NanI Sialidase Contributes to the Growth and Adherence of Clostridium perfringens Type F Strain F4969 in the Presence of Adherent Mucus

2021 ◽  
Author(s):  
Jihong Li ◽  
Mauricio A. Navarro ◽  
Francisco A. Uzal ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Gastrointestinal Diseases ◽  
Intestinal Colonization ◽  
Null Mutant ◽  
Mucus Layer ◽  
Mucus Production ◽  
Ht29 Cells ◽  
Mouse Small Intestine ◽  
Better Than

Clostridium perfringens type F strains causing non-foodborne human gastrointestinal diseases (NFD) typically produce NanI sialidase as their major secreted sialidase. Type F NFDs can persist for several weeks, indicating their pathogenesis involves intestinal colonization, including vegetative cell growth and adherence, with subsequent sporulation that fosters enterotoxin production and release. We previously reported that NanI contributes to type F NFD strain adherence to, and growth using, Caco-2 cells. However, Caco-2 cells make minimal amounts of mucus, which is significant because the intestines are coated with adherent mucus. Therefore, it was important to assess if NanI contributes to the growth and adherence of type F NFD strains in the presence of adherent mucus. Consequently, the current study first demonstrated greater growth of nanI -carrying vs. non- nanI -carrying type F strains in the presence of HT29-MTX-E12 cells, which produce an adherent mucus layer, vs. their parental HT29 cells, which make minimal mucus. Demonstrating the specific importance of NanI for this effect, type F NFD strain F4969 or a complementing strain grew and adhered better than an isogenic nanI null mutant in the presence of HT29-MTX-E12 cells vs HT29 cells. Those effects involved mucus production by MTX-E12 cells since mucus reduction using N-acetyl-cysteine reduced F4969 growth and adherence. Consistent with those in vitro results, NanI contributed to growth of F4969 in the mouse small intestine. By demonstrating a growth and adherence role for NanI in the presence of adherent mucus, these results further support NanI as a potential virulence factor during type F NFDs.

scholarly journals NanI Sialidase Is an Important Contributor toClostridium perfringensType F Strain F4969 Intestinal Colonization in Mice

2018 ◽  
Vol 86 (12) ◽  
Author(s):  
Mauricio A. Navarro ◽  
Jihong Li ◽  
Bruce A. McClane ◽  
Eleonora Morrell ◽  
Juliann Beingesser ◽  
...  
Keyword(s):  
Small Intestine ◽  
Food Poisoning ◽  
Extracellular Enzyme ◽  
Gastrointestinal Diseases ◽  
Intestinal Colonization ◽  
Null Mutant ◽  
Content Type ◽  
Intestinal Segments

ABSTRACTClostridium perfringenstype F (formerly enterotoxigenicC. perfringenstype A) strains produce an enterotoxin (CPE) to cause acute cases of food poisoning and chronic nonfoodborne human gastrointestinal diseases (NFD), e.g., antibiotic-associated diarrhea (AAD). NFD strains also produce NanI sialidase, an extracellular enzyme that releases sialic acids from sialyated host macromolecules. Recentin vitrostudies suggested that NanI may contribute to NFD strain intestinal colonization by enhancing the adherence of such strains to intestinal cells and promoting their bacterial growth using generated sialic acid as an energy source. The current study tested this hypothesis by developing a mouse intestinal colonization model involving clindamycin pretreatment to produce conditions mimicking those during AAD. In this model, the type F NFD strain F4969 persisted for at least 4 days in the small intestine, cecum, and colon. When clindamycin-pretreated mice were challenged by oral gavage with equivalent numbers of F4969 bacteria or its isogenicnanInull mutant, significantly lower numbers of thenanImutant were recovered from all intestinal segments, and it was completely cleared from the small intestine by day 4. Complementation of the mutant to restore NanI production also promoted colonization. When the samenanInull mutant strain was coinoculated into the mouse model together with ananI-producing strain, the numbers of this mutant were restored to wild-type F4969 levels in all intestinal segments. This result suggests that sialidases produced by other bacteria might also provide some support forC. perfringensintestinal colonization. Collectively, thesein vivofindings identify NanI to be the first known significant contributor to chronic intestinal colonization by NFD strains.

