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

IMPORTANCE: The genome of the strain Ligilactobacillus salivarius IBB3154 was sequenced, and transcriptome analysis was carried out at two different temperatures, allowing the determination of gene expression levels in response to environmental changes (temperature). Genes with higher expression at 42°C were identified. The use of a reporter gene (ß- glucuronidase) did not confirm the transcriptomic results; it was found that the promoters of the genes sasA1 and sasA2 were active in the presence of bile salts. This opens up new opportunities for the overexpression of genes of other bacterial species in Ligilactobacillus cells in the intestinal environment.

Structure and functions of a multireplicon genome of Antarctic Psychrobacter sp. ANT_H3: characterization of the genetic modules suitable for the construction of the plasmid-vectors for cold-active bacteria.

Among Psychrobacter spp., there are several multireplicon strains, carrying more than two plasmids. Psychrobacter sp. ANT_H3 carries as many as 11 extrachromosomal replicons, which is the highest number in Psychrobacter spp. Plasmids of this strain were subjected to detailed genomic analysis, which enables an insight into the structure and functioning of this multireplicon genome. The replication and conjugal transfer modules of ANT_H3 plasmids were analyzed functionally to discover their potential for being used as building blocks for the construction of novel plasmid-vectors for cold-active bacteria. It was shown that two plasmids have a narrow host range as they were not able to replicate in species other than Psychrobacter, while remaining plasmids had a wider host range and were functional in various Alpha- and Gammaproteobacteria. Moreover, it was confirmed that mobilization modules of seven plasmids were functional, i.e., could be mobilized for conjugal transfer by the RK2 conjugation system. Auxiliary genes were also distinguished in ANT_H3 plasmids, including these encoding putative DNA-protecting protein DprA, multidrug efflux SMR transporter of EmrE family, glycine cleavage system T protein, MscS small-conductance mechanosensitive channel protein, and two type II restriction-modification systems. Finally, all genome-retrieved plasmids of Psychrobacter spp. were subjected to complex genome- and proteome-based comparative analyses showing that Antarctic replicons are significantly different from plasmids from other locations.

Nonlinear operation of an FP laser with PT symmetry active medium.

In this paper, an analysis of the nonlinear laser operation in an active medium made of a parity time (PT) symmetric structure placed in a Fabry-Perot (FP) resonator is demonstrated for the first time. The FP mirrors' reflection coefficients and phases, the PT symmetric structure period, primitive cell number, and the gain and loss saturation effects are taken into account in a presented theoretical model. The modified transfer matrix method is used to obtain characteristics of laser output intensity. Numerical results show that the selection of the appropriate phase of the FP resonator's mirrors makes it possible to obtain different levels of the output intensity. Moreover, for certain value of a ratio of the grating period to the operating wavelength, it is possible to obtain the bistability effect.

Differential Localization and Functional Specialization of parS Centromere-Like Sites in repABC Replicons of Alphaproteobacteria.

Partitioning systems ensure the stable inheritance of bacterial low-copy-number replicons, such as chromosomes, chromids, and megaplasmids. These loci consist of two genes encoding partition proteins A and B, and at least one parS centromere-like sequence. In chromids and megaplasmids, partitioning systems are often located in the vicinity of replication systems. An extreme example of this co-localization are alphaproteobacterial repABC replicons, where the partition (repAB) and replication (repC) genes form a single operon, with parS sequences usually positioned in close proximity to these genes. In this study, we characterized a more complex repABC system found in Paracoccus aminophilus (Rhodobacterales) megaplasmid pAMI4 (438 kb). Besides the repABC operon with a single parS site, this replicon has a 2-kb non-coding locus positioned 11.5 kb downstream of repC, which contains three additional parS repeats (3parS). We demonstrated that 3parS is bound by partition protein B in vitro and is essential for proper pAMI4 partitioning in vivo. In search of similar loci, we conducted a comparative analysis of parS distribution in other repABC replicons. This revealed different patterns of parS localization in Rhodobacterales and Rhizobiales. However, in both these taxonomic orders, parS sites are almost always located inside or close to the repABC operon. No other 3parS-like loci were found in the closest relatives of pAMI4. Another evolutionarily-independent example of such a locus was identified as a conserved feature in chromosome 2 of Allorhizobium vitis and related replicons. IMPORTANCE The repABC replication/partitioning loci are widespread in extrachromosomal replicons of Alphaproteobacteria. They are evolutionarily diverse, subject to multi-layer self-regulation, and are responsible for the maintenance of different types of replicons, such as plasmids (e.g., Agrobacterium pTi and pRi tumorigenic and rhizogenic plasmids), megaplasmids (e.g., Sinorhizobium pSymA and pSymB) and essential chromids (e.g., secondary chromosomes of Agrobacterium, Brucella and Rhodobacter). In this study, we functionally analyzed an atypical partition-related component of repABC systems, the 3parS locus, found in the P. aminophilus megaplasmid pAMI4. We also identified parS centromere-like site distribution patterns in different groups of repABC replicons and found other unrelated 3parS-like loci, which had been overlooked. Our findings raise questions concerning the biological reasons for differential parS distribution, which may reflect variations in repABC operon regulation as well as different replication and partition modes of replicons belonging to the repABC family.

