Biology subjects

Chong, K. K. L.

Publications and source records attributed to Chong, K. K. L..

13 recordsLinked to original sources

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 faecalis-derived lactic acid facilitates persistent and polymicrobial wound infections by suppressing macrophage activation

Macrophage activation is essential for innate immunity, and its suppression enables pathogen persistence. We show that Enterococcus faecalis suppresses macrophage activation through lactic acid-mediated extracellular acidification. Mutants lacking lactate dehydrogenase (ldh), and thus unable to acidify the environment, fail to inhibit NF-{kappa}B. E. faecalis-derived lactic acid acts via MCT-1 and GPR81 through two distinct but complementary mechanisms that culminate in the reduction of NF-{kappa}B activity. Lactic acid acts through MCT-1 to inhibit ERK and STAT3 phosphorylation, leading to reduced STAT3 binding to the Myd88 promoter, and reduced MyD88 protein levels. Lactic acid signaling to GPR81 mediates the phosphorylation of the transcriptional factor YAP, ultimately attenuating NF-{kappa}B signaling. In a murine wound infection model, this lactic acid-driven immunosuppressive niche enables prolonged E. faecalis persistence and enhances the fitness of co-infecting bacteria such as Escherichia coli. These findings reveal how bacterial lactic acid subverts innate immunity to support chronic and polymicrobial infections. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/635924v2_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@2e1565org.highwire.dtl.DTLVardef@17eaf85org.highwire.dtl.DTLVardef@356139org.highwire.dtl.DTLVardef@821581_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- E. faecalis lactate dehydrogenase mutants fail to acidify the environment and cannot suppress NF-{kappa}B signaling. - Lactic acid is necessary and sufficient to drive NF-{kappa}B suppression in macrophages. - Lactic acid NF-{kappa}B suppression is relayed through two complementary routes: MCT- 1 transport and the lactate sensor GPR81. - MCT-1-dependent signaling blunts ERK/STAT3 phosphorylation, lowering MyD88 levels; whereas GPR81 drives YAP phosphorylation, reducing NF-{kappa}B activation. - E. faecalis-derived lactic acid drives immunosuppression, potentiates persistence, and promotes multi-species wound infection in vivo.

microbiology↗

bioRxiv2023

The HtrA chaperone monitors sortase-assembled pilus biogenesis in Enterococcus faecalis

Sortase-assembled pili contribute to virulence in many Gram-positive bacteria. In Enterococcus faecalis, the endocarditis and biofilm-associated pilus (Ebp) is polymerized on the membrane by sortase C (SrtC) and attached to the cell wall by sortase A (SrtA). In the absence of SrtA, polymerized pili remain anchored to the membrane (i.e. off-pathway). Here we show that the high temperature requirement A (HtrA) bifunctional chaperone/protease of E. faecalis is a quality control system that clears aberrant off-pathway pili from the cell membrane. In the absence of HtrA and SrtA, accumulation of membrane-bound pili leads to cell envelope stress and partially induces the regulon of the ceftriaxone resistance-associated CroRS two-component system, which in turn causes hyper-piliation and cell morphology alterations. Inactivation of croR in the {Delta}srtA{Delta}htrA background partially restores the observed defects of the {Delta}srtA{Delta}htrA strain, supporting a role for CroRS in the response to membrane perturbations. Moreover, absence of SrtA and HtrA decreases basal tolerance of E. faecalis against cephalosporins and daptomycin. The link between HtrA, pilus biogenesis and the CroRS two-component system provides new insights into the E. faecalis response to endogenous membrane perturbations. Author summaryTo explore the role of the HtrA chaperone/protease in E. faecalis off-pathway pilus clearance, we deleted htrA in an E. faecalis OG1RF {Delta}srtA strain known to retain polymerized pili on the cell membrane. Cells in the {Delta}srtA{Delta}htrA background are hyper-piliated, possess altered morphology, and are more susceptible to cell envelope-targeting antibiotics as compared to the parent OG1RF strain. RNA sequencing of the {Delta}srtA{Delta}htrA strain revealed transcriptional changes reminiscent of a membrane stress response. This response was pilus-dependent and contained several members of the CroR regulon. Inactivation of the response regulator CroR in the {Delta}srtA{Delta}htrA background restored (at least partially) piliation and cell morphology but not antibiotic susceptibility, linking CroR for the first time to pilus biogenesis and endogenous cell envelope stress.

