Modeling and Simulation of Bacterial Outer Membranes with Lipopolysaccharides and Capsular Polysaccharides.

Capsule is one of the common virulence factors in Gram-negative bacteria protecting pathogens from host defenses and consists of long-chain capsular polysaccharides (CPS) anchored in the outer membrane (OM). Elucidating structural properties of CPS is important to understand its biological functions as well as the OM properties. However, the outer leaflet of the OM in current simulation studies is represented exclusively by LPS due to the complexity and diversity of CPS. In this work, representative Escherichia coli CPS, KLPS (a lipid A-linked form) and KPG (a phosphatidylglycerol-linked form), are modeled and incorporated into various symmetric bilayers with co-existing LPS in different ratios. All-atom molecular dynamics simulations of these systems have been conducted to characterize various bilayer properties. Incorporation of KLPS makes the acyl chains of LPS more rigid and ordered, while incorporation of KPG makes them less ordered and flexible. These results are consistent with the calculated area per lipid (APL) of LPS, in which the APL of LPS becomes smaller when KLPS is incorporated, whereas it gets larger when KPG is included. Torsional analysis reveals that the influence of the CPS presence on the conformational distributions of the glycosidic linkages of LPS is small, and minor differences are also detected for the inner and outer regions of the CPS. Combined with previously modeled enterobacterial common antigens (ECAs) in the form of mixed bilayers, this work provides more realistic OM models as well as the basis for characterization of interactions between the OM and OM proteins.

Molecular detection and characterization of the endosymbiont Wolbachia in the European hedgehog flea, Archaeopsylla erinacei.

Wolbachia, the endosymbiont of arthropods and onchocercid nematodes is present in many medically important insect species, being also considered for the indirect control of parasitic ones. Archaeopsylla erinacei is a flea species infesting hedgehogs acting as vector of Rickettsia felis, Bartonella henselae, and Rickettsia helvetica, thus having public health relevance. The Wolbachia surface protein (wsp) and 16S rRNA genes were used to determine the presence, prevalence and molecular typing of Wolbachia in this flea species collected in two regions of southern Italy. Of the 45 fleas tested (n = 16 males, 35.6%; n = 29 females, 64.4%), 43 (95.6%; 95% CI: 84.8-99.2) scored positive for Wolbachia, of which 15 (33.3%) and 28 (62.2%) were males and females, respectively. The sex-wise prevalence of this endosymbiont was almost equal in both sexes (males 93.8%; 95% CI: 69.5-99.7; females 96.7%; 95% CI: 83.1-99.8). Single locus sequence analysis (SLST) of Wolbachia revealed two sequence types for 16S rRNA gene, named as wAr_15227 and wAr_15234, which came from two different areas, equally distributed in male and female fleas, whilst only one sequence type was identified for wsp gene. The phylogenetic analysis placed the two 16S rRNA sequence types in paraphyletic clades belonging to the supergroup A and B, respectively. Whilst, the tree of wsp gene clustered the corresponding sequence in the same clade including those of Wolbachia supergroup A. In MLST analyses, both Wolbachia sequence types clustered in a monophyletic clade with Drosophila nikananu (wNik) and Drosophila sturtevanti (wStv) from supergroup A. ClonalFrame analysis revealed a recombination event in the wAr_15234 strain which came from Apulia region. Scientific knowledge of the presence/prevalence of Wolbachia among medically important fleas, may contribute to develop an alternative biological method for the vector control.

Biophysical characterization of the homodimers of HomA and HomB, outer membrane proteins of Helicobacter pylori.

Helicobacter pylori is a Gram-negative bacterium that causes chronic inflammations in the stomach area and is involved in ulcers, which can develop into gastric malignancies. H. pylori attaches and colonizes to the human epithelium using some of their outer membrane proteins (OMPs). HomB and HomA are the most studied OMPs from H. pylori as they play a crucial role in adherence, hyper biofilm formation, antibiotic resistance and are also associated with severe gastric malignancies. The role of HomA and HomB in pathogenesis concerning their structure and function has not been evaluated yet. In the present study, we explored the structural aspect of HomA and HomB proteins using various computational, biophysical and small-angle X-ray scattering (SAXS) techniques. Interestingly, the in-silico analysis revealed that HomA/B consists of 8 discontinuous N and C terminal ß-strands forming a small ß-barrel, along with a large surface-exposed globular domain. Further, biophysical experiments suggested that HomA and HomB are dimeric and most likely the cysteine residues present on surface-exposed loops participate in protein-protein interactions. Our study provides essential structural information of unexplored proteins of the Hom family that can help in a better understanding of H. pylori pathogenesis.

