Outer membrane protein A (OmpA) of Shigella flexneri 2a, induces protective immune response in a mouse model

Background

In our earlier studies 34 kDa outer membrane protein (OMP) of Shigella flexneri 2a has been identified as an efficient immunostimulant.

Key Results

In the present study MALDI-TOF MS analysis of the purified 34 kDa OMP of Shigella flexneri 2a shows considerable sequence homology (Identity 65%) with the OmpA of S. flexneri 2a. By using the specific primers, the gene of interest has been amplified from S. flexneri 2a (N.Y-962/92) genomic DNA, cloned in pET100/D-TOPO® vector and expressed using induction with isopropyl thiogalactoside (IPTG) for the first time. Immunogenicity and protective efficacy of the recombinant OmpA has been evaluated in an intranasally immunized murine pulmonary model. The recombinant protein induces significantly enhanced protein specific IgG and IgA Abs in both mucosal and systemic compartments and IgA secreting cells in the systemic compartment (spleen). The mice immunized with OmpA have been protected completely from systemic challenge with a lethal dose of virulent S. flexneri 2a. Immunization with the protein causes mild polymorphonuclear neutrophil infiltration in the lung, without inducing the release of large amounts of proinflammatory cytokines.

Conclusion

These results suggest that the OmpA of S. flexneri 2a can be an efficacious mucosal immunogen inducing protective immune responses. Our findings also demonstrate that antibodies and Th1 immune response may be associated with the marked protective efficacy of immunized mice after intranasal shigellae infection.     

LPS Unmasking of Shigella flexneri Reveals Preferential Localisation of Tagged Outer Membrane Protease IcsP to Septa and New Poles

The Shigella flexneri outer membrane (OM) protease IcsP (SopA) is a member of the enterobacterial Omptin family of proteases which cleaves the polarly localised OM protein IcsA that is essential for Shigella virulence. Unlike IcsA however, the specific localisation of IcsP on the cell surface is unknown. To determine the distribution of IcsP, a haemagglutinin (HA) epitope was inserted into the non-essential IcsP OM loop 5 using Splicing by Overlap Extension (SOE) PCR, and IcsP^HA was characterised. Quantum Dot (QD) immunofluorescence (IF) surface labelling of IcsP^HA was then undertaken. Quantitative fluorescence analysis of S. flexneri 2a 2457T treated with and without tunicaymcin to deplete lipopolysaccharide (LPS) O antigen (Oag) showed that IcsP^HA was asymmetrically distributed on the surface of septating and non-septating cells, and that this distribution was masked by LPS Oag in untreated cells. Double QD IF labelling of IcsP^HA and IcsA showed that IcsP^HA preferentially localised to the new pole of non-septating cells and to the septum of septating cells. The localisation of IcsP^HA in a rough LPS S. flexneri 2457T strain (with no Oag) was also investigated and a similar distribution of IcsP^HA was observed. Complementation of the rough LPS strain with rmlD resulted in restored LPS Oag chain expression and loss of IcsP^HA detection, providing further support for LPS Oag masking of surface proteins. Our data presents for the first time the distribution for the Omptin OM protease IcsP, relative to IcsA, and the effect of LPS Oag masking on its detection.     

Outer Membrane Proteins A (OmpA) and X (OmpX) Are Essential for Basolateral Invasion of Cronobacter sakazakii

