scholarly journals Peritrophic matrix-degrading proteins are dispensable virulence factors in a virulent Melissococcus plutonius strain

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Keiko Nakamura ◽  
Kayo Okumura ◽  
Mariko Harada ◽  
Mariko Okamoto ◽  
Masatoshi Okura ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Gene Knockout ◽  
Midgut Epithelium ◽  
Bacterial Disease ◽  
Bacterial Cells ◽  
Peritrophic Matrix ◽  
Knockout Mutant ◽  
Chitin Binding Domain ◽  
Melissococcus Plutonius ◽  
Highly Virulent

AbstractEuropean foulbrood (EFB) caused by Melissococcus plutonius is a major bacterial disease of honey bees. Strains of the causative agent exhibit genetic heterogeneity, and the degree of virulence varies among strains. In bee larvae orally infected with the highly virulent strains, ingested bacterial cells colonize the larval midgut and proliferate within the sac of the peritrophic matrix (PM), a barrier lining the midgut epithelium. However, the barrier is degraded during the course of infection, and M. plutonius cells eventually directly interact with the midgut epithelium. As M. plutonius possesses genes encoding putative PM-degrading proteins (enhancin, a chitin-binding domain-containing protein and endo-α-N-acetylgalactosaminidase), we constructed PM-degrading protein gene-knockout mutants from a highly virulent M. plutonius strain and investigated their role in the pathogenesis of EFB. In larvae infected with the triple-knockout mutant, which has no PM-degrading protein genes, M. plutonius that proliferated in the larval midguts was confined to the sac of the PM. However, the midgut epithelial cells degenerated over time, and the mutant killed approximately 70–80% of bee brood, suggesting that although the PM-degrading proteins are involved in the penetration of the PM by M. plutonius, they are not indispensable virulence factors in the highly virulent M. plutonius strain.

scholarly journals Transcription Factor STE12α Has Distinct Roles in Morphogenesis, Virulence, and Ecological Fitness of the Primary Pathogenic Yeast Cryptococcus gattii

2006 ◽  
Vol 5 (7) ◽  
pp. 1065-1080 ◽  
Author(s):  
Ping Ren ◽  
Deborah J. Springer ◽  
Melissa J. Behr ◽  
William A. Samsonoff ◽  
Sudha Chaturvedi ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Gene Knockout ◽  
Natural Habitat ◽  
Cryptococcus Gattii ◽  
Human Neutrophils ◽  
Knockout Mutant ◽  
Pathogenic Yeast ◽  
Ecological Fitness

ABSTRACT Cryptococcus gattii is a primary pathogenic yeast, increasingly important in public health, but factors responsible for its host predilection and geographical distribution remain largely unknown. We have characterized C. gattii STE12α to probe its role in biology and pathogenesis because this transcription factor has been linked to virulence in many human and plant pathogenic fungi. A full-length STE12α gene was cloned by colony hybridization and sequenced using primer walk and 3′ rapid amplification of cDNA ends strategies, and a ste12αΔ gene knockout mutant was created by URA5 insertion at the homologous site. A semiquantitative analysis revealed delayed and poor mating in ste12αΔ mutant; this defect was not reversed by exogenous cyclic AMP. C. gattii parent and mutant strains showed robust haploid fruiting. Among putative virulence factors tested, the laccase transcript and enzymatic activity were down regulated in the ste12αΔ mutant, with diminished production of melanin. However, capsule, superoxide dismutase, phospholipase, and urease were unaffected. Similarly, Ste12 deficiency did not cause any auxotrophy, assimilation defects, or sensitivity to a large panel of chemicals and antifungals. The ste12αΔ mutant was markedly attenuated in virulence in both BALB/c and A/Jcr mice models of meningoencephalitis, and it also exhibited significant in vivo growth reduction and was highly susceptible to in vitro killing by human neutrophils (polymorphonuclear leukocytes). In tests designed to simulate the C. gattii natural habitat, the ste12αΔ mutant was poorly pigmented on wood agar prepared from two tree species and showed poor survival and multiplication in wood blocks. Thus, STE12α plays distinct roles in C. gattii morphogenesis, virulence, and ecological fitness.