Molecular detection of gyrA and mexA genes in Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa is one of the types of bacteria that arises resistance toward fluoroquinolos antibiotics remarkably in recent years. METHODS: Fifty P. aeruginosa isolates were isolated from one hundred clinical samples, investigated the antibiogram activity toward eight different groups of antibiotics. Screening about gyrA gene was done by conventional PCR further more qualitative gene expression of mexA gene was done by using Real-time PCR in 22 MDR isolates, furthermore Relative gene expression analysis of gyrA and mexA was done. RESULTS: The rate of P. aeruginosa isolates was (41.6%) from total clinical samples, the antibiogram test showed high resistance toward Ceftazidime, Ciprofloxacin, Levofloxacin and Gentamicin (100%), while the sensitivity was observed towards colistin (100%). Screening of gyrA that was achieved by PCR technique showed 22 positive isolates. Furthermore, the 22 isolates appeared high expression level of the efflux pump resistance gene mexA and gyrA gene compared with housekeeping gene rspL gene within fold change ranging (0.18-36 and 1-28.84 respectively) with a mean of 18.46 ct and 18.59 (respectively). CONCLUSIONS: All P. aeruginosa isolates were MDR with high level of efflux pump expression of mexA gene as well as gyrA gene.

Paper-based ELISA diagnosis technology for human brucellosis based on a multiepitope fusion protein.

BACKGROUND: Brucellosis, as a serious zoonotic infectious disease, has been recognized as a re-emerging disease in the developing countries worldwide. In china, the incidence of brucellosis is increasing each year, seriously threatening the health of humans as well as animal populations. Despite a quite number of diagnostic methods currently being used for brucellosis, innovative technologies are still needed for its rapid and accurate diagnosis, especially in area where traditional diagnostic is unavailable. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a total of 22 B cell linear epitopes were predicted from five Brucella outer membrane proteins (OMPs) using an immunoinformatic approach. These epitopes were then chemically synthesized, and with the method of indirect ELISA (iELISA), each of them displayed a certain degree of capability in identifying human brucellosis positive sera. Subsequently, a fusion protein consisting of the 22 predicted epitopes was prokaryotically expressed and used as diagnostic antigen in a newly established brucellosis testing method, nano-ZnO modified paper-based ELISA (nano-p-ELISA). According to the verifying test using a collection of sera collected from brucellosis and non-brucellosis patients, the sensitivity and specificity of multiepitope based nano-p-ELISA were 92.38% and 98.35% respectively. The positive predictive value was 98.26% and the negative predictive value was 91.67%. The multiepitope based fusion protein also displayed significantly higher specificity than Brucella lipopolysaccharide (LPS) antigen. CONCLUSIONS: B cell epitopes are important candidates for serologically testing brucellosis. Multiepitope fusion protein based nano-p-ELISA displayed significantly sensitivity and specificity compared to Brucella LPS antigen. The strategy applied in this study will be helpful to develop rapid and accurate diagnostic method for brucellosis in human as well as animal populations.

Co-delivery of hesperidin and clarithromycin in a nanostructured lipid carrier for the eradication of Helicobacter pylori in vitro.

Effective and precise eradication of Helicobacter pylori (H. pylori) is the most promising approach to avoid H. pylori-related gastrointestinal disorders. The present study was conducted to demonstrate the efficacy of the co-delivery of hesperidin (Hesp) and clarithromycin (CLR) in nanostructured lipid carriers (NLCs) against H. pylori. We have produced a new delivery system by combining bioflavonoid Hesp and CLR NLCs to address the failure in single antibiotic therapies. Briefly, a blend of solid lipid, liquid lipid, and surfactant was used. Homogeneous NLCs with all the formulations showed a nano size and surface-negative charge and presented high in vitro stability and slow release of the drug even after 24 h. Bioimaging studies by scanning electron microscopy, transmission electron microscopy, and imaging flow cytometry indicated that NLCs interacted with the membrane by adhering to the outer cell membrane and disrupted the membrane that resulted in the leakage of cytoplasmic contents. The prepared NLCs provide sustained and controlled drug release that can be used to increase the rate of H. pylori eradication.