microbiology↗

bioRxiv2023

An essential protease, FtsH, influences daptomycin resistance acquisition in Enterococcus faecalis

Daptomycin is a last-line antibiotic commonly used to treat vancomycin resistant Enterococci, but resistance evolves rapidly and further restricts already limited treatment options. While genetic determinants associated with clinical daptomycin resistance (DAPR) have been described, information on factors affecting the speed of DAPR acquisition is limited. The multiple peptide resistance factor (MprF), a phosphatidylglycerol modifying enzyme involved in cationic antimicrobial resistance, is linked to DAPR in pathogens such as methicillin-resistant Staphylococcus aureus. Since Enterococcus faecalis encodes two paralogs of mprF and clinical DAPR mutations do not map to mprF, we hypothesized that functional redundancy between the paralogs prevents mprF-mediated resistance and masks other evolutionary pathways to DAPR. Here we performed in vitro evolution to DAPR in mprF mutant background. We discovered that the absence of mprF results in slowed DAPR evolution and is associated with inactivating mutations in ftsH resulting in the depletion of the chaperone repressor HrcA. We also report that ftsH is essential in the parental, but not in the {Delta}mprF, strain where FtsH depletion results in growth impairment in the parental strain, a phenotype associated with reduced glycolysis and reduced ability for metabolic reduction. This presents FtsH and HrcA as enticing targets for developing anti-resistance strategies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=198 SRC="FIGDIR/small/551240v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@12ff3adorg.highwire.dtl.DTLVardef@127044aorg.highwire.dtl.DTLVardef@8ab082org.highwire.dtl.DTLVardef@14a4afc_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

Depleting cationic lipids involved in antimicrobial resistance drives adaptive lipid remodeling in Enterococcus faecalis

The bacterial cell membrane is an interface for cell envelope synthesis, protein secretion, virulence factor assembly and a target for host cationic antimicrobial peptides (CAMPs). To resist CAMP killing, several Gram-positive pathogens encode the multiple peptide resistance factor (MprF) enzyme that covalently attaches cationic amino acids to anionic phospholipids in the cell membrane. While E. faecalis encodes two mprF paralogs, MprF2 plays a dominant role in conferring resistance to killing by the CAMP human {beta}-defensin 2 (hBD-2) in E. faecalis strain OG1RF. The goal of the current study is to understand the broader lipidomic and functional roles of E. faecalis mprF. We analyzed the lipid profiles of parental wild type and mprF mutant strains and show that while {triangleup}mprF2 and {triangleup}mprF1 {triangleup}mprF2 mutants completely lacked cationic lysyl-phosphatidylglycerol (L-PG), the {triangleup}mprF1 mutant synthesized [~]70% of L-PG compared to the parent. Unexpectedly, we also observed a significant reduction of PG in {triangleup}mprF2 and {triangleup}mprF1 {triangleup}mprF2. In the mprF mutants, particularly {triangleup}mprF1 {triangleup}mprF2, the decrease in L-PG and PG is compensated by an increase in the phosphorus-containing lipid, GPDGDAG, and D-ala-GPDGDAG. These changes were accompanied by a downregulation of de novo fatty acid biosynthesis and an accumulation of long-chain acyl-acyl carrier proteins (long-chain acyl-ACPs), suggesting that the suppression of fatty acid biosynthesis was mediated by the transcriptional repressor FabT. Growth in chemically defined media lacking fatty acids revealed severe growth defects in the {triangleup}mprF1 {triangleup}mprF2 mutant strain, but not the single mutants, which was partially rescued through supplementation with palmitic and stearic acids. Changes in lipid homeostasis correlated with lower membrane fluidity, impaired protein secretion, and increased biofilm formation in both {triangleup}mprF2 and {triangleup}mprF1 {triangleup}mprF2, compared to wild type and {triangleup}mprF1. Collectively, our findings reveal a previously unappreciated role for mprF in global lipid regulation and cellular physiology, which could facilitate the development of novel therapeutics targeting MprF. Significance StatementThe cell membrane plays a pivotal role in protecting bacteria against external threats, such as antibiotics. Cationic phospholipids such as lysyl-phosphatidyglycerol (L-PG) resist the action of cationic antimicrobial peptides through electrostatic repulsion. Here we demonstrate that L-PG depletion has several unexpected consequences in Enterococcus faecalis, including a reduction of phosphatidylglycerol (PG), enrichment of a phosphorus-containing lipid, reduced fatty acid synthesis accompanied by an accumulation of long-chain acyl-acyl carrier proteins (long chain acyl-ACPs), lower membrane fluidity, and impaired secretion. These changes are not deleterious to the organism as long as exogenous fatty acids are available for uptake from the culture medium. Our findings suggest an adaptive mechanism involving compensatory changes across the entire lipidome upon removal of a single phospholipid modification. Such adaptations must be considered when devising antimicrobial strategies that target membrane lipids.

