Invited review: Application of omics tools to understanding probiotic functionality

J. Dairy Sci. 94:4753–4765 doi:10.3168/jds.2011-4384 © American Dairy Science Association®, 2011.

Invited review: Application of omics tools to understanding probiotic functionality J. L. Baugher and T. R. Klaenhammer1 Department of Food, Bioprocessing, and Nutrition Sciences, North Carolina State University, Raleigh 27695

ABSTRACT

The human gut microbiota comprises autochthonous species that colonize and reside at high levels permanently and allochthonous species that originate from another source and are transient residents of the human gut. The interactions between bacteria and the human host can be classified as a continuum from symbiosis and commensalism (mutualism) to pathogenesis. Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit on the host. Recent advances in omics tools and sequencing techniques have furthered our understanding of probiotic functionality and the specific interactions between probiotics and their human hosts. Although it is known that not all probiotics use the same mechanisms to confer benefits on hosts, some specific mechanisms of action have been revealed through omic investigations. These include competitive exclusion, bacteriocin-mediated protection against intestinal pathogens, intimate interactions with mucin and the intestinal epithelium, and modulation of the immune system. The ability to examine fully sequenced and annotated genomes has greatly accelerated the application of genetic approaches to elucidate many important functional roles of probiotic microbes. Key words: probiotics, omics, function, mechanism INTRODUCTION

Humans and their collective microbiomes have been termed “superorganisms” because of their close symbiotic relationships (Gill et al., 2006). The human microbiome contains an estimated 100 trillion microbes, 10 times as many cells as the human body, and may contain up to 100-fold more unique genes than the human complement (Ley et al., 2006). With the vast majority of the bacteria residing within the gastrointestinal tract, the composition of the human gut microbiota includes autochthonous species that colonize and reside Received March 22, 2011. Accepted June 2, 2011. 1 Corresponding author: [email protected]

at high levels permanently; normal species that can vary greatly in number and may be sporadically absent; pathogenic species that are periodically acquired from the environment; and allochthonous species that originate from another source and are transient residents of the human gut (Backhed et al., 2005). The interactions between bacteria and the human host involve a diverse microbiota that occupy varied environments and represent a continuum from symbiosis and commensalism (mutualism) to pathogenesis. Among the variable microbial components of the human gut microbiota are health-promoting, mucosaadherent species. Probiotics are “live microorganisms, which when administered in adequate amounts confer a health benefit on the host” (FAO/WHO, 2001). Because of their health benefits, probiotic bacteria are now a vital component in the multi-billion dollar dairy and functional foods industry. Probiotic bacteria can be added as commercial probiotic starter cultures or selected in naturally fermented foods; the best studied and most widely used commercial probiotic species belong to the genera Bifidobacterium and Lactobacillus (Felis and Dellagio, 2007; Kleerebezem and Vaughan, 2009). Although not all probiotic cultures confer identical benefits on hosts, the potential mechanisms of action include competitive exclusion, maintenance of barrier function, metabolic and antimicrobial effects, enhancement of a balanced microbial flora, modulation of signal transduction, and immunomodulation of innate or adaptive immunity (Sherman et al., 2009). Specific roles and benefits of probiotics in the gastrointestinal tract (GIT; Table 1) include protection against infection (Corr et al., 2007), lowered cold and influenza-like symptoms in children (Leyer et al., 2009), lowering of blood cholesterol levels (Ataie-Jafari et al., 2009), and suppression of allergic asthma (Aumeunier et al., 2010). Despite strong evidence for the functional claims of probiotics, poor characterization of the specific molecular mechanisms by which these probiotic microbes elicit health benefits underlies the skepticism of the validity of these findings in the biomedical community. Recent advances in omics and integrated functional genomic analyses involving transcriptomics, proteomics, secretomics, metabolomics, and interactomics have ac-

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Table 1. Role and benefits of probiotics in the gastrointestinal tract1 Benefits of probiotic bacteria

Reference

Protection against infection Lowered incidence of diarrhea Lowered levels of cold and influenza-like symptom in children and reduction of missed school days Antimicrobial activity Competitive exclusion of pathogens Immune tolerance Suppression of allergic asthma and autoimmune diabetes Reduction in colorectal cancer biomarkers Return to pre-antibiotic baseline flora Epithelial barrier function Increased cellular immunity (e.g., increased natural killer cell activity) Increased humoral response (e.g., IgA secretion) Lowering of blood cholesterol levels Reduction in irritable bowel disease symptoms Suppression of inflammatory autoimmune disorders Delivery of therapeutics

Corr et al., 2007 Lonnermark et al., 2010 Leyer et al., 2009 Ryan et al., 2009 Lee et al., 2003 van Baarlen et al., 2009 Aumeunier et al., 2010 Rafter et al., 2007 Engelbrektson et al., 2009 Mennigen et al., 2009 Takeda and Okumura, 2007 Viljanen et al., 2005 Ataie-Jafari et al., 2009 Macfarlane et al., 2009 Kwon et al., 2010 Wells and Mercenier, 2008

