- CRISPR-Cas9 Toolkit for Actinomycete Genome Editing. [Journal Article]
- MMMethods Mol Biol 2018; 1671:163-184
- Bacteria of the order Actinomycetales are one of the most important sources of bioactive natural products, which are the source of many drugs. However, many of them still lack efficient genome editin...
Bacteria of the order Actinomycetales are one of the most important sources of bioactive natural products, which are the source of many drugs. However, many of them still lack efficient genome editing methods, some strains even cannot be manipulated at all. This restricts systematic metabolic engineering approaches for boosting known and discovering novel natural products. In order to facilitate the genome editing for actinomycetes, we developed a CRISPR-Cas9 toolkit with high efficiency for actinomyces genome editing. This basic toolkit includes a software for spacer (sgRNA) identification, a system for in-frame gene/gene cluster knockout, a system for gene loss-of-function study, a system for generating a random size deletion library, and a system for gene knockdown. For the latter, a uracil-specific excision reagent (USER) cloning technology was adapted to simplify the CRISPR vector construction process. The application of this toolkit was successfully demonstrated by perturbation of genomes of Streptomyces coelicolor A3(2) and Streptomyces collinus Tü 365. The CRISPR-Cas9 toolkit and related protocol described here can be widely used for metabolic engineering of actinomycetes.
- Clinicopathologic and Microbiologic Findings Associated with Emphysematous Cystitis in 27 Dogs. [Journal Article]
- JAJ Am Anim Hosp Assoc 2017 Nov/Dec; 53(6):313-320
- This is a retrospective case series of 27 dogs with emphysematous cystitis. Medical records from two veterinary teaching hospitals from 1992 to 2014 were reviewed. The aims of the study were to deter...
This is a retrospective case series of 27 dogs with emphysematous cystitis. Medical records from two veterinary teaching hospitals from 1992 to 2014 were reviewed. The aims of the study were to determine imaging findings, common underlying disease processes, and prevalent bacterial species and their antimicrobial susceptibility patterns in dogs with emphysematous cystitis. The most common lower urinary tract sign was hematuria. Gas was detected in the wall and lumen of the urinary bladder in 14 of 27 dogs (51.9%), in only the wall of the bladder in 9 of 27 dogs (33%), and in only the lumen of the bladder in 4 of 27 dogs (14.8%). Comorbid diseases were identified in all but one case. The most common comorbid disease processes were diabetes mellitus in 33% of dogs, neurologic disease in 26% of dogs, and adrenal disease in 19% of dogs. Bacterial isolates included Escherichia coli, Enterococcus spp., Klebsiella pneumoniae, Proteus mirabilis, Streptococcus spp., and Actinomyces spp. Enterococcus spp. were always isolated in mixed infections with gas-producing bacterial species. During the period of study, most isolates were predicted to be susceptible to beta-lactam drugs, but updated veterinary breakpoints suggest that fluoroquinolones or trimethoprim-sulfamethoxazole would be more appropriate choices for empiric therapy.
- Evaluation of the bioactive extract of actinomyces isolated from the Egyptian environment against aflatoxin B1-induce cytotoxicity, genotoxicity and oxidative stress in the liver of rats. [Journal Article]
- FCFood Chem Toxicol 2017; 105:241-255
- This study aimed to determine the bioactive compounds of actinomyces (ACT) isolated from the Egyptian environment (D-EGY) and to evaluate their protective activity against AFB1 in female Sprague-Dawl...
This study aimed to determine the bioactive compounds of actinomyces (ACT) isolated from the Egyptian environment (D-EGY) and to evaluate their protective activity against AFB1 in female Sprague-Dawley rats. Six groups of animals were treated orally for 3 weeks included: C, the control group, T1, AFB1-treated group (80 μg/kg b.w), T2 and T3, the groups received ACT extract at low (25 mg/kg b.w) or high (50 mg/kg b.w) doses, T4 and T5, the groups received AFB1 plus the low or high dose of ACT extract. Blood, bone marrow and tissue samples were collected for different analyses and histological examination. The results revealed the identification of 40 components, representing 99.98%. Treatment with AFB1 disturbs liver function parameters, oxidative stress markers, antioxidant gene expressions, DNA fragmentation and induced severe histological changes. ACT extract at the low or high doses did not induce significant changes in all the tested parameters or histological picture of the liver. Moreover, ACT extract succeeded to induce a significant protection against the toxicity of AFB1. It could be concluded that the bioactive compounds in ACT are promise candidate for the development of food additive or drugs for the protection and treatment of liver disorders in the endemic area.
- Role of microbiological culture and polymerase chain reaction (PCR) of actinomyces in medication-related osteonecrosis of the jaw (MRONJ). [Journal Article]
- JCJ Craniomaxillofac Surg 2017; 45(3):357-363
- We hypothesized that local infection plays a critical role in the pathogenesis of medication-related osteonecrosis of the jaw (MRONJ). Recent developments in molecular methods have revolutionized new...
