Modified vaginal lactobacilli expressing fluorescent and luminescent proteins for more effective monitoring of their release from nanofibers, safety and cell adhesion

Abstract

Electrospun nanofibers offer a highly promising platform for the delivery of vaginal lactobacilli, providing an innovative approach to preventing and treating vaginal infections. To advance the application of nanofibers for the delivery of lactobacilli, tools for studying their safety and efficacy in vitro need to be established. In this study, fluorescent (mCherry and GFP) and luminescent (NanoLuc luciferase) proteins were expressed in three vaginal lactobacilli (Lactobacillus crispatus, Lactobacillus gasseri and Lactobacillus jensenii) and a control Lactiplantibacillus plantarum with the aim to use this technology for close tracking of lactobacilli release from nanofibers and their adhesion on epithelial cells. The recombinant proteins influenced the growth of the bacteria, but not their ability to produce hydrogen peroxide. Survival of lactobacilli in nanofibers immediately after electrospinning varied among species. Bacteria retained fluorescence upon incorporation into PEO nanofibers, which was vital for evaluation of their rapid release. In addition, fluorescent labelling facilitated efficient tracking of bacterial adhesion to Caco-2 epithelial cells, while luminescence provided important quantitative insights into bacterial attachment, which varied from 0.5 to 50% depending on the species. The four lactobacilli in dispersion or in nanofibers were not detrimental for the viability of Caco-2 cells, and did not demonstrate hemolytic activity highlighting the safety profiles of both bacteria and PEO nanofibers. To summarize, this study contributes to the development of a promising delivery system, tailored for local administration of safe vaginal lactobacilli.

Keywords: Bioluminescence; Caco-2 cells; Electrospinning; Fluorescent proteins; NanoLuc luciferase; Nanofibers; Vaginal lactobacilli.

Fig. 1

Growth curves of engineered lactobacilli expressing mCherry (red lines), Nluc (dark yellow lines) and GFP (green lines). NT strains (black lines) were used as control

Fig. 2

Scanning electron microscopy of incorporated engineered and non-engineered vaginal lactobacilli into PEO nanofibers. The samples were imaged at a magnification of 20,000x. The scale bar in the bottom left represents 1 μm

Fig. 3

Representative confocal microscopy images of mCherry-labelled lactobacilli L. plantarum, L. gasseri and L. jensenii and GFP-labelled L. crispatus incorporated into PEO nanofibers. Empty PEO nanofibers are shown for comparison

Fig. 4

Kinetics of release of vaginal lactobacilli from PEO nanofibers determined by measuring fluorescence (a), and the influence of electrospinning on the viability of fluorescent lactobacilli (b). Percentage of the released bacteria was determined based on the highest overall fluorescent signal. Viability in dispersion was normalized to dry mass to enable comparison with viability in nanofibers. Lpl, Lactiplantibacillus plantarum (red); Lga, Lactobacillus gasseri (brown); Lje, Lactobacillus jensenii (blue); Lcr, Lactobacillus crispatus (green); D, dispersion; N, nanofibers. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student’s t test) relative to bacterial dispersion

Fig. 5

(a) Hemolytic test of bacteria (Lactiplantibacillus plantarum (LPL); Lactobacillus gasseri (LGA); Lactobacillus jensenii (LJE); Lactobacillus crispatus (LCR)), PEO suspension (S) and PEO nanofibers (NF). Percentage of live Caco-2 cells after 2, 4, 6 and 24 h incubation with bacterial dispersion (b) or nanofibers (c). Caco-2 cells were incubated with Lactiplantibacillus plantarum (red); Lactobacillus gasseri (brown); Lactobacillus jensenii (blue); Lactobacillus crispatus (green). Controls are shown in black (full line – no addition, dotted line – nanofibers without bacteria). ANOVA with multiple pairwise comparison was performed for each time point. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001; relative to control (Caco-2 cells without bacteria or nanofibers)

Fig. 6

(a) Autoaggregation of Lactiplantibacillus plantarum (red); Lactobacillus gasseri (brown); Lactobacillus jensenii (blue); Lactobacillus crispatus (green) monitored over a period of 6 h. Surface hydrophobicity (b) of Lactiplantibacillus plantarum (Lpl); Lactobacillus gasseri (Lga); Lactobacillus jensenii (Lje); Lactobacillus crispatus (Lcr) shown as a percentage and determined through extraction with n-hexadecane

Fig. 7

Z-stack of confocal images of Caco-2 cells with fluorescent lactobacilli adhered to the top of the layer

Fig. 8

Luminescence intensity of engineered vaginal lactobacilli as a function of bacterial concentration. Lactiplantibacillus plantarum (red circles), Lactobacillus gasseri (brown circles), Lactobacillus jensenii (blue circles), Lactobacillus crispatus (green circles). Non-transformed bacteria (black diamonds) and phosphate-buffered saline (grey triangles) were used as controls

Fig. 9

Luminescence intensity of vaginal lactobacilli adhered to Caco-2 cells following their addition in dispersion or in nanofibers. NT (non-transformed species), Caco-2 cells without bacteria and PBS (phosphate-buffered saline) were used as controls. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student’s t tests)