Cystine-mediated oxidative defence in Lactobacillus reuteri BR11

Lactobacillus reuteri BR11 possesses an abundant cystine uptake (Cyu) ABC-transporter that was previously found to be involved in a novel mechanism of oxidative defence mediated by cystine. The current study aimed to elucidate this mechanism with a focus on the role of the co-transcribed cystathionine ã-lyase (Cgl). Growth studies of wild-type L. reuteri BR11 and mutants inactivated in cgl and the cystine-binding protein encoding gene cyuC showed that in contrast to the Cyu transporter, whose inactivation led to growth arrest in aerated cultures, Cgl is not crucial for oxidative defence. However, the role of Cgl in oxidative defence became apparent in the presence of severe oxidative damage and cysteine deprivation. Cysteine was found to be protective against oxidative stress, and the action of Cgl in both cysteine biosynthesis and degradation poses a seemingly futile pathway that deprives the intracellular cysteine pool. To further characterise the relationship between Cgl activity and cysteine and their roles in oxidative defence, enzymatic assays were performed on purified Cgl, and intracellular concentrations of cysteine, cystathionine and methionine were determined. Cgl was highly active towards cystine and cystathionine and less active towards cysteine in vitro, suggesting the main function of Cgl to be cysteine biosynthesis. Cysteine was found at high concentrations in the cell, but the levels were not significantly affected by inactivation of cgl or growth under aerobic conditions. It was concluded that both anabolic and catabolic activities of Cgl towards cysteine contribute to oxidative defence, the former by maintaining an intracellular reservoir of thiol analogous to glutathione, and the latter by producing H2S which is readily secreted, thus creating a reducing extracellular environment. The significance of the Cyu transporter to the physiology of L. reuteri BR11 prompted a phylogenetic study to determine its presence in bacteria. Orthologs of the Cyu transporter that are closest matches to the Cyu transporter are only limited to several species of Lactobacillus and Leuconostoc. Outside the Lactobacillales order, the closest matching orthologs belong to Proteobacteria, and there are more orthologs in Proteobacteria than non-Lactobacillales Firmicutes, suggesting that the Cyu transporter locus was present in the ancestor of the Proteobacteria and Firmicutes, and over evolutionary time has been lost or diverged in many Firmicutes. The clustering of the Cyu transporter locus with a gene encoding a Cgl family protein is even rarer. It was only found in L. reuteri, Lactobacillus vaginalis, Weissella paramesenteroides, the Lactobacillus casei group, and several Campylobacter sp. An accompanying phylogenetic study of L. reuteri BR11 using multi-locus sequence analysis showed that L. reuteri BR11 had diverged from more than 100 strains of L. reuteri isolated from various hosts and geographical locations. However, comparison with other Lactobacillus species supported the current classification of BR11 as L. reuteri. The most closely related species to L. reuteri is L. vaginalis or Lactobacillus antri, depending on the housekeeping gene used for analysis. The close evolutionary relationship of L. vaginalis to L. reuteri and the high degree of sequence identity between the cgl-cyuABC loci in both species suggest that the Cyu system is highly likely to perform similar functions in L. vaginalis. In search of other genes that function in oxidative defence, a number of mutants which were inactivated in genes that confer increased resistance to oxidative stress in other bacteria were constructed. The genes targeted were ahpC (peroxidase component of the alkyl hydroperoxide reductase system), tpx (thiol peroxidase), osmC (osmotically induced protein C), mntH (Mn2+/Fe2+ transporter), gshA (ã-glutamylcysteine synthetase) and msrA (methionine sulfoxide reductase). The ahpC and mntH mutants had slightly lower minimum inhibitory concentrations of organic peroxides, suggesting these genes might be involved in resistance to organic peroxides in L. reuteri. However, none of the mutants exhibited growth defects in aerated cultures, in stark contrast to the cyuC mutant. This may be due to compensatory functions of other genes, a hypothesis which cannot be tested until a robust protocol for constructing markerless multiple gene deletion mutants in L. reuteri is developed. These results highlight the importance of the Cyu transporter in oxidative defence and provide a foundation for extending the research of this system in other bacteria.

