Recombinant Fusion Protein Vaccine Containing FliC and FliD Protects Mice Against Clostridioides Difficile Infection

Bacterial flagella are involved in infection through their roles in host-cell adhesion, cell invasion, auto-agglutination, colonization, and formation of biofilms, as well as in the regulation and secretion of non-flagellar bacterial proteins involved in the virulence process. In this study, we constructed a fusion protein vaccine (FliCD) containing Clostridiodes difficile flagellar proteins FliC and FliD. Immunization of mice with FliCD induce potent IgG antibody responses against FliCD and protected mice against C. difficile infection and decrease C. difficile spores and toxin levels in the feces after infection. Furthermore, we found anti-FliCD serum protected mice against CDI and decreased C. difficile spores and toxin levels in the feces after C. difficile infection. Finally, we found that anti-FliCD serum inhibited the binding of C. difficile vegetative cells to HCT8 cells. These results imply that FliCD fusion protein may represent an effective vaccine candidate for the prevention from C. difficile infection (CDI).

Brucella and Its Hidden Flagellar System

Brucella, a Gram-negative bacterium with a high infective capacity and a wide spectrum of hosts in the animal world, is found in terrestrial and marine mammals, as well as amphibians. This broad spectrum of hosts is closely related to the non-classical virulence factors that allow this pathogen to establish its replicative niche, colonizing epithelial and immune system cells, evading the host’s defenses and defensive response. While motility is the primary role of the flagellum in most bacteria, in Brucella, the flagellum is involved in virulence, infectivity, cell growth, and biofilm formation, all of which are very important facts in a bacterium that to date has been described as a non-motile organism. Evidence of the expression of these flagellar proteins that are present in Brucella makes it possible to hypothesize certain evolutionary aspects as to where a free-living bacterium eventually acquired genetic material from environmental microorganisms, including flagellar genes, conferring on it the ability to reach other hosts (mammals), and, under selective pressure from the environment, can express these genes, helping it to evade the immune response. This review summarizes relevant aspects of the presence of flagellar proteins and puts into context their relevance in certain functions associated with the infective process. The study of these flagellar genes gives the genus Brucella a very high infectious versatility, placing it among the main organisms in urgent need of study, as it is linked to human health by direct contact with farm animals and by eventual transmission to the general population, where flagellar genes and proteins are of great relevance.

Role of the flagellar hook in the structural development and antibiotic tolerance of Pseudomonas aeruginosa biofilms

Pseudomonas aeruginosa biofilms exhibit an intrinsic resistance to antibiotics and constitute a considerable clinical threat. In cystic fibrosis, a common feature of biofilms formed by P. aeruginosa in the airway is the occurrence of mutants deficient in flagellar motility. This study investigates the impact of flagellum deletion on the structure and antibiotic tolerance of P. aeruginosa biofilms, and highlights a role for the flagellum in adaptation and cell survival during biofilm development. Mutations in the flagellar hook protein FlgE influence greatly P. aeruginosa biofilm structuring and antibiotic tolerance. Phenotypic analysis of the flgE knockout mutant compared to the wild type (WT) reveal increased fitness under planktonic conditions, reduced initial adhesion but enhanced formation of microcolony aggregates in a microfluidic environment, and decreased expression of genes involved in exopolysaccharide formation. Biofilm cells of the flgE knock-out mutant display enhanced tolerance towards multiple antibiotics, whereas its planktonic cells show similar resistance to the WT. Confocal microscopy of biofilms demonstrates that gentamicin does not affect the viability of cells located in the inner part of the flgE knock-out mutant biofilms due to reduced penetration. These findings suggest that deficiency in flagellar proteins like FlgE in biofilms and in cystic fibrosis infections represent phenotypic and evolutionary adaptations that alter the structure of P. aeruginosa biofilms conferring increased antibiotic tolerance.

Down in the pond: Isolation and characterization of a new Serratia marcescens strain (LVF3) from the surface water near frog’s lettuce (Groenlandia densa)