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Modified from O’Flaherty and Klaenhammer (2010).

celerated the research into deciphering specific mechanisms for commensal and probiotic functionality within the GIT. Since the first bacterial genome (Haemophilus influenzae) was completely sequenced in 1995, sequencing technology has experienced an exponential increase in processing speed, at significantly lowered costs, yielding more than 1,300 completed bacterial genome sequences (National Center for Biotechnology Information; http://www.ncbi.nlm.nih.gov/). With increasing interest in the benefits and the applications of lactic acid bacteria and probiotic microbes, the Lactic Acid Bacteria Genome Consortium (LABGC) and numerous food and pharmaceutical companies have sequenced a broad range of health-promoting and industrially relevant strains (Liu et al., 2005). The first probiotic microbe to be sequenced was Bifidobacterium longum NCC2705 (Schell et al., 2002), followed shortly by a procession of important lactic acid bacterial strains, including Lactobacillus plantarum WCSF1 (Kleerebezem et al., 2003), Streptococcus thermophilus CNRZ1066 (Bolotin et al., 2004), Lactobacillus acidophilus NCFM (Altermann et al., 2005), Lactobacillus casei ATCC334 (Makarova et al., 2006), Lactobacillus gasseri ATCC33323 (Makarova et al., 2006), Lactobacillus salivarius UCC118 (Claesson et al., 2006), and Lactobacillus rhamnosus GG (Kankainen et al., 2009). The availability of rapid, cost-effective sequencing technology has fostered a new area, termed “probiogenomics,” which has significantly expanded our knowledge about the evolution of commensal and probiotic bacteria and their genetic diversity, and in some cases has elucidated the molecular basis for health-promoting functions. Understanding intimate microbe–microbe and host–microbe interactions within the GIT has been facilitated by the integration of probiogenomics and functional genomic analyses of human gene expression within the gut (Ventura et al., 2009). These new omics technologies allow simultaneJournal of Dairy Science Vol. 94 No. 10, 2011

ous analysis of great numbers of genes and proteins from both host and microbe (Joyce et al., 2006). The focus of this review is to highlight landmark studies that have applied integrated omics tools to further the understanding of probiotic functionality and the specific interactions between probiotics and their human hosts. Metagenomic Sequencing of Gut Microbiota

Fecal samples of 124 individuals of European (Nordic and Mediterranean) origin were collected to establish a catalog of nonredundant human intestinal microbial genes (Qin et al., 2010). Utilizing Illumina (San Diego, CA; formerly Solexa) genome analyzer short-read, metagenomic-based sequencing, the gene set was found to be 150 times larger than the human gene complement. With over 99% of the genes in the set being bacterial, the cohort of the study included between 1,000 and 1,150 prevalent bacterial species, with each individual containing at least 160 of such bacterial species. By developing an extensive nonredundant catalog of the bacterial genes, vital functions for a bacterium to thrive in a gut context were identified (“minimal gut genome”). Within the minimal gut genome, 2 major gene types were identified: housekeeping genes, required for carbon metabolism, amino acid synthesis, and protein complexes, and genes specific to life within the GIT (Qin et al., 2010). The most commonly shared gut-specific functions of members of the microbiome community were functions promoting interactions with the host epithelium such as adherence to collagen, fibrinogen, and fibronectin, and sugar metabolism (Qin et al., 2010). Interestingly, within the fecal samples of the cohort, the sequences for the lactic acid bacterium Streptococcus thermophilus were among the 57 most frequent

INVITED REVIEW: OMICS TOOLS AND PROBIOTIC FUNCTIONALITY

species found in the human microbiome. Streptococcus thermophilus, used in yogurt and cheese manufacturing, is considered the second most widely used starter culture in the manufacturing of dairy products (Hols et al., 2005). Beneficial functions performed by Strep. thermophilus include efficiently breaking down lactose and activating host CD4+ and CD8+ lymphocytes that stimulate IFN-γ production in tissue-cultured cells (Aattouri and Lemonnier, 1997). Thermophilin 9, a class II bacteriocin produced by Strep. thermophilus LMD-9, displays an inhibitory spectrum that is effective against related gram-positive bacteria, including pathogens such as Listeria monocytogenes (Fontaine and Hols, 2008). A significant difference has been reported between the microbiota of patients with inflammatory bowel disease (IBD) and healthy individuals (Manichanh et al., 2006). The link between individuals’ health status (healthy, Crohn disease, and ulcerative colitis) and species abundance was tested, using an Illumina-based bacterial profiling. A clear differentiation between patients of each health status confirmed the hypothesis that a significant difference existed in the microbiota of IBD patients and healthy individuals. A major Firmicute representative of the gut microbiota, Fecalibacterium prausnitzii (member of the Clostridium leptum group) was found in abundance in each individual of the cohort who did not have IBD symptoms (Qin et al., 2010). The presence of F. prausnitzii could be crucial to the homeostasis of the gut because a species reduction was consistently associated with increased susceptibility of the gut mucosa in patients suffering from IBD and infectious colitis (Sokol et al., 2009). Orally administering either F. prausnitzii cells or supernatant reduced the severity of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in mice and stabilized the overall dysbiosis of the gut. Secreted metabolites found in the supernatant effectively blocked nuclear factor-κB activation and other inflammatory cytokines (Sokol et al., 2008). The following are specific examples of omics applications that have revealed important mechanisms underlying activities elicited by probiotic microbes, allowing for the validation and enhancement of conferred host benefits. Antimicrobial Activity

