High purity and recovery of native filamentous hemagglutinin (FHA) from Bordetella pertussis using affinity chromatography.

Filamentous hemagglutinin (FHA) is a critical adhesion molecule produced by Bordetella pertussis (BP), the causative agent of highly contagious respiratory infection known as whooping cough. FHA plays a pivotal role in the pathogenesis of whooping cough and is a key component of acellular pertussis vaccines (aPV). However, conventional purification methods for FHA often involve labor-intensive processes and result in low purity and recovery rates. Therefore, this study explores the use of monoclonal and polyclonal antibodies as specific tools to achieve highly pure and efficient FHA purification. To generate FHA-specific antibodies, polyclonal antibodies were produced by immunizing sheep and monoclonal antibodies (MAbs) were generated by immunizing mice with recombinant and native FHA. The MAbs were selected based on affinity, isotypes, and specificity, which were assessed through ELISA and Western blot assays. Two immunoaffinity columns, one monoclonal and one polyclonal, were prepared for FHA antigen purification. The purity and recovery rates of these purifications were determined using ELISA, SDS-PAGE, and immunoblotting. Furthermore, the MAbs were employed to develop an ELISA assay for FHA antigen concentration determination. The study's findings revealed that immunoaffinity column-based purification of FHA resulted in a highly pure antigen with recovery rates of approximately 57% ± 6.5% and 59% ± 7.9% for monoclonal and polyclonal columns, respectively. Additionally, the developed ELISA exhibited appropriate reactivity for determining FHA antigen concentration. This research demonstrates that affinity chromatography is a viable and advantageous method for purifying FHA, offering superior purity and recovery rates compared to traditional techniques. This approach provides a practical alternative for FHA purification in the context of aPV development.

Expression, purification, and characterization of pneumococcal PsaA-PspA fusion protein.

Streptococcus pneumoniae is a gram-positive bacterial pathogen causing invasive pneumonia, meningitis, otitis media, and bacteremia. Owing to the current pitfalls of polysaccharide and polysaccharide-conjugate vaccines, protein vaccines are considered promising candidates against pneumonia. Pneumococcal surface protein A (PspA) and pneumococcal surface adhesin A (PsaA) are virulence proteins showing good immunogenicity and protective effects against S. pneumoniae strains in mice. In this study, we expressed the fusion protein PsaA-PspA, which consists of PsaA and the N-terminal region of PspA family 1 and 2, in Escherichia coli. We describe a novel and effective method to purify PsaA-PspA using hydroxyapatite and two-step chromatography. After determining the optimal induction conditions and a series of purification steps, we obtained PsaA-PspA fusion protein with over 95% purity at a final yield of 22.44% from the starting cell lysate. The molecular weight of PsaA-PspA was approximately 83.6 kDa and its secondary structure was evaluated by circular dichroism. Immunization with the purified protein induced high levels of IgG antibodies in mice. Collectively, these results demonstrate that our purification method can effectively produce high-purity PsaA-PspA fusion protein with biological activity and chemical integrity, which can be widely applied to the purification of other PspA subclass proteins.

Immunodominant proteins P1 and P40/P90 from human pathogen Mycoplasma pneumoniae.

Mycoplasma pneumoniae is a bacterial human pathogen that causes primary atypical pneumonia. M. pneumoniae motility and infectivity are mediated by the immunodominant proteins P1 and P40/P90, which form a transmembrane adhesion complex. Here we report the structure of P1, determined by X-ray crystallography and cryo-electron microscopy, and the X-ray structure of P40/P90. Contrary to what had been suggested, the binding site for sialic acid was found in P40/P90 and not in P1. Genetic and clinical variability concentrates on the N-terminal domain surfaces of P1 and P40/P90. Polyclonal antibodies generated against the mostly conserved C-terminal domain of P1 inhibited adhesion of M. pneumoniae, and serology assays with sera from infected patients were positive when tested against this C-terminal domain. P40/P90 also showed strong reactivity against human infected sera. The architectural elements determined for P1 and P40/P90 open new possibilities in vaccine development against M. pneumoniae infections.

