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The 104-kDa Listeria adhesion protein (LAP) in Listeria monocytogenes is involved in binding to various mammalian cell lines. However, the receptor that interacts with LAP in eukaryotic cells is unknown. In this study, scanning immunoelectron microscopy qualitatively demonstrated greater binding capacity of wild-type (WT) L. monocytogenes strain (F4244) than a LAP-deficient mutant strain (KB208) to Caco-2 cells. The goal of this study was identification of the host cell receptor for LAP. Using a Western blot ligand overlay assay, we identified a protein of 58 kDa to be the putative receptor for LAP from Caco-2 cells. N-terminal sequencing and subsequent database search identified this protein as heat shock protein 60 (Hsp60). Modified immunoseparation with protein A-Sepharose beads bound to the LAP-specific monoclonal antibody H7 (MAb-H7) and a sequential incubation with LAP preparation and Caco-2 lysate confirmed the receptor to be the same 58-kDa protein. Western blot analysis with anti-Hsp60 MAb of whole-cell adhesion between Caco-2 and WT also revealed the receptor protein to be a 58-kDa protein, thus corroborating the identification of Hsp60 as a host cell receptor for LAP. Furthermore, the anti-Hsp60 antibody also caused approximately 74% reduction in binding of L. monocytogenes WT to Caco-2 cells, whereas a control antibody, C11E9, had no effect on binding. The adhesion mechanism of L. monocytogenes to eukaryotic cells is a complex process, and identification of Hsp60 as a receptor for LAP adds to the list of previously discovered ligand-receptor modules that are essential to achieve successful adhesion.

Supplementation of infant formulas with prebiotic ingredients continues the effort to mimic functional properties of human milk. In this double-blind, controlled, 28-day study, healthy term infants received control formula (control group; n ‫؍‬ 25) or control formula supplemented with polydextrose (PDX) and galactooligosaccharide (GOS) (4 g/liter) (PG4 group; n ‫؍‬ 27) or with PDX, GOS, and lactulose (LOS) (either 4 g/liter [PGL4 group; n ‫؍‬ 27] or 8 g/liter [PGL8 group; n ‫؍‬ 25]). A parallel breast-fed group (BF group) (n ‫؍‬ 30) was included. Stool characteristics, formula tolerance, and adverse events were monitored. Fecal bacterial subpopulations were evaluated by culture-based selective enumeration (Enterobacteriaceae), quantitative real-time PCR (Clostridium clusters I, XI, and XIV, Lactobacillus, and Bifidobacterium), and fluorescence in situ hybridization (FISH) (Bifidobacterium). Fecal bacterial community profiles were examined by using 16S rRNA gene PCR-denaturing gradient gel electrophoresis. The daily stool consistency was significantly softer or looser in the BF group than in all of the groups that received formula. The formulas were well tolerated, and the incidences of adverse events did not differ among feeding groups. Few significant changes in bacterial subpopulations were observed at any time point. The bacterial communities were stable; individual profiles tended to cluster by subject rather than by group. Post hoc analysis, however, demonstrated that the bacterial community profiles for subjects in the BF, PG4, PGL4, and PGL8 groups that first received formula at a younger age were less stable than the profiles for subjects in the same groups that received formula at an older age, but there was no difference for the control group. These data indicate that formulas containing PDX, GOS, and LOS blends are more likely to influence gut microbes when administration is begun in early infancy and justify further investigation of the age-related effects of these blends on fecal microbiota.Nondigestible food ingredients called prebiotics pass into the lower gastrointestinal tract and, by definition, may be selectively metabolized by mutualistic microorganisms, such as Lactobacillus spp. and Bifidobacterium spp., which in turn contribute to improved host health (12, 34). After lactose and lipids, oligosaccharides, which have prebiotic activity, are the third largest component of human breast milk (5 to 10 g/liter), and there are as many as 200 distinct molecular structures (5, 26). Lactobacilli and bifidobacteria are the predominant bacteria in the intestinal microbiota of breast-fed infants, whereas infants who receive cow's milk-based infant formulas, which naturally contain low levels of oligosaccharides, often have higher concentrations of potentially pathogenic bacteria, such as Enterobacteriaceae and clostridia, in their intestinal microbiota (4,15,17).Clinical investigations of infant formulas supplemented with galactooligosaccharide (GOS) and fructooligosaccharide (FOS) at a range of concent...

