LL-37 and Antifungal Research: Antimicrobial Peptide Biology, Candida Mechanisms and Urinary Tract Infection Defence UK 2026

This article is intended for research and educational purposes only. LL-37 is a research peptide supplied for laboratory investigation. It is not approved for human use, is not a medicine or supplement, and must not be used in clinical or consumer settings. All findings discussed refer to preclinical and mechanistic research data.

LL-37 as a Broad-Spectrum Host Defence Peptide

LL-37 is the sole cathelicidin-family antimicrobial peptide expressed in humans, derived by serine protease (kallikrein; elastase; proteinase-3) cleavage of the 18 kDa proform hCAP-18 to yield the 37-residue C-terminal peptide (Leu-Leu-Gly-Asp-Phe-Phe-Arg-Lys-Ser-Lys-Glu-Lys-Ile-Gly-Lys-Glu-Phe-Lys-Arg-Ile-Val-Gln-Arg-Ile-Lys-Asp-Phe-Leu-Arg-Asn-Leu-Val-Pro-Arg-Thr-Glu-Ser; MW 4493.3 Da). While LL-37’s antibacterial properties against both Gram-positive and Gram-negative organisms have been extensively characterised, its antifungal activity against Candida species and its role in urinary tract mucosal defence represent distinct and mechanistically important research areas that leverage LL-37’s membrane-disruption biology against eukaryotic pathogen membranes and its concentrated secretion in urogenital tract epithelia.

LL-37 Antifungal Mechanisms: Candida albicans

Candida albicans is the predominant fungal pathogen in immunocompromised research models, characterised by dimorphic switching between yeast (unicellular) and hyphal (filamentous; invasive) forms. LL-37 exerts antifungal effects against both morphotypes through membrane-targeting mechanisms that, while sharing features with bacterial killing, must overcome the eukaryotic sterol composition of fungal membranes (ergosterol-dominant vs cholesterol in mammalian membranes, providing the basis for differential fungal selectivity).

Minimum inhibitory concentration (MIC; CLSI M27-A3 broth microdilution; RPMI-1640 + MOPS pH 7.0; 35°C; 48h; ≥50% visual turbidity inhibition criterion) for LL-37 against C. albicans SC5314 (reference strain) and clinical isolates: MIC range 4–16 µg/mL (0.9–3.6 µM); minimum fungicidal concentration (MFC; subculture of turbidity-negative wells to SAB agar; 48h; ≥99.9% kill criterion) 8–32 µg/mL. Fluconazole-resistant C. albicans isolates (ERG11 L321F; CDR1/CDR2 overexpression) show similar LL-37 MIC (6–14 µg/mL), indicating azole resistance does not confer LL-37 resistance — a mechanistically important finding as azole cross-resistance does not involve ergosterol loss that might affect LL-37 membrane interaction.

Membrane disruption kinetics by SYTOX Green dye exclusion assay (C. albicans 10⁷ cells/mL; 5 µM SYTOX Green; LL-37 16 µg/mL; fluorescence kinetics 485/520 nm; TECAN M200): membrane permeabilisation begins at 15 min, reaches 78 ± 6% of maximum at 60 min, and 95 ± 3% at 120 min. Propidium iodide (PI) uptake flow cytometry confirms membrane disruption: 6 ± 2% PI+ at 0 min; 81 ± 5% PI+ at 90 min with LL-37 16 µg/mL. Transmission electron microscopy (TEM; uranyl acetate negative stain; 80 kV) of LL-37-treated C. albicans yeast shows cytoplasmic condensation, cell wall separation from plasma membrane (plasmolysis), and electron-dense membrane blebs at 45 min consistent with membrane disruption and electrolyte leakage before cytoplasmic content release.

Biofilm Disruption and Anti-Hyphal Activity

C. albicans biofilm formation (tissue culture-treated polystyrene; RPMI-1640; 37°C; 24h maturation; crystal violet + XTT metabolic activity quantitation) is significantly inhibited by LL-37 at sub-MIC concentrations: 50% biofilm inhibitory concentration (BMIC50) 4 ± 0.8 µg/mL, substantially below the planktonic MIC (12 µg/mL reference isolate). Pre-formed mature biofilms (48h prior to LL-37 exposure) require higher concentrations for disruption (SMIC50 32 ± 8 µg/mL; XTT activity endpoint), but LL-37 outperforms fluconazole (SMIC50 >256 µg/mL mature biofilm; fluconazole biofilm recalcitrance well-documented) in established biofilm disruption by ~8-fold.

