Cell Wall Anchoring of the Campylobacter Antigens to Lactococcus lactis

Campylobacter jejuni is the most frequent cause of human food-borne gastroenteritis and chicken meat is the main source of infection. Recent studies showed that broiler chicken immunization against Campylobacter should be the most efficient way to lower the number of human infections by this pathogen. Induction of the mucosal immune system after oral antigen administration should provide protective immunity to chickens. In this work we tested the usefulness of Lactococcus lactis, the most extensively studied lactic acid bacterium, as a delivery vector for Campylobacter antigens. First we constructed hybrid protein – CjaA antigen presenting CjaD peptide epitopes on its surface. We showed that specific rabbit anti-rCjaAD serum reacted strongly with both CjaA and CjaD produced by a wild type Campylobacter jejuni strain. Next, rCjaAD and CjaA were fused to the C-terminus of the L. lactis YndF containing the LPTXG motif. The genes expressing these proteins were transcribed under control of the L. lactis Usp45 promoter and their products contain the Usp45 signal sequences. This strategy ensures a cell surface location of both analysed proteins, which was confirmed by immunofluorescence assay. In order to evaluate the impact of antigen location on vaccine prototype efficacy, a L. lactis strain producing cytoplasm-located rCjaAD was also generated. Animal experiments showed a decrease of Campylobacter cecal load in vaccinated birds as compared with the control group and showed that the L. lactis harboring the surface-exposed rCjaAD antigen afforded greater protection than the L. lactis producing cytoplasm-located rCjaAD. To the best of our knowledge, this is the first attempt to employ LAB (Lactic Acid Bacteria) strains as a mucosal delivery vehicle for chicken immunization. Although the observed reduction of chicken colonization by Campylobacter resulting from vaccination was rather moderate, the experiments showed that LAB strains can be considered as an alternative vector to deliver heterologous antigens to the bird immune system. Additionally, the analysis of the structure and immunogenicity of the generated rCjaAD hybrid protein showed that the CjaA antigen can be considered as a starting point to construct multiepitope anti-Campylobacter vaccines

In vitro characteristics of Lactobacillus spp. strains isolated from the chicken digestive tract and their role in the inhibition of Campylobacter colonization

Campylobacter jejuni/coli infections are the leading cause of bacterial diarrheal illnesses in humans. Many epidemiological studies indicate that improperly prepared meat from chickens that carry a high load of Campylobacter in their intestinal tracts is the key source of human infections. LAB, mainly members of the Lactococcus and Lactobacillus genera, increasingly have been tested as vehicles for the delivery of heterologous bacterial or viral antigens to animal mucosal immune systems. Thus, the objective of this study was to isolate, identify, and characterize Lactobacillus spp. strains isolated from chickens bred in Poland. Their ability to decrease the level of bird gut colonization by C. jejuni strain was also analyzed. First, the influence of the different chicken rearing systems was evaluated, especially the effect of diets on the Lactobacillus species that colonize the gut of chickens. Next, selected strains were analyzed in terms of their anti-Campylobacter activity in vitro; potential probiotic traits such as adhesion properties, bile and low pH tolerance; and their ability to grow on a defined carbon source. Given that improperly prepared chicken meat is the main source of human infection by Campylobacter, the selected strains were also assessed for their ability to inhibit Campylobacter colonization in the bird's intestine. These experiments revealed enormous physiological diversity among the Lactobacillus genus strains. Altogether, our results showed that L. plantarum strains isolated from the digestive tracts of chickens bred in Poland displayed some probiotic attributes in vitro and were able to decrease the level of bird gut colonization by C. jejuni strain. This suggests that they can be employed as vectors to deliver Campylobacter immunodominant proteins to the bird's immune system to strengthen the efficacy of in ovo vaccination

Chicken Anti-Campylobacter Vaccine – Comparison of Various Carriers and Routes of Immunization

Campylobacter spp, especially the species Campylobacter jejuni, are important human enteropathogens responsible for millions of cases of gastro-intestinal disease worldwide every year. C. jejuni is a zoonotic pathogen, and poultry meat that has been contaminated by microorganisms is recognized as a key source of human infections. Although numerous strategies have been developed and experimentally checked to generate chicken vaccines, the results have so far had limited success. In this study, we explored the potential use of non-live carriers of Campylobacter antigen to combat Campylobacter in poultry. First, we assessed the effectiveness of immunization with orally or subcutaneously delivered GEM (Gram-positive Enhancer Matrix) particles carrying two Campylobacter antigens: CjaA and CjaD. These two immunization routes using GEMs as the vector did not protect against Campylobacter colonization. Thus, we next assessed the efficacy of in ovo immunization using various delivery systems: GEM particles and liposomes. The hybrid protein CjaAD, which is CjaA presenting CjaD epitopes on its surface, was employed as a model antigen. We found that CjaAD administered in ovo at embryonic development day 18 by both delivery systems resulted in significant levels of protection after challenge with a heterologous Campylobacter jejuni strain. In practice, in ovo chicken vaccination is used by the poultry industry to protect birds against several viral diseases. Our work showed that this means of delivery is also efficacious with respect to commensal bacteria such as Campylobacter. In this study, we evaluated the protection after one dose of vaccine given in ovo. We speculate that the level of protection may be increased by a post-hatch booster of orally delivered antigens

New strategies to combat infecious diseases - antivirulence drugs.