Cronobacter sakazakii is an opportunistic pathogen that actively invades host eukaryotic cells. To identify invasion factors responsible for the intestinal translocation of C. sakazakii, we constructed for the first time outer membrane protein X (OmpX) and A (OmpA) deletion mutants using the lambda Red recombination system. The ompX and ompA deletion mutants showed significantly reduced invasion of human enterocyte-like epithelial Caco-2 and human intestinal epithelial INT-407 cells, and significantly fewer mutant cells were recovered from the livers and spleens of rat pups. Furthermore, compared with intact target cells, the invasion and initial association potentials of the mutants increased at a rate similar to that of the wild type in tight-junction-disrupted target cells, suggesting that OmpX and OmpA are involved in basolateral invasion by C. sakazakii. This is the first report of C. sakazakii virulence determinants that are essential for basolateral invasion and that may be critical for the virulence of C. sakazakii.Enterobacter sakazakii is an emerging pathogen associated with several outbreaks of meningitis and local necrotizing enterocolitis in premature infants (2, 28, 37). There was considerable diversity among E. sakazakii isolates (13, 14), and the original taxonomic name of E. sakazakii was reclassified as Cronobacter spp., which included Cronobacter sakazakii (13, 14). Therefore, C. sakazakii will be used throughout this paper. Although the incidence of Cronobacter infection is rare, the mortality rate is as high as 33 to 80% (11, 27, 32, 39). Even when infants survive Cronobacter infection, they often experience serious sequelae, including brain abscesses, developmental delay, and impairment of sight and hearing (8). Premature infants, whose immune systems are not fully developed, may be at high risk for Cronobacter infection (26).Very little is known about the mechanisms of pathogenicity and the virulence determinants of the genus Cronobacter. Adhesion of Cronobacter spp. to eukaryotic cells showed two distinct patterns, i.e., a diffuse pattern and the formation of localized clusters, which was nonfimbrial (21). Pagotto et al. (29) reported that the genus Cronobacter produced enterotoxins and was lethal on intraperitoneal injection into suckling mice at levels as low as 10⁵ CFU per mouse. The genus Cronobacter interacts with and damages intestinal epithelial cells, which results in intestinal injury and villus disruption (12). In addition, the cell-bound zinc-containing metalloprotease encoded by zpx caused rounding of Chinese hamster ovary (CHO) cells (19), which may be important in dissemination of the pathogen into the systemic circulation. Furthermore, Townsend et al. (36) showed that Cronobacter can persist within rat macrophages.As an oral pathogen causing a systemic infection, C. sakazakii must translocate from the intestinal lumen into the blood circulation. The genus Cronobacter is capable of actively invading various epithelial and endothelial cells of human and animal origin (17, 25, 31). Kim and Loessner (17) reported that the active invasion of human intestinal Caco-2 cells by C. sakazakii requires de novo bacterial protein synthesis and the host cell cytoskeleton and that the invasion efficiency of C. sakazakii was enhanced in the absence of cellular tight junctions. With regard to the virulence determinants related to Cronobacter penetration of the host cells, Mohan Nair and Venkitanarayanan (25) and Singamsetty et al. (31) reported that outer membrane protein A (OmpA) of Cronobacter plays an important role in the invasion of human intestinal epithelial INT-407 cells and human brain microvascular endothelial cells (HBMECs); invasion was dependent on both microfilaments and microtubules in INT-407 cells but only on microtubule condensation in HBMECs. Obviously, bacterial translocation in the intestines is multifactorial, and more detailed studies are needed to gain a better understanding of C. sakazakii pathogenesis.Outer membrane protein X (OmpX) of C. sakazakii was identified in this study. Previously, OmpX in other bacteria was shown to be involved in the invasion of host cells (7, 18), neutralizing host defense mechanisms, and bacterial defense against the complement systems of the host (10, 38).In this study, we report for the first time a successful application of the lambda Red recombination system to construct in-frame OmpX and/or OmpA deletion mutants in C. sakazakii. We further report that both outer membrane proteins (OMPs) of C. sakazakii, OmpX and OmpA, play critical roles in its invasion through not only the apical side, but also the basolateral side, of the host cells. We also show that OmpX and OmpA are responsible for C. sakazakii translocation into the deeper organs (i.e., liver and spleen).     