Serratia marcescens is a species that belongs to the family of Yersiniaceae. This family comprises taxa representing opportunistic human- and phytopathogens but also plant growth-promoting rhizobacteria (PGPR). This study describes a novel Gram-negative strain (LVF3R) of the species Serratia marcescens. The strain was characterized genomically, morphologically, and physiologically. In addition, the potential of the isolate to act as a host strain to assess the diversity of Serratia associated phages in environmental samples was explored. Average nucleotide identity analysis revealed that LVF3R belongs to the species Serratia marcescens. In silico analysis and ProphageSeq data resulted in the identification of one prophage, which is capable of viral particle formation. Electron microscopy showed cells of a rod-shaped, flagellated morphotype. The cells revealed a length and width of 1–1.6 μm and 0.8 μm, respectively. LVF3R showed optimal growth at 30 C and in the presence of up to 2% (w/v) NaCl. It exhibited resistances to ampicillin, erythromycin, oxacillin, oxytetracycline, rifampicin, tetracycline, and vancomycin. Genome data indicate that strain S. marcescens LVF3R is a potential PGPR strain. It harbors genes coding for indole acetic acid (IAA) biosynthesis, siderophore production, plant polymer degradation enzymes, acetoin synthesis, flagellar proteins, type IV secretion system, chemotaxis, phosphorous solubilization, and biofilm formation.

Novel Na+/H+ antiporter (NapA) regulates the motility in Helicobacter pylori

Na+/H+ antiporter plays an important role in maintaining cellular homeostasis by regulating osmotic pressure and intracellular pH. It plays an important role in maintaining cellular homeostasis. In Helicobacter pylori, whole genome sequencing has revealed the presence of two types of Na+/H+ antiporter. A gene (nhaA) homologous to the Na+/H+ antiporter of Escherichia coli has been investigated and its function has been analyzed. However, another gene homologous to the Na+/H+ antiporter of Enterococcus hirae (napA) is not yet known in detail. In this study, we investigated the function of this gene (napA in H. pylori). First, to confirm the genetic presence of napA in 20 H. pylori clinical isolates, PCR analysis was performed, and the napA gene was confirmed in all strains. The amount of Na+ extrusion was measured by atomic absorption spectroscopy. The results showed that the Na+ concentration was decreased in the wild-type strain compared to the napA mutant strain. In addition, there was a significant dose-dependent difference in CFU of Na+ concentration in the napA mutant strain compared to the wild-type strain. We examined whether the napA gene is related to motility using both wild-type and napA mutant strains. As a result, in the motility agar test, the bacterial motility observed in the wild-type strain was not observed in the napA mutant strain. However, no difference in flagellar proteins was observed by SDS-PAGE analysis. These results suggest that the napA gene of H. pylori may regulate homeostasis by extruding Na+ and may also regulate motility.

Evaluation of Structural Changes and Molecular Mechanism Induced by High Hydrostatic Pressure in Enterobacter sakazakii

The contamination of infant milk and powder with Enterobacter sakazakii poses a risk to human health and frequently caused recalls of affected products. This study aims to explore the inactivation mechanism of E. sakazakii induced by high hydrostatic pressure (HHP), which, unlike conventional heat treatment, is a nonthermal technique for pasteurization and sterilization of dairy food without deleterious effects. The mortality of E. sakazakii under minimum reaction conditions (50 MPa) was 1.42%, which was increased to 33.12% under significant reaction conditions (400 MPa). Scanning electron microscopy (SEM) and fluorescent staining results showed that 400 MPa led to a loss of physical integrity of cell membranes as manifested by more intracellular leakage of nucleic acid, intracellular protein and K+. Real-time quantitative PCR (RT-qPCR) analysis presents a downregulation of three functional genes (glpK, pbpC, and ompR), which were involved in cell membrane formation, indicating a lower level of glycerol utilization, outer membrane protein assembly, and environmental tolerance. In addition, the exposure of E. sakazakii to HHP modified oxidative stress, as reflected by the high activity of catalase and super oxide dismutase. The HHP treatment lowered down the gene expression of flagellar proteins (fliC, flgI, fliH, and flgK) and inhibited biofilm formation. These results determined the association of genotype to phenotype in E. sakazakii induced by HHP, which was used for the control of food-borne pathogens.

Evolutionary dynamics of sex-biased gene expression in a young XY system: Insights from the brown algae

