Matti Karp

Implication of an Outer Surface Lipoprotein in Adhesion of Bifidobacterium bifidum to Caco-2 Cells

ABSTRACT We found that the human intestinal isolate Bifidobacterium bifidum MIMBb75 strongly adhered to Caco-2 cells. Proteinase K and lithium chloride treatments showed that proteins play a key role in MIMBb75 adhesion to Caco-2 cells. By studying the cell wall-associated proteins, we identified a surface protein, which we labeled BopA. We purified the protein chromatographically and found that it functioned as an adhesion promoter on Caco-2 cells. In silico analysis of the gene coding for this protein and globomycin experiments showed that BopA is a cysteine-anchored lipoprotein expressed as a precursor polypeptide. A database search indicated that BopA appears to function biologically as an oligopeptide/tripeptide-solute-binding protein in the ABC transport system. We discovered a protein corresponding to BopA and its gene in eight other highly adherent B. bifidum strains. Finally, we found that B. bifidum MIMBb75 and BopA affected the production of interleukin-8 in Caco-2 epithelial cells. BopA is the first protein described to date to be directly involved in the adhesion of bifidobacteria to Caco-2 cells and to show immunomodulatory activity.

Hydrogen production from glycerol using halophilic fermentative bacteria

Glycerol-based hydrogen production by the halophilic bacteria Halanaerobium saccharolyticum subspecies saccharolyticum and senegalensis was studied as batch experiments. The main metabolites of glycerol fermentation of both strains were hydrogen, carbon dioxide, and acetate. Subspecies saccharolyticum also produced 1,3-propanediol (1,3-PD), butyrate, and ethanol. The highest hydrogen yields were achieved with 2.5g/l glycerol and 150g/l salt at pH 7.4 (subsp. saccharolyticum, yield 0.6mol/mol glycerol) and at pH 7.0 (subsp. senegalensis, yield 1.6mol/mol glycerol). The hydrogen yield of subsp. senegalensis has potential for practical applications after scale-up and bioprocess optimizations and metabolic engineering after genome-wide sequencing could be applied to improve the yield of subsp. saccharolyticum.

Oral Bacteria as Potential Probiotics for the Pharyngeal Mucosa

ABSTRACT The research described here was aimed at the selection of oral bacteria that displayed properties compatible with their potential use as probiotics for the pharyngeal mucosa. We included in the study 56 bacteria newly isolated from the pharynges of healthy donors, which were identified at the intraspecies level and characterized in vitro for their probiotic potential. The experiments led us to select two potential probiotic bacterial strains (Streptococcus salivarius RS1 and ST3) and to compare them with the prototype oral probiotic S. salivarius strain K12. All three strains efficiently bound to FaDu human epithelial pharyngeal cells and thereby antagonized Streptococcus pyogenes adhesion and growth. All were sensitive to a variety of antibiotics routinely used for the control of upper respiratory tract infections. Immunological in vitro testing on a FaDu layer revealed different responses to RS1, ST3, and K12. RS1 and ST3 modulated NF-κB activation and biased proinflammatory cytokines at baseline and after interleukin-1β (IL-1β) induction. In conclusion, we suggest that the selected commensal streptococci represent potential pharyngeal probiotic candidates. They could display a good degree of adaptation to the host and possess potential immunomodulatory and anti-inflammatory properties.

Improved Triacylglycerol Production in Acinetobacter baylyi ADP1 by Metabolic Engineering

