Engineering of Vaginal Lactobacilli to Express Fluorescent Proteins Enables the Analysis of Their Mixture in Nanofibers

Abstract

Lactobacilli are a promising natural tool against vaginal dysbiosis and infections. However, new local delivery systems and additional knowledge about their distribution and mechanism of action would contribute to the development of effective medicine. This will be facilitated by the introduction of the techniques for effective, inexpensive, and real-time tracking of these probiotics following their release. Here, we engineered three model vaginal lactobacilli (Lactobacillus crispatus ATCC 33820, Lactobacillus gasseri ATCC 33323, and Lactobacillus jensenii ATCC 25258) and a control Lactobacillus plantarum ATCC 8014 to express fluorescent proteins with different spectral properties, including infrared fluorescent protein (IRFP), green fluorescent protein (GFP), red fluorescent protein (mCherry), and blue fluorescent protein (mTagBFP2). The expression of these fluorescent proteins differed between the Lactobacillus species and enabled quantification and discrimination between lactobacilli, with the longer wavelength fluorescent proteins showing superior resolving power. Each Lactobacillus strain was labeled with an individual fluorescent protein and incorporated into poly (ethylene oxide) nanofibers using electrospinning, as confirmed by fluorescence and scanning electron microscopy. The lactobacilli retained their fluorescence in nanofibers, as well as after nanofiber dissolution. To summarize, vaginal lactobacilli were incorporated into electrospun nanofibers to provide a potential solid vaginal delivery system, and the fluorescent proteins were introduced to distinguish between them and allow their tracking in the future probiotic-delivery studies.

Influence of Excipient Composition on Survival of Vaginal Lactobacilli in Electrospun Nanofibers

“…The mean diameter of PEO nanofibers without bacteria was 166 ± 41 nm, while the addition of alginate and alginate/sucrose to PEO significantly increased diameter to 229 ± 27 nm and 241 ± 79 nm, respectively. The incorporation of lactobacilli cells was observed with specific thickenings of the nanofibers, as shown previously [23]. Lactobacilli decreased the mean diameter of nanofibers, which is contrary to our previous findings [23].…”

“…The incorporation of lactobacilli cells was observed with specific thickenings of the nanofibers, as shown previously [23]. Lactobacilli decreased the mean diameter of nanofibers, which is contrary to our previous findings [23]. This may be due to the use of a different electrospinning machine (Fluidnatek LE100; BioInicia SL, Valencia, Spain) or higher conductivity of the sample due to higher ion concentration retained after the lactobacilli preparation procedure.…”

“…Due to different growth kinetics and different cell concentration in the stationary phase, the three species were grown in different volumes (L. gasseri in 400 mL, L. crispatus in 800 mL, and L. jensenii in 200 mL) to yield a comparable number of cells for viability studies and electrospinning. Dispersions of lactobacilli in polymer solutions were prepared as previously described [19,20,23] with some modifications as follows. The species were grown until reaching their maximal concentration in the stationary phase and were centrifuged at 4900× g for 10 min at 4 • C (Rotanta 460R, Hettich, Tuttlingen, Germany).…”

“…Incorporating probiotic bacteria into nanofibers has gained a lot of interest in recent years [19][20][21][22]. However, only a few studies described the encapsulation of vaginal probiotics [23][24][25]. Three different vaginal Lactobacillus species (L. crispatus, L. gasseri, and L. jensenii) were genetically modified to express fluorescent proteins with different spectral properties and incorporated into polyethylene oxide (PEO) nanofibers [23].…”

“…However, only a few studies described the encapsulation of vaginal probiotics [23][24][25]. Three different vaginal Lactobacillus species (L. crispatus, L. gasseri, and L. jensenii) were genetically modified to express fluorescent proteins with different spectral properties and incorporated into polyethylene oxide (PEO) nanofibers [23]. Vaginal isolate Lactobacillus acidophilus was incorporated into nanofibers using polyvinyl alcohol and polyvinylpyrrolidone with different molecular masses.…”