scholarly journals Identification of an Important Orphan Histidine Kinase for the Initiation of Sporulation and Enterotoxin Production byClostridium perfringensType F Strain SM101

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
John C. Freedman ◽  
Jihong Li ◽  
Eric Mi ◽  
Bruce A. McClane
Keyword(s):  
Clostridium Perfringens ◽  
Histidine Kinase ◽  
Insertional Mutagenesis ◽  
Food Poisoning ◽  
Gastrointestinal Diseases ◽  
Kinase Domain ◽  
Block Type ◽  
Null Mutant ◽  
Content Type

ABSTRACTClostridium perfringenstype F strains cause a common human foodborne illness and many cases of nonfoodborne human gastrointestinal diseases. Sporulation plays two critical roles during type F enteric disease. First, it produces broadly resistant spores that facilitate type F strain survival in the food and nosocomial environments. Second, production ofC. perfringensenterotoxin (CPE), the toxin responsible for causing the enteric symptoms of type F diseases, is restricted to cells in the process of sporulation. While later steps in the regulation ofC. perfringenssporulation have been discerned, the process leading to phosphorylation of Spo0A, the master early regulator of sporulation and consequent CPE production, has remained unknown. Using an insertional mutagenesis approach, the current study identified the orphan histidine kinase CPR0195 as an important factor regulatingC. perfringenssporulation and CPE production. Specifically, a CPR0195 null mutant of type F strain SM101 made 103-fold fewer spores than its wild-type parent and produced no detectable CPE. In contrast, a null mutant of another putativeC. perfringensorphan histidine kinase (CPR1055) did not significantly affect sporulation or CPE production. Studies using aspoIIAoperon promoter-driven reporter plasmid indicated that CPR0195 functions early during sporulation, i.e., prior to production of sporulation-associated sigma factors. Furthermore,in vitrostudies showed that the CPR0195 kinase domain can autophosphorylate and phosphorylate Spo0A. These results support the idea of CPR0195 as an important kinase that initiatesC. perfringenssporulation by directly phosphorylating Spo0A. This kinase could represent a novel therapeutic target to blockC. perfringenssporulation and CPE production during type F disease.IMPORTANCEClostridium perfringenstype F enteric diseases, which include a very common form of food poisoning and many cases of antibiotic-associated diarrhea, develop when type F strains sporulate and produceC. perfringensenterotoxin (CPE) in the intestines. Spores are also important for transmission of type F disease. Despite the importance of sporulation for type F disease and the evidence thatC. perfringenssporulation begins with phosphorylation of the Spo0A transcriptional regulator, the kinase phosphorylating Spo0A to initiate sporulation and CPE production had not been ascertained. In response, the current report now provides identification of an orphan histidine kinase named CPR0195 that can directly phosphorylate Spo0A. Results using a CPR0195 null mutant indicate that this kinase is very important for initiatingC. perfringenssporulation and CPE production. Therefore, the CPR0195 kinase represents a potential target to block type F disease by interfering with intestinalC. perfringenssporulation and CPE production.

scholarly journals Characterization of increased mucus production of HT29-MTX-E12 cells grown under Semi-Wet interface with Mechanical Stimulation

PLoS ONE ◽  
2021 ◽  
Vol 16 (12) ◽  
pp. e0261191
Author(s):  
Janneke Elzinga ◽  
Benthe van der Lugt ◽  
Clara Belzer ◽  
Wilma T. Steegenga
Keyword(s):  
Cell Line ◽  
Mechanical Stimulation ◽  
Cell Metabolism ◽  
Lactate Production ◽  
Intestinal Cell ◽  
Mucus Layer ◽  
Mucus Production ◽  
Intestinal Mucus ◽  
Cells Cultured

