scholarly journals Haemophilus influenzae HP1 Bacteriophage Encodes a Lytic Cassette with a Pinholin and a Signal-Arrest-Release Endolysin

2020 ◽  
Vol 21 (11) ◽  
pp. 4013
Author(s):  
Monika Adamczyk-Popławska ◽  
Zuzanna Tracz-Gaszewska ◽  
Przemysław Lasota ◽  
Agnieszka Kwiatek ◽  
Andrzej Piekarowicz
Keyword(s):  
Haemophilus Influenzae ◽  
Gene Product ◽  
Transmembrane Protein ◽  
In Silico Analysis ◽  
Viable Cell ◽  
Cell Lysis ◽  
Gene Products ◽  
E Coli ◽  
Myoviridae Family ◽  
Silico Analysis

HP1 is a temperate bacteriophage, belonging to the Myoviridae family and infecting Haemophilus influenzae Rd. By in silico analysis and molecular cloning, we characterized lys and hol gene products, present in the previously proposed lytic module of HP1 phage. The amino acid sequence of the lys gene product revealed the presence of signal-arrest-release (SAR) and muraminidase domains, characteristic for some endolysins. HP1 endolysin was able to induce lysis on its own when cloned and expressed in Escherichia coli, but the new phage release from infected H. influenzae cells was suppressed by inhibition of the secretion (sec) pathway. Protein encoded by hol gene is a transmembrane protein, with unusual C-out and N-in topology, when overexpressed/activated. Its overexpression in E. coli did not allow the formation of large pores (lack of leakage of β-galactosidase), but caused cell death (decrease in viable cell count) without lysis (turbidity remained constant). These data suggest that lys gene encodes a SAR-endolysin and that the hol gene product is a pinholin. HP1 SAR-endolysin is responsible for cell lysis and HP1 pinholin seems to regulate the cell lysis and the phage progeny release from H. influenzae cells, as new phage release from the natural host was inhibited by deletion of the hol gene.

scholarly journals Two Distinct Modes of Lysis Regulation in Campylobacter Fletchervirus and Firehammervirus Phages

Viruses ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 1247
Author(s):  
Athina Zampara ◽  
Stephen J. Ahern ◽  
Yves Briers ◽  
Lone Brøndsted ◽  
Martine Camilla Holst Sørensen
Keyword(s):  
Signal Peptide ◽  
In Silico ◽  
Protein Domains ◽  
In Silico Analysis ◽  
Cell Lysis ◽  
E Coli ◽  
Sec Pathway ◽  
Gene Functions ◽  
Lytic Genes ◽  
Silico Analysis

Campylobacter phages are divided into two genera; Fletchervirus and Firehammervirus, showing only limited intergenus homology. Here, we aim to identify the lytic genes of both genera using two representative phages (F352 and F379) from our collection. We performed a detailed in silico analysis searching for conserved protein domains and found that the predicted lytic genes are not organized into lysis cassettes but are conserved within each genus. To verify the function of selected lytic genes, the proteins were expressed in E. coli, followed by lytic assays. Our results show that Fletchervirus phages encode a typical signal peptide (SP) endolysin dependent on the Sec-pathway for translocation and a holin for activation. In contrast, Firehammervirus phages encode a novel endolysin that does not belong to currently described endolysin groups. This endolysin also uses the Sec-pathway for translocation but induces lysis of E. coli after overexpression. Interestingly, co-expression of this endolysin with an overlapping gene delayed and limited cell lysis, suggesting that this gene functions as a lysis inhibitor. These results indicate that Firehammervirus phages regulate lysis timing by a yet undescribed mechanism. In conclusion, we found that the two Campylobacter phage genera control lysis by two distinct mechanisms.

scholarly journals Identifying new COVID-19 receptor Neuropilin-1 in severe Alzheimer’s diseases patients group brain using genome-wide association study approach

