Engineering Components of the Lactobacillus S-Layer for Biotherapeutic Applications

Abstract

Lactic acid bacteria (LAB) are frequently harnessed for the delivery of biomolecules to mucosal tissues. Several species of Lactobacillus are commonly employed for this task, of which a subset are known to possess surface-layers (S-layers). S-layers are two-dimensional crystalline arrays of repeating proteinaceous subunits that form the outermost coating of many prokaryotic cell envelopes. Their periodicity and abundance have made them a target for numerous biotechnological applications. In the following review, we examine the multi-faceted S-layer protein (Slp), and its use in both heterologous protein expression systems and mucosal vaccine delivery frameworks, through its diverse genetic components: the strong native promoter, capable of synthesizing as many as 500 Slp subunits per second; the signal peptide that stimulates robust secretion of recombinant proteins; and the structural domains, which can be harnessed for both cell surface display of foreign peptides or adhesion enhancement of a host bacterium. Although numerous studies have established vaccine platforms based on one or more components of the Lactobacillus S-layer, this area of research still remains largely in its infancy, thus this review is meant to not only highlight past works, but also advocate for the future usage of Slps in biotherapeutic research.

Keywords: CRISPR; Lactobacillus; S-layer; biotherapeutic; mucosal vaccine; probiotic.

FIGURE 1

Simplified depiction of the genetic components comprising the Slp protein and how they can be harnessed for heterologous protein expression systems. (A) The genetic building blocks of an S-layer gene, including the promoter, secretion signal peptide, N-terminal self-assembly domain, and C-terminal peptidoglycan anchor. Each can be harnessed together or separately for antigen and biotherapeutic delivery applications. (B) The Slp promotor induces the production of high levels of target protein which will accumulate within the cytoplasm of the host bacterium. (C) The Slp secretion signal yields robust secretion of antigen into the extracellular environment. (D) Cell surface display of a foreign peptide can be achieved via fusion to an Slp anchoring domain (N- or C- depending on species). (E) The highest levels of antigen expression and cell surface display can be obtained via direct integration into the Slp, but epitope size is extremely limited in order to maintain array formation. (F) A novel display strategy utilizing S-layer associated protein (SLAP) fusions which theoretically enable the surface display of much larger proteins.

FIGURE 2

Proposed strategies for CRISPR-based genome editing in relevant S-layer-forming lactobacilli. (A) Antigen integration into L. acidophilus SlpA via an exogenous Type II system employing a Cas9 nickase variant which introduces a guide RNA-targeted single-stranded break. Unlike wild-type Cas9 which generates blunt double-stranded breaks (DSB), nickases cut only one strand of the DNA, permitting genome editing in bacteria deficient in DSB repair (Chiang et al., 2016; Song et al., 2017). (B) Antigen integration into L. crispatus CbsA using the endogenous Type I system which consists of the CRISPR-associated complex for antiviral defense (Cascade) and the signature Cas3 nuclease (Barrangou, 2015).