BackgroundTo ensure the suitability of an infant formula as the sole source of nutrition or provide benefits similar to outcomes in breastfed infants, advancements in formula composition are warranted as more research detailing the nutrient composition of human milk becomes available. This study was designed to evaluate growth and tolerance in healthy infants who received one of two investigational cow’s milk-based formulas with adjustments in carbohydrate, fat, and calcium content and supplemented with a prebiotic blend of polydextrose (PDX) and galactooligosaccharides (GOS) or GOS alone.MethodsIn this multi-center, double-blind, parallel-designed, gender-stratified prospective study 419 infants were randomized and consumed either a marketed routine cow’s milk-based infant formula (Control; Enfamil® LIPIL®, Mead Johnson Nutrition, Evansville, IN) (n = 142) or one of two investigational formulas from 14 to 120 days of age. Investigational formulas were supplemented with 4 g/L (1:1 ratio) of a prebiotic blend of PDX and GOS (PDX/GOS; n = 139) or 4 g/L of GOS alone (GOS; n = 138). Anthropometric measurements were taken at 14, 30, 60, 90, and 120 days of age. Daily recall of formula intake, tolerance, and stool characteristics was collected during study weeks 1 and 2 and 24-h recall was collected at 60, 90, and 120 days of age. Medically-confirmed adverse events were recorded throughout the study.ResultsThere were no group differences in growth rate from 14 to 120 days of age. Discontinuation rates were not significantly different among study groups. No differences in formula intake or infant fussiness or gassiness were observed. During study weeks 1 and 2 and at 60 days of age stool consistency ratings were higher (i.e. softer stools) for infants in the PDX/GOS and GOS groups versus Control and remained higher at 120 days for the PDX/GOS group (all P < 0.05). The overall incidence of medically-confirmed adverse events was similar among groups.ConclusionsInvestigational routine infant formulas supplemented with 4 g/L of either a prebiotic blend of PDX and GOS or GOS alone were well-tolerated and supported normal growth. Compared to infants who received the unsupplemented control formula, infants who received prebiotic supplementation experienced a softer stooling pattern similar to that reported in breastfed infants.Trial registrationClinicalTrials.gov Identifier: NCT00712608

Listeria adhesion protein (LAP) is an important adhesion factor in Listeria monocytogenes and interacts with its cognate receptor, mammalian heat shock protein 60 (Hsp60). The genetic identity of LAP was determined to be alcohol acetaldehyde dehydrogenase (Aad). A recombinant Escherichia coli strain expressing aad confirmed the involvement of Aad in adhesion to Caco-2 cells. Binding kinetics (ka) of recombinant LAP (rLAP) to Hsp60 was examined in a surface plasmon resonance sensor and was determined to be 5.35 x 10(8) M(-1) s(-1) and it was equivalent to the binding of anti-Hsp60 antibody (ka = 2.15 x 10(9) M(-1) s(-1)) to Hsp60. In contrast, Internalin B, an adhesion/invasion protein from L. monocytogenes, used as a control, had binding kinetics (ka) of only 2.9 x 10(6) M(-1) s(-1). The KD value of rLAP was 1.68 x 10(-8) M, which was significantly lower than Internalin B (KD = 6.5 x 10(-4) M). These results suggest that Hsp60 has significantly higher avidity for anti-Hsp60 antibody and LAP than Internalin B. In summary, LAP is identified as an alcohol acetaldehyde dehydrogenase and binding of recombinant E. coli to Caco-2 cells or rLAP to Hsp60 protein was found to be highly specific.