Studies of Virulence Factors of Avian Pathogenic Escherichia coli in Avian Colibacillosis by In vitro Method and PCR

2021 ◽  
Author(s):  
Ayushi Singh ◽  
Daljeet Chhabra ◽  
Rakhi Gangil ◽  
Rakesh Sharda ◽  
Ravi Sikrodia ◽  
...  
Keyword(s):  
Virulence Factors ◽  
Molecular Detection ◽  
Bacterial Disease ◽  
Avian Pathogenic Escherichia Coli ◽  
E Coli ◽  
Pathological Conditions ◽  
Biofilm Production ◽  
Pathogenic Escherichia Coli ◽  
Stx1 Gene

Background: Avian colibacillosis is considered as major cause of morbidity and mortality in poultry. It is a common bacterial disease of poultry and many virulence factors of E. coli are associated with the disease. The current study was aimed to investigate the presence of some virulence factors of E. coli isolated from the cases of colibacillosis.Methods: In present study, total 150 samples (liver, heart, lungs, air sacs and feaces) of chicken exhibiting pathological conditions of colibacillosis were collected from various poultry farms (organized and backyard) situated in and around Mhow and Indore cities. E.coli was isolated and identified from the samples on the basis of cultural characteristics and biochemical test. All E. coli isolates were further subjected to evaluate the presence of virulence factors such as biofilm production, haemolysis, invasiveness and molecular detection of fimH and stx1 gene.Result: Out of these 51.33% of incidence of E. coli was recorded. E. coli O84 and O149 serotypes were found most prevalent. Out of 77 isolates, 46 (59.7%) and 45 (58.4%) were positive for biofilm formation by tube method and modified CRA method, respectively. All E. coli isolates were showing invasiveness in congo red binding assay while none of the isolates was found haemolytic. Molecular detection revealed the presence of fimH (508bp) gene in 33.3% of tested samples while stx1 gene could not be detected in any isolates.

scholarly journals The Isolation DNA Chromosomal DNA from Local Isolate of Aeromonas hydrophila Bacteria from East Java

2018 ◽  
Vol 6 (1) ◽  
Author(s):  
Retno Sri Wahjuni ◽  
M. Gandul Atik Yuliani
Keyword(s):  
Aeromonas Hydrophila ◽  
Economic Loss ◽  
Dna Purification ◽  
Bacterial Disease ◽  
Bacterial Cells ◽  
Chromosomal Dna ◽  
Fish Farms ◽  
Antigenic Protein ◽  
Ulcer Disease ◽  
Improved Sanitation

Aeromoniasis is a bacterial disease caused by Aeromonas hydrophila that affects fish and shrimps in ponds and aquariums. This is an ulcer disease that causes petechiae in scales of fish and may be fatal. It can cause economic loss if it is not treated with medication accompanied with improved sanitation. It affects numerous freshwater fish farms in East Java. A previous study has characterized an antigenic protein derived from the outer membrane protein of A. hydrophila. In order to perform sequencing of the DNA coding for this protein, we first conducted a study for isolating DNA fragments of A. hydrophila via four stages: cultivation and harvesting of bacterial cells, cell lysis, DNA purification, and the concentration of chromosomal DNA. The results will be used as a predictive immunogenic determinant by the method by Kolaskar and Tongaonkar. Keywords: Aeromonas hydrophila, chromosomal DNA, immunogenic determinant

scholarly journals Construction of an Alpha Toxin Gene Knockout Mutant of Clostridium perfringens Type A by Use of a Mobile Group II Intron

2005 ◽  
Vol 71 (11) ◽  
pp. 7542-7547 ◽  
Author(s):  
Yue Chen ◽  
Bruce A. McClane ◽  
Derek J. Fisher ◽  
Julian I. Rood ◽  
Phalguni Gupta
Keyword(s):  
Clostridium Perfringens ◽  
Gene Knockout ◽  
Antibiotic Resistance Genes ◽  
Group Ii Intron ◽  
Toxin Gene ◽  
No Production ◽  
Alpha Toxin ◽  
Knockout Mutant ◽  
New Strategy ◽  
Group Ii

ABSTRACT In developing Clostridium perfringens as a safe vaccine vector, the alpha toxin gene (plc) in the bacterial chromosome must be permanently inactivated. Disrupting genes in C. perfringens by traditional mutagenesis methods is very difficult. Therefore, we developed a new strategy using group II intron-based Target-Tron technology to inactivate the plc gene in C. perfringens ATCC 3624. Western blot analysis showed no production of alpha toxin protein in the culture supernatant of the plc mutant. Advantages of this technology, such as site specificity, relatively high frequency of insertion, and introduction of no antibiotic resistance genes into the chromosome, could facilitate construction of other C. perfringens mutants.