Blood pressure lowering effects of Lactobacillus helveticus fermented milk containing bioactive peptides Ile-Pro-Pro and Val-Pro-Pro; mechanistic, kinetic and clinical studies

The purpose of the present study was to evaluate the effects of Lactobacillus helveticus fermented milk (peptide milk) containing the casein-derived tripeptides Isoleucyl-prolyl-proline (Ile-Pro-Pro) and Valyl-prolyl-proline (Val-Pro-Pro) on blood pressure and vascular function in hypertensive subjects. The peptide milk lowered systolic and diastolic blood pressure in long-term use in hypertensive subjects when blood pressure was measured by using 24-hour ambulatory blood pressure measurement (ABPM). The blood pressure lowering effect was seen with the dose of 50 mg of tripeptides, and a tendency for lowering blood pressure was also observed when the dose was 5 mg. No adverse effects compared to the placebo group were reported or detected in laboratory analysis. The effect of the peptide milk on arterial stiffness was shown using two different methods, the ambulatory arterial stiffness index (AASI) and pulse wave analysis (PWA). According to the AASI, arterial stiffness was significantly reduced in the peptide milk group compared to the baseline level, but the difference was not significant compared to the placebo group. PWA showed that the peptide milk reduced arterial stiffness significantly compared to the placebo group. Endothelium-independent relaxation (nitroglycerin) and endothelium-dependent relaxation (salbutamol) did not differ between the groups. The blood pressure lowering mechanisms of the tripeptides and the kinetics of Ile-Pro-Pro were investigated using spontaneously hypertensive rats (SHR) and Sprague-Dawley rats. Previous studies have suggested that the blood pressure lowering effect of the tripeptides Ile-Pro-Pro and Val-Pro-Pro is based on angiotensin-converting enzyme inhibition, but the present findings did not agree with these previous studies. It was shown in SHR that calcium, potassium and magnesium may also have an important role in attenuating the development of hypertension as part of the peptide milk effect. In addition, the present study suggests indirectly that improved endothelial nitric oxide release capacity is not the mechanism by which peptide milk mediates its favourable circulatory effects. The kinetics of Ile-Pro-Pro were studied using adult Sprague-Dawley rats. The results showed that orally administered Ile-Pro-Pro is absorbed at least partly intact from the gastrointestinal tract. Radiolabelled Ile-Pro-Pro was distributed in different tissues and considerable radioactivity levels were found in tissues related to the renin-angiotensin system (RAS), adrenals, aorta and kidneys. Ile-Pro-Pro does not bind to plasma proteins, and therefore it is possible that its blood pressure lowering effect is mediated by free Ile-Pro-Pro. In conclusion, consumption of the peptide milk lowers blood pressure and reduces arterial stiffness in hypertensive subjects. Ile-Pro-Pro can be absorbed partly intact from the gastrointestinal tract and might accumulate in tissues related to the RAS. The precise blood pressure lowering mechanism of peptide milk remains to be studied.

Surface Proteins of Lactobacillus crispatus: Adhesive Properties and Cell Wall Anchoring

Bacterial surface-associated proteins are important in communication with the environment and bacteria-host interactions. In this thesis work, surface molecules of Lactobacillus crispatus important in host interaction were studied. The L. crispatus strains of the study were known from previous studies to be efficient in adhesion to intestinal tract and ECM. L. crispatus JCM 5810 possess an adhesive surface layer (S-layer) protein, whose functions and domain structure was characterized. We cloned two S-layer protein genes (cbsA; collagen-binding S-layer protein A and silent cbsB) and identified the protein region in CbsA important for adhesion to host tissues, for polymerization into a periodic layer as well as for attachment to the bacterial cell surface. The analysis was done by extensive mutation analysis and by testing His6-tagged fusion proteins from recombinant Escherichia coli as well as by expressing truncated CbsA peptides on the surface of Lactobacillus casei. The N-terminal region (31-274) of CbsA showed efficient and specific binding to collagens, laminin and extracellular matrix on tissue sections of chicken intestine. The N-terminal region also contained the information for formation of periodic S-layer polymer. This region is bordered at both ends by a conserved short region rich in valines, whose substitution to leucines drastically affected the periodic polymer structure. The mutated CbsA proteins that failed to form a periodic polymer, did not bind collagens, which indicates that the polymerized structure of CbsA is needed for collagen-binding ability. The C-terminal region, which is highly identical in S-layer proteins of L. crispatus, Lactobacillus acidophilus and Lactobacillus helveticus, was shown to anchor the protein to the bacterial cell wall. The C-terminal CbsA peptide specifically bound to bacterial teichoic acid and lipoteichoic acids. In conclusion, the N-terminal domain of the S-layer protein of L. crispatus is important for polymerization and adhesion to host tissues, whereas the C-terminal domain anchors the protein to bacterial cell-wall teichoic acids. Lactobacilli are fermentative organisms that effectively lower the surrounding pH. While this study was in progress, plasminogen-binding proteins enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were identified in the extracellular proteome of L. crispatus ST1. In this work, the cell-wall association of enolase and GAPDH were shown to rely on pH-reversible binding to the cell-wall lipoteichoic acids. Enolase from L. crispatus was functionally compared with enolase from L. johnsonii as well as from pathogenic streptococci (Streptococcus pneumoniae, Streptococcus pyogenes) and Staphylococcus aureus. His6-enolases from commensal lactobacilli bound human plasminogen and enhanced its activation by human plasminogen activators similarly to, or even better than, the enolases from pathogens. Similarly, the His6-enolases from lactobacilli exhibited adhesive characteristics previously assigned to pathogens. The results call for more detailed analyses of the role of the host plasminogen system in bacterial pathogenesis and commensalism as well of the biological role and potential health risk of the extracellular proteome in lactobacilli.

