enterococcus faecium colonization and persistence in a model of diabetic wound infection

Chronic wound infections are a common comorbidity of diabetes mellitus and can progress to amputation if untreated, yet effective strategies to manage these infections are limited. Commensals such as Enterococcus faecium and Staphylococcus epidermidis can transition into opportunistic pathogens when host defenses are compromised, underscoring the complexity of chronic wound microbiology. E. faecium, particularly vancomycin-resistant strains, are an understudied, clinically important cause of chronic diabetic wound infections. Using a low-dose streptozocin-induced diabetic mouse model, we characterized E. faecium wound infection dynamics and identified differences in colonization and clearance compared to non-diabetic animals. At eight hours post infection (hpi), control mice exhibited higher E. faecium wound colony forming units (CFU) than diabetic mice, but cleared the infection more efficiently, resulting in similar CFU by 24 hpi. By contrast, diabetic mice showed impaired clearance, with elevated CFU persisting through 72 hpi. In mixed species infection with S. epidermidis, S. epidermidis CFU increased at 72 hpi while E. faecium CFU remained comparable to single species infection. Despite strong initial cytokine and neutrophil responses, E. faecium persisted in all wounds. Sustained neutrophil recruitment at 72 hpi occurred only in diabetic mice, whereas macrophage accumulation increased from 24 to 72 hpi in all wounds, including sterile controls. Histological analysis showed epithelial hyper-thickening in both groups, indicating that diabetes and E. faecium each contribute to impaired wound healing. This study establishes a diabetic mouse model of E. faecium wound infection and suggests that E. faecium modulates innate immune responses to persist in the wound bed.

microbiology↗

bioRxiv2025

Enterococcus faecalis persists and replicates intracellularly within neutrophils

Chronic wound infection is a major global public health issue, with Enterococcus faecalis among the most commonly isolated pathogens from such wounds. Neutrophils are short-lived immune cells critical for host defence, yet E. faecalis-neutrophil interactions are poorly understood. Here, we show that instead of eliminating E. faecalis, neutrophils provide a niche for intracellular persistence and replication, potentially prolonging infection and inflammation at the wound site. In murine wound beds and ex vivo wound cells, intracellular E. faecalis was detected in recruited neutrophils at 24 h post-infection (h p.i). Unexpectedly, extended infection did not induce neutrophil death. Rather, E. faecalis infection significantly prolonged the lifespans of both murine and human neutrophils in vitro compared to uninfected controls. Quantification of intracellular CFU revealed that E. faecalis were phagocytosed regardless of opsonisation and persisted intracellularly through to 24 h p.i. This finding was confirmed via transmission electron microscopy and confocal microscopy. Blinded quantification and fluorescent D-amino acid staining, which marks newly synthesised bacterial peptidoglycan, revealed active replication within murine neutrophils between 6-18 h p.i., followed by a predominately persistent phase between 18-24 h p.i. Infected murine neutrophils remained immunologically active, secreting pro-inflammatory and chemoattractant cytokines. These findings highlight an underappreciated intracellular lifestyle for E. faecalis that may underly its ability to persist in chronic wounds and contribute to biofilm-associated infections.

immunology↗

bioRxiv2025

Fsr quorum sensing system restricts biofilm growth and activates inflammation in enterococcal infective endocarditis

Infective endocarditis (IE) is a life-threatening biofilm-associated infection, yet the factors driving biofilm formation remain poorly understood. Here, we identified the Fsr quorum sensing (QS) system of Enterococcus faecalis as a potent negative regulator of IE pathogenesis. Using microfluidic and in vivo models, we show that Fsr is induced in late IE when bacteria become shielded from blood flow. Deleting Fsr altered biofilm metabolism and promoted robust biofilm growth and gentamicin tolerance in vivo. Furthermore, Fsr inactivation attenuated inflammation by disrupting IL-1{beta} cleavage and activation via the Fsr-regulated gelatinase (gelE), allowing biofilm to grow unchecked by the immune system. Consistent with our pre-clinical findings, analysis of two IE patient cohorts linked naturally occurring Fsr-deficient E. faecalis to prolonged bacteremia. Overall, our findings provide insights into the role of QS in biofilm growth, persistence, and immune evasion in enterococcal IE.

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↗