microbiology↗

bioRxiv2022

Purine and carbohydrate availability drive Enterococcus faecalis fitness during wound infection

Enterococcus faecalis is commonly isolated from a variety of wound types. Despite its prevalence, the pathogenic mechanisms of E. faecalis during wound infection are poorly understood. Using a mouse wound infection model, we performed in vivo E. faecalis transposon sequencing and RNA sequencing to identify fitness determinants that are crucial for replication and persistence of E. faecalis during wound infection. We found that E. faecalis purine biosynthesis genes are important for bacterial replication during the early stages of wound infection, a time when purine metabolites are rapidly consumed by E. faecalis within wounds. We also found that the E. faecalis MptABCD phosphotransferase system (PTS), involved in the import of galactose and mannose, is crucial for E. faecalis persistence within wounds of both healthy and diabetic mice, especially when carbohydrate availability changes throughout the course of infection. During in vitro growth with mannose as the sole carbohydrate source, shikimate and purine biosynthesis genes were downregulated in the OG1RF {Delta}mptD mutant compared to the isogenic wild-type strain, suggesting a link between mannose transport, shikimate, and purine biosynthesis. Together, our results suggest that dynamic and temporal microenvironment changes at the wound site affects pathogenic requirements and mechanisms of E. faecalis and raise the possibility of lowering exogenous purine availability and/or targeting galactose/mannose PTS to control wound infections. IMPORTANCEAlthough E. faecalis is a common wound pathogen, its pathogenic mechanisms during wound infection are unexplored. Here, combining a mouse wound infection model with in vivo transposon and RNA sequencing approaches, we identified the E. faecalis purine biosynthetic pathway and galactose/mannose MptABCD phosphotransferase system as essential for E. faecalis acute replication and persistence during wound infection, respectively. The essentiality of purine biosynthesis and the MptABCD PTS is driven by the rapid consumption of purine metabolites by E. faecalis during acute replication and changing carbohydrate availability during the course of wound infection. Overall, our findings reveal the importance of the wound microenvironment in E. faecalis wound pathogenesis and how these metabolic pathways can be targeted to better control wound infections.

microbiology↗

bioRxiv2022

Enterococcus faecalis and Staphylococcus aureus mixed species infection attenuates pathogen-specific neutrophil responses and impairs bacterial clearance

Enterococcus faecalis is an opportunistic pathogen that is frequently co-isolated with other microbes in catheterized urinary tract infections and chronically infected wounds. While E. faecalis can subvert the host immune response and promote the survival of other microbes via interbacterial synergy, little is known about the impact of immune suppression mediated by E. faecalis and how E. faecalis impacts the survival of co-infecting microbes. We hypothesized that E. faecalis can attenuate neutrophil-mediated responses in mixed-species infection to promote survival of the co-infecting species. Here, we show that E. faecalis and Staphylococcus aureus mono-species infection activates intracellular ROS production and NET formation, respectively, enabling effective neutrophil-mediated control of the microbial infection. Growth of both bacterial species was enhanced during co-infection in neutrophils in vitro and in wounds in vivo. E. faecalis reduced S. aureus-induced NET formation and S. aureus suppressed E. faecalis-induced intracellular ROS production. When the species ratios were skewed, the neutrophil reaction profile resembled that elicited by the more abundant species, favoring enhanced survival of the less abundant species. These findings highlight the complexity of the immune response to polymicrobial infections and show that attenuated pathogen-specific immune responses contribute to microbial survival in the mammalian host.