Outer membrane protein A (OmpA) of Shigella flexneri 2a links innate and adaptive immunity in a TLR2-dependent manner and involvement of IL-12 and nitric oxide

We determine that OmpA of Shigella flexneri 2a is recognized by TLR2 and consequently mediates the release of proinflammatory cytokines and activates NF-κB in HEK 293 cells transfected with TLR2. We also observe that in RAW macrophages TLR2 is essential to instigate the early immune response to OmpA via NF-κB activation and secretion of cytokines and NO. Consistent with these results, TLR2 knockdown using siRNA abolishes the initiation of immune responses. Processing and presentation of OmpA depend on TLR2; MHCII presentation of the processed antigen and expression of CD80 significantly attenuated in TLR2 knockdown macrophages. The optimum production of IFN-γ by the macrophages:CD4(+) T cells co-culture depends on both TLR2 activation and antigen presentation. So, TLR2 is clearly recognized as a decisive factor in initiating host innate immune response to OmpA for the development of CD4(+) T cell adaptive response. Furthermore, we demonstrate in vivo that intranasal immunization of mice with OmpA selectively enhances the release of IFN-γ and IL-2 by CD4(+) T cells. Importantly, OmpA increases the level of IFN-γ production in Ag-primed splenocytes. The addition of neutralizing anti-IL-12p70 mAb to cell cultures results in the decreased release of OmpA-enhanced IFN-γ by Ag-primed splenocytes. Moreover, coincubation with OmpA-pretreated macrophages enhances the production of IFN-γ by OmpA-primed CD4(+) T cells, representing that OmpA may enhance IFN-γ expression in CD4(+) T cells through the induction of IL-12 production in macrophages. These results demonstrate that S. flexneri 2a OmpA may play a critical role in the development of Th1 skewed adaptive immune response.     

Shigella flexneri 3a Outer Membrane Protein C Epitope Is Recognized by Human Umbilical Cord Sera and Associated with Protective Activity

Shigella flexneri 3a is one of the five major strains of the Shigella genus responsible for dysentery, especially among children, in regions of high poverty and poor sanitation. The outer membrane proteins (OMP) of this bacterium elicit immunological responses and are considered a prime target for vaccine development. When injected into mice they elicit a protective immunological response against a lethal dose of the pathogen. The OMPs from S. flexneri 3a were isolated and resolved by two-dimension-SDS-PAGE. Two 38-kDa spots were of particular interest since in our earlier studies OMPs of such molecular mass were found to interact with umbilical cord sera. These two spots were identified as OmpC by ESI-MS/MS spectrometry. By DNA sequencing, the ompC gene from S. flexneri 3a was identical to ompC from S. flexneri 2a [Gene Bank: 24113600]. A 3D model of OmpC was built and used to predict B-cell type (discontinuous) antigenic epitopes. Six epitopes bearing the highest score were selected and the corresponding peptides were synthesized. Only the peptides representing loop V of OmpC reacted strongly with the umbilical cord serum immunoglobulins. To determine which amino acids are essential for the antigenic activity of the epitope, the loop V was scanned with a series of dodecapeptides. The peptide RYDERY was identified as a minimal sequence for the loop V epitope. Truncation at either the C- or N-terminus rendered this peptide inactive. Apart from C-terminal tyrosine, substitution of each of the remaining five amino acids with glycine, led to a precipitous loss of immunological activity. This peptide may serve as a ligand in affinity chromatography of OmpC-specific antibodies and as a component of a vaccine designed to boost human immune defenses against enterobacterial infections.     

Outer membrane protein A (OmpA) binds to and activates human macrophages

Outer membrane protein (Omp)A is highly represented and conserved in the Enterobacteriaceae family. Using a recombinant OmpA from Klebsiella pneumoniae (P40), we have analyzed the interaction between OmpA and macrophages. We report that Alexa488-labeled P40 binds (at 4 degrees C) to murine and human macrophages in a dose-dependent manner and is rapidly internalized (at 37 degrees C). No binding or internalization of the Alexa488-labeled glycophorin A control protein is observed under the same conditions. Furthermore, P40 up-regulates the production of IL-1beta, IL-8, IL-10, IL-12, and TNF-alpha by human macrophages and of NO by the RAW 264.7 murine macrophage cell line. P40 also synergizes with IFN-gamma and suboptimal concentrations of LPS to up-regulate the production of these mediators. In conclusion, P40 binds to and activates macrophages. These data suggest that recognition of OmpA by macrophages may be an initiating event in the antibacterial host response.     