The sex-dependent regulation of gene expression is considered to be the underlying cause of often extensive, sexually dimorphic traits between males and females. Although the nature and degree of sex-biased gene expression has been well-documented in several animal and plant systems, far less is known about the commonality, conservation, recruitment mechanisms and evolution of sex-biased genes in more distant eukaryotic groups. Brown algae are of particular interest for empirical studies on the evolution of sex-biased gene expression, as they have been evolving independently from animals and plants for over one billion years. Here we focus on two brown algal dioecious species, Fucus serratus and Fucus vesiculosus, where male heterogamety (XX/XY) has recently emerged. Using RNA-seq, we study sex-biased gene expression and discuss different evolutionary forces responsible for the evolution of sex-biased genes. We find that both species evolved masculinized transcriptomes, with sex-biased genes allocated mainly to male reproductive tissue, but virtually absent in vegetative tissues. Conserved male-biased genes were enriched in functions related to gamete production, along with sperm competition and include two flagellar proteins under positive selection. In contrast to female-biased genes, which show high turnover rates, male-biased genes reveal remarkable conservation of bias and expression levels between the two species. As observed in other XY systems, male-biased genes also display accelerated rates of coding sequence evolution compared to female-biased or unbiased genes. Our results imply that evolutionary forces affect male and female sex-biased genes differently on structural and regulatory levels. Similarly to evolutionary distant plant and animal lineages, sex-biased gene expression in Fucus evolved during the transition to dioecy to resolve intra-locus sexual conflict arising from anisogamy.

Exploration of Targeting Mechanisms of Cinnamon Essential Oil Against Water Borne Multiple Drug Resistant Isolate Aeromonas Hydrophila C4

Introduction:Cinnamon is dietary part of almost every Indian, hence there is no harm to consume it or apply it as antibacterial agent. Herein, we have observed that 0.5, 0.6 and 0.7 % concentrations cinnamon essential oil sufficient to control A. hydrophila within 5hrs of exposure. Cinnamaldehyde of is major component of used essential oil which has targeted the Flgn, Fij and Flja protein of flagella flowed by inhibition of swimming motility. Cinnamaldehyde was associated with damaging of cell membrane and leakage of electrolytes in extracellular environment.Aim of the study:In the current study cinnamon essential oil has been used to control the growth of multiple drug resistant A. hydrophila. A. hydrophila was isolated from well water system, and it was imipenem resistant bacteria as well. Materials and Methods: Cinnamon essential oil was characterized by using HPLC and FTIR. Antibacterial killing mechanism was determined by using electrical conductivity test, cytotoxic test by MTT assay, cell wall integrity test and motility test. Results:Cinnamon essential oil has potential to control the growth of A. hydrophila, and complete inhibition of cells was observed within 5hrs of exposure to 0.7% concentration of essential oil. By motility test and molecular docking it has been confirmed that for killing of bacterial cells, cinnamaldehyde of cinnamon essential oil targeting the cell membrane proteins as well as flagellar proteins Flgn, Fij and Flja.

Condition-Specific Molecular Network Analysis Revealed That Flagellar Proteins Are Involved in Electron Transfer Processes of Shewanella piezotolerans WP3

Because of the ability to metabolize a large number of electron acceptors such as nitrate, nitrite, fumarate, and metal oxides, Shewanella species have attracted much attention in recent years. Generally, the use of these electron acceptors is mainly achieved through electron transfer proteins and their interactions which will dynamically change across different environmental conditions in cells. Therefore, functional analysis of condition-specific molecular networks can reveal biological information on electron transfer processes. By integrating expression data and molecular networks, we constructed condition-specific molecular networks for Shewanella piezotolerans WP3. We then identified condition-specific key genes and studied their potential functions with an emphasis on their roles in electron transfer processes. Functional module analysis showed that different flagellar assembly modules appeared under these conditions and suggested that flagellar proteins are important for these conditions. We also identified the electron transfer modules underlying these various environmental conditions. The present results could help with screening electron transfer genes and understanding electron transfer processes under various environmental conditions for the Shewanella species.

In silico designing of vaccine candidate against Clostridium difficile

Clostridium difficile is a spore-forming gram-positive bacterium, recognized as the primary cause of antibiotic-associated nosocomial diarrhoea. Clostridium difficile infection (CDI) has emerged as a major health-associated infection with increased incidence and hospitalization over the years with high mortality rates. Contamination and infection occur after ingestion of vegetative spores, which germinate in the gastro-intestinal tract. The surface layer protein and flagellar proteins are responsible for the bacterial colonization while the spore coat protein, is associated with spore colonization. Both these factors are the main concern of the recurrence of CDI in hospitalized patients. In this study, the CotE, SlpA and FliC proteins are chosen to form a multivalent, multi-epitopic, chimeric vaccine candidate using the immunoinformatics approach. The overall reliability of the candidate vaccine was validated in silico and the molecular dynamics simulation verified the stability of the vaccine designed. Docking studies showed stable vaccine interactions with Toll‐Like Receptors of innate immune cells and MHC receptors. In silico codon optimization of the vaccine and its insertion in the cloning vector indicates a competent expression of the modelled vaccine in E. coli expression system. An in silico immune simulation system evaluated the effectiveness of the candidate vaccine to trigger a protective immune response.