Overview of Lab Projects - Therapeutic Microbiology Laboratory
- Functional Genomics of Probiotic Immunomodulation
- Immunomodulation by Probiotics
- Mechanisms of Probiotic-Mediated Cytokine Suppression in Macrophages
- Biofilms Formed by Probiotics and Bacterial Cell:Cell Signaling
Functional Genomics of Probiotic Immunomodulation
Functional genomics approaches are being used to investigate mechanisms by which probiotic Lactobacillus species confer anti-inflammatory effects in the mammalian intestine. Current studies include identifying genes which regulate or directly confer immunomodulatory effects on mammalian macrophages or intestinal epithelial cells. A recent review nicely summarized comparative genomics of probiotic lactobacilli (Makarova, PNAS, 2006).
Lactobacillus reuteri genomes are being sequenced in collaboration with the Human Genome Sequencing Center (HGSC) at Baylor (Fig. 1). Four human-derived strains are being sequenced by a combination of 454 and Sanger chemistry in order to achieve high-throughput sequencing with maximal coverage. The availability of multiple bacterial genomes from a single probiotic species has provided fertile opportunities for comparative genomics and global approaches to understanding molecular mechanisms of probiotic functions.
Ample sequence data is being assembled with software tools from the HGSC followed by gap closure and annotation using automated approaches. These genomes are approximately 2 megabases, with about 1900 to 2150 genes depending on the strains. A first-generation of strain-specific microarrays have been fabricated for 2 different L. reuteri strains with 1864 or 1889 genes, and represented by 60-mer oligonucleotides. Comparative gene expression profiling of L. reuteri strains has yielded sets of genes that are differentially expressed in various conditions and with various stresses (Fig. 2).
Transcriptional regulators have been identified as differentially expressed genes and may control regulons important for probiotic functions, survival in the mammalian host, and stress tolerance. Transcirptional repressors and activators have been identified in lactic acid bacteria (LAB), and these regulators may affect gene expression important for secretion of immunomodulatory factors. Transporter and permease genes have also been identified in L. reuteri and other LAB, and these gene products may regulate secretion of immunoregulatory and antimicrobial factors mediating cell:cell signaling. Additionally genes involved in cell wall biosynthesis are being explored for roles in immunomodulation and stress tolerance.
In order to complement functional genomics, targeted mutagenesis strategies are being applied in L. reuteri and other lactobacilli so that candidate genes of interest can be knocked out. Randomized mutagenesis approaches are currently in development. Ultimately, insertional mutagenesis strategies will provide many opportunities to study gene function and molecular mechanisms of probiosis.
Immunomodulation by Probiotics
Biochemical strategies are being pursued in order to isolate and purify factors secreted by Lactobacillus species that may regulate the innate immune system. The secreted factors that modulate innate immunity or "immunomodulins" may be comprised of a variety of organic molecules including polypeptides and polysaccharides.
In order to complement the functional genomics studies, the entire secretome of Lactobacillus L. reuteri is being studied by multistage mass spectrometry (Fig. 3),High Performance Liquid Chromatography (HPLC) and Nuclear Magnetic Resonance (NMR).
Figure 3. Multistage mass spectrometry of products secreted by L. reuteri
In addition to polypeptides, carbohydrates and other organic molecules are being pursued by a combination of approaches. Genetic approaches including insertional mutagenesis may yield insights into the biosynthesis and secretion of non-polypeptide factors that may regulate innate immune responses in the oral and gastrointestinal mucosa. Mass spectrometry and will be applied to characterize both polypeptide and carbohydrate components of the secretome. In addition to characterization of potential immunomodulins, comprehensive studies of the secretome will be pursued in order to understand probiotic molecules with diverse beneficial functions. Molecules with roles as nutrients, antimicrobial compounds, or intercellular signaling molecules may be studied as possible mediators of probiotic functions.
Mechanisms of Probiotic-Mediated Cytokine Suppression in Macrophages
In order to test the "probiosis by direct immunomodulation" concept, we selected mouse-derived L. paracasei and L. reuteri strains on the basis of TNF-α-inhibitory activity toward macrophages (Peña, 2004). Murine intestinal Lactobacillus isolates (Peña, 2004) were screened for probiotic activity, as defined by inhibition of TNF-α production in LPS-stimulated murine macrophages. Of 29 lactobacilli isolated from the non-colitic mice (Fig. 4) (Swiss-Webster Mice), six (21%) L. reuteri/L. paracasei strains displayed TNF-α inhibitory effects (>50%) with LPS-stimulated macrophages (Peña, 2004). The magnitude of inhibition of TNF-α production varied among selected isolates, indicating potency differences among different candidate probiotic isolates (Fig. 4) (Peña, 2004). In contrast, none of the 29 lactobacilli recovered from IL-10-deficient mice (IL-10 K/O Mice) demonstrated TNF-inhibitory activity (Fig. 4) (Peña, 2004).
