Nasal administration of Lactococcus lactis improves local and systemic immune responses against Streptococcus pneumoniae

Lactococcus lactis NZ9000 is a non-pathogenic non-invasive bacterium extensively used for the delivery of antigens and cytokines at the mucosal level. However, there are no reports concerning the per se immunomodulatory capacity of this strain. The aim of the present study was to investigate the intrinsic immunostimulating properties of the nasal administration of L. lactis NZ9000 in a pneumococcal infection model. Mice were preventively treated with L. lactis (2, 5 or 7 days with 10(8) cells/day per mouse) and then challenged with Streptococcus pneumoniae. The local and the systemic immune responses were evaluated. Our results showed that nasal administration of L. lactis for 5 days (LLN5d) increased the clearance rate of S. pneumoniae from lung and prevented the dissemination of pneumococci into blood. This effect coincided with an upregulation of the innate and specific immune responses in both local and systemic compartments. LLN5d increased phagocyte activation in lung, blood and bone marrow, determined by NBT and peroxidase tests. Anti-pneumococcal immunoglobulin (Ig)A in bronchoalveolar lavages (BAL) and IgG in BAL and serum were increased in the LLN5d group. Lung tissue injury was reduced by LLN5d treatment as revealed by histopathological examination and albumin concentration and lactate dehydrogenase activity in BAL. The adjuvant effect of L. lactis in our infection model would be an important advantage for its use as a delivery vehicle of pneumococcal proteins and nasal immunization with recombinant L. lactis emerges as an effective route of vaccination for both systemic and mucosal immunity against pneumococcal infection.     

Comparison of Giardia lamblia and Giardia muris cyst inactivation by ozone.

Inactivation of Giardia lamblia and Giardia muris cysts was compared by using an ozone demand-free 0.05 M phosphate buffer in bench-scale batch reactors at 22 degrees C. Ozone was added to each trial from a concentrated stock solution for contact times of 2 and 5 min. The viability of the control and treated cysts was evaluated by using the C3H/HeN mouse and Mongolian gerbil models for G. muris and G. lamblia, respectively. The resistance of G. lamblia to ozone was not significantly different from that of G. muris under the study conditions, contrary to previously reported data that suggested G. lamblia was significantly more sensitive to ozone than G. muris was. The simple Ct value for 2 log unit inactivation of G. lamblia was 2.4 times higher than the Ct value recommended by the Surface Water Treatment Rule.     

The ultrastructure of the cyst wall of Giardia lamblia

Giardiasis is the most common human protozoal infection. In their cystic phase, giardias are protected from the environment by a filamentous cyst wall made up of carbohydrates, proteins, and by two outer membranes separated from the plasma membrane of the parasite by a peripheral space. The present transmission electron microscope observations of G. lamblia cysts of human origin suggest that the extracellular peritrophic space originates from the growth, elongation, and fusion of large cytoplasmic vacuoles. As the large clear vacuoles grew in size, flattening against the inner face of the plasma membrane, they formed a single vacuole that surrounded the body of the parasite, eventually forming two outer membranes. In mature Giardia cysts, the original plasma membrane of the trophozoite becomes the outermost membrane of the cyst wall (CM1). The large vacuoles form a second membrane surrounding the cyst (CM2), and also form a third membrane (CM3), that becomes the new plasma membrane of the trophozoite. During excystation CM1 and CM2 attach to each other and fragment, leaving abundant membrane residues in the peritrophic space. Knowledge of the biochemical composition and functional properties of the complex outer membranous system of G. lamblia cysts here described will be of use to understand the survival of Giardia cysts in the environment, a major factor responsible for the high prevalence of giardiasis worldwide.     

Antigenic variation and the murine immune response to Giardia lamblia

The protozoan parasite Giardia lamblia is an important causative agent of acute or chronic diarrhoea in humans and various animals. During infection, the parasite survives the hosts reactions by undergoing continuous antigenic variation of its major surface antigen, named VSP (variant surface protein). The VSPs form a unique family of cysteine-rich proteins that are extremely heterogeneous in size. The relevance of antigenic variation for the survival in the host has been most successfully studied by performing experimental infections in a combined mother/offspring mouse system and by using the G. lamblia clone GS/M-83-H7 (human isolate) as model parasite. In-vivo antigenic variation of G. lamblia clone GS/M-83-H7 is characterised by a diversification of the intestinal parasite population into a complex mixture of different variant antigen types. It could be shown that maternally transferred lactogenic anti-VSP IgA antibodies exhibit cytotoxic activity on the Giardia variant-specific trophozoites in suckling mice, and thus express a modulatory function on the proliferative parasite population characteristics. Complementarily, in-vitro as well as in-vivo experiments in adult animals indicated that non-immunological factors such as intestinal proteases may interfere into the process of antigen variation in that they favour proliferation of those variant antigen-type populations which resist the hostile physiological conditions within the intestine. These observations suggest that an interplay between immunological and physiological factors, rather than one of these two factor alone, modulates antigenic diversification of a G. lamblia population within an experimental murine host and thus influences the survival rate and strategy of the parasite.     