Detection of Fibronectin-Binding Proteins of Streptococcus pyogenes Using Ligand Blot Analysis.

Streptococcus pyogenes utilizes extracellular cellular matrix (ECM) proteins to adhere to human tissues and internalize into host cells. Fibronectin (Fn) is one of the most abundant ECM proteins and targeted by a wide variety of secreted Fn-binding proteins (Fbps) of S. pyogenes. However, prior to detailed kinetic analysis of that binding process, evaluations of the ability of S. pyogenes strains to bind to Fn as well as interactions of target molecules with Fn are required. In this chapter, we present routine procedures for ligand blot analysis with labeled human Fn, using bacterial cell wall extracts prepared by either enzymatic digestion of cells or extraction with a denaturing agent.

Analyzing the adhesion mechanism of FnBPA, a surface adhesin from Staphylococcus aureus on its interaction with nanoparticle.

Staphylococcus aureus expresses many Microbial Surface Recognizing Adhesive Matrix Molecules (MSCRAMM's) to recognize host extracellular matrix (ECM) molecules to initiate colonization. The MSCRAMM, fibronectin binding protein A (FnBPA), is an important adhesin for S. aureus infection. FnBPA also binds with fibrinogen (Fg) by using a unique ligand binding mechanism called dock, lock and latch. Nanoparticles, especially nanosilver particles have been widely used in a variety of biomedical applications which includes disease diagnosis and treatment, drug delivery and implanted medical device coating. In a biological system, when protein molecules encounter nanoparticle, they can be absorbed onto its surface which results in the formation of protein corona. In the present study, we have analysed the fibrinogen binding ability of rFnBPA(189-512) in the presence of silver nanoparticles by employing techniques like gel shift assay, Western blot, size exclusion chromatography, enzyme-linked immunosorbent assay, bio-layer interferometry and circular dichroism spectroscopy. The results indicate that rFnBPA(189-512) is unable to bind to Fg in the presence of a nanoparticle. This could be due to the inaccessibility of the Fg binding site and conformational change in rFnBPA(189-512). With nanoparticles, rFnBPA(189-512) undergoes significant structural changes as the ß-sheet content has drastically reduced to 10% from the initial 60% at higher concentration of the nanoparticle. Pathogenic bacteria interact with its surrounding environment through their surface molecules which includes MSCRAMMs. Therefore MSCRAMMs play an important role when bacteria encounter nanoparticles. The results of the present study suggest that the orientation of the protein during the absorption on the surface of a nanoparticle as well as the concentration of the nanoparticle, will dictate the function of the absorbed protein and in this case the Fg binding property of rFnBPA(189-512).

The adhesive PitA pilus protein from the early dental plaque colonizer Streptococcus oralis: expression, purification, crystallization and X-ray diffraction analysis.

PitA is the putative tip adhesin of the pilus islet 2 (PI-2)-encoded sortase-dependent pilus in the Gram-positive Streptococcus oralis, an opportunistic pathogen that often flourishes within the diseased human oral cavity. Early colonization by S. oralis and its interaction with Actinomyces oris seeds the development of oral biofilm or dental plaque. Here, the PI-2 pilus plays a vital role in mediating adherence to host surfaces and other bacteria. A recombinant form of the PitA adhesin has now been produced and crystallized. Owing to the large size (∼100 kDa), flexibility and complicated folding of PitA, obtaining diffraction-quality crystals has been a challenge. However, by the use of limited proteolysis with α-chymotrypsin, the diffraction quality of the PitA crystals was considerably enhanced to 2.16 Šresolution. These crystals belonged to space group P1, with unit-cell parameters a = 61.48, b = 70.87, c = 82.46 Å, α = 80.08, ß = 87.02, γ = 87.70°. The anomalous signal from the terbium derivative of α-chymotrypsin-treated PitA crystals prepared with terbium crystallophore (Tb-Xo4) was sufficient to obtain an interpretable electron-density map via terbium SAD phasing.

Featured Species-Specific Loops Are Found in the Crystal Structure of Mhp Eno, a Cell Surface Adhesin From Mycoplasma hyopneumoniae.

