Development of a Novel Anti-Adhesive Vaccine Against Pseudomonasaeruginosa Targeting the C-terminal Disulfide Loop of the Pilin Protein.

Type IV pili (T4P) are major virulence factors of Pseudomonas aeruginosa (P. aeruginosa) that are associated with primary adhesion, biofilm formation and twitching motility. This study focuses on the introduction of a novel biologically active subunit vaccine derived from the disulfide loop (DSL) of P. aeruginosa pilin. We investigated the expression of the novel PilA in-frame with pET26b vector, which contains three domains, that each domain contains three tandem repeats. The flexible (GGGGS) and (GGGGS)3 linkers were linked between the three tandem repeats and each pilA domain, respectively. The recombinant construct (pET26b/pilA) was transformed and expressed in Escherichia coli BL21 (DE3). The reactivity of specific antiserum against PilA was assessed by ELISA method. The biological activities of this candidate vaccine were evaluated by western blotting, opsonophagocytosis and twitching inhibition assays. The pET26b/pilA plasmid was confirmed by enzymatic digestion. The purified PilA protein was confirmed by immunoblot analysis. The checkerboard titration showed that the optimal dilution of the antibody to react with antigen was 1:8. The results of opsonophagocytosis assay revealed that the antibodies raised against PilA promoted phagocytosis of the PAO1 and 6266E strains to some extent (17.5% and 16.3%, respectively), so the twitching inhibition test confirmed this result. Taken together, these are the preliminary results based on a first chimerical structure failure to induce antibodies that promote the opsonization and eradication of the pathogen. Therefore, the biological activity of the PilA protein showed that it should be introduced with other proteins or target antigens against P. aeruginosa in the future studies.

Construction, expression, purification and characterization of secretin domain of PilQ and triple PilA-related disulfide loop peptides fusion protein from Pseudomonas aeruginosa.

OBJECTIVES: Infection with Pseudomonas aeruginosa has been a long-standing obstacle for clinical therapy due to the complexity of the genetics and pathogenesis, as well for widespread resistance to antibiotics, thus attaching great importance to explore effective vaccines for prevention and treatment. This paper focuses on the introduction of novel Pseudomonas aeruginosa type IV pili (T4P)-based fusion protein containing the secretin domain of PilQ and tandem PilA-related peptides. MATERIALS AND METHODS: We surveyed the expression of the PilQ380-705-PilA fusion protein in-frame with pET26b vector in which a rigid linker was used between two polypeptides and flexible linkers were inserted between the three tandem repeats and each pilA domains. The transformants were expressed in Escherichia coli BL21. The reactivity of specific antisera to the fusion protein was assessed by ELISA. The biological activities of this candidate vaccine were evaluated by western blotting, opsonophagocytosis, and twitching inhibition assays. RESULTS: The fusion protein was purified in high yield by osmotic shock method using HisTrap affinity column. The protein was confirmed by immunoblot analysis. The checkerboard titration showed that the optimal dilution of the antibody to react with antigen is 1:128. Results of opsonophagocytosis assay revealed that the antibodies elevated to the fusion protein promoted phagocytosis of the PAO1 and 6266E strains, so that the twitching immobilization test confirmed these results. CONCLUSION: Due to excellent killing activity mediated by opsonic antibodies and efficient immobilization of the strains, it seems that PilQ380-705-PilA fusion protein could be a reliable candidate vaccine against P. aeruginosa infection.

Preparation of Pseudomonas aeruginosa alginate-flagellin immunoconjugate.