BackgroundTriacylglycerols are used in various purposes including food applications, cosmetics, oleochemicals and biofuels. Currently the main sources for triacylglycerol are vegetable oils, and microbial triacylglycerol has been suggested as an alternative for these. Due to the low production rates and yields of microbial processes, the role of metabolic engineering has become more significant. As a robust model organism for genetic and metabolic studies, and for the natural capability to produce triacylglycerol, Acinetobacter baylyi ADP1 serves as an excellent organism for modelling the effects of metabolic engineering for energy molecule biosynthesis.ResultsBeneficial gene deletions regarding triacylglycerol production were screened by computational means exploiting the metabolic model of ADP1. Four deletions, acr1, poxB, dgkA, and a triacylglycerol lipase were chosen to be studied experimentally both separately and concurrently by constructing a knock-out strain (MT) with three of the deletions. Improvements in triacylglycerol production were observed: the strain MT produced 5.6 fold more triacylglycerol (mg/g cell dry weight) compared to the wild type strain, and the proportion of triacylglycerol in total lipids was increased by 8-fold.ConclusionsIn silico predictions of beneficial gene deletions were verified experimentally. The chosen single and multiple gene deletions affected beneficially the natural triacylglycerol metabolism of A. baylyi ADP1. This study demonstrates the importance of single gene deletions in triacylglycerol metabolism, and proposes Acinetobacter sp. ADP1 as a model system for bioenergetic studies regarding metabolic engineering.

Expression of luciferase genes from different origins in Bacillus subtilis.

SummaryA group of vectors for luciferase expression in Bacillus subtilis was constructed. So far, only bacterial luciferases have been expressed in Bacillus, but in this study we wanted also to express genes encoding eukaryotic luciferases to perform direct comparisons of the light levels produced by the two different systems in B. subtilis. The vectors constructed can replicate both in Escherichia coli and B. subtilis, and the luciferase expression is strictly regulated due to the dual plasmid system used. Nearly a 100-fold increase in light production compared to previous results was achieved when genes encoding bacterial luciferase were inserted into the constructs and transformed into B. subtilis. An additional tenfold increase in light production was obtained when luciferase genes from the North American firefly (Photinus pyralis) or a click beetle (Pyrophorus plagiophtalamus) were introduced in a similar fashion into B. subtilis. Measurement of the light emission was performed without disruption of bacterial cells in a real-time manner, which is a common feature when working with all of these constructions. Structures of the shuttle vector constructs and results from light emission measurements are presented.

S-layer protein mediates the stimulatory effect of Lactobacillus helveticus MIMLh5 on innate immunity.

ABSTRACT The ability to positively affect host health through the modulation of the immune response is a feature of increasing importance in measuring the probiotic potential of a bacterial strain. However, the identities of the bacterial cell components involved in cross talk with immune cells remain elusive. In this study, we characterized the dairy strain Lactobacillus helveticus MIMLh5 and its surface-layer protein (SlpA) using in vitro and ex vivo analyses. We found that MIMLh5 and SlpA exert anti-inflammatory effects by reducing the activation of NF-κB on the intestinal epithelial Caco-2 cell line. On the contrary, MIMLh5 and SlpA act as stimulators of the innate immune system by triggering the expression of proinflammatory factors tumor necrosis factor alpha and COX-2 in the human macrophage cell line U937 via recognition through Toll-like receptor 2. In the same experiments, SlpA protein did not affect the expression of the anti-inflammatory cytokine interleukin-10. A similar response was observed following stimulation of macrophages isolated from mouse bone marrow or the peritoneal cavity. These results suggest that SlpA plays a major role in mediating bacterial immune-stimulating activity, which could help to induce the hosts defenses against and responses toward infections. This study supports the concept that the viability of bacterial cells is not always essential to exert immunomodulatory effects, thus permitting the development of safer therapies for the treatment of specific diseases according to a paraprobiotic intervention.

A novel biosensor for the detection of zearalenone family mycotoxins in milk

In this study, a method for detecting estrogenic mycotoxin residues in milk was developed utilizing bioluminescent whole-cell biosensors. Milk products of various compositions were spiked with the estrogenic mycotoxins zearalenone and its metabolites zearalanone, alpha-zearalanol, beta-zearalanol, alpha-zearalenol and beta-zearalenol. The estrogenic response was detected by a whole-cell biosensor based on a genetically modified Saccharomyces cerevisiae strain that in the presence of an estrogenic compound produces firefly luciferase-enzyme and further light emission within a system provided with D-luciferin substrate. The results show that the yeast sensor reacts to mycotoxins with typical sigmoidal response at nanomolar concentrations. The response differs in different milk products with regard to the fat content of the milk. Due to short assay time of less than 3h and automation the approach can be used as a bioavailability and activity screening method prior to more detailed chemical analysis.