Enolase is an evolutionarily conserved enzyme involved in the processes of glycolysis and gluconeogenesis. Mycoplasma hyopneumoniae belongs to Mycoplasma, whose species are wall-less and among the smallest self-replicating bacteria, and is an important colonizing respiratory pathogen in the pig industry worldwide. Mycoplasma hyopneumoniae enolase (Mhp Eno) expression is significantly increased after infection and was previously found to be a virulence factor candidate. Our studies show that Mhp Eno is a cell surface-localized protein that can adhere to swine tracheal epithelial cells (STECs). Adhesion to STECs can be specifically inhibited by an Mhp Eno antibody. Mhp Eno can recognize and interact with plasminogen with high affinity. Here, the first crystal structure of the mycoplasmal enolase from Mycoplasma hyopneumoniae was determined. The structure showed unique features of Mhp Eno in the S3/H1, H6/S6, H7/H8, and H13 regions. All of these regions were longer than those of other enolases and were exposed on the Mhp Eno surface, making them accessible to host molecules. These results show that Mhp Eno has specific structural characteristics and acts as a multifunctional adhesin on the Mycoplasma hyopneumoniae cell surface.

Introduction of a C-terminal hexa-lysine tag increases thermal stability of the LacDiNac binding adhesin (LabA) exodomain from Helicobacter pylori.

Helicobacter pylori is a pathogenic microorganism infecting approximately 50% of the global population, and establishes life-long colonization despite the hostile stomach environment. H. pylori employs a wide range of outer membrane proteins (adhesins) for epithelial attachment, which specifically bind to glycans or non-carbohydrate structures expressed on the gastric epithelium. A recently described adhesin from H. pylori is LabA, named after its ability to bind to a disaccharide present in gastric mucus (LacdiNAc-specific adhesin). Here, we describe the recombinant expression of LabA from H. pylori strains J99 and 26695 in E. coli. High yields of recombinant LabA were obtained using periplasmic expression. We found that the addition of a C-terminal hexalysine (6K) tag enhanced the thermal stability of LabA without affecting its secondary structure, using differential scanning fluorimetry and circular dichroism spectroscopy. In contrast to our previous report for another H. pylori adhesin (BabA), the 6K tag did not enhance recombinant protein yield or solubility. Both versions of LabA, with or without the 6K tag, were expressed and isolated from the periplasmic space of Escherichia coli, with a surprisingly high yield of at least 40 mg/L for each independent preparation, following a two-step purification protocol. The proteins were analyzed with mass spectrometry (MS). Unlike its reported effect on stability of BabA, the 6K tag did not appear to protect the N-term of recombinant LabA from partial periplasmic degradation.

Cloning, Expression, and Purification of Recombinant Neisseria gonorrhoeae Proteins.

Modern DNA recombinant techniques and major advances in genetic engineering have resulted in the development of bacterial expression systems that guarantee an unlimited supply of valuable proteins that have potential clinical or industrial use, but which are often limited by their low natural availability. This chapter provides the reader with a general scheme to clone, express, and purify native histidine (His)-tagged proteins in the desired quantity and quality required for its intended use, and reviews the most important factors affecting the production of recombinant proteins in a soluble form. Alternative methods for purification of insoluble recombinant proteins under denaturing conditions are also discussed. An optimized protocol to successfully purify native Neisseria gonorrhoeae Adhesin Complex Protein (Ng-ACP; NGO1981) is used as a technical example for the processes, which could potentially be applied to any gonococcal recombinant protein of interest.

Production and characterization of recombinant P1 adhesin essential for adhesion, gliding, and antigenic variation in the human pathogenic bacterium, Mycoplasma pneumoniae.