Mesoporous Silica as Sorbents and Enzymatic Nanoreactors for Microbial Membrane Proteomics.

The membrane proteins of microbes are at the forefront of host and parasite interactions. Having a general view of the functions of microbial membrane proteins is vital for many biomedical studies on microbiota. Nevertheless, due to the strong hydrophobicity and low concentration of membrane proteins, it is hard to efficiently enrich and digest the proteins for mass spectrometry analysis. Herein, we design an enzymatic nanoreactor for the digestion of membrane proteins using methylated well-ordered hexagonal mesoporous silica (Met-SBA-15). The material can efficiently extract hydrophobic membrane proteins and host the proteolysis in nanopores. The performance of the enzymatic nanoreactor is first demonstrated using standard hydrophobic proteins and then validated using membrane proteins extracted from Escherichia coli (E. coli) or a mixed bacterial sample of eight strains. Using the nanoreactor, 431 membrane proteins are identified from E. coli, accounting for 38.5% of all membrane proteins of the species, which is much more than that by the widely used in-solution digestion protocol. From the mixed bacterial sample of eight strains, 1395 membrane proteins are identified using the nanoreactor. On the contrary, the traditional in-solution proteolysis workflow only leads to the identification of 477 membrane proteins, demonstrating that the Met-SBA-15 can be offered as an excellent tool for microbial membrane proteome research and is expected to be used in human microbiota studies, e.g. host-microbe interactions.

Phos-Tag Fluorescent Gel Staining for the Quantitative Detection of His- and Asp-Phosphorylated Proteins.

We describe a standard protocol for phosphate-affinity fluorescent gel staining that uses a fluorophore-labeled dizinc(II) complex of a derivative of the phosphate-binding tag molecule Phos-tag to detect His- and Asp-phosphorylated proteins separated by SDS-PAGE. The procedure permits the quantitative monitoring of phosphorylated histidine kinases (His-phosphoproteins) and their cognate phosphorylated response regulators (Asp-phosphoproteins) in bacterial two-component signaling transduction systems. The total time required for each gel staining operation is about 2 h at room temperature.

Separation of Glycosylated OmpA-Like Proteins from Porphyromonas gingivalis and Tannerella forsythia.

OmpA-like proteins located in the outer bacterial membrane are potential virulence factors from the major periodontal pathogens Porphyromonas gingivalis and Tannerella forsythia. Our previous studies have shown that OmpA-like proteins are glycosylated by O-linked N-acetylglucosamine (O-GlcNAc) and are strongly reactive to wheat germ agglutinin (WGA) lectin, which shows sugar specificity to GlcNAc. Utilizing this property, we have developed a separation method for OmpA-like proteins by affinity chromatography using WGA lectin-agarose. The purity of enriched native OmpA-like proteins were confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Brilliant Blue (CBB) staining. More importantly, the purified OmpA-like proteins formed a unique trimeric structure keeping their bioactivity intact. In this chapter, we describe a detailed procedure to separate OmpA-like proteins, which may be used to further progress the biological studies of OmpA-like proteins.

Molecular detection and identification of piroplasms (Babesia spp. and Theileria spp.) and Anaplasma phagocytophilum in questing ticks from northwest Spain.

Anaplasma phagocytophilum and some piroplasm species are pathogens mainly transmitted by Ixodes ricinus. Considering that this tick species is predominant in north-western Spain, individual specimens (652 nymphs, 202 females and 202 males) and 23 larval pools were processed to determine the prevalence of these pathogens in questing I. ricinus from that region. Additionally, Dermacentor marginatus, Dermacentor reticulatus, Ixodes frontalis and Ixodes acuminatus were individually analysed. The groESL operon as well as the 16S rRNA and msp2 genes of Anaplasma were analysed. Similarly, piroplasms were identified at the 18S rRNA gene and the ITS1 of Babesia spp. and Theileria spp. Babesia venatorum (1.5%), A. phagocytophilum (0.7%), Babesia microti (0.3%) and Theileria sp. OT3 (0.2%) were detected in I. ricinus. A single I. frontalis (8.3%) tested positive to A. phagocytophilum. Although a low percentage of I. ricinus were infected with A. phagocytophilum and piroplasms, a potentially human pathogenic variant of A. phagocytophilum was detected, and both Babesia species found were zoonotic. Since the vector of Theileria sp. OT3 remains unknown, further investigations are needed to unravel the role of I. ricinus in the transmission of this piroplasm.