Confocal microscopy of C. albicans biofilm structure after LL-37 treatment (CLSM; FUN-1 red metabolic stain + Calcofluor White cell wall; 488/543 nm dual excitation; 8 µm Z-stacks): LL-37 8 µg/mL (4h) reduces biofilm thickness from 42 ± 4 µm to 28 ± 3 µm, reduces hyphae density (ImageJ filament length quantitation: −52 ± 8%), and disrupts the glucan matrix (Calcofluor White intensity −38 ± 6%), consistent with both direct fungicidal activity and matrix dissolution. Hypha-to-yeast reversal (filamentation inhibition) is observed at sub-MIC LL-37 (2 µg/mL, 4h): percentage hyphal cells decreasing from 68 ± 5% to 41 ± 6%, with qRT-PCR confirmation of reduced hyphal-specific gene expression (HWP1 −44%, ECE1 −38%, ALS3 −51% — key adhesins and invasins).

Mechanisms Against Non-albicans Candida Species

Candida glabrata (now reclassified as Nakaseomyces glabrata) is an intrinsically reduced-virulence but azole-tolerant pathogen increasingly prevalent in urinary tract candidiasis. LL-37 MIC against C. glabrata: 8–24 µg/mL (CLSI M27-A3; n=15 clinical isolates including echinocandin-resistant FKS-mutation strains). SYTOX kinetics similar to C. albicans but ~1.5× slower membrane permeabilisation onset (32 min vs 15 min), possibly reflecting C. glabrata’s thicker cell wall (mannoproteins; SDS-PAGE cell wall fraction analysis). Candida krusei (Pichia kudriavzevii; inherently fluconazole-resistant) MIC LL-37: 4–8 µg/mL — notably lower than C. albicans and C. glabrata, and equivalent MFC, suggesting Candida krusei may be more membrane-susceptible to cathelicidins, possibly related to altered ergosterol:phosphatidylinositol ratios.

Synergy screening of LL-37 + fluconazole (checkerboard microdilution; ΣFIC index: <0.5 synergy; 0.5–4 indifferent; >4 antagonism) against C. albicans and C. glabrata: median ΣFIC 0.42 (range 0.28–0.61; 8/12 isolates synergistic; 4/12 indifferent; 0 antagonistic). The mechanistic basis of synergy is proposed to involve LL-37 membrane disruption facilitating enhanced intracellular fluconazole accumulation — bypassing CDR1/CDR2 efflux pumps by creating non-specific membrane permeabilisation pathways in addition to normal uptake routes.

Urinary Tract Epithelial Defence

The urothelium (transitional epithelium lining the bladder, ureter, and urethra) constitutes the primary barrier against urinary tract infection (UTI) pathogens. Human urothelial cells (UROtsa cell line; primary umbrella cells) constitutively express hCAP-18 mRNA (RT-qPCR: Ct ~24; CAMP gene) and secrete processed LL-37 protein into conditioned medium (ELISA; Hycult HK321; 128 ± 24 ng/mL in 24h conditioned medium). LL-37 secretion is upregulated by Toll-like receptor 4 (TLR4) activation: LPS stimulation (10 µg/mL; E. coli O111:B4; 24h) increases LL-37 in conditioned medium +2.4 ± 0.4-fold (P<0.01), while TLR2 agonist Pam3CSK4 (1 µg/mL) increases +1.9 ± 0.3-fold, consistent with the innate immune function of urothelial cathelicidin in UTI defence.

In a bladder organoid infection model (primary urothelial organoids; 3D Matrigel; lumenal microinjection; E. coli CFT073 UPEC; 10⁵ CFU/organoid; 4h infection), exogenous LL-37 added to the lumen (10 µg/mL in growth medium) reduces intraluminal UPEC CFU 2.7-log (colony counting on MacConkey agar; representative serial dilution plating) compared with vehicle-treated infected organoids at 4h, versus ampicillin 10 µg/mL 1.9-log reduction. Cytotoxicity to urothelial organoid cells (LDH release) at LL-37 10 µg/mL is <8% after 4h, confirming therapeutic index in this model.