2021 ◽  
Author(s):  
Key-Hwan Lim ◽  
Sumin Yang ◽  
Sung-Hyun Kim ◽  
Jae-Yeol Joo
Keyword(s):  
In Silico ◽  
Genome Wide Association Study ◽  
Transmembrane Protein ◽  
In Silico Analysis ◽  
Therapeutic Drug ◽  
Rna Seq ◽  
Total Rna ◽  
Neuropilin 1 ◽  
Silico Analysis ◽  
Brain Tissues

Abstract Background Numerous studies have been conducted on different aspects of the COVID-19 (coronavirus disease 2019) pandemic, which is caused by SARS-CoV-2, since its emergence in late 2019. Mutual relations among SARS-CoV-2 and neuro-pathophysiological phenomena are continuously being demonstrated, and several underlying diseases, such as those in the elderly, are positively correlated with susceptibility to SARS-CoV-2 infection. The expression of angiotensin converting enzyme 2 (ACE2), which is required for SARS-CoV-2 infection, was recently demonstrated to be increased in Alzheimer’s disease (AD) patients. Methods Recent preclinical studies have shown that Neuropilin-1 (NRP1), which is a transmembrane protein with roles in neuronal development, axonal outgrowth, and angiogenesis, also plays a role in the infectivity of SARS-CoV-2. Thus, we hypothesized that NRP1 may be upregulated in AD patients and that a correlation between AD and SARS-CoV-2 NRP1-mediated infectivity may exist. We used an AD mouse model that mimics AD and performed high throughput total RNA-seq with brain tissue and whole blood. For quantification of NPR1 in AD, brain tissues and blood were subjected to western blotting and RT-qPCR analysis. In silico analysis for NRP1 expression in AD patients has been performed on the human hippocampus data sets (GSE4226, GSE1297). Results Many cases of severe symptom of COVID-19 are concentrated in elderly group who have complications such as diabetes, degenerative disease, and brain disorders. Total RNA-seq analysis showed that Nrp1 gene was commonly overexpressed in AD model. Similar to ACE2, NRP1 protein also strongly expressed in the AD brain tissues. Interestingly, in silico analysis revealed that the level of expression for NRP1 was distinct at age and AD progression. Conclusions Given that the NRP1 is highly expressed in AD, it will be important to understand and predict that NRP1 may a risk factor for SARS-CoV-2 infection in AD patients. This will support to development of potential therapeutic drug to reduce SARS-CoV-2 transmission.

scholarly journals A Novel Thermostable Keratinase from Deinococcus geothermalis with Potential Application in Feather Degradation

2021 ◽  
Vol 11 (7) ◽  
pp. 3136
Author(s):  
Yin Tang ◽  
Leizhou Guo ◽  
Mingming Zhao ◽  
Yuan Gui ◽  
Jiahui Han ◽  
...  
Keyword(s):  
Thermal Stability ◽  
High Efficiency ◽  
In Silico Analysis ◽  
Maximum Activity ◽  
Feather Degradation ◽  
Good Tolerance ◽  
E Coli ◽  
Thermophilic Species ◽  
Mature Domain ◽  
Silico Analysis

Keratinase can specifically attack disulfide bridges in keratin to convert them from complex to simplified forms. Keratinase thermal stability has drawn attention to various biotechnological industries. In this study, a keratinase DgeKer was identified from a slightly thermophilic species, D. geothermalis. The in silico analysis showed that DgeKer is composed of signal peptide, N-terminal propeptide, mature domain, and C-terminal extension. DgeKer and its C-terminal extension-truncated enzyme (DgeKer-C) were cloned and expressed in E. coli. The purified DgeKer and DgeKer-C showed maximum activity at 70 °C and pH 9–The thermal stability assay (60 °C) showed that the half-life value of DgeKer and DgeKer-C were 103.45 min and 169.10 min, respectively. DgeKer and DgeKer-C were stable at the range of pH from 9 to 11 and showed good tolerance to some metal ions, surfactants and organic solvent. Furthermore, DgeKer could degrade feathers at 70 °C for 60 min. However, the medium became turbid with obvious softening of barbules after being treated with DgeKer-C, which might be due to C-terminal extension. In summary, a thermostable keratinase DgeKer with high efficiency degradation of feathers may have great potential in industry.

