Regulation of type III secretion in enterohaemorrhagic Escherichia coli

Enterohaemorrhagic Escherichia coli (EHEC) strains are associated with gastrointestinal and severe systemic disease in humans. EHEC O157:H7 is the most common serotype causing human infections in North America and the UK. Human infections mainly originate from cattle, through either direct contact with infected animals or indirectly through contamination of food or water with animal faeces. From the sequencing of EHEC O157 strains, it is clear that the genomes contain multiple prophages, many of them cryptic, which define this E. coli pathotype. These regions include the locus of enterocyte effacement (LEE) which is a critical horizontally acquired pathogenicity island and encodes a type III secretion system (T3SS). The T3SS translocates effector proteins into epithelial cells that enable tight attachment to these host cells and also modify innate responses and other cellular functions to promote persistence in the animal host. The T3SS is essential for the colonisation of cattle by EHEC O157 where it is localised to the terminal rectum. The regulation of T3S is complex with many regulators and environmental factors already identified. Previous work has demonstrated marked variation in the levels of T3S among EHEC O157 strains. The aim of this research was to further investigate the regulation of T3S towards two objectives: (1) to understand the localisation of EHEC O157 at the terminal rectum of cattle; (2) to understand the strain variation in T3S. (1) In relation to rectal and mucosal colonisation, established aerobic/anaerobic regulators were investigated including arcA, fnr, narX, narQ. Briefly, arcA, fnr, narX, narQ were deleted in an E. coli O157 strain ZAP198 by lambda red recombination. Apart from the fnr mutant which showed lower levels of T3S, the remaining mutants displayed similar T3S protein levels compared to the wild type strain. In addition, no significant changes in adherence and A/E lesion formation capacity were measured for the mutants following interaction with bovine epithelial cells. (2) Strain secretion variation was approached in two ways; the first was to control expression from the LEE1 operon, required for T3S expression, in order to both induce expression and examine the importance of downstream regulation. The second was to investigate variation in T3S between different phages types of EHEC O157. While attempts to construct an inducible T3SS were not successful, intermediate strains made in the process have been useful to dissect how regulators being studied in the laboratory control T3S. The main novel insights from the research have come from examining T3S in different EHEC O157 phage types. We found that the average level of T3S in PT 21/28 strains was lower than in PT 32 strains. Interestingly, most (90%) of PT 21/28 strains contained both Stx2 and Stx2c phages. In contrast, only 28% of PT 32 strains had both phages. Taken together, this raised the possibility that Stx phage integration might have a repressive impact on T3SS regulation in E.coli O157:H7. This hypothesis was addressed using a number of different approaches. Deletions of Stx phages were constructed and these had increased levels of T3S when compared to the parental strains. This phage regulation of T3SS was confirmed in an E. coli K12 background by examining an induced LEE1 reporter in the presence and absence of a transduced Stx2 phage. In addition, it was shown that deletion of the CII phage regulator led to increased T3S and may contribute to the Stx phage repression reported above. This work demonstrates for the first time that Stx phage integration represses T3S expression. It is proposed that this control may limit immune exposure of this critical colonisation factor and that the repression actually allows activation by prophage encoded regulators, including PchA/B, that co-ordinate T3S and non LEE-encoded effector expression to promote epithelial cell colonisation

A Highly Selective Colorimetric Sensor for Cysteine in Water Solution and Bovine Serum Albumin

A simple colorimetric sensor, 2-bromonaphthalene-1,4-dione, has been developed for the Cysteine detection. The sensor showed its best performance in a mixture of ethanol and HEPES (5 : 5, v/v) solution at pH of 7.0. The results of UV-vis and fluorescence indicated that 2-bromonaphthalene-1,4-dione was selective and sensitive for Cysteine detection without the interference of other amino acids (Cysteine, Alanine, Arginine, Aspartinie, Glutamine, Glycine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Proline, Serine, Threonine, Phenylalanine, Valine, Tryptophan, and Hydroxyproline). 2-Bromonaphthalene-1,4-dione also showed binding ability for Cysteine in bovine serum albumin and could be used as a potential colorimetric sensor among eighteen kinds of natural amino acids. Importantly, the recognition of CySH could be observed by naked eye

The role of nanotechnology-based approaches for clinical infectious diseases and public health