Lactobacillus clones with TNF-α inhibitory activity in vitro were evaluated by studying anti-inflammatory effects in a Helicobacter-exacerbated mouse colitis model (Peña, 2005). In a pilot study, both male (n=21) and female (n=18) mice were pre-colonized with a combination of two murine lactobacilli, L. paracasei 1602 and L. reuteri 6798, that had displayed anti-inflammatory activity with cultured macrophages. Lactobacillus colonization was followed by infection with the mouse enteric pathogen, H. hepaticus. After ten weeks, the ceco-colic junctions were semi-quantitatively graded for inflammation, hyperplasia and dysplasia by histopathologic examination (scale 0-4). When animals were stratified by gender, significant reductions were observed in ceco-colic lesion scores (inflammation, hyperplasia and dysplasia; P=0.032, 0.032 and 0.016, respectively) in female mice co-colonized with Lactobacillus and H. hepaticus versus animals that were mono-infected with H. hepaticus. In contrast, male mice co-colonized with murine Lactobacillus, did not yield statistically significant reduction in intestinal lesion grades.
Because female mice appeared to derive the greatest benefit from probiotic therapy, we performed a follow-up study with female mice only (n=36) using the same co-colonization and infection protocol. As in the pilot study, uninfected animals exhibited normal ceco-colic morphology (Fig. 4) while those mono-infected with H. hepaticus (Fig. 4; Hh only panel) developed moderate to severe typhlocolitis characterized by infiltration of lymphocytes and macrophages, with fewer granulocytes, in the mucosa and submucosa. Reactive epithelial changes included hyperplasia with crypt elongation. Lesions were partially abrogated in mice treated with L. paracasei and L. reuteri (Fig. 4; Lp1602/Lr6798 + Hh panel) and completely prevented in mice pre-colonized with L. paracasei only) (Fig. 4; Lp1602 + Hh panel). Although the degree of hyperplasia was similar with the L. paracasei /L. reuteri combination (Lp1602/Lr6798 + Hh panel), intestinal inflammation was significantly reduced in these animals (Peña, 2005).
Probiotic bacteria can suppress pro-inflammatory cytokines by mucosal immune cells including macrophages and intestinal epithelial cells. Pro-inflammatory cytokines such as tumor necrosis factor - alpha (TNF-a) and interleukin-8 (IL-8) are regulated by transcriptional and post-transcriptional mechanisms. The challenge is to elucidate how specific probiotic strains suppress the secretion of pro-inflammatory cytokines by intestinal epithelial cells and macrophages.
Figure 5. Macrophages as Targets for Immunoprobiotic Action
The main cell types of interest in the laboratory are myeloid cells of the monocytoid or macrophage lineage (Fig 5). We are also primarily interested in intestinal epithelial cells. Murine and human cells are studied in the laboratory so that mouse models of human disease and human cell biology can be explored in parallel. Factors of interest secreted by probiotics include polypeptides and polysaccharides and their effects on mammalian innate immune cell signaling pathways. Commensal-associated microbial patterns (CAMPs) may contribute to the abilities of the intestinal microbiota to quench or downregulate immune responses and promote tolerance.
Mouse and human intestinal epithelial cell (IEC) lines are studied in transwell cultures in order to assess the effects of probiotics on barrier function and chemokine production. Probiotic strains suppress IL-8 production by human IECs and investigations in the lab have explored mechanisms of IL-8 suppression. Transwell cultures may be stimulated by different agonists including LPS and TNF and effects may be explored with different agonists and probiotic molecules.
Cell culture models have been developed that enable selective stimulation of toll-like receptor 2 (TLR2) or toll-like receptor 4 (TLR4) signaling pathways in human macrophages. Selective pattern recognition receptor stimulation provides opportunities to examine proximal signaling steps regulated by probiotics. Macrophage activation (with TLR4 agonists such as LPS or TLR2 agonists such as lipopeptides) may test the ability of probiotics to regulate responses to different pathogenic features. Key nodes in signaling pathways are being explored with kinase assays and immunoblots. Mouse and human microarrays are also being used to assess signaling pathways modulated by probiotic agents (Fig. 6).