Mycoplasma pneumoniae forms an attachment organelle at one cell pole, binds to the host cell surface, and glides via a unique mechanism. A 170-kDa protein, P1 adhesin, present on the organelle surface plays a critical role in the binding and gliding process. In this study, we obtained a recombinant P1 adhesin comprising 1476 amino acid residues, excluding the C-terminal domain of 109 amino acids that carried the transmembrane segment, that were fused to additional 17 amino acid residues carrying a hexa-histidine (6 × His) tag using an Escherichia coli expression system. The recombinant protein showed solubility, and chirality in circular dichroism (CD). The results of analytical gel filtration, ultracentrifugation, negative-staining electron microscopy, and small-angle X-ray scattering (SAXS) showed that the recombinant protein exists in a monomeric form with a uniformly folded structure. SAXS analysis suggested the presence of a compact and ellipsoidal structure rather than random or molten globule-like conformation. Structure model based on SAXS results fitted well with the corresponding structure obtained with cryo-electron tomography from a closely related species, M. genitalium. This recombinant protein may be useful for structural and functional studies as well as for the preparation of antibodies for medical applications.

Unexpected Prevalence of eae-Positive Escherichia coli in the Animas River, Durango, Colorado.

Since 2014, biology students at Fort Lewis College have studied the water quality of the Animas River in Durango, Colorado. Environmental microbiology and molecular biology techniques have been employed to study Escherichia coli isolates from the river and to define characteristics of the bacteria related to public health. E. coli was found in the river, as well as in culverts and tributary creeks that drain into the river within the Durango city limits. Concentrations of E. coli in the river occasionally exceeded the US EPA guideline of 126 CFU per 100 mL for recreational water use. Many of the E. coli isolates were able to be grown at 45 °C, an indication of mammalian origin. Unexpectedly, 8% of the isolates contained the intimin (eae) gene, a virulence gene characteristic of two pathotypes of E. coli, the enterohemorrhagic and enteropathogenic E. coli. Several isolates tested were resistant to multiple antibiotics commonly used in animal and human medicine. Further study is warranted to determine the source of these bacteria entering the Animas River, and to further characterize the possible disease potential of multi-antibiotic resistant and virulence gene-containing isolates found in a semi-rural/urban setting.

The Chlamydia trachomatis PmpD adhesin forms higher order structures through disulphide-mediated covalent interactions.

Chlamydia trachomatis (Ct) is the most common sexually transmitted bacterial pathogen, and the leading cause of infectious blindness worldwide. We have recently shown that immunization with the highly conserved antigenic passenger domain of recombinant Ct polymorphic membrane protein D (rPmpD) is protective in the mouse model of Ct genital tract infection, and previously, that ocular anti-rPmpD antibodies are elicited following vaccination. However, the mechanisms governing the assembly and structure-function relationship of PmpD are unknown. Here, we provide a biophysical analysis of this immunogenic 65 kDa passenger domain fragment of PmpD. Using differential cysteine labeling coupled with LC-MS/MS analysis, we show that widespread intra- and intermolecular disulphide interactions play important roles in the preservation of native monomeric secondary structure and the formation of higher-order oligomers. While it has been proposed that FxxN and GGA(I, L,V) repeat motifs in the Pmp21 ortholog in Chlamydia pneumoniae mediate self-interaction, no such role has previously been identified for cysteine residues in chlamydial Pmps. Further characterisation reveals that oligomeric proteoforms and rPmpD monomers adopt ß-sheet folds, consistent with previously described Gram-negative bacterial type V secretion systems (T5SSs). We also highlight adhesin-like properties of rPmpD, showing that both soluble rPmpD and anti-rPmpD serum from immunized mice abrogate binding of rPmpD-coated beads to mammalian cells in a dose-dependent fashion. Hence, our study provides further evidence that chlamydial Pmps may function as adhesins, while elucidating yet another important mechanism of self-association of bacterial T5SS virulence factors that may be unique to the Chlamydiaceae.

Expression of fibronectin-binding protein of L. acidophilus NCFM and in vitro refolding to adhesion capable native-like protein from inclusion bodies.