microbiology↗

bioRxiv2021

Escherichia coli BarA-UvrY regulates the pks island and kills Staphylococci via the genotoxin colibactin

Wound infections are often polymicrobial in nature and are associated with poor disease prognoses. Escherichia coli and Staphylococcus aureus are among the top five most cultured pathogens from wound infections. However, little is known about the polymicrobial interactions between E. coli and S. aureus during wound infections. In this study, we show that E. coli kills S. aureus both in vitro and in a mouse excisional wound model via the genotoxin, colibactin. We also show that the BarA-UvrY two component system (TCS) is a novel regulator of the pks island, which acts through the carbon storage global regulatory (Csr) system. Together, our data demonstrate the role of colibactin in inter-species competition and show that it is regulated by BarA-UvrY TCS, a previously uncharacterized regulator of the pks island.

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↗

bioRxiv2020

Enterococcus faecalis manganese exporter MntE alleviates manganese toxicity and is required for mouse gastrointestinal colonization

Bacterial pathogens encounter a variety of nutritional environments in the human host, including nutrient metal restriction and overload. Uptake of manganese (Mn) is essential for Enterococcus faecalis growth and virulence; however, it is not known how this organism prevents Mn toxicity. In this study, we examine the role of the highly conserved MntE transporter in E. faecalis Mn homeostasis and virulence. We show that inactivation of mntE results in growth restriction in the presence of excess Mn, but not other metals, demonstrating its specific role in Mn detoxification. Upon growth in the presence of excess Mn, an mntE mutant accumulates intracellular Mn, iron (Fe), and magnesium (Mg), supporting a role for MntE in Mn and Fe export, and a role for Mg in offsetting Mn toxicity. Growth of the mntE mutant in excess Fe also results in increased levels of intracellular Fe, but not Mn or Mg, providing further support for MntE in Fe efflux. Inactivation of mntE in the presence of excess iron also results in the upregulation of glycerol catabolic genes and enhanced biofilm growth, and addition of glycerol is sufficient to augment biofilm growth for both the mntE mutant and its wild type parental strain, demonstrating that glycerol availability significantly enhances biofilm formation. Finally, we show that mntE contributes to infection of the antibiotic-treated mouse gastrointestinal (GI) tract, suggesting that E. faecalis encounters excess Mn in this niche. Collectively, these findings demonstrate that the manganese exporter MntE plays a crucial role in E. faecalis metal homeostasis and virulence.

microbiology↗

bioRxiv2019

Sortase-assembled pili promote extracellular electron transfer and iron acquisition in Enterococcus faecalis biofilm