Probiotic cultures have long been considered to exert protective effects against pathogens via direct antagonism or competitive exclusion. Lactobacillus salivarius has been isolated from the intestinal mucosa of 9% of all humans sampled (Molin et al., 1993). Lactobacil-

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lus salivarius UCC118 was originally isolated from a sample of the terminal ileum (distal part of the small intestine) of a healthy participant. Studied for its probiotic benefits in humans and animal models (Dunne et al., 2001; Sheil et al., 2004), Lb. salivarius UCC118 has been shown to alleviate gastrointestinal infections such those caused by Citrobacter rodentium, Salmonella typhimurium and Helicobacter pylori, (Johnson-Henry et al., 2005; O’Hara et al., 2006; Ryan et al., 2008). Despite the health benefits of Lb. salivarius UCC118 being well established, the precise mechanisms by which this microbe confers these benefits to its host remain largely unresolved (Corr et al., 2007). The genomic sequence of Lb. salivarius UCC118 revealed a 1.83-Mb chromosome, a 242-kb megaplasmid (pMP118), and 2 smaller plasmids. The 242-kb megaplasmid is the largest plasmid extracted from a lactobacillus, but no genes encoded on this plasmid were deemed essential for the viability of the bacterium. The megaplasmid did encode a lactate dehydrogenase for d-lactate (cell wall precursor), a bifunctional acetaldehyde/alcohol dehydrogenase (only enzyme present to catalzye ethanol from acetyl-CoA), a choloylglycine hydrolase (bile-salt hydrolase), and a 2-component class IIb bacteriocin, Abp118 (Flynn et al., 2002; Claesson et al., 2006). The bacteriocin Abp-118 is small, heat-stable, class IIb bacteriocin, whose activity depends on the complementary activity of both peptides (Klaenhammer, 1993; Flynn et al., 2002). Genetic characterization of the 2-component bacteriocin found that Abp-118 is composed of Abp118α, which shows antimicrobial activity against Lis. monocytogenes, and Abp118β, which enhances that activity (Flynn et al., 2002). In a landmark study, the ability of Lb. salivarius UCC118, fed orally, to protect mice from infection by Lis. monocytogenes was evaluated. A 3-d pretreatment of mice with Lb. salivarius UCC118 conferred significant protection against listerial infection of the liver and spleen. A luciferase-based reporter system was integrated into the chromosome of Lis. monocytogenes EGDe, and luciferase expression was used to follow organ-specific colonization of the pathogen in murine liver and spleen (Corr et al., 2007) To investigate if the bacteriocin Abp-118 was the primary mediator of protection, a stable mutant of Lb. salivarius UCC118 unable to produce the bacteriocin was created. The mutant failed to provide protection to the mice when challenged with Lis. monocytogenes strains L028 and EGDe (Figure 1). This confirmed that protection against listerial infection was the result of bacteriocin production that directly antagonized Lis. monocytogenes in vivo (Corr et al., 2007). Journal of Dairy Science Vol. 94 No. 10, 2011

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Figure 1. The bacteriocin Abp-118 of Lactobacillus salivarius UCC118 was shown to be the primary mediator of protection to mice challenged with Listeria monocytogenes strains L028 and EGDe by evaluating the protective effects of placebo, UCC118, and UCC118 (Bac−). Letters above bars indicate statistically significant differences between all treatments. Reprinted with permission from Corr et al. (2007). Copyright © 2007 National Academy of Sciences, U.S.A.

Cell Surface Adherence Factors

Probiotic bacterial surface proteins have been implicated in the adherence to gastrointestinal epithelial cells (Granato et al., 1999), mucin (Rojas et al., 2002), and extracellular matrix (ECM) proteins such as fibrinogen, fibronectin, and fetuin (Styriak et al., 2003). Functional analyses of surface (S) layers have not yet definitively determined their significance in adherence and retention of probiotics within the GIT. Although the genetic mechanisms of adherence of enteropathogens have been well characterized, the genetic mechanisms underlying the adherence of probiotic microbes remain to be elucidated (Klaenhammer et al., 2005). Bacterial adherence structures comprise proteins or polysaccharides on the cell surface. Mucin-binding proteins (Mub) selectively adhere to the intestinal mucin glycoprotein. Originally isolated from an infant fecal sample in 1900 (Klaenhammer and Russell, 2000), Lactobacillus acidophilus is a probiotic strain that is used extensively throughout the food industry in dietary supplements and cultured yogurt products (Sanders and Journal of Dairy Science Vol. 94 No. 10, 2011