Lactobacillus bacteremia, with special focus on the safety of probiotic Lactobacillus rhamnosus GG

Lactobacilli are gram positive rods, which belong to normal oropharyngeal, gastrointestinal and urogenital flora. They are widely used in food industry and as food additives. Although their virulence is presumed to be very low, opportunistic bacteremic infections, especially in immunocompromised hosts, have been detected. In the present study, the possible effects of increasing probiotic use of Lactobacillus rhamnosus GG (LGG) on the occurrence of bacteremia due to lactobacilli was evaluated on population level. In Finland, 90 Lactobacillus bacteremia cases were reported to the National Infectious Disease Register maintained by National Public Health Institute, during 1995-2000. Their proportion of all bacteremia cases was on average 0.24%, corresponding to 0.3 cases/100 000 inhabitants annually. In the Helsinki University Central Hospital district the corresponding proportion of all bacteremia cases was observed during 1990-2000. Despite LGG intake increased six folded no increasing trend in the occurrence of lactobacilli bacteremia was seen. A total of 85 Lactobacillus sp. blood isolates collected from different human bacteremic cases were characterised and compared with the commercial probiotic LGG strain. In species characterisation 46 L. rhamnosus strains, 12 L. fermentum and L. casei strains each, three each of L. gasseri, L. salivarius and L. jensenii species, two L. curvatus, and one each of L. plantarum, L. sakei, L. zeae and L. reuteri species were detected. Nearly half of the L. rhamnosus findings turned out to be indistinguishable from the probiotic LGG strain. Common predisposing factors to Lactobacillus bacteremia were immunosuppression, prior prolonged hospitalisation and prior surgical interventions. Severe or fatal comorbidities were found in 82% of the patients. Mortality at one month was 26% and severe underlying diseases were a significant predictor of death (OR 15.8). Antimicrobial susceptibility of Lactobacillus strains was species dependent. The Lactobacillus isolates were generally susceptible to imipenem, piperacillin-tazobactam, clindamycin and erythromycin, whereas all other than L. gasseri and L. jensenii species were not at all susceptible to vancomycin. The susceptibility to cephalosporin varied greatly even within species why they might not be recommended for treatment of Lactobacillus infections. The effect and safety of probiotic LGG preparation in amelioration of gastric symptoms and diarrhea in HIV-infected patients was evaluated. No significant differences in gastrointestinal symptoms or diarrhoea in LGG treated patients compared to placebo could be found. LGG was well tolerated with no adverse effects including bacteremic outbreaks could be observed. The use of probiotic LGG can be regarded safe in this immunocompromised patient group.

