Flagellar Hook-basal Body Complexes Research Articles

An In-Silico Investigation to Design a Multi-Epitopes Vaccine against Multi-Drug Resistant Hafnia alvei.

Antimicrobial resistance has become a significant health issue because of the misuse of antibiotics in our daily lives, resulting in high rates of morbidity and mortality. Hafnia alvei is a rod-shaped, Gram-negative and facultative anaerobic bacteria. The medical community has emphasized H. alvei’s possible association with gastroenteritis. As of now, there is no licensed vaccine for H. alvei, and as such, computer aided vaccine design approaches could be an ideal approach to highlight the potential vaccine epitopes against this bacteria. By using bacterial pan-genome analysis (BPGA), we were able to study the entire proteomes of H. alvei with the aim of developing a vaccine. Based on the analysis, 20,370 proteins were identified as core proteins, which were further used in identifying potential vaccine targets based on several vaccine candidacy parameters. The prioritized vaccine targets against the bacteria are; type 1 fimbrial protein, flagellar hook length control protein (FliK), flagellar hook associated protein (FlgK), curli production assembly/transport protein (CsgF), fimbria/pilus outer membrane usher protein, fimbria/pilus outer membrane usher protein, molecular chaperone, flagellar filament capping protein (FliD), TonB-dependent hemoglobin /transferrin/lactoferrin family receptor, Porin (OmpA), flagellar basal body rod protein (FlgF) and flagellar hook-basal body complex protein (FliE). During the epitope prediction phase, different antigenic, immunogenic, non-Allergenic, and non-Toxic epitopes were predicted for the above-mentioned proteins. The selected epitopes were combined to generate a multi-epitope vaccine construct and a cholera toxin B subunit (adjuvant) was added to enhance the vaccine’s antigenicity. Downward analyses of vaccines were performed using a vaccine three-dimensional model. Docking studies have confirmed that the vaccine strongly binds with MHC-I, MHC-II, and TLR-4 immune cell receptors. Additionally, molecular dynamics simulations confirmed that the vaccine epitopes were exposed to nature and to the host immune system and interpreted strong intermolecular binding between the vaccine and receptors. Based on the results of the study, the model vaccine construct seems to have the capacity to produce protective immune responses in the host, making it an attractive candidate for further in vitro and in vivo studies.

Reverse Vaccinology and Immunoinformatic Assisted Designing of a Multi-Epitopes Based Vaccine Against Nosocomial Burkholderia cepacia.

Burkholderia cepacia is a Gram-negative nosocomial pathogen and is considered as a troublesome bacterium due to its resistance to many common antibiotics. There is no licensed vaccine available to prevent the pathogen infections, thus making the condition more alarming and warrant the search for novel therapeutic and prophylactic approaches. In order to identify protective antigens from pathogen proteome, substantial efforts are put forth to prioritized potential vaccine targets and antigens that can be easily evaluated experimentally. In this vaccine design investigation, it was found that B. cepacia completely sequenced proteomes available in NCBI genome database has a total of 28,966 core proteins. Out of total, 25,282 proteins were found redundant while 3,684 were non-redundant. Subcellular localization revealed that 18 proteins were extracellular, 31 were part of the outer membrane, 75 proteins were localized in the periplasm, and 23 were virulent proteins. Five proteins namely flagellar hook protein (FlgE), fimbria biogenesis outer membrane usher protein, Type IV pilus secretin (PilQ), cytochrome c4, flagellar hook basal body complex protein (FliE) were tested for positive for antigenic, non-toxic, and soluble epitopes during predication of B-cell derived T-cell epitopes. A vaccine peptide of 14 epitopes (joined together via GPGPG linkers) and cholera toxin B subunit (CTBS) adjuvant (joined to epitopes peptide via EAAAK linker) was constructed. Binding interaction of the modeled vaccine with MHC-I, MHC-II, and Toll-like receptor 4 (TLR-4) immune receptors was studied using molecular docking studies and further analyzed in molecular dynamics simulations that affirms strong intermolecular binding and stable dynamics. The maximum root mean square deviation (RMSD) score of complexes in the simulation time touches to 2 Å. Additionally, complexes binding free energies were determined that concluded robust interaction energies dominated by van der Waals. The total energy of each complex is < −190 kcal/mol. In summary, the designed vaccine showed promising protective immunity against B. cepacia and needs to be examined in experiments.