scholarly journals Mobilization of the nonconjugative virulence plasmid from hypervirulent Klebsiella pneumoniae

2021 ◽  
Vol 13 (1) ◽  
Author(s):  
Yanping Xu ◽  
Jianfeng Zhang ◽  
Meng Wang ◽  
Meng Liu ◽  
Guitian Liu ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
In Silico ◽  
In Silico Analysis ◽  
Conjugative Plasmid ◽  
Virulence Plasmid ◽  
E Coli ◽  
Virulence Plasmids ◽  
Carbapenem Resistant ◽  
Helper Plasmids ◽  
Silico Analysis

Abstract Background Klebsiella pneumoniae, as a global priority pathogen, is well known for its capability of acquiring mobile genetic elements that carry resistance and/or virulence genes. Its virulence plasmid, previously deemed nonconjugative and restricted within hypervirulent K. pneumoniae (hvKP), has disseminated into classic K. pneumoniae (cKP), particularly carbapenem-resistant K. pneumoniae (CRKP), which poses alarming challenges to public health. However, the mechanism underlying its transfer from hvKP to CRKP is unclear. Methods A total of 28 sequence type (ST) 11 bloodstream infection-causing CRKP strains were collected from Ruijin Hospital in Shanghai, China, and used as recipients in conjugation assays. Transconjugants obtained from conjugation assays were confirmed by XbaI and S1 nuclease pulsed-field gel electrophoresis, PCR detection and/or whole-genome sequencing. The plasmid stability of the transconjugants was evaluated by serial culture. Genetically modified strains and constructed mimic virulence plasmids were employed to investigate the mechanisms underlying mobilization. The level of extracellular polysaccharides was measured by mucoviscosity assays and uronic acid quantification. An in silico analysis of 2608 plasmids derived from 814 completely sequenced K. pneumoniae strains available in GenBank was performed to investigate the distribution of putative helper plasmids and mobilizable virulence plasmids. Results A nonconjugative virulence plasmid was mobilized by the conjugative plasmid belonging to incompatibility group F (IncF) from the hvKP strain into ST11 CRKP strains under low extracellular polysaccharide-producing conditions or by employing intermediate E. coli strains. The virulence plasmid was mobilized via four modes: transfer alone, cotransfer with the conjugative IncF plasmid, hybrid plasmid formation due to two rounds of single-strand exchanges at specific 28-bp fusion sites or homologous recombination. According to the in silico analysis, 31.8% (242) of the putative helper plasmids and 98.8% (84/85) of the virulence plasmids carry the 28-bp fusion site. All virulence plasmids carry the origin of the transfer site. Conclusions The nonconjugative virulence plasmid in ST11 CRKP strains is putatively mobilized from hvKP or E. coli intermediates with the help of conjugative IncF plasmids. Our findings emphasize the importance of raising public awareness of the rapid dissemination of virulence plasmids and the consistent emergence of hypervirulent carbapenem-resistant K. pneumoniae (hv-CRKP) strains.

scholarly journals Identifying new COVID-19 receptor Neuropilin-1 in severe Alzheimer’s diseases patients group brain using genome-wide association study approach

2021 ◽  
Author(s):  
Key-Hwan Lim ◽  
Sumin Yang ◽  
Sung-Hyun Kim ◽  
Jae-Yeol Joo
Keyword(s):  
In Silico ◽  
Genome Wide Association Study ◽  
Transmembrane Protein ◽  
In Silico Analysis ◽  
Therapeutic Drug ◽  
Rna Seq ◽  
Total Rna ◽  
Neuropilin 1 ◽  
Silico Analysis ◽  
Brain Tissues

