Identification and characterization of novel endolysins targeting Gardnerella vaginalis biofilms to treat bacterial vaginosis

Abstract

Bacterial vaginosis (BV) is a recurrent dysbiosis that is frequently associated with preterm birth, increased risk for acquisition of human immunodeficiency virus (HIV) and other sexually transmitted infections (STIs). The overgrowth of a key pathobiont, Gardnerella vaginalis, as a recalcitrant biofilm is central to the development of this dysbiosis. Overgrowth of vaginal biofilms, seeded by initial G. vaginalis colonization, leads to recurrent symptomatic BV which is poorly resolved by classically used antibiotics. In this light, the use of bacteriophages and/or their proteins, represents a promising alternative. Here we identify 84 diverse anti-Gardnerella endolysins across 7 protein families. A subset of 36 endolysin candidates were refactored and overexpressed in an E. coli BL21 (DE3) system and 5 biochemically and structurally diverse endolysins were fully characterized. Each candidate endolysin showed good lytic activity against planktonic G. vaginalis ATCC14018, as well as G. vaginalis clinical isolates. These endolysin candidates were assayed in biofilm prevention and disruption assays, with biofilm disruption at low microgram concentrations (5 μg/ml) observed. In addition to clonal G. vaginalis biofilms, endolysin candidates could also successfully disrupt polyspecies biofilms. Importantly, none of our candidates showed lytic activity against commensal lactobacilli present in the vaginal microbiota such as L. crispatus, L. jensenii, L. gasseri, and L. iners or against Atopobium vaginae (currently classified as Fannyhessa vaginae). The potency and selectivity of these novel endolysins constitute a promising alternative treatment to combat BV, avoiding problems associated with antibiotic resistance, while retaining beneficial commensal bacteria in the vaginal flora. The diverse library of candidates reported here represents a strong repository of endolysins for further preclinical development.

In vitro bacterial vaginosis biofilm community manipulation using endolysin therapy

“…Recombinant endolysins candidates were produced as previously reported [ 12 ]. In short, pCCB7.1, a pET30a(+) vector harbouring the codon optimised ccb 7.1 gene sequence, was transformed into E. coli BL21(DE3).…”

“…Given this mechanism of action, these enzymes have good promise as therapeutics to be harnessed by clinicians. Previous studies have shown these enzymes are capable of lysing Gram-positive bacteria which lack an outer lipopolysaccharide membrane when added exogenously to culture media and more complex models of disease [ [12] , [13] , [14] ]. Interestingly, endolysins have also shown promise as antimicrobial agents against Gram-negative bacteria [ 15 ], despite their diderm cell wall structure.…”

“…[ 14 ], capable of eliminating mono-species G. vaginalis biofilms [ 17 ] and significantly reducing G. vaginalis load in dual-species biofilm models [ 18 ]. Likewise, recent screening of Gardnerella genome sequences has identified a library of endolysin candidates which prevent G. vaginalis biofilm formation and degrade pre-formed mono- and dual-species biofilms [ 12 ]. This library comprised 84 endolysin candidates which are diverse at the sequence and structural levels, with 3 different enzymatic active domains and 4 cell wall binding domains, with convergent bioactivities.…”

“…Notably, both F. vaginae and P. bivia have been reported to influence Gardnerella biofilm-related gene expression in vitro [ 20 ], with the presence and persistence of M. curtisii associated with BV recurrence [ 5 ]. This biofilm model was initially assessed against current front-line antibiotics (metronidazole, clindamycin) and subsequently against a Gardnerella targeted phage endolysin candidate - CCB7.1 [ 12 ]. We sought to characterise the efficacy of this protein against our more complex polymicrobial system, lending further credence to the clinical utilisation of endolysin proteins.…”

Supporting the strategic pillars of translational research in biofilms

“…One of these approaches is based on the use of phage components such as endolysins. In a study by Arroyo-Moreno et al 9 the authors show that endolysins can disrupt Gardnerella vaginalis both in single species and polymicrobial biofilms with no effect on microorganisms from the commensal vaginal flora nor the emergence of resistance, offering a promising strategy to combat bacterial vaginosis. An alternative approach to identify novel antimicrobials is to search from them in microbiomes as they are a great resource for bioactive molecules which has been very much unexploited.…”