Designing a Recombinant Vaccine against Providencia rettgeri Using Immunoinformatics Approach.

Antibiotic resistance (AR) is the resistance mechanism pattern in bacteria that evolves over some time, thus protecting the bacteria against antibiotics. AR is due to bacterial evolution to make itself fit to changing environmental conditions in a quest for survival of the fittest. AR has emerged due to the misuse and overuse of antimicrobial drugs, and few antibiotics are now left to deal with these superbug infections. To combat AR, vaccination is an effective method, used either therapeutically or prophylactically. In the current study, an in silico approach was applied for the design of multi-epitope-based vaccines against Providencia rettgeri, a major cause of traveler’s diarrhea. A total of six proteins: fimbrial protein, flagellar hook protein (FlgE), flagellar basal body L-ring protein (FlgH), flagellar hook-basal body complex protein (FliE), flagellar basal body P-ring formation protein (FlgA), and Gram-negative pili assembly chaperone domain proteins, were considered as vaccine targets and were utilized for B- and T-cell epitope prediction. The predicted epitopes were assessed for allergenicity, antigenicity, virulence, toxicity, and solubility. Moreover, filtered epitopes were utilized in multi-epitope vaccine construction. The predicted epitopes were joined with each other through specific GPGPG linkers and were joined with cholera toxin B subunit adjuvant via another EAAAK linker in order to enhance the efficacy of the designed vaccine. Docking studies of the designed vaccine construct were performed with MHC-I (PDB ID: 1I1Y), MHC-II (1KG0), and TLR-4 (4G8A). Findings of the docking study were validated through molecular dynamic simulations, which confirmed that the designed vaccine showed strong interactions with the immune receptors, and that the epitopes were exposed to the host immune system for proper recognition and processing. Additionally, binding free energies were estimated, which highlighted both electrostatic energy and van der Waals forces to make the complexes stable. Briefly, findings of the current study are promising and may help experimental vaccinologists to formulate a novel multi-epitope vaccine against P. rettgeri.

Genetic analysis of assembly of the Salmonella enterica serovar Typhimurium type III secretion-associated needle complex.

Several pathogenic bacteria have evolved a specialized protein secretion system termed type III to secrete and deliver effector proteins into eukaryotic host cells. Salmonella enterica serovar Typhimurium uses one such system to mediate entry into nonphagocytic cells. This system is composed of more than 20 proteins which are encoded within a pathogenicity island (SPI-1) located at centisome 63 of its chromosome. A subset of these components form a supramolecular structure, termed the needle complex, that resembles the flagellar hook-basal body complex. The needle complex is composed of a multiple-ring cylindrical base that spans the bacterial envelope and a needle-like extension that protrudes from the bacterial outer surface. Although the components of this structure have been identified, little is known about its assembly. In this study we examined the effect of loss-of-function mutations in each of the type III secretion-associated genes encoded within SPI-1 on the assembly of the needle complex. This analysis indicates that the assembly of this organelle occurs in discrete, genetically separable steps. A model for the assembly pathway of this important organelle is proposed that involves a sec-dependent step leading to the assembly of the base substructure followed by a sec-independent process resulting in the assembly of the needle portion.

Flagellar assembly in Salmonella typhimurium: analysis with temperature-sensitive mutants

The process of flagellar assembly in Salmonella typhimurium was investigated by using temperature-sensitive mutants. The mutants were grown at the restrictive temperature and then at the permissive temperature, with radiolabel supplied in the first phase of the experiment and not the second, or vice versa. Flagellar hook-basal body complexes were then purified and analyzed by gel electrophoresis and autoradiography. The extent to which a given protein was labeled in the two phases of the experiment provided information as to whether it preceded or followed the block caused by the mutant protein. We conclude the following concerning flagellar assembly. The M-ring protein (FliF) is stably incorporated in the earliest stage detected, along with two previously unknown proteins, with apparent molecular masses of 23 and 26 kilodaltons, respectively, and possibly one of the switch components, FliG. Independent of that event and all other events, the P-ring and L-ring proteins (FlgI and FlgH) are synthesized and exported to the periplasm and outer membrane by the primary cellular export pathway. Rod assembly occurs by export (via the flagellum-specific pathway) of subunits of four proteins, FlgB, FlgC, FlgF, and FlgG, and their incorporation, probably in that order, into the rod structure; this stage requires the flhA and fliI genes, perhaps because they encode part of the export apparatus. Once rod assembly is complete, the FlgI and FlgH proteins assemble around the rod to form the P and L rings. The rod structure, which is only metastable while it is being constructed, becomes stable upon P-ring addition. Export (via the flagellum-specific pathway) and assembly of hook protein, hook-associated proteins, and filament protein then occur successively. A number of flagellar proteins, whose genetic origin and structural role are not yet known, were identified on the basis of their dependence on the flagellar master operon for expression.