Outer membrane protein A (OmpA) activates human epidermal Langerhans cells

Outer membrane protein (Omp)A is highly represented and conserved in the Enterobacteriaceae family. Using a recombinant OmpA from Klebsiella pneumoniae (kpOmpA), we have analysed the interaction between this bacterial cell wall protein and human Langerhans cells (LC), the antigen-presenting cells of the epidermis and mucosa. We showed that biotinylated kpOmpA binds to human LC freshly isolated from epidermis. kpOmpA up-regulated MHC class II, CD86 and CCR7 expression, enhanced migration in response to macrophage inflammatory protein-3beta (MIP-3beta) through a reconstituted basement membrane mimicking the prerequisite passage through the dermal-epidermal basement membrane on the way to lymph nodes. The allostimulatory function of kpOmpA-treated LC was more potent than that of untreated cells. Even though the proportion of LC which binds kpOmpA was shown to vary between individuals, our data indicate that kpOmpA binds to and activates LC, and suggest that recognition of OmpA by LC may be an initiating event in the antibacterial host response.     

Localization of the Outer Membrane Protein OmpA2 in Caulobacter crescentus Depends on the Position of the Gene in the Chromosome

The outer membrane of Gram-negative bacteria is an essential structure involved in nutrient uptake, protection against harmful substances, and cell growth. Different proteins keep the outer membrane from blebbing out by simultaneously interacting with it and with the cell wall. These proteins have been mainly studied in enterobacteria, where OmpA and the Braun and Pal lipoproteins stabilize the outer membrane. Some degree of functional redundancy exists between these proteins, since none of them is essential but the absence of two of them results in a severe phenotype. Caulobacter crescentus has a different strategy to maintain its outer membrane, since it lacks the Braun lipoprotein and Pal is essential. In this work, we characterized OmpA2, an OmpA-like protein, in this bacterium. Our results showed that this protein is required for normal stalk growth and that it plays a minor role in the stability of the outer membrane. An OmpA2 fluorescent fusion protein showed that the concentration of this protein decreases from the stalk to the new pole. This localization pattern is important for its function, and it depends on the position of the gene locus in the chromosome and, as a consequence, in the cell. This result suggests that little diffusion occurs from the moment that the gene is transcribed until the mature protein attaches to the cell wall in the periplasm. This mechanism reveals the integration of different levels of information from protein function down to genome arrangement that allows the cell to self-organize.     

The making of a gradient: IcsA (VirG) polarity in Shigella flexneri

The generation and maintenance of subcellular organization in bacteria is critical for many cell processes and properties, including growth, structural integrity and, in pathogens, virulence. Here, we investigate the mechanisms by which the virulence protein IcsA (VirG) is distributed on the bacterial surface to promote efficient transmission of the bacterium Shigella flexneri from one host cell to another. The outer membrane protein IcsA recruits host factors that result in actin filament nucleation and, when concentrated at one bacterial pole, promote unidirectional actin-based motility of the pathogen. We show here that the focused polar gradient of IcsA is generated by its delivery exclusively to one pole followed by lateral diffusion through the outer membrane. The resulting gradient can be modified by altering the composition of the outer membrane either genetically or pharmacologically. The gradient can be reshaped further by the action of the protease IcsP (SopA), whose activity we show to be near uniform on the bacterial surface. Further, we report polar delivery of IcsA in Escherichia coli and Yersinia pseudotuberculosis, suggesting that the mechanism for polar delivery of some outer membrane proteins is conserved across species and that the virulence function of IcsA capitalizes on a more global mechanism for subcellular organization.     