The intestinal mucus layer plays a crucial role in human health. To study intestinal mucus function and structure in vitro, the mucus-producing intestinal cell line HT29-MTX-E12 has been commonly used. However, this cell line produces only low amounts of the intestine-specific MUC2. It has been shown previously that HT29-MTX-E12 cells cultured under Semi-Wet interface with Mechanical Stimulation (SWMS) produced higher amounts of MUC2, concomitant with a thicker mucus layer, compared to cells cultured conventionally. However, it remains unknown which underlying pathways are involved. Therefore, we aimed to further explore the cellular processes underlying the increased MUC2 production by HT29-MTX-E12 cells grown under SWMS conditions. Cells grown on Transwell membranes for 14 days under static and SWMS conditions (after cell seeding and attachment) were subjected to transcriptome analysis to investigate underlying molecular pathways at gene expression level. Caco-2 and LS174T cell lines were included as references. We characterized how SWMS conditions affected HT29-MTX-E12 cells in terms of epithelial barrier integrity, by measuring transepithelial electrical resistance, and cell metabolism, by monitoring pH and lactate production per molecule glucose of the conditioned medium. We confirmed higher MUC2 production under SWMS conditions at gene and protein level and demonstrated that this culturing method primarily stimulated cell growth. In addition, we also found evidence for a more aerobic cell metabolism under SWMS, as shown previously for similar models. In summary, we suggest different mechanisms by which MUC2 production is enhanced under SWMS and propose potential applications of this model in future studies.

scholarly journals The Agr-Like Quorum-Sensing System Is Important for Clostridium perfringens Type A Strain ATCC 3624 To Cause Gas Gangrene in a Mouse Model

mSphere ◽  
2020 ◽  
Vol 5 (3) ◽  
Author(s):  
Mauricio A. Navarro ◽  
Jihong Li ◽  
Juliann Beingesser ◽  
Bruce A. McClane ◽  
Francisco A. Uzal
Keyword(s):  
Quorum Sensing ◽  
Mouse Model ◽  
Clostridium Perfringens ◽  
Regulatory System ◽  
Type A ◽  
Gas Gangrene ◽  
Null Mutant ◽  
Wild Type ◽  
Content Type

ABSTRACT Clostridium perfringens type A is involved in gas gangrene in humans and animals. Following a traumatic injury, rapid bacterial proliferation and exotoxin production result in severe myonecrosis. C. perfringens alpha toxin (CPA) and perfringolysin (PFO) are the main virulence factors responsible for the disease. Recent in vitro studies have identified an Agr-like quorum-sensing (QS) system in C. perfringens that regulates the production of both toxins. The system is composed of an AgrB membrane transporter and an AgrD peptide that interacts with a two-component regulatory system in response to fluctuations in the cell population density. In addition, a synthetic peptide named 6-R has been shown to interfere with this signaling mechanism, affecting the function of the Agr-like QS system in vitro. In the present study, C. perfringens type A strain ATCC 3624 and an isogenic agrB-null mutant were tested in a mouse model of gas gangrene. When mice were intramuscularly challenged with 106 CFU of wild-type ATCC 3624, severe myonecrosis and leukocyte aggregation occurred by 4 h. Similar numbers of an agrB-null mutant strain produced significantly less severe changes in the skeletal muscle of challenged mice. Complementation of the mutant to regain agrB expression restored virulence to wild-type levels. The burdens of all three C. perfringens strains in infected muscle were similar. In addition, animals injected intramuscularly with wild-type ATCC 3624 coincubated with the 6-R peptide developed less severe microscopic changes. This study provides the first in vivo evidence that the Agr-like QS system is important for C. perfringens type A-mediated gas gangrene. IMPORTANCE Clostridium perfringens type A strains produce toxins that are responsible for clostridial myonecrosis, also known as gas gangrene. Toxin production is regulated by an Agr-like quorum-sensing (QS) system that responds to changes in cell population density. In this study, we investigated the importance of this QS system in a mouse model of gas gangrene. Mice challenged with a C. perfringens strain with a nonfunctional regulatory system developed less severe changes in the injected skeletal muscle compared to animals receiving the wild-type strain. In addition, a synthetic peptide was able to decrease the effects of the QS in this disease model. These studies provide new understanding of the pathogenesis of gas gangrene and identified a potential therapeutic target to prevent the disease.