Basal-body-associated disks are additional structural elements of the flagellar apparatus isolated from Wolinella succinogenes.

The intact flagella of Wolinella succinogenes, a gram-negative, anaerobic bacterium with a single polar flagellum, were obtained by an improved procedure, introduced recently by Aizawa et al. (S.-J. Aizawa, G. E. Dean, C. J. Jones, R. M. Macnab, and S. Yamaguchi, J. Bacteriol. 161:836-849, 1985) for the flagellum of Salmonella typhimurium. Disks with a diameter of 130 +/- 30 nm, which were attached to the basal body of the isolated intact flagella, could be identified by electron microscopy as additional structural elements of the bacterial flagellar apparatus. In freeze-dried and metal-shadowed samples, two rings of the basal body were detected on one side and a terminal knob was located on the other side of the disks. Suspension of the flagellar apparatus in acidic solution dissociated the flagellar filaments, yielding hook-basal body complexes with and without the associated disks. If whole cells were subjected to low pH, double disks of the same diameter and with a central hole of about 13 nm could be isolated. Similar parallel disks could be seen also in negatively stained whole cells. When uranyl acetate was used for negative staining of the intact flagella, concentric rings were detected on the disks, similar to the concentric membrane rings found by Coulton and Murray (J. W. Coulton and R. G. E. Murray, J. Bacteriol. 136:1037-1049, 1978) on platelike arrays of proteins in outer membrane preparations of Aquaspirillum serpens. Because the disks of W. succinogenes can be isolated together with the flagellar hook-basal body complex, they appear to be basal-body-rather than secondary membrane-associated structures. It is possible that these disks are the bearing or stator of this rotary device.

Identification of the M-ring protein of the flagellar motor of Salmonella typhimurium.

The M ring is a substructure of the flagellar basal body of bacteria, which lies in the cytoplasmic membrane and is therefore close to the site where the energy of the transmembrane proton potential is converted into mechanical work of rotation of the motor. The protein from which this ring is constructed has not been identified. Flagellar hook-basal body complexes from Salmonella typhimurium were used as the immunogen for the preparation of monoclonal antibodies. An antibody obtained was directed against a major basal-body component, a 65-kDa protein that from mutant studies has been assigned as the product of the flaAII.1 gene. By immunoelectron microscopy, the antibody was observed to bind the innermost feature of the basal body: the cytoplasmic-facing surface of the M ring. We conclude that the 65-kDa protein is a component--probably the main component--of this important substructure of the flagellar motor.

Purification and characterization of the flagellar hook-basal body complex of Salmonella typhimurium.

The hook-basal body complex of Salmonella typhimurium, a major component of its flagellar apparatus, was subjected to detailed analysis by electron microscopy and gel electrophoresis. The study was facilitated by the development of an improved protocol for isolation of the complexes in high yield and purity. Nine proteins were identified with the structure. These proteins had apparent molecular weights of 65,000 (65K), 60K, 42K, 38K, 32K, 30K, 27K, 16K, and 14K. Small but reproducible shifts in the apparent molecular weights of specific proteins from conditionally nonflagellate mutants indicated the following gene-polypeptide correspondences: flaFV, 42K; flaFVI, 32K; flaFVII, 30K; flaFIX, 38K; flaAII.1, 65K. Several new morphological features of hook-basal body complexes were recognized, including a clawlike structure on the cytoplasm-proximal M ring and additional material at the cytoplasmic face of the M ring. Based on this study and the work of others, we suggest that the morphological features of the hook-basal body complex correspond to the following proteins: hook-filament junction, 60K; hook, 42K; rod, 30K and 32K; L ring and outer cylinder wall, 27K; P ring, 38K; S ring, unknown; M ring 65K.