Control of Shigella flexneri in Celebes black macaques (Macaca nigra)

Stool specimens collected systematically from a group of Celebes black macaques (Macaca nigra) with a high incidence of diarrhea were examined microbiologically. Numerous isolates of Shigella flexneri, Campylobacter jejuni and pathogenic Escherichia coli were recovered. Previous parasitology reports had revealed that the majority of the animals had Balantidium coli. Subsequently, the group was treated with trimethoprim-sulfamethoxazole, erythromycin and tetracycline. After treatment, Shigella flexneri was not detected in the stool of any animal for 1 year, and the clinical condition of the group was improved. Reduced recovery rates were obtained with other enteric pathogens.     

A Function of 40 kDa Outer Membrane Protein in Serratia marcescens

The function of one of the outer membrane proteins of Serratia marcescens was investigated. S. marcescens with an abundant 40 kDa outer membrane protein was induced to form spheroplast at a high rate in an isotonic medium in the presence of calcium, although the spheroplasts were generally fragile in the isotonic environment. The degree of spheroplast induction was correlated to the amount of the 40 kDa protein present in the membrane. In the 40 kDa proteinless mutant strains, the spheroplast induction rate was remarkably decreased. Autoradiography of the outer membrane revealed the presence of a calcium-binding protein as a radioactive band whose position coincided with the 40 kDa protein. These results suggest that the 40 kDa protein has an important role in maintaining the structural integrity of the cell wall against osmotic shock.     

Lipopolysaccharide O antigen chains mask IcsA (VirG) in Shigella flexneri

Shigella flexneri 2a strain 2457T lipopolysaccharide (LPS) has O antigen (Oag) chains with two modal lengths (S-type and VL-type), and has IcsA apparently located at one pole on its cell surface. Treatment of Y serotype derivatives of 2457T and RMA696 (2457T wzz(SF)) with Sf6 tailspike protein (TSP) resulted in hydrolysis of Oag chains, and an increase in detection of IcsA by indirect immunofluorescence staining on both the lateral and polar regions of the cell surface. Newly synthesised IcsA expressed from a pBAD promoter in a S. flexneri Y strain was also detected on both the lateral and polar regions of the cell when incubated with TSP prior to immunofluorescence staining. We conclude that IcsA is actually located on both lateral and polar regions of the S. flexneri cell surface, and that LPS Oag chains mask the presence of IcsA by hindering its detection with antibodies. These results have implications for the mechanism of IcsA export. They suggest that while IcsA export is predominantly targeted to the old cell pole, it can also occur on the lateral regions of the cell surface.     

Analysis of Alternative Splicing and Long Noncoding RNAs After the Edwardsiella anguillarum Infected the Immunized European Eels (Anguilla anguilla) Revealed the Role of Outer Membrane Protein A in OmpA Subunit Vaccine

Marine Biotechnology - Edwardsiella anguillarum is a bacterium that commonly infects cultivated eels. Outer membrane protein A (OmpA) emulsified with Freund’s adjuvant has been shown to be an...     

细菌外膜囊泡(OMV)研究进展

外膜囊泡(outer membrane vesicles,OMV)是在细菌生命活动中不断从细菌细胞表面脱离而形成的功能性囊泡,其内部含有蛋白质、脂质和核酸等成分,具有多种特殊的生物学功能,在细菌-细菌和细菌-宿主相互作用中起着关键作用.虽然大多数OMV的研究都是关于动物病原菌,但最近OMV在植物-细菌相作领域的作用已逐...     