scholarly journals LuxG Is a Functioning Flavin Reductase for Bacterial Luminescence

2007 ◽  
Vol 190 (5) ◽  
pp. 1531-1538 ◽  
Author(s):  
Sarayut Nijvipakul ◽  
Janewit Wongratana ◽  
Chutintorn Suadee ◽  
Barrie Entsch ◽  
David P. Ballou ◽  
...  
Keyword(s):  
Gene Knockout ◽  
Biochemical Properties ◽  
Flavin Mononucleotide ◽  
Flavin Reductase ◽  
Knockout Mutant ◽  
Prosthetic Group ◽  
Bacterial Luminescence ◽  
Luminous Bacteria

ABSTRACT The luxG gene is part of the lux operon of marine luminous bacteria. luxG has been proposed to be a flavin reductase that supplies reduced flavin mononucleotide (FMN) for bacterial luminescence. However, this role has never been established because the gene product has not been successfully expressed and characterized. In this study, luxG from Photobacterium leiognathi TH1 was cloned and expressed in Escherichia coli in both native and C-terminal His6-tagged forms. Sequence analysis indicates that the protein consists of 237 amino acids, corresponding to a subunit molecular mass of 26.3 kDa. Both expressed forms of LuxG were purified to homogeneity, and their biochemical properties were characterized. Purified LuxG is homodimeric and has no bound prosthetic group. The enzyme can catalyze oxidation of NADH in the presence of free flavin, indicating that it can function as a flavin reductase in luminous bacteria. NADPH can also be used as a reducing substrate for the LuxG reaction, but with much less efficiency than NADH. With NADH and FMN as substrates, a Lineweaver-Burk plot revealed a series of convergent lines characteristic of a ternary-complex kinetic model. From steady-state kinetics data at 4°C pH 8.0, Km for NADH, Km for FMN, and k cat were calculated to be 15.1 μM, 2.7 μM, and 1.7 s−1, respectively. Coupled assays between LuxG and luciferases from P. leiognathi TH1 and Vibrio campbellii also showed that LuxG could supply FMNH− for light emission in vitro. A luxG gene knockout mutant of P. leiognathi TH1 exhibited a much dimmer luminescent phenotype compared to the native P. leiognathi TH1, implying that LuxG is the most significant source of FMNH− for the luminescence reaction in vivo.

scholarly journals Cephalosporins target quorum sensing and suppress virulence of Pseudomonas aeruginosa in Caenorhabditis elegans infection model

2020 ◽  
Author(s):  
Lokender Kumar ◽  
Nathanael Brenner ◽  
John Brice ◽  
Judith Klein-Seetharaman ◽  
Susanta K. Sarkar
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Caenorhabditis Elegans ◽  
Quorum Sensing ◽  
Virulence Factors ◽  
Biofilm Formation ◽  
Host Cells ◽  
Infection Model ◽  
Host Immune System ◽  
Bacterial Cells

ABSTRACTPseudomonas aeruginosa utilizes a chemical social networking system referred to as quorum sensing (QS) to strategically co-ordinate the expression of virulence factors and biofilm formation. Virulence attributes damage the host cells, impair the host immune system, and protect bacterial cells from antibiotic attack. Thus, anti-QS agents may act as novel anti-infective therapeutics to treat P. aeruginosa infections. The present study was performed to evaluate the anti-QS, anti-biofilm, and anti-virulence activity of β-lactam antibiotics (carbapenems and cephalosporins) against P. aeruginosa. The anti-QS activity was quantified using Chromobacterium violaceum CV026 as a QS reporter strain. Our results showed that cephalosporins including cefepime (CP), ceftazidime (CF), and ceftriaxone (CT) exhibited potent anti-QS and anti-virulence activities against P. aeruginosa PAO1. These antibiotics significantly impaired motility phenotypes, decreased pyocyanin production, and reduced the biofilm formation by P. aeruginosa PAO1. In the present study, we studied isogenic QS mutants of PAO1: ΔLasR, ΔRhlR, ΔPqsA, and ΔPqsR and found that the levels of virulence factors of antibiotic-treated PAO1 were comparable to QS mutant strains. Molecular docking predicted high binding affinities of cephalosporins for the ligand-binding pocket of QS receptors (CviR, LasR, and PqsR). In addition, our results showed that the anti-microbial activity of aminoglycosides increased in the presence of sub-inhibitory concentrations (sub-MICs) of CP against P. aeruginosa PAO1. Further, utilizing Caenorhabditis elegans as an animal model for the in vivo anti-virulence effects of antibiotics, cephalosporins showed a significant increase in C. elegans survival by suppressing virulence factor production in P. aeruginosa. Thus, our results indicate that cephalosporins might provide a viable anti-virulence therapy in the treatment of infections caused by multi-drug resistant P. aeruginosa.

