Sortase-deficient lactobacilli: effect on immunomodulation and gut retention

Abstract

Surface proteins of probiotic microbes, including Lactobacillus acidophilus and Lactobacillus gasseri, are believed to promote retention in the gut and mediate host-bacterial communications. Sortase, an enzyme that covalently couples a subset of extracellular proteins containing an LPXTG motif to the cell surface, is of particular interest in characterizing bacterial adherence and communication with the mucosal immune system. A sortase gene, srtA, was identified in L. acidophilus NCFM (LBA1244) and L. gasseri ATCC 33323 (LGAS_0825). Additionally, eight and six intact sortase-dependent proteins were predicted in L. acidophilus and L. gasseri, respectively. Due to the role of sortase in coupling these proteins to the cell wall, DsrtA deletion mutants of L. acidophilus and L. gasseri were created using the upp-based counterselective gene replacement system. Inactivation of sortase did not cause significant alteration in growth or survival in simulated gastrointestinal juices. Meanwhile, both DsrtA mutants showed decreased adhesion to porcine mucin in vitro. Murine dendritic cells exposed to the DsrtA mutant of L. acidophilus or L. gasseri induced lower levels of pro-inflammatory cytokines TNF-a and IL-12, respectively, compared with the parent strains. In vivo co-colonization of the L. acidophilus DsrtA mutant and its parent strain in germ-free 129S6/SvEv mice resulted in a significant one-log reduction of the DsrtA mutant population. Additionally, a similar reduction of the DsrtA mutant was observed in the caecum. This study shows for the first time that sortase-dependent proteins contribute to gut retention of probiotic microbes in the gastrointestinal tract.

The S-layer Associated Serine Protease Homolog PrtX Impacts Cell Surface-Mediated Microbe-Host Interactions of Lactobacillus acidophilus NCFM

“…In vitro bacterial-DC co-incubation assays were performed as described previously (Johnson et al, 2013; Call et al, 2015). Cytokine measurements for IL-6, IL-10, and IL-12 were quantified using Single-Analyte ELISArray kits (Qiagen), according to the manufacturer’s instructions.…”

“…For L. acidophilus NCFM, the specificity of probiotic activity on the host has been characterized for numerous cell surface components, including lipoteichoic acid (Mohamadzadeh et al, 2011), sortase-dependent proteins (Call et al, 2015), an aggregation promoting factor (Goh and Klaenhammer, 2010), and a myosin-cross reactive protein (O’Flaherty and Klaenhammer, 2010b). Proteins at the Surface (S-) layer, which are non-covalently bound to the apical component of the cell wall peptidoglycan, are also of interest to the understanding of microbe-host interactions (Hynönen and Palva, 2013; Johnson and Klaenhammer, 2014).…”

Deletion of Lipoteichoic Acid Synthase Impacts Expression of Genes Encoding Cell Surface Proteins in Lactobacillus acidophilus

“…Many of these approaches have been extensively applied to investigate cell-surface constituents, as they are highly strain-specific and are uniquely positioned to effect host–microbe crosstalk (Buck et al, 2005; Kleerebezem et al, 2010). Predictive and functional genomics studies of L. acidophilus NCFM have revealed that surface layer (Slps) and surface layer associated proteins (SLAPs) (Buck et al, 2005; Johnson et al, 2013), sortase-dependent proteins (Call et al, 2015), and adhesion exoproteins mediate adhesion and immunomodulatory activities of L. acidophilus (Buck et al, 2005). L. acidophilus synthesizes three Slps, the dominant SlpA and minor components SlpB and SlpX (Goh et al, 2009).…”

AcmB Is an S-Layer-Associated β- N -Acetylglucosaminidase and Functional Autolysin in Lactobacillus acidophilus NCFM

“…Most notably, the probiotic activity of L. acidophilus is mediated by cell surface-associated components which interact with the host gastrointestinal mucosa and immune system (9–11). …”