The ability of Lactobacilli to adhere to host epithelial surface and intestinal tracts is important for colonization and persistence of bacteria in the host gut. Extracellular matrix components like fibronectin, mucin, collagen and other adhesion molecules serve as substratum for attachment of bacteria. However, the precise structure, function and mechanism of binding of microbial surface adhesion proteins such as Fibronectin-binding protein (FBP) with host molecules remains unclear. This is primarily due to limitations in high expression of these proteins in biologically active form. To study adhesion of its FBP (64 kDa), the fbp gene of L. acidophilus NCFM was cloned and expressed in E. coli. However, the fibronectin-binding protein expressed in soluble form could not be purified by Ni-NTA affinity chromatography possibly because of partially buried Histidine tag in the recombinant fusion protein. Therefore, the protein was expressed as inclusion bodies (IBs) at 37 °C and solubilized in urea followed by purification in denatured form by Ni-NTA affinity chromatography. The purified denatured protein was refolded in vitro to structurally stable and biologically active form. The conformational properties of the refolded protein were studied by circular dichroism, which showed prominence of α+ ß structural element. The refolded FBP also showed significant binding to human intestinal tissue sections. Our optimized refolding protocol from IBs of this recombinant probiotic FBP led into high amounts of biologically active protein. Our results help in increasing understanding of structure-function relation of surface adhesion proteins and host-microbial interactions.

Typical & atypical enteropathogenic Escherichia coli in diarrhoea & their role as carrier in children under five.

BACKGROUND & OBJECTIVES: Multidrug-resistant enteropathogenic Escherichia coli (EPEC) is responsible for a large number of cases of infantile diarrhoea in developing countries, causing failure in treatment with consequent health burden and resulting in a large number of deaths every year. This study was undertaken to determine the proportion of typical and atypical EPEC in under five children with diarrhoea and controls, their function as a carriage and to identify virulent genes associated with them. METHODS: During the study period, 120 stool samples including 80 from controls children were collected and analyzed for the presence of EPEC using standard bacteriological methods. Isolates were subjected to antimicrobial testing by disc diffusion method. Isolates confirmed as E. coli by phenotypic method were further tested for the presence of attaching and effacing (eae) and bundle-forming pilus (bfpA) genes by real-time SYBR Green-based polymerase chain reaction. RESULTS: All isolates were tested for the presence of EPEC. The frequency of typical EPEC was 20 and 16.25 per cent whereas the frequency of atypical EPEC strains was 5 and 23.75 per cent in patients and controls, respectively (PbfpA was seen in 45 and 18.75 per cent isolates of diarrhoeal patients and controls, respectively. INTERPRETATION & CONCLUSIONS: Our results showed that typical EPEC was a common cause of diarrhoea, but at the same time, atypical EPEC was emerging as colonizers in the intestine of children with and without diarrhoea in and around Delhi. Children can be considered asymptomatic carriers of these pathogens and can transmit them to other susceptible children. Adequate steps need to be taken to stop these strains from developing and spreading further.

TrmFO, a Fibronectin-Binding Adhesin of Mycoplasma bovis.

Mycoplasma bovis is an important pathogenic mycoplasma, causing the cattle industry serious economic losses. Adhesion is a crucial step in the mycoplasmas' infection and colonization process; fibronectin (Fn), an extracellular matrix glycoprotein, is a molecular bridge between the bacterial adhesins and host cell receptors. The present study was designed to characterize the Fn-binding ability of methylenetetrahydrofolate-tRNA-(uracil-5-)-methyltransferase (TrmFO) and its role in M. bovis cytoadherence. The trmFO (MBOV_RS00785) gene was cloned and expressed in E. coli BL21, and polyclonal antibodies against the recombinant TrmFO (rTrmFO) were raised in rabbits. Immunoblotting demonstrated that TrmFO was an immunogenic component, and the TrmFO expression was conserved in different M. bovis isolates. The mycoplasmacidal assay further showed that in the presence of complement, rabbit anti-recombinant TrmFO serum exhibited remarkable mycoplasmacidal efficacy. TrmFO was detected in both the M. bovis membrane and cytoplasm. By ligand dot blot and enzyme-linked immunosorbent assay (ELISA) binding assay, we found that rTrmFO bound Fn in a dose-dependent manner. Immunostaining visualized by confocal laser scanning microscopy showed that rTrmFO had capacity to adhere to the embryonic bovine lung (EBL) cells. In addition, the adhesion of M. bovis and rTrmFO to EBL cells could be inhibited by anti-rTrmFO antibodies. To the best of our knowledge, this is the first report to characterize the Fn-binding ability of TrmFO and its role in the bacterial adhesion to host cells.