We hypothesized that local infection plays a critical role in the pathogenesis of medication-related osteonecrosis of the jaw (MRONJ). Recent developments in molecular methods have revolutionized new approaches for the rapid detection of microorganisms including those difficult to culture. The aim of our study is to identify the bacterial profiles in MRONJ by microbiological culture and polymerase chain reactions (PCR). A retrospective analysis was performed on MRONJ patients from 2008 to 2014. The bacterial profile from MRONJ bone samples was determined using microbiological culture and PCR. Ninety five patients fulfilled the inclusion criteria with mean age of 69.85 ± 8.71 years. A female predilection was detected. The mandible was more commonly affected than maxilla. Tooth extraction was the frequent triggering factor. Breast cancer was the primary cause for administration and intravenous bisphosphonates were the most commonly administrated antiresorptive drugs. The majority of patients were classified as stage 2. Posterior teeth were most commonly affected. Based on bone culture results, the most common microorganism were both actinomyces and mixed flora. PCR confirmed the presence of actinomyces in 55 patients. Our data suggest that PCR might be an innovative method for detection of microorganisms difficult to culture using traditional microbiological techniques.
- Induction of antibody response in the oral cavity of dogs following intraocular (eye drop) immunization with Porphyromonas gingivalis cell lysate incorporated in pH-sensitive fusogenic polymer-modified liposomes. [Journal Article]
- JVJ Vet Med Sci 2017 Feb 14; 79(2):290-298
- Induction of mucosal immune responses against Porphyromonas gingivalis within the oral cavity of dogs was studied by immunizing with pH-sensitive fusogenic polymer (MGluPG)-modified liposome-associat...
Induction of mucosal immune responses against Porphyromonas gingivalis within the oral cavity of dogs was studied by immunizing with pH-sensitive fusogenic polymer (MGluPG)-modified liposome-associated cell lysate. Dogs immunized with P. gingivalis cell lysate-containing MGluPG-modified liposomes by intraocular (eye drop) route displayed significant levels of P. gingivalis cell lysate-specific serum IgG and IgA as well as mucosal IgA antibodies in saliva secretion. Serum and salivary antibodies generated by intraocularly immunized with MGluPG-modified liposome-associated P. gingivalis cell lysate revealed a significant aggregation activity against P. gingivalis, whereas serum and saliva from dogs receiving MGluPG-modified liposomes unentrapping P. gingivalis cell lysate did not show the aggregation activity against P. gingivalis. Furthermore, P. gingivalis-specific antibodies in saliva of immunized dogs inhibited the adherence of P. gingivalis to cultured HeLa cells. More importantly, salivary antibodies induced by intraocular immunization with P. gingivalis cell lysate-containing MGluPG-modified liposomes significantly inhibited the coaggregation of P. gingivalis with Actinomyces naeslundii and the cell damage activity of P. gingivalis against FaDu cells, an oral epithelial cell. These results suggest that intraocularly administered P. gingivalis cell lysate-containing MGluPG-modified liposomes should be an effective mucosal vaccine against P. gingivalis infection in dogs and may be an important tool for the prevention of periodontitis.
- Discovery of Ibomycin, a Complex Macrolactone that Exerts Antifungal Activity by Impeding Endocytic Trafficking and Membrane Function. [Journal Article]
- CCCell Chem Biol 2016 Nov 17; 23(11):1383-1394
- Natural products are invaluable historic sources of drugs for infectious diseases; however, the discovery of novel antimicrobial chemical scaffolds has waned in recent years. Concurrently, there is a...
Natural products are invaluable historic sources of drugs for infectious diseases; however, the discovery of novel antimicrobial chemical scaffolds has waned in recent years. Concurrently, there is a pressing need for improved therapeutics to treat fungal infections. We employed a co-culture screen to identify ibomycin, a large polyketide macrolactone that has preferential killing activity against Cryptococcus neoformans. Using chemical and genome methods, we determined the structure of ibomycin and identified the biosynthetic cluster responsible for its synthesis. Chemogenomic profiling coupled with cell biological assays link ibomycin bioactivity to membrane function. The preferential activity of ibomycin toward C. neoformans is due to the ability of the compound to selectively permeate its cell wall. These results delineate a novel antifungal agent that is produced by one of the largest documented biosynthetic clusters to date and underscore the fact that there remains significant untapped chemical diversity of natural products with application in antimicrobial research.
- Anti-plaque effect of a synergistic combination of green tea and Salvadora persica L. against primary colonizers of dental plaque. [Journal Article]
- AOArch Oral Biol 2016; 70:117-124
- CONCLUSIONS: Combination between Gt and Sp aqueous extracts exhibited synergistic anti-plaque activity, and could be used as a useful active agent to produce oral health care products.
- Genome-wide identification and characterization of macrolide glycosyltransferases from a marine-derived Bacillus strain and their phylogenetic distribution. [Journal Article]
- EMEnviron Microbiol 2016; 18(12):4770-4781
- Clarifying glycosyltrasferases (GTs) function is of significance for the development of GT inhibitors as drugs, and the use of GTs to glycodiversify small molecules in the search of drug leads. While...