Probiotic Escherichia coli Nissle 1917-derived outer membrane vesicles enhance immunomodulation and antimicrobial activity in RAW264.7 macrophages.

BACKGROUND: Probiotic Escherichia coli Nissle 1917 (EcN) has been widely studied for the treatment of intestinal inflammatory diseases and infectious diarrhea, but the mechanisms by which they communicate with the host are not well-known. Outer membrane vesicles (OMVs) are produced by Gram-negative bacteria and deliver microbial molecules to distant target cells in the host, which play a very important role in mediating bacteria-host communication. Here, we aimed to investigate whether EcN-derived OMVs (EcN_OMVs) could mediate immune regulation in macrophages. RESULTS: In this study, after the characterization of EcN_OMVs using electron microscopy, nanoparticle tracking and proteomic analyses, we demonstrated by confocal fluorescence microscopy that EcN_OMVs could be internalized by RAW 264.7 macrophages. Stimulation with EcN_OMVs at appropriate concentrations promoted proliferation, immune-related enzymatic activities and phagocytic functions of RAW264.7 cells. Moreover, EcN_OMVs induced more anti-inflammatory responses (IL-10) than pro-inflammatory responses (IL-6 and TNF-α) in vitro, and also modulated the production of Th1-polarizing cytokine (IL-12) and Th2-polarizing cytokine (IL-4). Treatments with EcN_OMVs effectively improved the antibacterial activity of RAW 264.7 macrophages. CONCLUSIONS: These findings indicated that EcN_OMVs could modulate the functions of the host immune cells, which will enrich the existing body of knowledge of EVs as an important mechanism for the communication of probiotics with their hosts.

1H, 13C and 15 N NMR assignments of solubility tag protein Msyb of Escherichia coli.

Biochemical and structural characterizations of a protein are the prerequisite for the further understanding of its biological role and potential applications. The expression of recombinant protein is almost unavoidable to produce the amount of the protein required for these studies, especially at the industrial level. Escherichia coli is the single most used system for recombinant protein expression and the first choice for a trial expression. Besides the inherited defects of its prokaryotic origin, the E. coli system has problems like low protein solubility and formation of inclusion bodies. To improve the solubility while assisting correct folding of the target protein, fusing a tag protein prior to its N-terminus is one of the common approaches. GST, MBP, Trx and SUMO proteins are among the most used tags by providing different advantages during recombinant protein expression. Msyb, a small and acidic protein native to E. coli, is another example that could improve the solubility of the target protein. While the biophysical and biochemical properties of these common tag proteins have been studied to a great extent, Msyb protein remains largely uncharacterized. Here, using solution-state NMR, our near-complete resonance assignment of Msyb provides a basis for future structure determination which would help to expand its usage as a common tag protein.

The emm-Cluster Typing System.

emm-cluster typing system allows to classify most Streptococcus pyogenes variants into 48 different emm clusters. The system correlates nicely with the host serum binding capacities of the M proteins and has been used in epidemiological surveys, strain selection, and vaccine development. Here we describe the allocation of the emm cluster based on the emm-typing defining region.

Detergent Titration as an Efficient Method for NMR Resonance Assignments of Membrane Proteins in Lipid-Bilayer Nanodiscs.