Enterococcus faecalis sortase-assembled endocarditis and biofilm-associated pili (Ebp) are a virulence factor implicated in enterococcal biofilm-associated infections and gastrointestinal colonization. We previously showed that E. faecalis biofilm metabolism is influenced by extracellular electron transfer (EET) under iron-rich conditions, raising the question of whether Ebp pili also play a role in EET. Here, we report a novel role of Ebp pili in E. faecalis adhesion to the iron oxides magnetite, goethite and hematite, where the EbpA tip adhesin contributes to this interaction. Adhesion by Ebp pili is conditionally important for EET to iron oxides, as pilus mutants are attenuated in EET under non-static growth conditions. In alignment with the established role of EET in redox homeostasis, we find that EET to ferricyanide supports E. faecalis anaerobic growth on glycerol. Further, in an antibiotic-treated mouse gastrointestinal colonization model, we show that E. faecalis mutants deficient in EET poorly colonize the intestinal niche. Taken together, our findings suggest that Ebp pili can influence E. faecalis metabolic fitness by promoting EET to iron oxides, raising new questions of how Ebp pili shape E. faecalis interactions with environmental ecosystems. Furthermore, the important role of EET in E. faecalis colonization of the dysbiotic gastrointestinal environment highlights the need for further inquiry into how EET contributes to E. faecalis microbial pathogenesis. ImportanceIn this study, we explored the interplay between extracellular electron transfer (EET) and an Enterococcus faecalis biofilm factor, the endocarditis and biofilm-associated pili (Ebp). We demonstrate that Ebp pili have a novel role in adhesion to iron oxides, which consequently promotes EET to iron oxides under non-static conditions. Along with our findings that E. faecalis EET can be coupled to anaerobic cell growth, our results point to a potential ecological role of Ebp pili in natural environments, outside of its established function in adhesion to host ligands. We provide the first evidence of the contribution of EET to E. faecalis colonization of the antibiotic-treated murine intestinal niche, which adds to the limited experimental evidence linking EET and microbial pathogenesis, as well as highlights the need for further studies of EET in bacterial pathogens.

microbiology↗

bioRxiv2017

Enterococcus faecalis Promotes Innate Immune Suppression And Polymicrobial Catheter-Associated Urinary Tract Infection

Enterococcus faecalis, a member of the human gastrointestinal microbiota, is an opportunistic pathogen associated with hospital-acquired wound, bloodstream, and urinary tract infections. E. faecalis can subvert or evade immune-mediated clearance, although the mechanisms are poorly understood. In this study, we examined E. faecalis-mediated subversion of macrophage activation. We observed that E. faecalis actively prevents NF-{kappa}B signaling in mouse RAW264.7 macrophages in the presence of Toll-like receptor agonists and during polymicrobial infection with Escherichia coli. E. faecalis and E. coli co-infection in a mouse model of catheter-associated urinary tract infection (CAUTI) resulted in a suppressed macrophage transcriptional response in the bladder compared to E. coli infection alone. Finally, we demonstrated that co-inoculation of E. faecalis with E. coli into catheterized bladders significantly augmented E. coli CAUTI. Taken together, these results support that E. faecalis suppression of NF-{kappa}B-driven responses in macrophages promotes polymicrobial CAUTI pathogenesis.\n\nAuthor SummarySynergistic polymicrobial infections can contribute to both disease severity and persistence. Enterococcus faecalis and Escherichia coli are frequently co-isolated from polymicrobial urinary tract infections. Immunomodulation by co-infecting microbes can result in a more permissive environment for pathogens to establish infection. Presently, we do not yet understand how these microbes overcome host immunity to establish polymicrobial infections. To address this, we investigated how the immunosuppressive function of E. faecalis can contribute to acute infection. We defined that E. faecalis is able to suppress macrophages in vitro, despite the presence of E. coli. We also demonstrated E. faecalis ability to augment E. coli titers in vivo to establish kidney infection. Our findings raise the prospect that E. faecalis can alter host immunity to increase susceptibility to other uropathogens.

microbiology↗

bioRxiv2017

Enterococcus faecalis Modulates Immune Activation And Slows Healing During Wound Infection

Enterococcus faecalis is one of most frequently isolated bacterial species in wounds yet little is known about its pathogenic mechanisms in this setting. Here, we used a mouse wound excisional model to characterize the infection dynamics of E. faecalis and show that infected wounds result in two different states depending on the initial inoculum. Low dose inocula were associated with short term, low titer colonization whereas high dose inocula were associated with acute bacterial replication and long term persistence. High dose infection and persistence were also associated with immune cell infiltration, despite suppression of some inflammatory cytokines and delayed wound healing. During high dose infection, the multiple peptide resistance factor (MprF) which is involved in resisting immune clearance, contributes to E. faecalis fitness. These results comprehensively describe a mouse model for investigating E. faecalis wound infection determinants, and suggest that both immune modulation and resistance contribute to persistent, non-healing wounds.

microbiology↗