Abstract Background Numerous studies have been conducted on different aspects of the COVID-19 (coronavirus disease 2019) pandemic, which is caused by SARS-CoV-2, since its emergence in late 2019. Mutual relations among SARS-CoV-2 and neuro-pathophysiological phenomena are continuously being demonstrated, and several underlying diseases, such as those in the elderly, are positively correlated with susceptibility to SARS-CoV-2 infection. The expression of angiotensin converting enzyme 2 (ACE2), which is required for SARS-CoV-2 infection, was recently demonstrated to be increased in Alzheimer’s disease (AD) patients.Methods Recent preclinical studies have shown that Neuropilin-1 (NRP1), which is a transmembrane protein with roles in neuronal development, axonal outgrowth, and angiogenesis, also plays a role in the infectivity of SARS-CoV-2. Thus, we hypothesized that NRP1 may be upregulated in AD patients and that a correlation between AD and SARS-CoV-2 NRP1-mediated infectivity may exist. We used an AD mouse model that mimics AD and performed high throughput total RNA-seq with brain tissue and whole blood. For quantification of NPR1 in AD, brain tissues and blood were subjected to western blotting and RT-qPCR analysis. In silico analysis for NRP1 expression in AD patients has been performed on the human hippocampus data sets (GSE4226, GSE1297).Results Many cases of severe symptom of COVID-19 are concentrated in elderly group who have complications such as diabetes, degenerative disease, and brain disorders. Total RNA-seq analysis showed that Nrp1 gene was commonly overexpressed in AD model. Similar to ACE2, NRP1 protein also strongly expressed in the AD brain tissues. Interestingly, in silico analysis revealed that the level of expression for NRP1 was distinct at age and AD progression.Conclusions Given that the NRP1 is highly expressed in AD, it will be important to understand and predict that NRP1 may a risk factor for SARS-CoV-2 infection in AD patients. This will support to development of potential therapeutic drug to reduce SARS-CoV-2 transmission.

Camelus dromedarius glucose transporter 4: in silico analysis, cloning, expression, purification and characterisation in E. coli

2017 ◽  
Vol 123 (4) ◽  
pp. 254-264
Author(s):  
Anwar Ahmed ◽  
Mohammed Arshad ◽  
Ajamaluddin Malik ◽  
Shama Parveen ◽  
Abdulrahman M. Alsenaidy
Keyword(s):  
In Silico ◽  
Glucose Transporter ◽  
In Silico Analysis ◽  
Camelus Dromedarius ◽  
Glucose Transporter 4 ◽  
E Coli ◽  
Silico Analysis

scholarly journals In Silico Analysis, Cloning and Expression of Recombinant CD166 in E. coli BL21 (DE3) as a Marker for Detection and Treatment of Colorectal Cancer

2017 ◽  
Vol 06 (01) ◽  
Author(s):  
Vahid Marmari ◽  
Habibollah Mahmoodzadeh ◽  
Hassan Dana ◽  
Ghanbar Mahmoodi Chalbatani ◽  
Ali Mazraeh ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
In Silico ◽  
In Silico Analysis ◽  
Cloning And Expression ◽  
E Coli ◽  
Silico Analysis

scholarly journals Analisis In-Silico Protein Tiol-Disulfida Isomerase Bacillus sp. RP1

2012 ◽  
Vol 12 (1) ◽  
pp. 43 ◽  
Author(s):  
Vanda S Kamu ◽  
Jemmy Abidjulu ◽  
Maureen Kumaunang
Keyword(s):  
Protein Folding ◽  
Gene Product ◽  
Active Site ◽  
In Silico ◽  
In Silico Analysis ◽  
Bacillus Sp ◽  
Disulfide Isomerase ◽  
Disulfide Oxidoreductase ◽  
Silico Analysis ◽  
Bonding Site

