Biology subjects

Choo, P. Y.

Publications and source records attributed to Choo, P. Y..

5 recordsLinked to original sources

bioRxiv2025

Adaptation of Enterococcus faecalis to intestinal mucus revealed by a human colonic organoid model

The human gastrointestinal tract hosts a diverse population of microorganisms that have a significant impact on host health. Among this population, Enterococcus faecalis (Ef) represents a common member of intestinal microbiota colonizing humans early in life, but which can also opportunistically infect its host. Despite its importance in human health, investigations of its physiological adaptation to the mucosal environment remain limited. Building on recent advances in tissue engineering, we here leverage human colonic organoids (colonoids) to investigate the Efs mechanisms of mucosal surface colonization across space and time. Using high-resolution microscopy, we visualized Ef growth within the natively formed colonic mucus layer in colonoids. Leveraging a custom perfusion chamber, we tracked Ef growth within the mucus of live colonoids over time under flow, which revealed specific colonization strategies, including biofilm-like microcolony formation. To identify Ef fitness determinants in this niche, we implemented transposon insertion sequencing (Tn-seq) in the natively formed mucus of live colonoids. This approach revealed a large fitness rearrangement compared to typical liquid culture, mainly involving metabolic activity and regulatory response during mucosal colonization, as well as factors that may contribute to colony formation at the mucosal surface. Altogether, our results show important physiological and biophysical adaptation of Ef to the mucosal surface that are not captured by in vitro conditions and that cannot be revealed in vivo at high resolution.

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

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

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

Spatial and temporal localization of cell wall associated pili in Enterococcus faecalis

Enterococcus faecalis relies upon a number of cell wall-associated proteins for virulence. One virulence factor is the sortase-assembled endocarditis and biofilm associated pilus (Ebp), an important factor for biofilm formation in vitro and in vivo. The current paradigm for sortase-assembled pilus biogenesis in Gram-positive bacteria is that the pilus sortase covalently links pilus monomers prior to recognition, while the housekeeping sortase cleaves at the LPXTG motif within the terminal pilin subunit, and subsequently attaches assembled pilus fiber to the growing cell wall at sites of new cell wall synthesis. While the cell wall anchoring mechanism and polymerization of Ebp is well characterized, less is known about the spatial and temporal deposition of this protein on the cell surface. We followed the distribution of Ebp and peptidoglycan (PG) throughout the E. faecalis cell cycle via immunofluorescence microscopy and fluorescent D-amino acids (FDAA) staining. Surprisingly, cell surface Ebp did not co-localize with newly synthesized PG. Instead, surface-anchored Ebp was localized to the cell hemisphere but never at the septum where new cell wall is deposited. In addition, the older hemisphere of the E. faecalis diplococcus were completely saturated with Ebp, while Ebp appeared as two foci directly adjacent to the nascent septum in the newer hemisphere. A similar localization pattern was observed for another cell wall anchored substrate by sortase A, aggregation substance (AS), suggesting that this may be a general rule for all SrtA substrates in E. faecalis. When cell wall synthesis was inhibited by ramoplanin, an antibiotic that binds and sequesters lipid II cell wall precursors, new Ebp deposition at the cell surface was not disrupted. These data suggest an alternative paradigm for sortase substrate deposition in E. faecalis, in which Ebp are anchored directly onto un-crosslinked cell wall, independent of new PG synthesis.

microbiology↗