Transcription factor activation can be assessed by protein/DNA arrays, phospho-ELISAs, and reporter gene assays. The relative importance of MAP kinases and NF-?B signaling are being explored with multiple approaches in murine and human cell lines. Cytokine mRNA levels are being explored by quantitative real-time RT-PCR using hydrolysis probes. Secreted cytokines are being tested by quantitative ELISAs or spectrally addressable liquid bead arrays on a Luminex platform (Fig. 7).
Our laboratory can screen probiotic compounds with high throughput 96-well plate assays and mammalian cells in small culture volumes. Many different probiotic strains can be screened in parallel for effects on different cytokines. In addition to screens for inhibitory effects on pro-inflammatory cytokines, we are interested in screening for up-regulation of anti-inflammatory or immunosuppressive cytokines. High-throughput screens can be combined with functional genomics approaches by insertional mutagenesis to explore the roles of specific probiotic Lactobacillus genes in immunomodulation.
Figure 8. Biology of Probiotic Lactobacillus as Planktonic Cells and Biofilms
Biofilms Formed by Oral Probiotics and Bacterial Cell:Cell Signaling
Probiotic and commensal bacteria form microbial communities (Figs. 8, 9) composed of millions of cells in the mammalian oral cavity and gastrointestinal tract. Biofilms composed of mixed species highlight the importance of structured communities dependent on sophisticated patterns of intercellular signaling between bacterial species and between bacteria and mammalian host.
Figure 9. Scanning Electron Micrograph of L. reuteri
Quorum sensing (QS) refers to cell density-dependent intercellular signaling among bacteria by small, secreted signal molecules or bacterial "hormones". Homoserine lactones (autoinducer type-1 or AI-1) and peptides are produced by gram-negative and gram-positive bacteria, respectively, and function as autoinducers (AI) to stimulate expression of QS-dependent gene networks. The LuxS synthetase represents a key enzyme involved in a third QS mechanism that is dependent on the production of autoinducer type-2 (AI-2) (Fig. 10). AI-2 represents a collection of furanones that transmit signals through specific receptors and functions as a widespread interspecies communication system among bacteria.
The luxS gene is now recognized as a critical mediator of signals in quorum sensing and modulator of virulence properties in pathogenic bacteria. LuxS is essential for the synthesis of AI-2, thereby facilitating intercellular communication among bacterial species. AI-2 is important in mixed species biofilms of the oral microbiota including the species Streptococcus gordonii and Porphyromonas gingivalis. Mixed microcolonies containing P. gingivalis and S. gordonii luxS mutants demonstrate altered aggregative behavior with respect to wild-type colonies. AI-2 produced via LuxS may be important for transmitting signals across species and genus boundaries in mixed species biofilms.
Probiotic Lactobacillus species including L. reuteri have the luxS gene and secreted AI-2. The role of AI-2 in probiotic Lactobacillus biology remains unclear although luxS appears to modulate biofilm formation. Our laboratory studies biofilm formation by probiotic strains using scanning electron microscopy (Fig. 9), microtiter plate assays with absorbance spectrophotometry, and flow cells. Flow cells enable the studies of biofilm formation using continuous flow with fresh media and routine sampling to be performed for dynamic studies. Studies with luxS knockout mutants of L. reuteri and S. mutans enable us to explore the roles of LuxS in biofilm formation and probiotic:oral pathogen interactions. The role of luxS in intercellular communication among probiotic bacteria is being studied in the context of bacterial biofilms and planktonic cultures.
Intercellular communication is also being studied between different probiotic strains and between probiotics and pathogens using a two-chambered vessel system (Fig. 11). A semi-permeable membrane separates two chambers, and samples can be withdrawn to sample cells and media from each chamber. Finally, stress challenge studies of probiotics are being pursued to explore stress tolerance of probiotic bacteria including exposure to virulence factors of known oral or enteric pathogens.
Functional genomics approaches including applications of gene expression profiling and insertional (knockout) mutagenesis represent key strategies for increasing our understanding of how probiotic strains communicate with each other, bacterial pathogens, and the host. Fluorescence in situ hybridization (FISH) approaches (Fig. 12) and denaturing HPLC methods are being applied to studies of mixed bacterial populations and investigations of probiotics in animal models. By combining molecular and physiologic approaches, our laboratory seeks to gain a greater understanding of bacterial:mammalian crosstalk as a two-way street.
Figure 12. Fluorescence in situ Hybridization (FISH) of Lactobacillus reuteri