Klaenhammer, 2001). Genomic analysis of Lb. acidophilus NCFM revealed 13 putative proteins that contain one or more mucin-binding domains, 3 of which contain the necessary components for a complete Mub protein: a signal peptide sequence and a LPxTG cell surface anchor (Klaenhammer, 2010). Encoded by LBA1392, a 4,326-residue Mub protein is the largest encoded protein within the Lb. acidophilus NCFM genome (Altermann et al., 2005). A conserved putative fibronectin (dimeric ECM glycoprotein)-binding protein (Fbp) has been found in all Lactobacillus genomes (Goh and Klaenhammer, 2009) and Lb. acidophilus NCFM was shown to contain FbpA (Altermann et al., 2005). Three putative S-layer proteins (Slp), proteinaceous subunits that abundantly coat cell surfaces of many eubacteria and archaea, were identified and characterized in the Lb. acidophilus NCFM genome (SlpA, SlpB, and SlpX; Goh et al., 2009). To determine the importance of genes that are potentially vital for NCFM adherence to the human GIT, predicted adherence factors were individually inactivated to determine the effect of single gene products on NCFM adherence. Insertionally inactivated mutants included FbpA, a mucin-binding protein (encoded by LBA1392), and the dominant surface layer protein (SlpA) (Buck et al., 2005). Each knockout mutant was tested for its abilities to adhere in vitro to Caco-2 intestinal epithelial cells (Figure 2). A significant reduction in adherence was observed in the Fbp mutant (76%), mucin-binding protein mutant (65%), and in the Slp mutant (84%). Functional genomic studies of the Lb. acidophilus NCFM adherence factors demonstrated that the organism’s ability to adhere to intestinal epithelial cells can be attributed to multiple cell surface structures, and no single protein is likely responsible for the adherence properties of the cell (Buck et al., 2005; Klaenhammer et al., 2005). Highly homologous surface proteins and adherence factors are found throughout Lactobacillus and other probiotic species. Lactobacillus reuteri 100-23, a persistent resident of the nonsecretory epithelium of the forestomach and gut in mice, encodes an approximately 185-kDa large surface protein (Lsp) vital for adherence to the host epithelium (Walter et al., 2005). Mutational phenotypic analysis showed that Lsp has a vital role in initiating adherence to the epithelium. Sequence analysis of Lsp revealed a high degree of similarity to Lactobacillus johnsonii NCC 533 and Lactobacillus gasseri ATCC 33323 adherence proteins, as well as sequence similarity to other gram-positive genes associated with epithelial adherence and biofilm production (Walter et al., 2005). Genomic sequencing of Lactobacillus rhamnosus GG revealed a pilus as a major cell surface component

INVITED REVIEW: OMICS TOOLS AND PROBIOTIC FUNCTIONALITY

Figure 2. Percentages of adhesion properties of Lactobacillus acidophilus NCFM and insertionally inactivated mutants of fibronectinbinding protein A (FbpA), mucin-binding protein (Mub), and surface layer protein A (SlpA), enumerated microscopically. Error bars represent 1 SD. Reprinted with permission from Buck et al. (2005). Copyright © American Society for Microbiology, Applied Environmental Microbiology (2005) 71:8344–8351.

potentially involved in mucin adherence. Originally isolated from a healthy human intestinal sample, Lb. rhamnosus GG has been extensively studied and used as a probiotic strain in a variety of functional foods (Bernardeau et al., 2006; Guarino et al., 2009). When incubated together, Lb. rhamnosus and Lactobacillus casei Shirota have been shown to competitively exclude and displace 8 strains of Escherichia coli and Salmonella spp. from adhering to human intestinal mucus glycoproteins and Caco-2 cells (Lee et al., 2003). Pretreatment with probiotic Lb. rhamnosus GG results in a significant reduction in the ability of Escherichia coli O157:H7 (enterohemorrhagic E. coli) to change cell morphology, lower electrical resistance, increase dextran permeability, and manipulate the distribution and expression of tight junction proteins claudin-1 and ZO-1 of the mucosal epithelial cell barrier that makes up the GIT (Johnson-Henry et al., 2008). Several studies have compared the adhesion properties of Lb. rhamnosus GG (a probiotic microbe) and Lb. rhamnosus LC705 (an adjunct starter culture in dairy products; Suomalainen and Mayra-Makinen, 1999) and concluded that Lb. rhamnosus GG was more adherent to human cells than Lb. rhamnosus LC705 (Jacobsen et al., 1999; Tuomola et al., 2000). To investigate the molecular mechanisms possibly involved in adherence to intestinal cells and mucin, the complete sequences of Lb. rhamnosus GG and Lb. rhamnosus LC705 were