Given the high incidence of infection and the growing resistance of bacterial and viral infections to the traditional antiseptic, the need for novel antiseptics is critical. Therefore, novel approaches are urgently required to reduce the activity of bacterial and viral infections. Nanotechnology is increasingly being exploited for medical purposes and is of significant interest in eliminating or limiting the activity of various pathogens. Due to the increased surface-to-volume ratio of a given mass of particles, the antimicrobial properties of some naturally occurring antibacterial materials, such as zinc and silver, increase as particle size decreases into the nanometer regime. However, the physical structure of a nanoparticle and the way it interacts with and penetrates the bacteria also appear to provide unique bactericidal mechanisms. To measure the efficacy of nanoparticles (diameter 100 nm) as antimicrobial agents, it is necessary to comprehend the range of approaches for evaluating the viability of bacteria; each of them has its advantages and disadvantages. The nanotechnology-based disinfectants and sensors for SARS-CoV-2 provide a roadmap for creating more effective sensors and disinfectants for detecting and preventing coronaviruses and other infections. Moreover, there is an increasing role of nanotechnology-based approaches in various infections, including wound healing and related infection, nosocomial infections, and various bacterial infections. To meet the demand for patient care, nanotechnology-based disinfectants need to be further advanced with optimum approaches. Herein, we review the current burden of infectious diseases with a focus on SARS-CoV-2 and bacterial infection that significantly burdens developed healthcare systems and small healthcare communities. We then highlight how nanotechnology could aid in improving existing treatment modalities and diagnosis of those infectious agents. Finally, we conclude the current development and future perspective of nanotechnology for combating infectious diseases. The overall goal is to update healthcare providers on the existing role and future of nanotechnology in tackling those common infectious diseases

Effects of Probiotic Bacillus as an Alternative of Antibiotics on Digestive Enzymes Activity and Intestinal Integrity of Piglets

The previous study in our team found that supplementation of probiotic Bacillus amyloliquefaciens (Ba) instead of antibiotics promote growth performance of piglets. Hence, the present study was carried out to further demonstrate the effect of Ba replacement of antibiotics on digestive and absorption enzyme activity and intestinal microbiota population of piglets. A total of 90 piglets were selected and divided into three groups: G1 group was fed with basal diet supplemented with 150 mg/Kg aureomycin, G2 group was fed with 1 × 108 cfu/Kg Ba and half dose of aureomycin, G3 group was used the diet with 2 × 108cfu/Kg Ba replaced aureomycin. Each treatment had three replications of 10 pigs per pen. Results indicated that Ba replacement significantly increased the activities of amylase, disaccharides and Na+/K+-ATPase. And chymotrypsin activity in different section of intestine was dramatically enhanced in half replacement of aureomycin with Ba. Moreover, Ba replacement maintained the intestinal integrity with the significantly decreased activity of DAO compared with aureomycin group. Besides, supplementation with Ba increased the β-diversity of intestinal microbiota. Taken together, the current study indicated that diet supplementation with Ba instead of aureomycin increased the growth performance of piglets by improving the digestive and absorb enzyme activities, enhancing the intestinal integrity and regulating the population of intestinal micrbiota

Identification of a novel prophage regulator in Escherichia coli controlling the expression of type III secretion

This study has identified horizontally acquired genomic regions of enterohaemorrhagic Escherichia coli O157:H7 that regulate expression of the type III secretion (T3S) system encoded by the locus of enterocyte effacement (LEE). Deletion of O-island 51, a 14.93 kb cryptic prophage (CP-933C), resulted in a reduction in LEE expression and T3S. The deletion also had a reduced capacity to attach to epithelial cells and significantly reduced E. coli O157 excretion levels from sheep. Further characterization of O-island 51 identified a novel positive regulator of the LEE, encoded by ecs1581 in the E. coli O157:H7 strain Sakai genome and present but not annotated in the E. coli strain EDL933 sequence. Functionally important residues of ECs1581 were identified based on phenotypic variants present in sequenced E. coli strains and the regulator was termed RgdR based on a motif demonstrated to be important for stimulation of gene expression. While RgdR activated expression from the LEE1 promoter in the presence or absence of the LEE-encoded regulator (Ler), RgdR stimulation of T3S required ler and Ler autoregulation. RgdR also controlled the expression of other phenotypes, including motility, indicating that this new family of regulators may have a more global role in E. coli gene expression