Clarifying glycosyltrasferases (GTs) function is of significance for the development of GT inhibitors as drugs, and the use of GTs to glycodiversify small molecules in the search of drug leads. While many Actinomyces natural-product GTs had been functionally characterized, our understanding towards Bacillus natural-product GTs is so far very limited. Herein, genome-wide identification of macrolide GT genes from marine-derived Bacillus methylotrophicus B-9987 revealed the presence of three macrolide GT genes bmmGT1-3. While bmmGT1 was previously revealed to be involved in the biosynthesis of trans-acyltransferase (AT) polyketides compounds macrolactins (MLNs) and bacillaenes (BAEs), the functions of bmmGT2 and bmmGT3 were probed, demonstrating that they are capable to biochemically catalyze glycosylation of MLNs and BAEs as well but interestingly with different regioselectivity, affording four new MLNs analogs. Notably, further genome mining revealed that the orthologs of these three macrolide GT genes showed a regular distribution in the subtilis- and the cereus-clade Bacillus strains; interestingly, bmmGT1 orthologs only occurred in the subtilis-clade Bacillus, and they were also found in the genomes of Streptomyces strains, suggesting their close phylogenetic relationship. These results provide the first significant insight into the important roles of Bacillus macrolide GTs in the biology of the species.
- Disulfide-Bond-Forming Pathways in Gram-Positive Bacteria. [Review]
- JBJ Bacteriol 2016; 198(5):746-54
- Disulfide bonds are important for the stability and function of many secreted proteins. In Gram-negative bacteria, these linkages are catalyzed by thiol-disulfide oxidoreductases (Dsb) in the peripla...
Disulfide bonds are important for the stability and function of many secreted proteins. In Gram-negative bacteria, these linkages are catalyzed by thiol-disulfide oxidoreductases (Dsb) in the periplasm. Protein oxidation has been well studied in these organisms, but it has not fully been explored in Gram-positive bacteria, which lack traditional periplasmic compartments. Recent bioinformatics analyses have suggested that the high-GC-content bacteria (i.e., actinobacteria) rely on disulfide-bond-forming pathways. In support of this, Dsb-like proteins have been identified in Mycobacterium tuberculosis, but their functions are not known. Actinomyces oris and Corynebacterium diphtheriae have recently emerged as models to study disulfide bond formation in actinobacteria. In both organisms, disulfide bonds are catalyzed by the membrane-bound oxidoreductase MdbA. Remarkably, unlike known Dsb proteins, MdbA is important for pathogenesis and growth, which makes it a potential target for new antibacterial drugs. This review will discuss disulfide-bond-forming pathways in bacteria, with a special focus on Gram-positive bacteria.
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- A New Screen for Tuberculosis Drug Candidates Utilizing a Luciferase-Expressing Recombinant Mycobacterium bovis Bacillus Calmette-Guéren. [Journal Article]
- PlosPLoS One 2015; 10(11):e0141658
- Tuberculosis (TB) is a serious infectious disease caused by a bacterial pathogen. Mortality from tuberculosis was estimated at 1.5 million deaths worldwide in 2013. Development of new TB drugs is nee...
Tuberculosis (TB) is a serious infectious disease caused by a bacterial pathogen. Mortality from tuberculosis was estimated at 1.5 million deaths worldwide in 2013. Development of new TB drugs is needed to not only to shorten the medication period but also to treat multi-drug resistant and extensively drug-resistant TB. Mycobacterium tuberculosis (Mtb) grows slowly and only multiplies once or twice per day. Therefore, conventional drug screening takes more than 3 weeks. Additionally, a biosafety level-3 (BSL-3) facility is required. Thus, we developed a new screening method to identify TB drug candidates by utilizing luciferase-expressing recombinant Mycobacterium bovis bacillus Calmette-Guéren (rBCG). Using this method, we identified several candidates in 4 days in a non-BSL-3 facility. We screened 10,080 individual crude extracts derived from Actinomyces and Streptomyces and identified 137 extracts which possessed suppressive activity to the luciferase of rBCG. Among them, 41 compounds inhibited the growth of both Mtb H37Rv and the extensively drug-resistant Mtb (XDR-Mtb) strains. We purified the active substance of the 1904-1 extract, which possessed strong activity toward rBCG, Mtb H37Rv, and XDR-Mtb but was harmless to the host eukaryotic cells. The MIC of this substance was 0.13 μg/ml, 0.5 μg/ml, and 2.0-7.5 μg/ml against rBCG, H37Rv, and 2 XDR-strains, respectively. Its efficacy was specific to acid-fast bacterium except for the Mycobacterium avium intracellular complex. Mass spectrometry and nuclear magnetic resonance analyses revealed that the active substance of 1904-1 was cyclomarin A. To confirm the mode of action of the 1904-1-derived compound, resistant BCG clones were used. Whole genome DNA sequence analysis showed that these clones contained a mutation in the clpc gene which encodes caseinolytic protein, an essential component of an ATP-dependent proteinase, and the likely target of the active substance of 1904-1. Our method provides a rapid and convenient screen to identify an anti-mycobacterial drug.