Controlling the Growth of the Skin Commensal Staphylococcus epidermidis Using d -Alanine Auxotrophy

Abstract

Using live microbes as therapeutic candidates is a strategy that has gained traction across multiple therapeutic areas. In the skin, commensal microorganisms play a crucial role in maintaining skin barrier function, homeostasis, and cutaneous immunity. Alterations of the homeostatic skin microbiome are associated with a number of skin diseases. Here, we present the design of an engineered commensal organism, Staphylococcus epidermidis, for use as a live biotherapeutic product (LBP) candidate for skin diseases. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. We therefore constructed an auxotrophic strain of S. epidermidis that requires exogenously supplied d-alanine. The S. epidermidis NRRL B-4268 Δalr1 Δalr2 Δdat strain (SEΔΔΔ) contains deletions of three biosynthetic genes: two alanine racemase genes, alr1 and alr2 (SE1674 and SE1079), and the d-alanine aminotransferase gene, dat (SE1423). These three deletions restricted growth in d-alanine-deficient medium, pooled human blood, and skin. In the presence of d-alanine, SEΔΔΔ colonized and increased expression of human β-defensin 2 in cultured human skin models in vitro. SEΔΔΔ showed a low propensity to revert to d-alanine prototrophy and did not form biofilms on plastic in vitro. These studies support the potential safety and utility of SEΔΔΔ as a live biotherapeutic strain whose growth can be controlled by d-alanine. IMPORTANCE The skin microbiome is rich in opportunities for novel therapeutics for skin diseases, and synthetic biology offers the advantage of providing novel functionality or therapeutic benefit to live biotherapeutic products. The development of novel bacterial strains whose growth can be controlled without the use of antibiotics or genetic elements conferring antibiotic resistance enables modulation of therapeutic exposure and improves safety. This study presents the design and in vitro evidence of a skin commensal whose growth can be controlled through d-alanine. The basis of this strain will support future clinical studies of this strain in humans.

Engineering a “detect and destroy” skin probiotic to combat methicillin-resistant Staphylococcus aureus

“…Finally, an important consideration for genetically engineered microbial therapeutics are methods of biocontainment, preventing the transmission of the heterologous organism from the target site or from person to person, e.g., auxotrophy, foreign nucleotides and amino acid usage, and kill switches have been proposed or utilized [ 46 , 47 ]. While understanding the underlying biology of colonization of exogenous strains will be a critical next step, we hope that this work provides an example of a potential initial design of a “detect and destroy” probiotic approach for mitigation of S .…”

Development of synthetic biotics as treatment for human diseases

“…Several classes of effectors have been engineered into bacteria, including enzyme pathways that metabolize disease-causing toxins ( 9 ), production of protein effectors ( 10 ), and production of small-molecule effectors ( 11 ). Synthetic biotics have been engineered for application to the skin ( 12 ) and for intra-tumoral injection in oncology ( 11 , 13 ), but the area of most active research is the development of synthetic biotics designed to work from the gut lumen ( 10 , 14 ), as shown in Table 1 . Due to the active cross-talk between the gastrointestinal (GI) tract, metabolic pathways, the nervous system and the immune system, oral synthetic biotic medicines can be designed to treat GI tract–specific as well as systemic diseases.…”

Disorders of the skin microbiome in atopic dermatitis and psoriasis

“…Комменсальные микроорганизмы играют важную роль в поддержании работы иммунной системы кожи, целостности эпидермального барьера и гомеостаза микробного сообщества [20]. Для сохранения оптимальных условий локального микроокружения и доступа к питательным веществам резидентные микроорганизмы должны ограничивать колонизацию кожи патогенной флорой.…”

“…[3]. Коагулаз-негативные стафилококки являются гетерогенной группой микроорганизмов, включающей 38 видов бактерий, отличительной особенностью которых является отсутствие энзима коагулазы [20,22]. Одним из наиболее изученных представителей данной группы является S. epidermidis.…”

“…Микроорганизм может быть изолирован из различных анатомических зон, включая «сухие», «влажные», «себорейные» зоны, а также кожу стоп [3,22]. Колонизация кожи S. epidermidis происходит в младенческом возрасте, что позволяет микроорганизму оказывать влияние на развитие иммунной системы кожи новорожденного [20].…”