Gingipain of Porphyromonas gingivalis manipulates M1 macrophage polarization through C5a pathway.

Gingipains secreted by Porphyromonas gingivalis (P. gingivalis, Pg) play an important role in maintaining macrophage infiltrating. And, this study is to evaluate effects of gingipain on M1 macrophage polarization after exposure to Porphyromonas gingivalis (P. gingivalis, Pg) and if these effects are through complement component 5a (C5a) pathway. Mouse RAW264.7 macrophages were exposed to gingipain extracts, Escherichia coli lipopolysaccharides (Ec-LPS), Pg-LPS with or without the C5aR antagonist: PMX-53 for 24 h. Then, gene expressions and protein of IL-12, IL-23, iNOS, IL-10, TNF-α, IL-1ß, and IL-6 were determined by qRT-PCR and ELISA assays. Surface markers CD86 for M1 and CD206 for M2 were also evaluated by flow cytometry. The results show that gingipain extracts alone increased expressions of IL-12, IL-23, iNOS, TNF-α, IL-1ß, and IL-6, but not IL-10. Gingipain extracts plus Ec-LPS decreased expressions of IL-12, IL-23, iNOS, TNF-α, IL-1ß, and IL-6 in which Ec-LPS induced increase. For gingipain extracts plus Pg-LPS-treated RAW264.7, macrophages, gingipain extracts enhanced expressions of IL-12 and IL-23 in which Pg-LPS induced increase, but not iNOS and IL-10 while gingipain extracts decreased expressions of TNF-α, IL-1ß, and IL-6 in which Pg-LPS induced increase. Interestingly, PMX-53 increased expressions of IL-12, IL-23, and iNOS when RAW264.7 macrophages were treated with gingipain extracts plus Ec-LPS or Pg-LPS and PMX-53, while PMX-53 decreased expressions of TNF-α, IL-1ß, and IL-6. Changes of CD86-positive macrophages were consistent with cytokine changes. Our data indicate that gingipain is a critical regulator, more like a promoter to manipulate M1 macrophage polarization in order to benefit P. gingivalis infection through the C5a pathway.

Expression of recombinant truncated domains of mucus-binding (Mub) protein of Lactobacillus plantarum in soluble and biologically active form.

Mucins amount to 70% of total proteins present in mammalian mucus and serve as important substrata for bacterial adhesion. In probiotic bacteria such as Lactobacillus plantarum, surface adhesion proteins mediate its adhesion to mucus and adhesion is pivotal in bi-directional host-microbe interactions. Mucus binding (Mub) proteins are a group of bacterial surface adhesion proteins that bind to mucin proteins. The structural framework and functional role of these proteins needs immediate attention but is poorly understood because of their large size, low yield and lack of highly purified protein. The lp_1643 gene of L. plantarum encodes a large Mub protein of 240 kDa and has six mucus binding (Mub) domains in tandem. In this study, the fragment of lp_1643 containing the last two domains with their preceding spacers herein referred to as Mubs5s6 was cloned and expressed in E. coli for probing its functional role in the adhesion of L. plantarum. The protein was expressed with a solubility enhancing maltose binding protein (MBP) fusion tag, yet the MBP-Mubs5s6 protein expressed majorly (>90%) as biologically insoluble inclusion bodies. Thus, extensive optimization of culture conditions was carried out to achieve high level soluble expression (∼70%) of Mubs5s6 protein from its initial low level of solubility. The recombinant protein was purified up to homogeneity by affinity chromatography. Recombinant MBP-Mubs5s6 protein showed strong adhesion potential by binding with human intestinal tissue sections. Our results show a step-by-step hierarchical approach to improve the solubility of difficult-to-express extracellular surface proteins while retaining high functional viability.