A proteomic view of probiotic Lactobacillus rhamnosus GG

Lactobacillus rhamnosus GG is a probiotic bacterium that is known worldwide. Since its discovery in 1985, the health effects and biology of this health-promoting strain have been researched at an increasing rate. However, knowledge of the molecular biology responsible for these health effects is limited, even though research in this area has continued to grow since the publication of the whole genome sequence of L. rhamnosus GG in 2009. In this thesis, the molecular biology of L. rhamnosus GG was explored by mapping the changes in protein levels in response to diverse stress factors and environmental conditions. The proteomics data were supplemented with transcriptome level mapping of gene expression. The harsh conditions of the gastro-intestinal tract, which involve acidic conditions and detergent-like bile acids, are a notable challenge to the survival of probiotic bacteria. To simulate these conditions, L. rhamnosus GG was exposed to a sudden bile stress, and several stress response mechanisms were revealed, among others various changes in the cell envelope properties. L. rhamnosus GG also responded in various ways to mild acid stress, which probiotic bacteria may face in dairy fermentations and product formulations. The acid stress response of L. rhamnosus GG included changes in central metabolism and specific responses related to the control of intracellular pH. Altogether, L. rhamnosus GG was shown to possess a large repertoire of mechanisms for responding to stress conditions, which is a beneficial character of a probiotic organism. Adaptation to different growth conditions was studied by comparing the proteome level responses of L. rhamnosus GG to divergent growth media and to different phases of growth. Comparing different growth phases revealed that the metabolism of L. rhamnosus GG is modified markedly during shift from the exponential to the stationary phase of growth. These changes were seen both at proteome and transcriptome levels and in various different cellular functions. When the growth of L. rhamnosus GG in a rich laboratory medium and in an industrial whey-based medium was compared, various differences in metabolism and in factors affecting the cell surface properties could be seen. These results led us to recommend that the industrial-type media should be used in laboratory studies of L. rhamnosus GG and other probiotic bacteria to achieve a similar physiological state for the bacteria as that found in industrial products, which would thus yield more relevant information about the bacteria. In addition, an interesting phenomenon of protein phosphorylation was observed in L. rhamnosus GG. Phosphorylation of several proteins of L. rhamnosus GG was detected, and there were hints that the degree of phosphorylation may be dependent on the growth pH.

Moonlighting Proteins of Lactobacillus crispatus : Extracellular Localization, Cell Wall Anchoring and Interactions with the Host

Moonlighting functions have been described for several proteins previously thought to localize exclusively in the cytoplasm of bacterial or eukaryotic cells. Moonlighting proteins usually perform conserved functions, e. g. in glycolysis or as chaperonins, and their traditional and moonlighting function(s) usually localize to different cell compartments. The most characterized moonlighting proteins in Grampositive bacteria are the glycolytic enzymes enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which function in bacteria-host interactions, e. g. as adhesins or plasminogen receptors. Research on bacterial moonlighting proteins has focused on Gram-positive bacterial pathogens, where many of their functions have been associated with bacterial virulence. In this thesis work I show that also species of the genus Lactobacillus have moonlighting proteins that carry out functions earlier associated with bacterial virulence only. I identified enolase, GAPDH, glutamine synthetase (GS), and glucose-6-phosphate isomerase (GPI) as moonlighting proteins of Lactobacillus crispatus strain ST1 and demonstrated that they are associated with cell surface and easily released from the cell surface into incubation buffer. I also showed that these lactobacillar proteins moonlight either as adhesins with affinity for basement membrane and extracellular matrix proteins or as plasminogen receptors. The mechanisms of surface translocation and anchoring of bacterial moonlighting proteins have remained enigmatic. In this work, the surface localization of enolase, GAPDH, GS and GPI was shown to depend on environmental factors. The members of the genus Lactobacillus are fermentative organisms that lower the ambient pH by producing lactic acid. At acidic pH enolase, GAPDH, GS and GPI were associated with the cell surface, whereas at neutral pH they were released into the buffer. The release did not involve de novo protein synthesis. I showed that purified recombinant His6-enolase, His6-GAPDH, His6-GS and His6-GPI reassociate with cell wall and bind in vitro to lipoteichoic acids at acidic pH. The in-vitro binding of these proteins localizes to cell division septa and cell poles. I also show that the release of moonlighting proteins is enhanced in the presence of cathelicidin LL- 37, which is an antimicrobial peptide and a central part of the innate immunity defence. I found that the LL-37-induced detachment of moonlighting proteins from cell surface is associated with cell wall permeabilization by LL-37. The results in this thesis work are compatible with the hypothesis that the moonlighting proteins of L. crispatus associate to the cell wall via electrostatic or ionic interactions and that they are released into surroundings in stress conditions. Their surface translocation is, at least in part, a result from their release from dead or permeabilized cells and subsequent reassociation onto the cell wall. The results of this thesis show that lactobacillar cells rapidly change their surface architecture in response to environmental factors and that these changes influence bacterial interactions with the host.