Microbial testing methods for detection of residual cleaning agents and disinfectants-prevention of ATP bioluminescence measurement errors in the food industry.

The ATP luminescence measurement is based on the presence of an enzymatic reaction and may significantly be affected by cleaning agents and disinfectants. In addition, disinfectants can also reduce the activity of the luciferase enzyme and also act as ATP-releasing agents. The agents disrupt the cell walls but preserve ATP in measurable form, and therefore correlation with culture methods can be poor. Therefore, if a rapid method is used to detect ATP, a control must be used for reliable results. The possible effect of disinfectants can be eliminated with a rapid test to minimize sources of error. In the present study a microbiological residue testing method that is nonspecific for residues was developed. The effects of a total of 38 commercial cleaning agents and disinfectants of various types were assessed using two microbiological methods, the Vibrio fischeri photobacteria test and Micrococcus luteus inhibition zone technique. The results show that the V. fischeri photobacteria test is very sensitive. This test can therefore be used for testing cleaning agent residues on surfaces in very small amounts. A small study was also carried out in a food factory to show applicability in processing facilities. The study showed, that a need for this type of method exists in food processing.

Halophilic anaerobic fermentative bacteria

In hypersaline environments bacteria are exposed to a high osmotic pressure caused by the surrounding high salt concentrations. Halophilic microorganisms have specific strategies for balancing the osmotic pressure and surviving in these extreme conditions. Halophilic fermentative bacteria form taxonomically and phylogenetically a coherent group mainly belonging to the order Halanaerobiales. In this review, halophilic anaerobic fermentative bacteria in terms of taxonomy and phylogeny, special characteristics, survival strategies, and potential for biotechnological applications in a wide variety of branches, such as production of hydrogen, are discussed.

A Dairy Bacterium Displays In Vitro Probiotic Properties for the Pharyngeal Mucosa by Antagonizing Group A Streptococci and Modulating the Immune Response

ABSTRACT The probiotic approach represents an alternative strategy in the prevention and treatment of infectious diseases, not only at the intestinal level but also at other sites of the body where the microbiota plays a role in the maintenance of physiological homeostasis. In this context, we evaluated in vitro the potential abilities of probiotic and dairy bacteria in controlling Streptococcus pyogenes infections at the pharyngeal level. Initially, we analyzed bacterial adhesion to FaDu hypopharyngeal carcinoma cells and the ability to antagonize S. pyogenes on FaDu cell layers and HaCat keratinocytes. Due to its promising adhesive and antagonistic features, we studied the dairy strain Lactobacillus helveticus MIMLh5, also through in vitro immunological experiments. First, we performed quantification of several cytokines and measurement of NF-κB activation in FaDu cells. MIMLh5 efficiently reduced the induction of interleukin-6 (IL-6), IL-8, and tumor necrosis factor alpha (TNF-α), in a dose-dependent manner. After stimulation of cells with IL-1β, active NF-κB was still markedly lowered. Nevertheless, we observed an increased secretion of IL-6, gamma interferon (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) under these conditions. These effects were associated with the ability of MIMLh5 to enhance the expression of the heat shock protein coding gene hsp70. In addition, MIMLh5 increased the GM-CSF/G-CSF ratio. This is compatible with a switch of the immune response toward a TH1 pathway, as supported by our observation that MIMLh5, once in contact with bone marrow-derived dendritic cells, triggered the secretion of TNF-α and IL-2. In conclusion, we propose MIMLh5 as a potential probiotic bacterium for the human pharynx, with promising antagonistic and immunomodulatory properties.