Reversible interruption of Giardia lamblia cyst wall protein transport in a novel regulated secretory pathway

To survive in the environment and infect a new host, Giardia lamblia secretes an extracellular cyst wall using a poorly understood pathway. The two cyst wall proteins (CWPs) form disulphide-bonded heterodimers and are exported via novel encystation-specific secretory vesicles (ESVs). Exposure of eukaryotic cells to dithiothreitol (DTT) blocks the formation of disulphide bonds in nascent proteins that accumulate in the endoplasmic reticulum (ER) and induces an unfolded protein response (UPR). Proteins that have exited the ER are not susceptible. Exposure to DTT inhibits ESV formation by > 85%. Addition of DTT to encysting cells causes rapid ( t _1/2 < 10 min), reversible disappearance of ESVs, correlated with reduction of CWPs to monomers and reformation of CWP oligomers upon removal of DTT. Neither CWPs nor ESVs are affected by mercaptoethanesulphonic acid, a strong reducing agent that does not penetrate cells. DTT does not inhibit the overall protein secretory pathway, and recovery does not require new protein synthesis. We found evidence of protein disulphide isomerases in the ESV and the surface of encysting cells, in which they may catalyse initial CWP folding and recovery from DTT. This is the first suggestion of non-CWP proteins in ESVs and of enzymes on the giardial surface. DTT treatment did not stimulate a UPR, suggesting that Giardia may have diverged before the advent of this conserved form of ER quality control.     

Antigenic variation in Giardia lamblia and the host's immune response.

Giardia lamblia, a protozoan parasite of the small intestine of humans and other animals, undergoes surface antigenic variation. The antigens involved belong to a family of variant-specific surface proteins (VSPs), which are unique, cysteine-rich zinc finger proteins. The patterns of infection in humans and animals fail to show the expected cyclical waves of increasing and decreasing numbers of parasites expressing unique VSPs. Nevertheless, changes in VSP expression occur within the population in vivo owing to selection of VSPs by both immune and non-immune mechanisms. After inoculation of a single G. lamblia clone (able to persist in the absence of immune pressure) expressing one VSP (> or = 90%) into mice or humans, the original VSP continues to be expressed until 2 weeks post inoculation (p.i.), when many other VSPs gradually replace it. Selection by immune-mediated processes is suggested because switching occurs at the same time that humoral responses are first detected. In most mouse strains, switching also occurs at about two weeks. Almost all trophozoites are eliminated at three weeks (p.i.), but a barely detectable infection persists over months. In neonatal mice, apparent self-cure is delayed until the sixth or seventh week. Antigenic switching does not occur in adult or neonatal severe combined immunodeficiency disease (SCID) mice, but does occur in neonatal nude mice, thus implicating B-cell-mediated mechanisms in immune switching. Not all VSPs are expressed to the same degree in vivo. Some VSPs appear to be preferentially selected whereas others are eliminated on a non-immune basis. In infections in which immunity does not play a role, such as in SCID mice, and during the first week of infection in immunocompetent mice or gerbils, persisting VSPs are preferentially expressed and maintained whereas non-persisting VSPs are replaced within the first week of infection. The purpose of antigenic variation may be presentation of a wide assortment of VSPs to hosts, increasing the chance of a successful initial infection or reinfection. Immune selection of variants comes into play following biological selection.     

Oral immunization of BALB/c mice by intragastric delivery of Streptococcus gordonii-expressing Giardia cyst wall protein 2 decreases cyst shedding in challenged mice

Giardia lamblia (Giardia duodenalis or Giardia intestinalis) is a protozoan parasite of vertebrates with broad host specificity. Specific antibodies directed against cyst antigens can interfere with the cyst wall-building process. In this study, we engineered Streptococcus gordonii to express a 26 kDa fragment of cyst wall protein 2 (CWP2), containing a relevant B cell epitope, on the cell surface. This is the first report of S. gordonii expressing a protein of parasite origin. As S. gordonii was intended for intestinal delivery of CWP2, it was determined that this oral commensal bacterium is able to persist in the murine intestine for 30 days. Immunization with recombinant streptococci expressing the 26 kDa fragment resulted in higher antibody levels. Specific anti-CWP2 IgA antibodies were detected in fecal samples and anti-CWP2 IgG antibodies were detected in serum demonstrating the efficacy of S. gordonii for intragastric antigen delivery. In a pilot challenge experiment, immunized mice demonstrated a significant 70% reduction in cyst output.     