Enhanced Survival of Probiotic Lactobacillus acidophilus by Encapsulation with Nanostructured Polyelectrolyte Layers through Layer-by-Layer Approach

The encapsulation of probiotic Lactobacillus acidophilus through layer-by-layer self-assembly of polyelectrolytes (PE) chitosan (CHI) and carboxymethyl cellulose (CMC) has been investigated,to enhance its survival m adverse conditions encountered in the GI tract The survival of encapsulated cells in simulated gastric (SGF) and intestinal fluids (SIF) is significant when compared to nonencapsulated cells On sequential exposure to SGF and SIF fox 120 nun, almost complete death of free cells is observed However, for cells coated with three nanolayers of PEs (CHI/CMC/CHI) about 33 log % of the cells (6 log cfu/500 mg) survived under the same conditions The enhanced survival rate of encapsulated L acidophilus can be attributed to the impermeability of polyelectrolyte nanolayers to large enzyme molecules like pepsin, and pancreatin that cause proteolysis and to the stability of the polyelectrolyte nanolayers in gastric and intestinal pH The PE coating also serves to reduce viability losses during freezing and freeze- drying About 73 and 92 log % of uncoated and coated cells survived after freeze:drying, and the losses occurring between freezing and freeze-drying were found to be lower for coated cells

Influence of a fish bacterium Lactobacillus sp. on the production of swordtail xiphophorus helleri (Heckel 1848)

The influence of a fish gut bacterium Lactobacillus sp on the production of swordtail Xiphophorus helleri was studied for a period of one year. The Lactobacillus sp P21 produced bacteriocin-like inhibitory substance and exhibited wide spectrum of action against Aeromonas hydrophila, Bacillus spp, Pseudomonas spp and Citrobacter freundi in vitro. The growth performance of X. helleri reared in the presence of Lactobacillus P21 at 106/ml rearing water was better than the control. The total plate counts, total MRS agar counts and the counts of motile aeromonads, presumptive pseudomonads, lactose fermenters and lactose non-fermenters in the gut of probiotic group were comparatively low than the control. On day 60 the count of Lactobacillus sp P21 was observed to be log 5.28/g in the gut of X. helleri indicating colonization of this bacterium in the gastrointestinal tract. The fecundity of X. helleri was in the range of 9-134. On average, it produced from 39.42±18.72 fry/female in control group to 53.00±23.57 fry/female in probiotic group. The increase in average fecundity in probiotic group over the control group was about 25%. There existed significant difference between probiotic group and control in respect of average fecundity/female (p<0.02), average number of fry survived /female (p<0.006) and average number of fry dead/female (p<0.029). The results of the present study demonstrated that the rearing of X. helleri in probiotic-enriched water have growth inducing ability and favourably influenced the reproductive performance in terms of high fecundity, high fry survival, reduced fry mortality and reduced fry deformity.

Effect of feeding bioencapsulated Lactobacillus sp. in live Tubifex sp. on the growth performance of gold fish Carassius auratus (Linnaeus, 1758)

An attempt was made to feed bioencapsulate Lactobacillus sp. in live fish food organism Tubifex for use in the culture of gold fish Carassius auratus. The C. auratus fries when fed with bioencapsulated Lactobacillus sp. in Tubifex showed significant improvement in total wet weight gain (p<0.007) and FCR (p<0.01) compared to control. The specific growth rale and mean survival were slightly higher, although insignificantly (p>0.05) in bioencapsulated Tubifex fed group. None of the bacteriological parameters of the fish gut between the experimental and control groups differed significantly (p>0.05). Lactobacillus sp. was recorded at a level of log 5.11/g on the 90th day of experimentation. When the experimental C. auratus fries were infected with Pseudomonas fluorescents, the bioencapsulated Tubifex fed group resisted the infection. The survival was significantly higher (p<0.05) in bioencapsulated Tubifex fed group (44%) than in control (22%). The C. auratus fed with bioencapsulated Tubifex showed less (55%) signs of tail/fin rot. Likewise, a significant improvement in total wet weight gain (p<0.009), FCR (p<0.01) and SGR (p<0.04) of C. auratus brooder fed with bioencapsulated Tubifex was seen compared to control group fed with depurated Tubifex.