ANALISIS IN-SILICO PROTEIN TIOL-DISULFIDA ISOMERASE Bacillus sp. RP1 ABSTRAK Pelipatan protein membutuhkan bantuan molekul chaperone serta katalis pelipatan. Penelitian ini bertujuan untuk mengkarakterisasi produk gen tiol-disulfida oksidoreduktase dari sumber organisme termofilik Bacillus sp. RP1. Metode yang dilaksanakan untuk mencapai tujuan tersebut, adalah mengkarakterisasi produk gen yang dihasilkan dengan menggunakan analisis in-silico. Dari hasil penelitian diperoleh bahwa karakterisasi terhadap produk gen yang dihasilkan menunjukkan adanya tiga protein, yaitu Bdbdred, Bdbdox, dan Etda, yang memiliki motif tioredoksin dan DsbA, serta sisi aktif dan sisi pengikatan dengan atom Zn. Prediksi struktur ketiga protein tersebut menunjukkan kemiripan satu sama lain. Kata kunci: Bdbd, chaperone, DsbA, tiol-disulfida oksidoreduktase, tioredoksin ANALYSIS OF IN-SILICO TIOL DISULFIDE ISOMERASE PROTEIN Bacillus sp. RP1 ABSTRACT Protein folding is facilitated by chaperone molecule as well as folding catalysts. The aim of this research was to characterize the gene product of thiol-disulfide oxidoreductase gene from thermophylic organism Bacillus sp. RP1, by using in-silico analysis. The characteristic of the gene product indicated three proteins, i.e. Bdbdred, Bdbdox, and Etda, which have thioredoxin motif, DsbA motif, active site and bonding site with Zn. The structure predicted of these three proteins showed similarity among them. Keywords: Bdbd, chaperone, DsbA, thiol-disulfide oxidoreductase, thioredoxin

scholarly journals Human carriage of cefotaxime-resistant Escherichia coli in North-East India: an analysis of STs and associated resistance mechanisms

2019 ◽  
Author(s):  
Deepjyoti Paul ◽  
Dmitriy Babenko ◽  
Mark A Toleman
Keyword(s):  
Escherichia Coli ◽  
Core Genome ◽  
In Silico Analysis ◽  
Carbapenem Resistance ◽  
Resistance Mechanisms ◽  
E Coli ◽  
North East India ◽  
North East ◽  
The City ◽  
Silico Analysis

Abstract Objectives To determine the prevalence of Escherichia coli STs and associated resistance mechanisms carried by the community in North-East India. Methods E. coli (108) were isolated from sewage collected from 19 sites across the city of Silchar by plating on MacConkey agar with/without selection (50 mg/L cefotaxime). Species identification was confirmed by MALDI-TOF MS for 82 isolates. Common resistance mechanisms were determined by WGS of pooled E. coli isolates. PFGE combined with specific probes determined the presence of common resistance mechanisms in all isolates. Phylotypes, multilocus STs, core-genome multilocus STs, resistance genes and virulence genes were determined by in silico analysis of 38 genomes. Results and conclusions Analysis of isolates collected without selection (n = 33) indicated that cefotaxime resistance in E. coli was 42% (14/33) and estimated meropenem resistance at 9%. The remaining 58% (19/33) were additionally susceptible to ampicillin, trimethoprim, ciprofloxacin and aminoglycosides. The most common ST among the cefotaxime-resistant E. coli was ST167 (29%), followed by ST410 (17%) and ST648 (10%). E. coli ST131 was absent from the collection. Sixty-three isolates were resistant to cefotaxime and harboured blaCTX-M-15 [54% (34/63)] or blaCMY-42 [46% (29/63)], of which 10% (6/63) harboured both genes. Carbapenem resistance was due to blaNDM-5, found in 10/63 cefotaxime-resistant isolates, and/or blaOXA-181, found in 4/63 isolates. NDM-5 was encoded by IncX3 and/or IncFII plasmids and CMY-42 was mostly encoded by IncI plasmids. NDM-5 appears to have replaced NDM-1 in this region and CMY-42 appears to be in the process of replacing CTX-M-15.

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