Delivery of Toxins and Effectors by Bacterial Membrane Vesicles.

Pathogenic bacteria interact with cells of their host via many factors. The surface components, i.e., adhesins, lipoproteins, LPS and glycoconjugates, are particularly important in the initial stages of colonization. They enable adhesion and multiplication, as well as the formation of biofilms. In contrast, virulence factors such as invasins and toxins act quickly to damage host cells, causing tissue destruction and, consequently, organ dysfunction. These proteins must be exported from the bacterium and delivered to the host cell in order to function effectively. Bacteria have developed a number of one- and two-step secretion systems to transport their proteins to target cells. Recently, several authors have postulated the existence of another transport system (sometimes called "secretion system type zero"), which utilizes extracellular structures, namely membrane vesicles (MVs). This review examines the role of MVs as transporters of virulence factors and the interaction of toxin-containing vesicles and other protein effectors with different human cell types. We focus on the unique ability of vesicles to cross the blood-brain barrier and deliver protein effectors from intestinal or oral bacteria to the central nervous system.

Interplay between DsbA1, DsbA2 and C8J_1298 Periplasmic Oxidoreductases of Campylobacter jejuni and Their Impact on Bacterial Physiology and Pathogenesis.

The bacterial proteins of the Dsb family catalyze the formation of disulfide bridges between cysteine residues that stabilize protein structures and ensure their proper functioning. Here, we report the detailed analysis of the Dsb pathway of Campylobacter jejuni. The oxidizing Dsb system of this pathogen is unique because it consists of two monomeric DsbAs (DsbA1 and DsbA2) and one dimeric bifunctional protein (C8J_1298). Previously, we showed that DsbA1 and C8J_1298 are redundant. Here, we unraveled the interaction between the two monomeric DsbAs by in vitro and in vivo experiments and by solving their structures and found that both monomeric DsbAs are dispensable proteins. Their structures confirmed that they are homologs of EcDsbL. The slight differences seen in the surface charge of the proteins do not affect the interaction with their redox partner. Comparative proteomics showed that several respiratory proteins, as well as periplasmic transport proteins, are targets of the Dsb system. Some of these, both donors and electron acceptors, are essential elements of the C. jejuni respiratory process under oxygen-limiting conditions in the host intestine. The data presented provide detailed information on the function of the C. jejuni Dsb system, identifying it as a potential target for novel antibacterial molecules.

Lactic Acid Bacteria - A Promising Tool for Controlling Chicken Campylobacter Infection.