Shewanella putrefaciens mtrB Encodes an Outer Membrane Protein Required for Fe(III) and Mn(IV) Reduction

Iron and manganese oxides or oxyhydroxides are abundant transition metals, and in aquatic environments they serve as terminal electron acceptors for a large number of bacterial species. The molecular mechanisms of anaerobic metal reduction, however, are not understood. Shewanella putrefaciens is a facultative anaerobe that uses Fe(III) and Mn(IV) as terminal electron acceptors during anaerobic respiration. Transposon mutagenesis was used to generate mutants of S. putrefaciens, and one such mutant, SR-21, was analyzed in detail. Growth and enzyme assays indicated that the mutation in SR-21 resulted in loss of Fe(III) and Mn(IV) reduction but did not affect its ability to reduce other electron acceptors used by the wild type. This deficiency was due to Tn5 inactivation of an open reading frame (ORF) designated mtrB. mtrB encodes a protein of 679 amino acids and contains a signal sequence characteristic of secreted proteins. Analysis of membrane fractions of the mutant, SR-21, and wild-type cells indicated that MtrB is located on the outer membrane of S. putrefaciens. A 5.2-kb DNA fragment that contains mtrB was isolated and completely sequenced. A second ORF, designated mtrA, was found directly upstream of mtrB. The two ORFs appear to be arranged in an operon. mtrA encodes a putative 10-heme c-type cytochrome of 333 amino acids. The N-terminal sequence of MtrA contains a potential signal sequence for secretion across the cell membrane. The amino acid sequence of MtrA exhibited 34% identity to NrfB from Escherichia coli, which is involved in formate-dependent nitrite reduction. To our knowledge, this is the first report of genes encoding proteins involved in metal reduction.     

Survival of Shigella in Sewage: II. Effect of Glycerol on Shigella flexneri and Shigella Bacteriophage1

Glycerol (30%) inhibited or delayed the adsorption of Shigella bacteriophage on its host organism, S. flexneri II; glycerol also inhibited or delayed the burst of phage, whether or not adsorption was carried out in the presence of glycerol. Studies of the mechanisms of these effects showed that viscosity and osmotic shock probably were not responsible for either phenomenon. The inhibition of adsorption, however, was proportional to the concentration of glycerol, and appeared to be a function of the hydroxyl groups on the glycerol molecule. The inhibition of burst seemed to be related to the osmotic pressure outside the bacterial cells.     

Overexpression and topology of the Shigella flexneri O-antigen polymerase (Rfc/Wzy)

Lipopolysaccharides (LPS), particularly the O-antigen component, are one of many virulence determinants necessary for Shigella flexneri pathogenesis. O-antigen biosynthesis is determined mostly by genes located in the rfb region of the chromosome. The rfc/wzy gene encodes the O-antigen polymerase, an integral membrane protein, which polymerizes the O-antigen repeat units of the LPS. The wild-type rfc/wzy gene has no detectable ribosome-binding site (RBS) and four rare codons in the translation initiation region (TIR). Site-directed mutagenesis of the rare codons at positions 4, 9 and 23 to those corresponding to more abundant tRNAs and introduction of a RBS allowed detection of the rfc/wzy gene product via a T7 promoter/polymerase expression assay. Complementation studies using the rfc/wzy constructs allowed visualization of a novel LPS with unregulated O-antigen chain length distribution, and a modal chain length could be restored by supplying the gene for the O-antigen chain length regulator (Rol/Wzz) on a low-copy-number plasmid. This suggests that the O-antigen chain length distribution is determined by both Rfc/Wzy and Rol/Wzz proteins. The effect on translation of mutating the rare codons was determined using an Rfc::PhoA fusion protein as a reporter. Alkaline phosphatase enzyme assays showed an approximately twofold increase in expression when three of the rare codons were mutated. Analysis of the Rfc/Wzy amino acid sequence using TM-PREDICT indicated that Rfc/Wzy had 10–13 transmembrane segments. The computer prediction models were tested by genetically fusing C-terminal deletions of Rfc/Wzy to alkaline phosphatase and β-galactosidase. Rfc::PhoA fusion proteins near the amino-terminal end were detected by Coomassie blue staining and Western blotting using anti-PhoA serum. The enzyme activities of cells with the rfc/wzy fusions and the location of the fusions in rfc/wzy indicated that Rfc/Wzy has 12 transmembrane segments with two large periplasmic domains, and that the amino- and carboxy-termini are located on the cytoplasmic face of the membrane.