Comparison of cell wall proteinases from Lactococcus lactis subsp. cremoris AC1 and Lactococcus lactis subsp. lactis NCDO 763

Summary The cell wall proteinases of Lactococcus lactis subsp. lactis NCDO 763 and L. lactis subsp. cremoris AC1 hydrolyse -casein with a similar specificity even though some quantitative differences can be observed for a few degradation products analysed by reverse phase HPLC and sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The main peptides soluble in 1.1% trifluoroacetic acid and liberated by the two proteinases were identified and have been found to be the same for the two enzymes. They are located in two areas of the -casein sequence (53–93 and the C-terminal part: 129–209) and they include bitter tasting or physiologically active fragments. No narrow specificity was observed for these proteinases. However, glutamine and serine residues are more frequently encountered in position P1 and P1 of the sensitive peptide bond and the close environment (position P2 to P4 and P2 to P4) of the cleaved bond is mainly hydrophobic.     

Comparison of cell wall proteinases from Lactococcus lactis subsp. cremoris AC1 and Lactococcus lactis subsp. lactis NCDO 763

Summary Cell wall-associated proteinases were isolated from Lactococcus lactis subsp. cremoris AC1 and subsp. lactis NCDO 763 in order to compare their specificities towards different caseins. Two purification strategies were applied. Cells grown in casein-free M17 medium were a suitable starting material for purification, since electrophoretic purity could be achieved after one chromatographic step. Both enzymes has an apparent molecular mass of about 145000 daltons as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Electrophoresis and reversed phase HPLC patterns of hydrolysates of _s1-, _s2-, -, and K-caseins indicated that both proteinases had a similar specificity. The enzyme of L. lactis subsp. lactis split _s1- and _s2-caseins more extensively than that of L. lactis subsp. cremoris.     

Enhancement of recombinant streptokinase production in Lactococcus lactis by suppression of acid tolerance response

Lactococcus lactis is a potential host for production of recombinant proteins, especially of therapeutic importance. However, in glucose-grown cultures, lowering of pH due to accumulation of lactic acid and the concomitant induction of acid tolerance response (ATR) may affect the recombinant protein produced. In this work, we have analyzed the effect of culture pH and the associated ATR on production of recombinant streptokinase. Streptokinase gene was cloned and expressed as a secretory protein in L. lactis under the control of P170 promoter. It was found to undergo degradation to form inactive products leading to low productivity. The extent of degradation and productivity of streptokinase was greatly influenced by the development of ATR, which was dependent on the pH of the culture and initial phosphate concentration of the medium. It was found that high pH and high initial phosphate concentration leads to suppression of ATR and this results in at least 2.5-fold increase in streptokinase productivity and significant decrease in degradation of streptokinase.     

Optimization of fed-batch production of the model recombinant protein GFP in Lactococcus lactis

Optimization of recombinant protein production using lactic acid bacteria (LAB) remains an important obstacle on the road to realizing LAB as oral vaccine delivery vehicles. Despite this, there have been few published investigations to explore the higher limits of LAB recombinant protein expression in fed-batch fermentations. In this study, results from response surface experiments suggested an optimal set of conditions for expression of green fluorescent protein (GFP), a model recombinant protein, in bench-scale, fed-batch Lactococcus lactis IL1403 fermentations. The 48 4-L fed-batch fermentations in this set of experiments, along with preliminary studies, investigated the effects of pH, temperature, hemin concentration, concentration of the nisin inducer per cell, and time of induction. Cell densities in this data set ranged from 2.9 to 7.4 g/L and maximum GFP expression per cell ranged from 0.1 to 4.4 relative fluorescence units (RFU)/g. The optimal 4-L, fed-batch fermentation process found here yields growth and protein expression values that dramatically improve upon results from traditional test tube and flask processes. Relative to the traditional process, the experimental optimum conditions yield 4.9 times the cell density, 1.6 times the protein per cell mass, and 8 times the total protein concentration. Unexpectedly, experiments also revealed that the compound hemin, known previously to improve growth and survival of Lactococcus lactis (L. lactis), negatively impacted recombinant protein production when added in concentrations from 5 to 20 microg/mL with this strain. The improvement in protein expression over traditional processes demonstrated here is an important step toward commercial development of LAB for oral delivery of recombinant vaccines and therapeutic proteins.     

Recognition of cyclonucleoside lesions by the Lactococcus lactis FPG protein

Several purine and pyrimidine cyclonucleosides were found to be not recognized by several Escherichia coli and yeast DNA N-glycosylases. Interestingly, a non covalent complex was observed between the Lactoccocus lactis formamidopyrimidine-DNA glycosylases (Fpg-Ll) and the cyclonucleosides. This may provide new information on the mechanism involved in the activity of the latter enzyme.