Since 2005, campylobacteriosis has been the most common zoonotic disease in Europe. The main reservoir of pathogenic Campylobacter strains is broilers, which makes raw and undercooked poultry meat two major sources of disease. Infection in chicken flocks is most often asymptomatic, despite a high level of colonization reaching 106-109cfu/g in animal ceca. It is widely believed that controlling the level of colonization of the birds' digestive tract by pathogenic strains is a good way to increase food safety. Many treatments have been proposed to combat or at least reduce the level of colonization in animals reservoirs: probiotics, bacteriophages, vaccines, and anti-Campylobacter bacteriocins. This review focuses on the effects of Campylobacter infection on the chicken microbiome and colonization control strategies using probiotics (mostly lactic acid bacteria, LAB), which are live microorganisms included in the diet of animals as feed additives or supplements. Probiotics are not only an alternative to antibiotics, which were used for years as animal growth promoters, but they also constitute an effective protective barrier against excessive colonization of the digestive system by pathogenic bacteria, including Campylobacter. Moreover, one of the many beneficial functions of probiotics is the ability to manipulate the host's microbiota. Recently, there have also been some promising attempts to use lactic acid bacteria as a delivery system of oral vaccine against Campylobacter. Recombinant LAB strains induce primarily a mucosal immune response against foreign antigens, accompanied by at most a low-level immune response against carrier strains. Since the main barrier against the invasion of pathogens in the gastrointestinal tract is the intestinal mucosal membrane, the development of effective oral vaccines to protect animals against enteric infection is very reasonable.

FRAX prognostic and intervention thresholds in the management of major bone fractures in hemodialysis patients: A two-year prospective multicenter cohort study.

PURPOSE: The usefulness of FRAX in predicting major bone fractures in patients with end-stage kidney disease on maintenance hemodialysis treatment has been confirmed in previous studies. For meaningful clinical use, the prognostic and intervention FRAX thresholds need to be established. METHODS: The primary aim of our study was to calculate the optimal cut-off point of FRAX for the best prediction of an increased bone fracture risk in dialysis patients and additionally, to propose its intervention threshold, indicating the need for antifracture pharmacological treatment. The study included 718 hemodialysis patients, who were followed up for two years. Thirty low-energy major bone fractures were diagnosed during the study period. We used the Polish version of FRAX (without the DXA examination) and some particular variables of the FRAX calculator. The optimal cut-off point for prediction of an increased major bone fracture risk was based on the analysis of the sensitivity and specificity curves of FRAX. RESULTS: The analysis revealed FRAX >5% (sensitivity of 70.0%, specificity of 69.8%) as the prognostic threshold for major bone fractures. Its sensitivity for bone fracture prediction was significantly higher, but specificity lower than those of FRAX ≥10%, used in general Polish population. The reason for this can be an underestimation of bone fracture risk with FRAX in dialysis patients. CONCLUSIONS: We conclude that the FRAX prognostic threshold for identification of an increased risk of major bone fractures in hemodialysis patients is >5%. We propose to use this specific value of FRAX as an intervention threshold for pharmacological antifracture treatment in hemodialysis patients.

Influence of Environmental and Genetic Factors on Proteomic Profiling of Outer Membrane Vesicles from Campylobacter jejuni.

The proteomes of outer membrane vesicles (OMVs) secreted by C. jejuni 81-176 strain, which was exposed to oxygen or antibiotic stress (polymyxin B), were characterized. We also assessed the OMVs production and their content in two mutated strains - ∆dsbI and ∆htrA. OMVs production was significantly increased under the polymyxin B stress and remained unaltered in all other variants. Interestingly, the qualitative load of OMVs was constant regardless of the stress conditions or genetic background. However, certain proteins exhibited notable quantitative changes, ranging from 4-fold decrease to 10-fold increase. Up- or downregulated proteins (e.g. major outer membrane protein porA, iron ABC transporter, serine protease- htrA, 60 kDa chaperonin-groL, enolase) represented various cell compartments (cytoplasm, periplasm, and membrane) and exhibited various functions; nevertheless, one common group was noted that consisted of components of flagellar apparatus, i.e., FlaA/B, FlgC/E, which were mostly upregulated. Some of these proteins are the putative substrates of DsbI protein. Further investigation of the regulation of C. jejuni OMVs composition and their role in virulence will allow a better understanding of the infectious process of C. jejuni.The proteomes of outer membrane vesicles (OMVs) secreted by C. jejuni 81­176 strain, which was exposed to oxygen or antibiotic stress (polymyxin B), were characterized. We also assessed the OMVs production and their content in two mutated strains ­ ∆dsbI and ∆htrA. OMVs production was significantly increased under the polymyxin B stress and remained unaltered in all other variants. Interestingly, the qualitative load of OMVs was constant regardless of the stress conditions or genetic background. However, certain proteins exhibited notable quantitative changes, ranging from 4-fold decrease to 10-fold increase. Up- or downregulated proteins (e.g. major outer membrane protein porA, iron ABC transporter, serine protease- htrA, 60 kDa chaperonin-groL, enolase) represented various cell compartments (cytoplasm, periplasm, and membrane) and exhibited various functions; nevertheless, one common group was noted that consisted of components of flagellar apparatus, i.e., FlaA/B, FlgC/E, which were mostly upregulated. Some of these proteins are the putative substrates of DsbI protein. Further investigation of the regulation of C. jejuni OMVs composition and their role in virulence will allow a better understanding of the infectious process of C. jejuni.