Mucoid strains of Pseudomonas aeruginosa are closely associated with chronic pulmonary infections. In this report we describe a straightforward approach to conjugate high molecular weight alginate to type b-flagellin (FLB) and investigation of its bioactivity. The conjugation process was performed by using ADH and EDAC. The endotoxin was eliminated from the candidate vaccine by LPS removal resin followed by LAL test. The bioconjugate molecules were verified by simultaneously determination of polysaccharide/protein content followed by gel filtration chromatography and FTIR spectroscopy. Groups of eight BALB/c mice were injected intranasally with 5 µg (per each nostril) of purified alginate, FLB and conjugated alginate-FLB with two week intervals. The functional activity of the vaccine was evaluated by ELISA and opsonophagocytosis tests. Vaccination with the alginate-FLB conjugate induced a significant (P = 0.0033) rise in alginate specific IgG in mice. At all dilution ranges, the opsonic activity of the conjugate vaccine antisera was significantly higher than alginate alone (61.9% vs. 17.3% at 1:4 dilution; P = 0.0067). The alginate-FLB conjugate could elicit high specific antibodies titer against alginate by improving its immunogenicity. In addition, the antisera raised against conjugate vaccine act as a suitable opsonin for phagocytosis of the mucoid strains of P. aeruginosa.

High Yield Overexpression, Refolding, Purification and Characterization of Pseudomonas aeruginosa Type B-Flagellin: An Improved Method Without Sonication.

Pseudomonas aeruginosa as an opportunistic pathogen is a significant cause of acute and chronic infections in patients with compromised defenses. This bacterium is motile via a single polar flagellum made of polymerized flagellin subunits differentiated into two major serotypes: A and B. flagellin plays an important role as a virulence factor in the adhesion, colonization and invasion of P. aeruginosa into host epithelial cells. To develop a functional vaccine that can be used in practical application to prevent and treat infection, type B-flagellin was produced as recombinant protein. In this work, the fliC gene was introduced into a pET28a vector and expressed in Escherichia coli BL21 (DE3). The expressed recombinant protein was purified by a modified method without sonication using a HisTrap affinity column. The functional activities of produced flagellin were confirmed by ELISA, western blot analysis, motility inhibition assay and opsonophagocytosis test. The purification process of the type B-flagellin was lead to a high yield. The produced recombinant type B-flagellin showed high biological activity in all of these standard assays. In conclusions, this report provides the new protocol to efficiently obtain the type B-flagellin with high biological activity and immunogenicity. This immunogen can be introduced as an adjuvant or vaccine in the future study.

Haarlem 3 is the predominant genotype family in multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis in the capital of Iran: A 5-year survey.

The objective of this study was to further understand the genetic diversity of multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis isolates prevalent in Tehran, the capital city of Iran. From January 2010 to March 2015, a total of 723 M. tuberculosis strains were isolated from patients with pulmonary tuberculosis (TB). A total of 23 MDR, pre-XDR and XDR M. tuberculosis isolates were genotyped by spoligotyping and 24-loci mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) typing. The results showed that the MDR, pre-XDR and XDR M. tuberculosis strains mainly belonged to the Haarlem 3 genotype (11/23; 47.8%), followed by the Beijing family (9/23; 39.1%). In addition, the 23 strains were clustered into 21 genotypes using a 24-loci MIRU-VNTR. In conclusion, Haarlem 3 genotype was the predominant genotype among the isolates from MDR-TB cases in this study, which could be of special concern.

High prevalence of Mycobacterium tuberculosis mixed infection in the capital of moderate tuberculosis incidence country.

OBJECTIVE: Recent studies using molecular epidemiological techniques have demonstrated mixed infection with multiple strains of Mycobacterium tuberculosis especially in countries with high tuberculosis (TB) burden. We aimed to determine the prevalence of mixed infection among patients with TB in the capital of Iran as a country with moderate incidence rate. METHODS: Samples were collected randomly from January 2011 to December 2013 in Tehran, capital of Iran. A total of 75 M. tuberculosis isolates were genotyped by 24 loci mycobacterial interspersed repetitive unit-variable number tandem repeat typing (MIRU-VNTR) for screening the mixed infection. RESULTS: Twenty patients (20/75) were identified with mixed infection, and the estimated rate of mixed infection was 26.6%. Thirteen out of the 24 loci were able to detect the mixed infection in our study. CONCLUSIONS: Mixed infections occur at high prevalence among studied Iranian TB patients. Further research is inevitable to evaluate the association of mixed infection and disease progression and treatment.