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compared. Within the Lb. rhamnosus GG genome, genes encoding 2 different pilus fibers (spaCBA) and (spaFED) were identified, whereas the spaCBA operon was not found in Lb. rhamnosus LC705. The genes for spaCBA were found on an island of genes that secrete 3 LPxTG-like pilins and a pilin-dedicated sortase (Kankainen et al., 2009). The pilus was predicted to be a heterotrimer composed of the major pilin or the pilus backbone, the minor pilin that covers the pilus backbone, and another minor pilin that contains adherence properties (Mandlik et al., 2008; Proft and Baker, 2009). Using immunoblotting with anti-SpaC antibodies, the physical presence of the pili were confirmed (Figure 3). Immunogold electron micrograph shows an average of 10 to 50 pili per cell located predominantly at the poles, with a pilus length of up to 1 μm. Pilin subunit SpaC was located primarily at the tip of the pilus, as well as sporadically throughout the pilus structure. When Lb. rhamnosus GG cells were pretreated with antiserum to SpaC, a 10-fold reduction in the overall adherence of the cells to mucus was observed.. An insertional mutant of Lb. rhamnosus GG (ΩspaC) further confirmed that expression of the SpaC protein was required for mucus binding by pili (Kankainen et al., 2009). Interestingly, the Spa-encoded proteins showed considerable similarity to pili from Enterococcus faecium, also a commensal resident of the GIT that binds mucin (Kankainen et al., 2009). The mucin-binding predisposition of Lb. rhamnosus GG pilus fibers provided support for a probiotic mechanism of competitive exclusion and the potential for steric hindrance of adherence to the mucin that lines the epithelial cells of the host GIT (Chan et al., 1985; Reid et al., 1985; Isolauri et al., 2002; Lee et al., 2003). Stimulation of the Host Immune System

It is now well known that probiotic and commensal microbes can modulate host immune responses. However, the specific mechanisms by which probiotic and commensal bacteria modulate the immune responses of human epithelial cells has not been defined. Dendritic cells (DC), professional antigen-presenting cells, line the epithelium of the human GIT and regulate the host immune system in response to mucosally encountered antigens of the microbiota and viruses. Dendritic cells are responsible for recognizing exogenous and endogenous stimuli and responding to those signals by eliciting the correct innate and adaptive response or tolerance (Smits et al., 2005; van Vliet et al., 2007). Immature DC migrate from the bloodstream to specific compartments within the GIT to directly interact with structures of pathogenic and nonpathogenic bacteria Journal of Dairy Science Vol. 94 No. 10, 2011

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Figure 3. Transmission electron microscopy showing negatively stained immunogold-labeled anti-SpaC pili subunits of Lactobacillus rhamnosus GG. Reprinted with permission from Kankainen et al. (2009). Copyright © 2009 National Academy of Sciences, U.S.A.

and viruses that protrude through the mucosal barrier (Smits et al., 2005). Dendritic cells mature phenotypically and functionally in response to the external stimuli of the gastrointestinal environment by upregulating expression of a pattern recognition receptor on the cell surface, producing inflammatory chemokines and cytokines, and differentiating naive helper T cells into Th1 and Th2 cells (Mohamadzadeh et al., 2005; Kabelitz and Medzhitov, 2007). Toll-like receptors (TLR) and C-type lectins (CLR) are examples of pattern recognition receptors that recognize specific microbial molecular patterns of interactJournal of Dairy Science Vol. 94 No. 10, 2011

ing compounds such as carbohydrates, lipids, nucleic acids, and proteins (Weis et al., 1998). Toll-like receptors communicate information from interacting microbial compounds to DC through signaling cascades, eliciting cellular processes such as DC maturation and inducing proinflammatory cytokines (e.g., IL-12, IFNγ; Underhill and Ozinsky, 2002). Unlike TLR, CLR are formed without the induction of DC maturation and bind to mannose- and fucose-containing glycan ligands in a calcium-dependent manner (Engering et al., 2002). Strains of Lactobacillus plantarum contain carbohydrate-protein surface structures such as mannosespecific adhesins that have been shown to bind to human colonic cells (Adlerberth et al., 1996). Candidate mannose-specific adhesion genes of Lb. plantarum WCFS1 were investigated by gene-specific deletion and overexpression systems. The sortase-dependent cell surface protein Msa (mannose-specific adhesion) was identified as having carbohydrate-binding domains and is likely involved in the interaction of Lb. plantarum with the host GIT (Pretzer et al., 2005). Studying the adherence of Lb. acidophilus NCFM cell surface components to DC using a functional genomic approach revealed that the interaction between the receptor and ligand induced concentration-dependent production of IL-10 and low IL-12p70. Receptor–ligand interactions were studied to determine the specific molecular mechanisms that influence cytokine production and sequestering of differentiated helper T cells. Using an ELISA, the bacterium was shown to bind to a DCspecific ICAM-3-grabbing nonintegrin (DC-SIGN), a CLR that recognizes mannose- and fructose-containing glycans on microbial and viral surfaces. A knockout mutant in NCFM of the major surface layer A protein (SlpA) showed a significant reduction in DC-SIGN binding (Konstantinov et al., 2008). The SlpA mutant incurred a chromosomal inversion causing the slpB gene (the normally silent gene located downstream and in the opposite orientation of the slpA gene) to invert directly behind the S-promoter and begin expression of SlpB protein (Boot et al., 1996). Binding of the SlpB-dominant strain (the SlpA knockout) with DC elicited a different response from the SlpA-dominant parent: increased production of proinflammatory cytokines IL-12p70, tumor necrosis factor-α (TNFα), and IL-1β. The parent NCFM strain was shown to stimulate T cells to produce more IL-4 than the SlpA-knockout mutant. Direct ligation of purified SlpA protein and DC-SIGN confirmed that the SlpA of Lb. acidophilus NCFM was the first probiotic DC-SIGN ligand identified and that it is directly involved in the immunomodulation of DC and helper T cells (Konstantinov et al., 2008). Probiotic stimulation of intestinal epithelium to produce cytokine TNFα has