Lipid bilayer nanodiscs are an attractive tool to study membrane proteins in a detergent-free lipid-bilayer environment. In the case of NMR studies, a sequence-specific resonance assignment is required in order to gain structural and functional insights with atomic resolution. Although NMR backbone assignments of membrane proteins in detergents are available, they are largely absent for membrane proteins in nanodiscs due to unfavorable relaxation properties of the slowly tumbling membrane protein-nanodisc complex. The necessary residue-specific reassignment of resonances in nanodiscs is therefore extremely time and sample consuming and represents the fundamental bottleneck in the application of nanodiscs for NMR studies. Here we present an elegant and fast solution to the problem. We show that a resonance assignment in detergent micelles can be transferred to a spectrum recorded in nanodiscs via detergent titration. The procedure requires that lipid-detergent exchange kinetics are in the fast exchange regime in order to follow linear and nonlinear peak shift trajectories with increasing detergent concentration. We demonstrate the feasibility of the approach on the 148-residue membrane protein OmpX. The titration method is then applied to VDAC, a 19-stranded ß-barrel with 283 residues, for which 67% of the detergent assignment could be transferred to the nanodisc spectrum. We furthermore show that this method also works for the largest currently assigned membrane protein, BamA with 398 residues. The method is applicable for backbone amide and side chain methyl groups and represents a time and cost-effective assignment method, for example, to investigate membrane protein allostery and drug binding in a more natural and detergent-free lipid bilayer.

Investigation of the effects of oxidative stress-inducing factors on culturing and productivity of Bordetella pertussis.

The stress response of Bordetella pertussis during fermentation was assessed by means of fluorescence-based techniques. During the manufacturing of vaccines, B. pertussis is subjected to stress during adaptation to a new environment and operating conditions in the bioreactor, which can have harmful consequences on growth and protein yield. In this study, stress was imposed by varying the percentage of dissolved oxygen (DO) and inoculum size, and by adding rotenone and hydrogen peroxide. In this study, fluorescence spectroscopy is used as a tool for measuring oxidative stress. High levels of DO during fed-batch operation had no detrimental effect on growth, but the specific productivity of pertactin (PRN) decreased. Cultures that were started with an inoculum size that was 10 times smaller than the control resulted in significantly less PRN as compared to controls where reduction was more significant in flasks as compared to bioreactors. A comparison of filtered to heat-sterilized media revealed that filtered media offered a protective effect against H2 O2 . Heat sterilization of the media might result in the destruction of components that offer protection against oxidative stress. Nonetheless, filter sterilization on its own would be insufficient for large-scale manufacturing. It should be emphasized that the effects of these stressors while investigating for other microorganisms have not been studied for B. pertussis.

Nanofabricated versatile electrochemical sensor for Leptospira interrogans detection.

In this report, a LipL32 gene based nanofabricated electrochemical sensor for the detection of Leptospira interrogans has been developed using carboxylated multiwalled carbon nanotubes with gold nanoparticles (c-MWCNTs/nanoAu) electrode and graphene quantum dots (GQDs). The c-MWCNTs/nanoAu electrode was linked to GQDs using 4-aminothiophenol (ATP). The surface modifications on the electrode surface were delineated using Raman spectroscopy and field emission scanning electron microscopy (FE-SEM). 5'-Amino (NH2) labeled single stranded DNA (ssDNA) probe was immobilized on the surface of c-MWCNTs/nanoAu/ATP/GQD composite electrode. The electrochemical changes of the developed sensor after hybridization with single stranded complementary DNA of L. interrogans were analyzed by differential pulse voltammetry (DPV) using 1 mM methylene blue. The sensor showed good linearity with complementary ssDNA concentration ranging from 0.37 to 12 ng/µl. The sensor exhibited high specificity to L. interrogans and showed good reproducibility when stored at 4°C.

Identification and characterization of OmpT-like proteases in uropathogenic Escherichia coli clinical isolates.

Bacterial colonization of the urogenital tract is limited by innate defenses, including the production of antimicrobial peptides (AMPs). Uropathogenic Escherichia coli (UPEC) resist AMP-killing to cause a range of urinary tract infections (UTIs) including asymptomatic bacteriuria, cystitis, pyelonephritis, and sepsis. UPEC strains have high genomic diversity and encode numerous virulence factors that differentiate them from non-UTI-causing strains, including ompT. As OmpT homologs cleave and inactivate AMPs, we hypothesized that UPEC strains from patients with symptomatic UTIs have high OmpT protease activity. Therefore, we measured OmpT activity in 58 clinical E. coli isolates. While heterogeneous OmpT activities were observed, OmpT activity was significantly greater in UPEC strains isolated from patients with symptomatic infections. Unexpectedly, UPEC strains exhibiting the greatest protease activities harbored an additional ompT-like gene called arlC (ompTp). The presence of two OmpT-like proteases in some UPEC isolates led us to compare the substrate specificities of OmpT-like proteases found in E. coli. While all three cleaved AMPs, cleavage efficiency varied on the basis of AMP size and secondary structure. Our findings suggest the presence of ArlC and OmpT in the same UPEC isolate may confer a fitness advantage by expanding the range of target substrates.