MICROPROPAGATION OF CHICKPEA FROM SHOOT TIP AND NODE SEGMENT

2018 ◽  
Vol 24 (2) ◽  
Author(s):  
J. D. BARSHILE
Keyword(s):  
Shoot Tip ◽  
Root Induction ◽  
Multiple Response ◽  
Ms Medium ◽  
Growth Responses ◽  
Rapid Multiplication ◽  
Micro Propagation ◽  
G 12 ◽  
Better Than

Present investigation was undertaken to standardize technique for in vitro micro-propagation of chickpea( Cicer arietinum ) cultivar Vishwas (Phule G 12). Micropropagation method for chickpea was established and this method enabled much more efficient propagation of plants. The present work was aimed at evolving a protocol for rapid multiplication of chickpea using micropropagation technique. Explants from shoot tip and node segment were cultured on MS medium supplemented with different concentrations of BAP and Kinetin (1.0 to 2.5 mg/l) and their growth responses like shooting were elucidated. The maximum multiple response was observed with 2 mg/l concentration of BAP from both types of explant. The highest number of shoots (12.5 ± 0.3) was achieved on MS medium with 2 mg/l BAP using node segments. The medium supplemented with 2 mg/l of BAP was found better than all other concentrations. Individual shoots were transferred to IBA and IAA (1.0-1.5 mg/l) for root induction. MS medium supplemented with 2 mg/l of IBA proved better for rooting. Rooted plantlets were successfully hardened in greenhouse and established in the pot.

scholarly journals CirBiTree: Citrullination Site Inference Based on a Fuzzy Neural Network and Flexible Neural Tree

2020 ◽  
Vol 2020 ◽  
pp. 1-8
Author(s):  
Chuandong Song ◽  
Haifeng Wang
Keyword(s):  
Neural Network ◽  
Fuzzy Neural Network ◽  
Classification Model ◽  
Peptide Sequence ◽  
Sequence Information ◽  
Post Translational Modification ◽  
Fuzzy Neural ◽  
Human Complex ◽  
Better Than

Emerging evidence demonstrates that post-translational modification plays an important role in several human complex diseases. Nevertheless, considering the inherent high cost and time consumption of classical and typical in vitro experiments, an increasing attention has been paid to the development of efficient and available computational tools to identify the potential modification sites in the level of protein. In this work, we propose a machine learning-based model called CirBiTree for identification the potential citrullination sites. More specifically, we initially utilize the biprofile Bayesian to extract peptide sequence information. Then, a flexible neural tree and fuzzy neural network are employed as the classification model. Finally, the most available length of identified peptides has been selected in this model. To evaluate the performance of the proposed methods, some state-of-the-art methods have been employed for comparison. The experimental results demonstrate that the proposed method is better than other methods. CirBiTree can achieve 83.07% in sn%, 80.50% in sp, 0.8201 in F1, and 0.6359 in MCC, respectively.

scholarly journals Natural killer T cell immunotherapy combined with oncolytic vesicular stomatitis virus or reovirus treatments differentially increases survival in mouse models of ovarian and breast cancer metastasis

2021 ◽  
Vol 9 (3) ◽  
pp. e002096
Author(s):  
Simon Gebremeskel ◽  
Adam Nelson ◽  
Brynn Walker ◽  
Tora Oliphant ◽  
Lynnea Lobert ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Vesicular Stomatitis Virus ◽  
Cell Activation ◽  
Tumor Burden ◽  
Oncolytic Viruses ◽  
Nkt Cell ◽  
Cell Immunotherapy ◽  
Vesicular Stomatitis ◽  
Better Than