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been shown to promote gut health by restoring epithelial barrier functions in vivo (Pagnini et al., 2010). Lactobacillus plantarum is commonly found on plants and is widely used in vegetable fermentations. With the entire genome sequenced, Lb. plantarum WCFS1 is accessible to genetic modifications and allows for specific bacterial compounds to be studied for possible interactions with the host immune system (Kleerebezem et al., 2003). Components of the cell wall of gram-positive bacteria, such as teichoic acids (TA) and especially lipoteichoic acids (LTA), have been shown to induce the production of cytokines within host cells (Morath et al., 2001). Lipoteichoic acids have been shown to stimulate TNFα in human macrophages and have been proposed to function in the adhesion of lactobacilli to epithelial cells (Sherman and Savage, 1986; Granato et al., 1999). Utilizing comparative genomic hybridization of Lb. plantarum strains, specific gene loci within Lb. plantarum WCFS1 were identified as candidate genes for the modulation of the host immune system. Subsequent gene deletions of the candidate genes confirmed that 3 bacteriocin and 1 transcriptional regulator loci have an immunomodulatory effect on the DC (Meijerink et al., 2010). To further elucidate the bioactive components of the probiotic cell surface that have immunomodulatory functions, the importance of the specific TA composition and the importance of LTA in probiotic functionality were studied in Lb. plantarum WCFS1 (Grangette et al., 2005). Using a suicide knockout vector, a single-step homologous recombination that disrupted the dlt operon (genes responsible for the d-alanylation of TA) was performed to produce a mutant strain with LTA deficient in d-alanine (Dlt− mutant). After peripheral blood mononuclear cells and monocytes were exposed to the Dlt− mutant and the parent strain, comparisons of the cytokine profiles showed that the Dlt− mutant caused a significant reduction in the secretion of proinflammatory cytokine IL-12 and greatly induced the secretion of the antiinflammatory cytokine IL-10 (Grangette et al., 2005). The proinflammatory ability of Lb. plantarum LTA was determined to be dependent on TLR2 (Grangette et al., 2005); TLR2 has been shown to stimulate the secretion of IL-12 in response to interactions with double-stranded RNA and a potential viral attack (Weiss et al., 2010). This functional genomics study showed that the specific changes to the composition of Lb. plantarum LTA could vary cytokine secretion and modulate the overall immune response (Grangette et al., 2005). To study the involvement of LTA in the induction and repression of intestinal inflammation, the phosphoglycerol transferase (key enzyme in LTA biosynthesis) of Lb. acidophilus NCFM was deleted. When exposed to

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the LTA-negative cells (NCK 2025), DC of mice showed a reduction in the release of cytokines IL-12 and TNFα, an increase in IL-10 secretion, and an overall reduction in immune response within the cells. The overall reduction in immune response was evident by the inability of DC to sequester and activate CD4+ T cells (Mohamadzadeh et al., 2011). When comparing the efficacy of strains NCK 2025 and NCFM to alleviate dextran sulfate sodium (DSS)-induced and pathogenic CD4+CD45RBhigh T cell-induced colitis, NCK 2025 was shown to significantly reduce the severity of colitis and effectively alleviated DSS-induced colitis via elicited IL-10 and CD4+FoxP3+ regulatory T cells to lessen the exacerbated inflammation of the epithelium. Intestinal samples collected from the experimental mice showed differences in the amelioration of DSS-induced colitis in the control (DSS-treated mice), NCFM-DSS–treated, and NCK2025-DSS–treated mice (Figure 4). These studies established that directed alterations and engineering of probiotic cell surface structures is a potential strategy for therapeutic treatment of colitis and other inflammatory gastrointestinal disorders (Grangette et al., 2005; Mohamadzadeh et al., 2011). Influence of Dairy Environment on Gene Expression