scholarly journals 6S-1 RNA Contributes to Sporulation and Parasporal Crystal Formation in Bacillus thuringiensis

2020 ◽  
Vol 11 ◽  
Author(s):  
Zhou Li ◽  
Li Zhu ◽  
Zhaoqing Yu ◽  
Lu Liu ◽  
Shan-Ho Chou ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Deletion Mutant ◽  
Gene Deletion ◽  
Gene Knockout ◽  
Single Gene ◽  
Crystal Formation ◽  
Bacterial Cells ◽  
Parasporal Crystal ◽  
6S Rna ◽  
Gene Deletion Mutant

6S RNA is a kind of high-abundance non-coding RNA that globally regulates bacterial transcription by interacting with RNA polymerase holoenzyme. Through bioinformatics analysis, we found that there are two tandem 6S RNA-encoding genes in the genomes of Bacillus cereus group bacteria. Using Bacillus thuringiensis BMB171 as the starting strain, we have explored the physiological functions of 6S RNAs, and found that the genes ssrSA and ssrSB encoding 6S-1 and 6S-2 RNAs were located in the same operon and are co-transcribed as a precursor that might be processed by specific ribonucleases to form mature 6S-1 and 6S-2 RNAs. We also constructed two single-gene deletion mutant strains ΔssrSA and ΔssrSB and a double-gene deletion mutant strain ΔssrSAB by means of the markerless gene knockout method. Our data show that deletion of 6S-1 RNA inhibited the growth of B. thuringiensis in the stationary phase, leading to lysis of some bacterial cells. Furthermore, deletion of 6S-1 RNA also significantly reduced the spore number and parasporal crystal content. Our work reveals that B. thuringiensis 6S RNA played an important regulatory role in ensuring the sporulation and parasporal crystal formation.

scholarly journals Acanthamoeba castellanii as a Screening Tool for Mycobacterium avium Subspecies paratuberculosis Virulence Factors with Relevance in Macrophage Infection

2020 ◽  
Vol 8 (10) ◽  
pp. 1571
Author(s):  
Ida L. Phillips ◽  
Jamie L. Everman ◽  
Luiz E. Bermudez ◽  
Lia Danelishvili
Keyword(s):  
Virulence Factors ◽  
Mycobacterium Avium ◽  
Screening Tool ◽  
Gene Knockout ◽  
Mycobacterium Avium Subspecies Paratuberculosis ◽  
Acanthamoeba Castellanii ◽  
Mammalian Host ◽  
Intracellular Growth ◽  
Macrophage Infection ◽  
Selection Of

The high prevalence of Johne’s disease has driven a continuous effort to more readily understand the pathogenesis of the etiological causative bacterium, Mycobacterium avium subsp. paratuberculosis (MAP), and to develop effective preventative measures for infection spread. In this study, we aimed to create an in vivo MAP infection model employing an environmental protozoan host and used it as a tool for selection of bacterial virulence determinants potentially contributing to MAP survival in mammalian host macrophages. We utilized Acanthamoeba castellanii (amoeba) to explore metabolic consequences of the MAP-host interaction and established a correlation between metabolic changes of this phagocytic host and MAP virulence. Using the library of gene knockout mutants, we identified MAP clones that can either enhance or inhibit amoeba metabolism and we discovered that, for most part, it mirrors the pattern of MAP attenuation or survival during infection of macrophages. It was found that MAP mutants that induced an increase in amoeba metabolism were defective in intracellular growth in macrophages. However, MAP clones that exhibited low metabolic alteration in amoeba were able to survive at a greater rate within mammalian cells, highlighting importance of both category of genes in bacterial pathogenesis. Sequencing of MAP mutants has identified several virulence factors previously shown to have a biological relevance in mycobacterial survival and intracellular growth in phagocytic cells. In addition, we uncovered new genetic determinants potentially contributing to MAP pathogenicity. Results of this study support the use of the amoeba model system as a quick initial screening tool for selection of virulence factors of extremely slow-grower MAP that is challenging to study.

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