Stale rosnąca liczba szczepów bakterii patogennych opornych na stosowane w terapiach ludzi antybiotyki stanowi zagrożenie dla ludzkości. Zgodnie z danymi Europejskiego Centrum ds. Zapobiegania i Kontroli Chorób tylko w Europie odnotowywanych jest rocznie około 25.000 zgonów z powodu chorób będącymi konsekwencją infekcji powodowanych przez mikroorganizmy oporne na antybiotyki. Pomiędzy rokiem 1930 a 1962 zidentyfikowano i wprowadzono do terapii ponad 20 klas antybiotyków. Od tego czasu zarejestrowano tylko dwie nowe klasy antybiotyków. Skuteczna walka z chorobami zakaźnymi wymaga opracowania i wprowadzenia na rynek nowych klas leków o odmiennych od antybiotyków mechanizmach działania. Praca przeglądowa prezentuje najnowsze osiągnięcia dotyczące identyfikacji nowej klasy leków antybakteryjnych, tzw. leków blokujących procesy wirulencji. Omówione są zarówno leki blokujące konkretne czynniki wirulencji, jak i te, których celem działania są globalne procesy zachodzące w komórkach, istotne także dla procesów patogenezy.The rapid emergence of resistant bacteria occurring in many parts of the world constitutes an increasing risk to public health. According to European Centre for Disease Prevention and Control (ECDC), in 2009 infections caused by a subset of resistant bacteria were responsible for about 25 000 deaths in Europe. The issue of resistance concerns both gram-positive and gram-negative pathogens that cause infections in the hospitals and in the community. The success in combat against infectious diseases depends upon development of effective anti-infective drugs. More than 20 novel classes of antibiotics were introduced into market between 1930 and 1962. Since then only two new classes of antibiotics have been approved for clinical use. This review presents recent advances toward the development of alternative medicines to classical antibiotics, antivirulence drugs, and highlights their benefits and disadvantages over conventional antibacterials. There are described both potential drugs aimed at single targets as well as those able to inhibit global cellular processes essential for virulence

Lactic acid bacteria--20 years exploring their potential as live vectors for mucosal vaccination.

Lactic acid bacteria (LAB) are a diverse group of Gram-positive, nonsporulating, low G + C content bacteria. Many of them have been given generally regarded as safe status. Over the past two decades, intensive genetic and molecular research carried out on LAB, mainly Lactococcus lactis and some species of the Lactobacillus genus, has revealed new, potential biomedical LAB applications, including the use of LAB as adjuvants, immunostimulators, or therapeutic drug delivery systems, or as factories to produce therapeutic molecules. LAB enable immunization via the mucosal route, which increases effectiveness against pathogens that use the mucosa as the major route of entry into the human body. In this review, we concentrate on the encouraging application of Lactococcus and Lactobacillus genera for the development of live mucosal vaccines. First, we present the progress that has recently been made in the field of developing tools for LAB genetic manipulations, which has resulted in the successful expression of many bacterial, parasitic, and viral antigens in LAB strains. Next, we discuss the factors influencing the efficacy of the constructed vaccine prototypes that have been tested in various animal models. Apart from the research focused on an application of live LABs as carriers of foreign antigens, a lot of work has been recently done on the potential usage of nonliving, nonrecombinant L. lactis designated as Gram-positive enhancer matrix (GEM), as a delivery system for mucosal vaccination. The advantages and disadvantages of both strategies are also presented

Lactic acid bacteria as a surface display platform for Campylobacter jejuni antigens.

BACKGROUND Food poisoning and diarrheal diseases continue to pose serious health care and socioeconomic problems worldwide. Campylobacter spp. is a very widespread cause of gastroenteritis. Over the past decade there has been increasing interest in the use of lactic acid bacteria (LAB) as mucosal delivery vehicles. They represent an attractive opportunity for vaccination in addition to vaccination with attenuated bacterial pathogens. METHODS We examined the binding ability of hybrid proteins to nontreated or trichloroacetic acid (TCA)-pretreated LAB cells by immunofluorescence and Western blot analysis. RESULTS In this study we evaluated the possibility of using GEM (Gram-positive enhancer matrix) particles of Lactobacillus salivarius as a binding platform for 2 conserved, immunodominant, extracytoplasmic Campylobacter jejuni proteins: CjaA and CjaD. We analyzed the binding ability of recombinant proteins that contain C. jejuni antigens (CjaA or CjaD) fused with the protein anchor (PA) of the L. lactis peptidoglycan hydrolase AcmA, which comprises 3 LysM motifs and determines noncovalent binding to the cell wall peptidoglycan. Both fused proteins, i.e. 6HisxCjaAx3LysM and 6HisxCjaDx3LysM, were able to bind to nontreated or TCA-pretreated L. salivarius cells. CONCLUSION Our results documented that the LysM-mediated binding system allows us to construct GEM particles that present 2 C. jejuni antigens

Genomic and transcriptomic analysis of Ligilactobacillus salivarius IBB3154—in search of new promoters for vaccine construction

Transcriptomic analysis of the genome sequenced Ligilactobacillus salivarius strain IBB3154 grown at two different temperatures (37°C vs 42°C) identified differentially expressed genes involved in metabolic pathways, osmoregulation, and surface protein expression. Two highly expressed genes, sasA1 and sasA2, which encode cell wall-anchored proteins belonging to the serine-rich repeat protein group, were found to be temperature-inducible. Moonlighting proteins with various functions, such as glyceraldehyde 3-phosphate dehydrogenase, fructose-bisphosphate aldolase, elongation factor Tu, and enolase, were highly expressed at both temperatures. The efficiency of promoters has been confirmed by the β-glucuronidase activity test; however, temperature dependence was not detected. We also found that the P sasA1 promoter retained its activity in the presence of bile salts. Knowledge of promoters that are highly active in L. salivarius cells can be used to produce strains that are carriers of immunogenic proteins