Despite the need for scientific understanding to provide regulatory substantiation of the benefits of probiotics used in foods, the effects of the food matrix and product formulation on the viability and functionality of probiotic microbes are relatively unknown (Sanders and Marco, 2010). To investigate the differences in gene expression and probiotic attributes of Lb. acidophilus NCFM when propagated in milk (the most common platform of probiotic delivery), whole-genome microarrays of the temporal gene expression of NCFM cells were performed. The Lb. acidophilus NCFM cells were propagated in 11% milk during the early, mid, and late logarithmic phases and the stationary phase to determine the potential effect of a dairy environment on probiotics (Azcarate-Peril et al., 2009). After analyzing the differences in gene expression during growth in milk, 21% of the 1,864 open reading frames were differentially expressed in at least one time point. The expression of genes involved in carbohydrate utilization were rapidly induced in early stages of growth and decreased over time, as the main carbohydrate sources became depleted. The decreased expression of carbohydrate utilization genes tended to coincide with increased expression of key components of carbon catabolite repression systems, which control the transcription of genes involved in the transport and catabolism of carbohydrates. Expression patterns of proteolytic genes increased consistently over time, Journal of Dairy Science Vol. 94 No. 10, 2011

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Figure 4. Intestinal samples collected from the experimental mice show the differences in the amelioration of dextran sulfate sodium (DSS)-induced colitis in the (B) control (DSS-treated mice); (C) Lactobacillus acidophilus NCFM (NCK56) DSS–treated mice, and D) Lb. acidophilus NCFM LTA-negative (NCK2025) DSS–treated mice. Panel A (from original; not shown here) showed the treatment schedule of the experimental mice and graphs of the percentage body weight change, fecal hemoccult blood positivity, disease activity index, and diarrhea score of each treatment throughout the experiment. Color version available in the online PDF. Reprinted with permission from Mohamadzadeh et al. (2011). Journal of Dairy Science Vol. 94 No. 10, 2011

whereas the 2 oligopeptide transporter genes, opp1 and opp2, showed 2 different maximum expression levels, one in the early logarithmic phase and the other in the stationary phase, respectively (Azcarate-Peril et al., 2009). Lactobacillus acidophilus NCFM showed temporal expression patterns peaking at 12 h (early stationary phase) for 16 peptidases. Peptidases such as PepF, which are able to hydrolyze large peptides, were shown to have high early expression, followed by decreased expression as other peptidases that hydrolyze smaller peptides began to increase in expression levels. Expression of stress-related (LacL) and quorum-sensing (luxS) genes were shown to increase as growth progressed, the number of cells increased, and lactic acid accumulated. Using NCFM mutants that contained insertionally inactivated genes, adherence assays were able to determine the possible importance of 2 NCFM genes (LBA1690 and LBA1524HPK) to promote mucosal and cell adherence during NCFM growth in milk.. One gene that was overexpressed during growth in milk was an aggregation-promoting factor (Apf; AzcaratePeril et al., 2009). Sequence analysis of Lb. acidophilus NCFM identified LBA0493 as a 696-bp apf gene that encodes a putative 21-kDa Apf protein (Altermann et al., 2005). Transcriptional studies determined the apf gene to be one of the most differentially upregulated (5 times the normalized value) genes in milk (AzcaratePeril et al., 2009). Aggregation-promoting factors and the genes that encode them have been characterized for several Lactobacillus species; some of these proteins have been shown to be major aggregation factors, whereas others are not directly involved in aggregation of bacteria. The Apf have been reported to influence coaggregation with specific pathogens (Schachtsiek et al., 2004), enhance conjugation efficiency (Reniero et al., 1992), and maintain cell shape similarly to S-layer proteins (Jankovic et al., 2003). Reverse transcription-quantitative PCR analysis was used to determine that the apf gene was most highly induced during the stationary phase. Mutational analysis of the apf gene was used to study the functional roles of Apf in Lb. acidophilus NCFM (Goh and Klaenhammer, 2010). An Δapf mutant was constructed using an uppbased counterselective gene replacement system, utilizing the upp-encoded uracil phosphoribosyltransferase (UPRTase) as a counterselection marker to positively select for single crossover plasmid integrants without the use of antibiotics for selective pressure (Goh et al., 2009). A comparison of the cell morphologies of Lb. acidophilus NCFM with and without the apf gene showed no detectable differences. The Δapf mutant also showed no effect on the sedimentation and autoaggregation of NCFM. However, inactivation of the apf locus increased susceptibility of NCFM to 2.5% oxgall

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(bile salt reduced survival), and 0.02% SDS (reduced survival and altered cell morphology). The Δapf mutant was shown to be more susceptible to simulated small-intestinal juice (4 log reduction after 3-h incubation) and to simulated gastric juice (1 log reduction after 1-h incubation). The adherence ability of Δapf mutant was examined on Caco-2 epithelial cells, mucin, and the ECM components collagen IV, laminin, and fibronectin. A reduction in adherence was observed for Caco-2 cells (30%), mucin (36.5%), and fibronectin (40%). Although the study did not show Apf to be an important factor in cell aggregation, the apf gene was demonstrated to be highly expressed in the dairy environment and to play an important role in promoting bile tolerance, survival in the GIT, and interactions with the mucus layer, epithelial cells, and fibronectin (Goh and Klaenhammer, 2010). Developing Novel Probiotic Functionality