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.

Evaluation of a protective effect of in ovo delivered Campylobacter jejuni OMVs.

Campylobacter jejuni is the most prevalent cause of a food-borne gastroenteritis in the developed world, with poultry being the main source of infection. Campylobacter jejuni, like other Gram-negative bacteria, constitutively releases outer membrane vesicles (OMVs). OMVs are highly immunogenic, can be taken up by mammalian cells, and are easily modifiable by recombinant engineering. We have tested their usefulness for an oral (in ovo) vaccination of chickens. Four groups of 18-day-old chicken embryos (164 animals) underwent injection of wt C. jejuni OMVs or modified OMVs or PBS into the amniotic fluid. The OMVs modifications relied on overexpression of either a complete wt cjaA gene or the C20A mutant that relocates to the periplasm. Fourteen days post-hatch chicks were orally challenged with live C. jejuni strain. Cecum colonization parameters were analyzed by two-way ANOVA with Tukey post-hoc test. The wtOMVs and OMVs with wtCjaA overexpression were found to confer significant protection of chicken against C. jejuni (p = 0.03 and p = 0.013, respectively) in comparison to PBS controls and are promising candidates for further in ovo vaccine development.

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 Gram-positive Enhancer Matrix (GEM) 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 rCjaAD, which is CjaA presenting CjaD epitopes on its surface, was employed as a model antigen. We found that rCjaAD administered in ovo at embryonic development day 18 by both delivery systems resulted in significant levels of protection after challenge with a heterologous C. 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.

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 C. 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 analyzed 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 Lactic Acid Bacteria (LAB) 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.

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.

Evaluation of the immunogenicity of Campylobacter jejuni CjaA protein delivered by Salmonella enterica sv. Typhimurium strain with regulated delayed attenuation in chickens.

Campylobacter spp. are regarded as the most common bacterial cause of gastroenteritis worldwide, and consumption of chicken meat contaminated by Campylobacter is considered to be one of the most frequent sources of human infection in developed countries. Here we evaluated the immunogenicity and protective efficacy of Salmonella Typhimurium χ9718 producing the Campylobacter jejuni CjaA protein as a chicken anti-Campylobacter vaccine. In this study chickens were orally immunized with a new generation S. Typhimurium strain χ9718 with regulated delayed attenuation in vivo and displaying delayed antigen expression. The immunization with the S. Typhimurium χ9718 strain producing C. jejuni CjaA antigen induced strong immune responses against CjaA in both serum IgY and intestinal IgA, however, it did not result in the significant reduction of intestinal colonization by Campylobacter strain. The low level of protection might arise due to a lack of T cell response. Our results demonstrated that a Salmonella strain with regulated delayed attenuation and displaying regulated delayed antigen expression might be an efficient vector to induce immune response against Campylobacter. It seems that an efficient anti-Campylobacter subunit vaccine should be multicomponent. Since S. Typhimurium χ9718 contains two compatible balanced-lethal plasmids, it can provide the opportunity of cloning several Campylobacter genes encoding immunodominant proteins. It may also be used as a delivery vector of eukaryotic genes encoding immunostimulatory molecules to enhance or modulate functioning of chicken immune system.