Effects of different rearing systems on growth, small intestinal morphology and selected indices of fermentation status in broilers.

A 3×2 factorial experiment was conducted to determine the effects of rearing system and stocking density on the growth performance, intestinal morphology and fermentation status of broilers. Broilers were kept on three rearing systems: floor litter rearing (FRS), plastic net rearing (NRS) and multilayer cage rearing system (CRS), each with two stocking densities (normal and high stocking densities). Results showed that on 7 to 28 days of age, body weight gain appeared as FRS > NRS > CRS. Whereas, CRS significantly enhanced the weight gain of broilers compared with the other systems subsequently. Broilers on FRS had higher counts of cecum Bifidobacteria and Lactobacilli at 28 days of age but had more Escherichia coli and less Bifidobacteria than CRS at 42 days of age. The FRS also decreased volatile fatty acid (VFA) concentration and jejunal villus height-to-crypt depth ratio at all ages. In conclusion, FRS appeared to benefit gut microorganisms during the early growing period along with high body weight gain of broilers, whereas this system might have a harmful effect on subsequent intestinal growth, as indicated by high E. coli, low Bifidobacteria count, low VFA concentration and villus height-to-crypt depth ratio along with low weight gain of broilers.

Use of Atomic Force Microscopy to Study the Multi-Modular Interaction of Bacterial Adhesins to Mucins.

The mucus layer covering the gastrointestinal (GI) epithelium is critical in selecting and maintaining homeostatic interactions with our gut bacteria. However, the molecular details of these interactions are not well understood. Here, we provide mechanistic insights into the adhesion properties of the canonical mucus-binding protein (MUB), a large multi-repeat cell-surface adhesin found in Lactobacillus inhabiting the GI tract. We used atomic force microscopy to unravel the mechanism driving MUB-mediated adhesion to mucins. Using single-molecule force spectroscopy we showed that MUB displayed remarkable adhesive properties favouring a nanospring-like adhesion model between MUB and mucin mediated by unfolding of the multiple repeats constituting the adhesin. We obtained direct evidence for MUB self-interaction; MUB-MUB followed a similar binding pattern, confirming that MUB modular structure mediated such mechanism. This was in marked contrast with the mucin adhesion behaviour presented by Galectin-3 (Gal-3), a mammalian lectin characterised by a single carbohydrate binding domain (CRD). The binding mechanisms reported here perfectly match the particular structural organization of MUB, which maximizes interactions with the mucin glycan receptors through its long and linear multi-repeat structure, potentiating the retention of bacteria within the outer mucus layer.

Development of a Sandwich ELISA for EHEC O157:H7 Intimin γ1.

Enterohemorrhagic Escherichia coli (EHEC) O157:H7 is a zoonotic pathogen of worldwide importance that causes foodborne infections in humans. Intimin gamma 1 (intimin γ1) is one of the most important outer membrane proteins required for EHEC's intimate adhesion to epithelial cells. Here, we generated a polyclonal antibody (pAb) and a monoclonal antibody (mAb) against intimin γ1 to develop a double antibody sandwich ELISA (DAS-ELISA) with increased sensitivity and specificity for measuring EHEC O157:H7. To achieve this goal, a rabbit pAb was used as a capture antibody, and a mouse mAb was a detection antibody. No cross-reactivity was observed with the other genera of pathogenic bacteria tested with the DAS-ELISA, which included Salmonella enteritidis, Shigella flexneri type 2, Listeria monocytogenes, Streptococcus suis type 2, and other 18 serotype E. coli. Detection limits of the DAS-ELISA were 1 × 103 CFU/mL for EHEC O157:H7 cultures, 1 × 104 CFU/g before enrichment, and 1 × 102 CFU/g after enrichment of contaminated samples. Field samples (n = 498) were tested using a previously established duplex-PCR method and compared to our DAS-ELISA. The DAS-ELISA had a specificity of 94.4%, a sensitivity of 91.5% and accuracy of 94.0% compared with duplex-PCR. The DAS-ELISA developed here can be applied to EHEC O157:H7 quantification in food, animal, and environmental samples.