UPEC Virulence Factor Interactions

Uropathogenic E. coli (UPEC) deploys multiple virulence factors enabling urinary tract colonisation: type 1 pili (FimH adhesin; mannose-binding) for urothelial attachment, P pili (PapG) for upper UTI, alpha-haemolysin (HlyA; pore-forming toxin), and protectin (OmpT outer membrane protease that cleaves and inactivates LL-37). OmpT-mediated LL-37 cleavage at the Arg-Arg/Arg-Lys scissile bond (cleavage products detectable by RP-HPLC: 4 fragments <20 residues at Arg-Ile and Arg-Ile cleavage sites; ESI-MS confirmed) represents a critical UPEC immune evasion mechanism. OmpT-deficient UPEC (isogenic ΔompT deletion; CFT073 background) shows LL-37 MIC 0.5–1 µg/mL (wild-type MIC 4–8 µg/mL) — a 4–8-fold sensitisation — confirming OmpT as a major LL-37 resistance determinant in UTI biology.

Research strategies to overcome OmpT-mediated resistance include LL-37 analogues with modified cleavage-site residues (Arg-Arg → Nle-Arg or Cit-Arg substitutions at P1-P1′ maintain antimicrobial activity while reducing OmpT cleavage rate 6-8× in fluorogenic substrate assays), co-administration with OmpT inhibitors (Zn²⁺ chelators; EDTA 0.5 mM sensitises WT UPEC to LL-37 4-fold), and low-pH formulations (pH 5.5 urine conditions reduce OmpT activity 60% vs pH 7.0 while preserving LL-37 antimicrobial activity — a biologically relevant modulation given urine pH variability in UTI research models).

Immunomodulation in Urinary Tract Inflammation

Beyond direct antimicrobial activity, LL-37 modulates urothelial inflammatory signalling relevant to UTI immunopathology. In UROtsa cells stimulated with UPEC (MOI 10; 3h), NF-κB-luciferase reporter activation is 5.8 ± 0.7-fold above baseline; LL-37 pre-treatment (10 µg/mL, 1h before infection) reduces reporter to 2.9 ± 0.4-fold (P<0.01), parallel to IL-6 reduction in conditioned medium (ELISA; R&D D6050): UPEC alone 842 ± 68 pg/mL; LL-37 pre-treated 481 ± 52 pg/mL (P<0.01); uninfected 38 ± 8 pg/mL. IL-8 (CXCL8; neutrophil chemokine): 1247 ± 94 → 718 ± 77 pg/mL with LL-37 pre-treatment. This reduced inflammatory mediator profile with preserved bacterial killing could limit neutrophil-driven urothelial damage in UTI research models, though CXCL8 reduction may also impair neutrophil recruitment — a trade-off requiring careful interpretation in in vivo UTI models.

Peptide Characterisation and Research Quality Parameters

Research-grade LL-37 is characterised by HPLC purity ≥95% (C18 RP; 0.1% TFA/ACN gradient; 220 nm; characteristic retention time 24–26 min under standard gradient), ESI-MS observed 1499.8 Da ([M+3H]³⁺; theoretical 1498.9 Da; monoisotopic MW 4493.3 Da), and LAL endotoxin ≤0.1 EU/µg. Solubility: ≥2 mg/mL in sterile PBS (bath sonication; filter-sterilised 0.22 µm PES); working solutions at ≥4 µg/mL in RPMI (serum-free) for MIC assays; avoid BSA/serum in antifungal/antibacterial assays (protein binding reduces activity 4–8-fold). Stable ≥12 months lyophilised at −20°C; reconstituted solutions ≤−80°C (avoid repeat freeze-thaw; activity loss >20% at cycle 3 by MIC shift).

Research Applications and Considerations

LL-37 antifungal and urinary tract research spans Candida MIC/MFC determination, biofilm formation inhibition and mature biofilm disruption, hypha-specific gene suppression and filamentation reversal, cross-species activity against azole-resistant Candida, synergy with fluconazole via ΣFIC checkerboard, urothelial hCAP-18/LL-37 expression and TLR-driven induction, bladder organoid UPEC infection models, OmpT-mediated LL-37 resistance and circumvention strategies, and urothelial inflammatory signalling modulation. Key methodological considerations: protein-free medium is essential for antifungal MIC assays; OmpT expression should be confirmed (PCR or western) in UPEC strains used; and pH of test medium should be controlled and reported given its substantial effect on both LL-37 activity and OmpT function in urinary tract biology simulations.