Campylobacter jejuni dsb gene expression is regulated by iron in a Fur-dependent manner and by a translational coupling mechanism.

BACKGROUND: Many bacterial extracytoplasmic proteins are stabilized by intramolecular disulfide bridges that are formed post-translationally between their cysteine residues. This protein modification plays an important role in bacterial pathogenesis, and is facilitated by the Dsb (disulfide bond) family of the redox proteins. These proteins function in two parallel pathways in the periplasmic space: an oxidation pathway and an isomerization pathway. The Dsb oxidative pathway in Campylobacter jejuni is more complex than the one in the laboratory E. coli K-12 strain. RESULTS: In the C. jejuni 81-176 genome, the dsb genes of the oxidative pathway are arranged in three transcriptional units: dsbA2-dsbB-astA, dsbA1 and dba-dsbI. Their transcription responds to an environmental stimulus - iron availability - and is regulated in a Fur-dependent manner. Fur involvement in dsb gene regulation was proven by a reporter gene study in a C. jejuni wild type strain and its isogenic fur mutant. An electrophoretic mobility shift assay (EMSA) confirmed that analyzed genes are members of the Fur regulon but each of them is regulated by a disparate mechanism, and both the iron-free and the iron-complexed Fur are able to bind in vitro to the C. jejuni promoter regions. This study led to identification of a new iron- and Fur-regulated promoter that drives dsbA1 gene expression in an indirect way. Moreover, the present work documents that synthesis of DsbI oxidoreductase is controlled by the mechanism of translational coupling. The importance of a secondary dba-dsbI mRNA structure for dsbI mRNA translation was verified by estimating individual dsbI gene expression from its own promoter. CONCLUSIONS: The present work shows that iron concentration is a significant factor in dsb gene transcription. These results support the concept that iron concentration - also through its influence on dsb gene expression - might control the abundance of extracytoplasmic proteins during different stages of infection. Our work further shows that synthesis of the DsbI membrane oxidoreductase is controlled by a translational coupling mechanism. The dba expression is not only essential for the translation of the downstream dsbI gene, but also Dba protein that is produced might regulate the activity and/or stability of DsbI.

Update on Campylobacter jejuni vaccine development for preventing human campylobacteriosis.

Campylobacteriosis constitutes a serious medical and socioeconomic problem worldwide. Rapidly increasing antibiotic resistance of bacterial strains compels us to develop alternative therapeutic strategies and to search for efficient immunoprophylactic methods. The vast majority of Campylobacter infections in developed countries occur as sporadic cases, mainly caused by eating undercooked Campylobacter-contaminated poultry. The most efficient strategy of decreasing the number of human Campylobacter infections is by implementing protective vaccinations for humans and/or chickens. Despite more than 10 years of research, an effective anti-Campylobacter vaccine has not been developed. This review highlights our increasing knowledge of Campylobacter interaction with host cells and focuses on recently published data describing the efficacy of anti-Campylobacter vaccine prototypes.

The decline of antibiotic era--new approaches for antibacterial drug discovery.

Infectious diseases still remain the main cause of human premature deaths; especially in developing countries. The emergence and spread of pathogenic bacteria resistant to many antibiotics (multidrug-resistant strains) have created the need for the development of novel therapeutic agents. Only two new classes of antibiotics of novel mechanisms of action (linezolid and daptomycin) have been introduced into the market during the last three decades. The recent progress in molecular biology and bacterial genome analysis has had an enormous impact on antibacterial drug research. This review presents new achievements in searching a new bacterial essential genes, a potential targets for antibacterial drugs. Application of metagenomics strategy is also shown. Some recent technologies aimed at development of anti-pathogenic drugs such as inhibitors of quorum sensing process or histidine kinases are also discussed. Extensive research efforts have provided many details concerning structure of bacterial proteins playing an important role in pathogenesis such as adherence proteins or toxins, what allowed searching for antitoxin drugs or drugs interfering with bacterial adhesion. As an example, the review focuses on anthrax therapies under development. Additionally, the article presents the progress in phage therapy; using bacteriophages or their products such as lysins in antibacterial therapy.