With increased knowledge of mechanisms of action and gene expression, probiotic cellular structures can be modified to potentially increase their protective functionality in the host. Effective vaccines confer protection to recipients against pathogenic microbes by potentiating antibody avidity and boosting the

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longevity and number of T cells. Although live attenuated pathogens such as Salmonella, Bordetella, and Listeria have been successfully used as vaccine vectors, the potential for virulent reversion within these strains limits their practical application (Roberts et al., 2000; Saklani-Jusforgues et al., 2003; Stevenson and Roberts, 2003). Once inside a host, Bacillus anthracis produces the anthrax toxin, which causes systemic infections with 100% mortality (Inglesby et al., 1999; Tournier and Mohamadzadeh, 2008). Multiple subcutaneous injections of aluminum hydroxide (alhydrogel) are currently used as the vaccination against B. anthracis infection; however, this vaccine regimen causes significant transient side effects (Pittman et al., 2002). Lactic acid bacteria have been studied as possible vaccine vectors because they are GRAS (“generally regarded as safe”) organisms that can survive passage through the GIT, induce regulated inflammatory responses against infections, increase IgA production, and activate monocytic lineages (Mohamadzadeh and Klaenhammer, 2008) . A novel oral vaccine strategy was developed, utilizing Lb. acidophilus to deliver expressed B. anthracis protective antigen (PA) via DC-targeting peptides (PA-DCpep fusion proteins) to DC residing in the submucosal layers of the GIT (Mohamadzadeh et al., 2009).

Figure 5. Experimental mice were fed PBS, Lactobacillus gasseri (empty vector), Lb. gasseri expressing Bacillus anthracis protective antigen fused to control peptide (PA-Ctrlpep), and Lb. gasseri expressing B. anthracis protective antigen fused to a dendritic cell-binding peptide (PADCpep) for 3 wk before challenge with B. anthracis (anthrax) Sterne. In the days following a lethal challenge of Sterne, the percentage survival for each group was monitored. Reprinted with permission from Mohamadzadeh et al. (2010). Journal of Dairy Science Vol. 94 No. 10, 2011

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The Lb. acidophilus PA-DCpep vaccine efficiency was evaluated in mice challenged with B. anthracis Sterne. Compared with the mice vaccinated with Lb. acidophilus expressing PA-control peptide or an empty vector, the Lb. acidophilus PA-DCpep induced robust protective immunity and comparable anti-PA titers and levels of IgA-expressing cells to that of the aluminum hydroxide vaccine. After monitoring for 40 d, the survival rates of the experimental groups were 80% for aluminum hydroxide vaccine, 75% for Lb. acidophilus PA-DCpep vaccine, 25% for Lb. acidophilus PA-Ctrlpep vaccine, and no survival for Lb. acidophilus (empty vector) and control groups (Mohamadzadeh et al., 2009). To improve the efficacy of the oral probiotic-based vaccine, PA-DCpep was inserted into a stable, highcopy θ-replicating plasmid (pTRKH2) and expressed in Lb. gasseri. The genetically accessible Lb. gasseri has been sequenced and extensively studied for its use as a human probiotic and commensal species of the human GIT and vagina (Mohamadzadeh et al., 2010). After oral administration of Lb. gasseri expressing PA-DCpep, the fusion proteins elicited an effective PA-neutralizing and T-cell mediated immune response that resulted in 100% survival to B. anthracis Sterne infections (Figure 5). An orally administered Lactobacillus-based vaccine offers many advantages to recipients, such as reduced transient side effects and no need for potentially harmful chemical coupling agents (Mohamadzadeh et al., 2010). The advancements in omics tools were responsible for the ability to direct genetic modifications in these probiotic lactobacilli. The development of probioticbased vaccines shows promise as a means to deliver safe and effective protective immunity against human pathogens. CONCLUSIONS

The recent advancements in sequencing technology and functional omics techniques have provided greater insights into the specific mechanisms underlying probiotic functionality. The developing comprehension of the human gut microbiome will allow proper characterization of probiotic effects on the commensal microbiota of humans in vivo. Identification of genes vital to probiotic functionality is providing researchers the ability to genetically tailor probiotics to meet the needs for specific applications. Recent studies have shown the effective use of probiotics in preventive medicine, maintaining the microbiota and host physiology of a healthy GIT, and the remediation of inflammatory bowel diseases, as well as the potential use of probiotic-based vaccine delivery systems. Future clinical trials are required to fully elucidate the therapeutic potential of probiotics in humans. Journal of Dairy Science Vol. 94 No. 10, 2011

ACKNOWLEDGMENTS

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