BackgroundOncolytic viruses reduce tumor burden in animal models and have generated promising results in clinical trials. However, it is likely that oncolytic viruses will be more effective when used in combination with other therapies. Current therapeutic approaches, including chemotherapeutics, come with dose-limiting toxicities. Another option is to combine oncolytic viruses with immunotherapeutic approaches.MethodsUsing experimental models of metastatic 4T1 breast cancer and ID8 ovarian peritoneal carcinomatosis, we examined natural killer T (NKT) cell-based immunotherapy in combination with recombinant oncolytic vesicular stomatitis virus (VSV) or reovirus. 4T1 mammary carcinoma cells or ID8 ovarian cancer cells were injected into syngeneic mice. Tumor-bearing mice were treated with VSV or reovirus followed by activation of NKT cells via the intravenous administration of autologous dendritic cells loaded with the glycolipid antigen α-galactosylceramide. The effects of VSV and reovirus on immunogenic cell death (ICD), cell viability and immunogenicity were tested in vitro.ResultsVSV or reovirus treatments followed by NKT cell activation mediated greater survival in the ID8 model than individual therapies. The regimen was less effective when the treatment order was reversed, delivering virus treatments after NKT cell activation. In the 4T1 model, VSV combined with NKT cell activation increased overall survival and decreased metastatic burden better than individual treatments. In contrast, reovirus was not effective on its own or in combination with NKT cell activation. In vitro, VSV killed a panel of tumor lines better than reovirus. VSV infection also elicited greater increases in mRNA transcripts for proinflammatory cytokines, chemokines, and antigen presentation machinery compared with reovirus. Oncolytic VSV also induced the key hallmarks of ICD (calreticulin mobilization, plus release of ATP and HMGB1), while reovirus only mobilized calreticulin.ConclusionTaken together, these results demonstrate that oncolytic VSV and NKT cell immunotherapy can be effectively combined to decrease tumor burden in models of metastatic breast and ovarian cancers. Oncolytic VSV and reovirus induced differential responses in our models which may relate to differences in virus activity or tumor susceptibility.

Factor-VIII Inhibitor in Less Severely Affected Haemophilic Patients

1975 ◽  
Author(s):  
E. G. D. Tuddenham ◽  
A. L. Bloom ◽  
J. C. Giddings ◽  
C. A. Barrett
Keyword(s):  
Factor Viii ◽  
Factor Viii Inhibitor ◽  
Factor Viii Level ◽  
Replacement Treatment ◽  
Viii Level ◽  
Viii Inhibitor ◽  
In Vivo Recovery ◽  
Better Than

The occurrence of factor VIII inhibitor in five mild or moderately affected liaemophilic patients is described. In four patients the inhibitor inactivated endogenous factor VIII an dtemporarily converted them to severely affected haemophiliacs with factor VIII level of 0%. In the fifth patient, a brother of one of the others, the inhibitor although more potent did not inactivate the patient’s own factor VIII and did not completely inactivate normal factor VIII in vitro. This patient responded to treatment with factor-VIII concentrate but the in-vivo recovery was reduced. The patient’s plasma was tested against a panel of normal donors but it inactivated factor VIII in each to a similar extent and no evidence for normal factor-VIII groups was obtained. In the other patients the response to replacement treatment was also better than that usually seen in severely affected haemophilic patients with inhibitor. In the two related patients the inhibitors have so far persisted but in the unrelated patients the inhibitors eventually disappeared and did not always recur with subsequent therapy. The incidence of factor- VIII inhibitor in less severe haemophiliacs (factor VIII > 3% ) in this centre is 6% suggesting that the complication is more frequent in this type of patient than hitherto recognised.