Electrochemical immunosensor based on gold-labeled monoclonal anti-LipL32 for leptospirosis diagnosis.

Leptospirosis is a critical human health problem in the tropical area, thus, a precise technique that can be used for point-of-care analysis is greatly required. This is the first report on electrochemical immunosensor based on gold-labeled monoclonal anti-LipL32 for rapid, simple and sensitive determination of LipL32. The sensor consisted of two LipL32-specific antibodies: an unlabeled capture primary antibody (Anti-1°Ab) and an electrochemically detectable gold-conjugated secondary antibody (Au-2°Ab). The Anti-1°Ab was immobilized onto the modified screen-printed graphene electrode (SPGE) to form the anti-LipL32 surface. The electrochemical signal response was determined by differential pulse voltammetry (DPV). In the presence of LipL32, the sensor displayed a significant increase in current response in a concentration-dependent manner, but no observable signal was detected in the absence of LipL32. The linearity between LipL32 concentration and the measured current was found in a range of 1-100 ng/mL, and the limit of detection (LOD) (3SDblank/Slope) and limit of quantitation (LOQ) (10SDblank/Slope) were found to be 0.28 and 0.93 ng/mL, respectively. This sensor was successfully applied to detect pathogenic Leptospira whole cell lysates samples with the satisfactory results. The promissing results suggested that this immunosensor might be an alternative tool for diagnosis of leptospirosis.

Measurement of Yersinia Translocon Pore Formation in Erythrocytes.

Many Gram-negative pathogens produce a type III secretion system capable of intoxicating eukaryotic cells with immune-modulating effector proteins. Fundamental to this injection process is the prior secretion of two translocator proteins destined for injectisome translocon pore assembly within the host cell plasma membrane. It is through this pore that effectors are believed to travel to gain access to the host cell interior. Yersinia species especially pathogenic to humans and animals assemble this translocon pore utilizing two hydrophobic translocator proteins-YopB and YopD. Although a full molecular understanding of the biogenesis, function and regulation of this translocon pore and subsequent effector delivery into host cells remains elusive, some of what we know about these processes can be attributed to studies of bacterial infections of erythrocytes. Herein we describe the methodology of erythrocyte infections by Yersinia, and how analysis of the resultant contact-dependent hemolysis can serve as a relative measurement of YopB- and YopD-dependent translocon pore formation.

Obtaining an ELISA test based on a recombinant protein of Chlamydia trachomatis.

Chlamydia trachomatis is considered as a public health problem due to its high prevalence and increased rates of gynecological disorders. The major outer membrane protein (MOMP) of this bacterium is the most abundant protein in its membrane and has been evaluated not only as a vaccine development candidate but also is used in many diagnostic tests. The MOMP weighs 69 kDa and contains four variable segments (VS 1-4) separated by constant regions. Several research groups have developed recombinant single-variable segments of MOMP expressed in Escherichia coli cytoplasm. But, all variable segments have been used minimally for the diagnosis of a chlamydial infection. In this experiment, the authors obtained the recombinant MOMP of C. trachomatis (rMOMP) in E. coli rMOMP and extracted, purified, and partially characterized it. This was later used to identify anti-Chlamydia trachomatis antibodies in sera of infertile patients by immunodetection assays, enzyme-linked immunosorbent assay (ELISA), and indirect immunofluorescence tests. The ELISA test showed high sensitivity and low specificity of 100 and 58.3%, respectively. The above results obtained were linked to the cross-reactivity of antibodies against C. pneumoniae or C. psittaci. Hence, an evaluation was performed to obtain an optimized test for the diagnosis of C. trachomatis infection.