Portable, battery-operated, low-cost, bright field and fluorescence microscope.

This study describes the design and evaluation of a portable bright-field and fluorescence microscope that can be manufactured for $240 USD. The microscope uses a battery-operated LED-based flashlight as the light source and achieves a resolution of 0.8 microm at 1000x magnification in fluorescence mode. We tested the diagnostic capability of this new instrument to identify infections caused by the human pathogen, Mycobacterium tuberculosis. Sixty-four direct, decontaminated, and serially diluted smears were prepared from sputa obtained from 19 patients suspected to have M. tuberculosis infection. Slides were stained with auramine orange and evaluated as being positive or negative for M. tuberculosis with both the new portable fluorescence microscope and a laboratory grade fluorescence microscope. Concordant results were obtained in 98.4% of cases. This highly portable, low cost, fluorescence microscope may be a useful diagnostic tool to expand the availability of M. tuberculosis testing at the point-of-care in low resource settings.

High-level rifampin resistance correlates with multiple mutations in the rpoB gene of pulmonary tuberculosis isolates from the Afghanistan border of Iran.

The aim of this study was to investigate the significance of multiple mutations in the rpoB gene as well as predominant nucleotide changes and their correlation with high levels of resistance to rifampin (rifampicin) in Mycobacterium tuberculosis isolates that were randomly collected from the sputa of 46 patients with primary and secondary cases of active pulmonary tuberculosis from the southern region (Afghanistan border) of Iran where tuberculosis is endemic. Drug susceptibility testing was performed using the CDC standard conventional proportional method. DNA extraction, rpoB gene amplification, and DNA sequencing analysis were performed. Thirty-five (76.09%) isolates were found to have multiple mutations (two to four) in the rpoB (beta-subunit) gene. Furthermore, we demonstrate that the combination of mutations with more prevalent nucleotide changes were observed in codons 523, 526, and 531, indicating higher frequencies of mutations among patients with secondary infection. In this study, 76.08% (n = 35) of all isolates found to have mutation combinations involving nucleotide changes in codons 523 (GGG-->GCG), 531 (TCG-->TTG or TTC), and 526 (CAC-->CGC, TTC, AAC, or CAA) demonstrated an association with higher levels of resistance to rifampin (MIC, >or=100 microg/ml).

RETRACTED: High level Rifampin Resistance Correlates with Multiple Mutations in the rpoB Gene of Pulmonary Tuberculosis Isolates from the Afghanistan Border of Iran.

This article has been retracted.

Chlamydia pneumoniae DNA is more frequent in advanced than in mild atherosclerosis lesions.

There is growing evidence of an association between Chlamydia pneumoniae infection and atherosclerosis. By using polymerase chain reaction (PCR), we detected the presence of C. pneumoniae DNA in 16 of 92 (17%) arterial specimens with severe atherosclerotic lesions, and in 3 of 109 (3%) such specimens with mild atherosclerotic lesions (p < 0.01) from 49 cases with an autopsy diagnosis of cardiac death and 5 patients who underwent vascular reconstructive surgery. 14 of the 54 cases (28%) were C. pneumoniae-positive in at least 1 vascular sample. 12 of the 14 (86%) PCR positive cases were aged 60 y or older. Normal pulmonary artery specimens from 24 autopsy cases, used as a methodological control, tested negative. The levels of low density lipoprotein cholesterol and triglycerides were significantly lower in the PCR-positive cases than in the PCR-negative cases (p < 0.05). Importantly, 11 of the 14 PCR-positive cases had only 1 risk factor for atherosclerotic cardiovascular disease, whereas all PCR-negative cases had multiple risk factors (p < 0.05). Our data support the idea that C. pneumoniae may be involved in the development of atherosclerosis in humans, especially in cases where classic risk factors are not identified to explain the incidence of atherosclerotic vascular disease.