scholarly journals In vitro and in vivo Evaluation of Ibuprofen Nanosuspensions for Enhanced Oral Bioavailability

2021 ◽  
Author(s):  
Mohsen Hedaya ◽  
Farzana Bandarkar ◽  
Aly Nada
Keyword(s):  
Particle Size ◽  
Tween 80 ◽  
Aqueous Solubility ◽  
In Vitro Dissolution ◽  
In Vivo Evaluation ◽  
Poorly Soluble ◽  
Extent Of Absorption ◽  
Better Than

Introduction: The objectives were to prepare, characterize and in vivo evaluate different ibuprofen (IBU) nanosuspensions prepared by ultra-homogenization, after oral administration to rabbits. Methods: The nanosuspensions produced by ultra-homogenization were tested and compared with a marketed IBU suspension for particle size, in vitro dissolution and in vivo absorption. Five groups of rabbits received orally 25 mg/kg of IBU nanosuspension, nanoparticles, unhomogenized suspension, marketed product and untreated suspension. A sixth group received 5 mg/kg IBU intravenously. Serial blood samples were obtained after IBU administration. Results: The formulated nanosuspensions showed significant decrease in particle size. Polyvinyl Pyrrolidone K30 (PP) was found to improve IBU aqueous solubility much better than the other tested polymers. Addition of Tween 80 (TW), in equal amount as PP (IBU: PP:TW, 1:2:2 w/w) resulted in much smaller particle size and better dissolution rate. The Cmax achieved were 14.8±1.64, 11.1±1.37, 9.01±0.761, 7.03±1.38 and 3.23±1.03 μg/ml and the tmax were 36±8.2, 39±8.2, 100±17.3, 112±15 and 105±17 min for the nanosuspension, nanoparticle, unhomogenized suspension, marketed IBU suspension and untreated IBU suspension in water, respectively. Bioavailability of the different formulations relative to the marketed suspension were the highest for nanosuspension> unhomogenized suspension> nanoparticles> untreated IBU suspension. Conclusion: IBU/PP/TW nanosuspensions showed enhanced in vitro dissolution as well as faster rate and higher extent of absorption as indicated from the higher Cmax, shorter tmax and larger AUC. The in vivo data supported the in vitro results. Nanosuspensions prepared by ultra-high-pressure-homogenization technique can be used as a good formulation strategy to enhance the rate and extent of absorption of poorly soluble drugs.

scholarly journals HopA1 Effector from Pseudomonas syringae pv syringae Strain 61 Affects NMD Processes and Elicits Effector-Triggered Immunity

2021 ◽  
Vol 22 (14) ◽  
pp. 7440
Author(s):  
Shraddha K. Dahale ◽  
Daipayan Ghosh ◽  
Kishor D. Ingole ◽  
Anup Chugani ◽  
Sang Hee Kim ◽  
...  
Keyword(s):  
Pseudomonas Syringae ◽  
Disease Susceptibility ◽  
Null Mutant ◽  
In Planta ◽  
Host Range Expansion ◽  
Unique System ◽  
Effector Triggered Immunity ◽  
Transcriptomic Changes ◽  
Immune Detection

Pseudomonas syringae-secreted HopA1 effectors are important determinants in host range expansion and increased pathogenicity. Their recent acquisitions via horizontal gene transfer in several non-pathogenic Pseudomonas strains worldwide have caused alarming increase in their virulence capabilities. In Arabidopsis thaliana, RESISTANCE TO PSEUDOMONAS SYRINGAE 6 (RPS6) gene confers effector-triggered immunity (ETI) against HopA1pss derived from P. syringae pv. syringae strain 61. Surprisingly, a closely related HopA1pst from the tomato pathovar evades immune detection. These responsive differences in planta between the two HopA1s represents a unique system to study pathogen adaptation skills and host-jumps. However, molecular understanding of HopA1′s contribution to overall virulence remain undeciphered. Here, we show that immune-suppressive functions of HopA1pst are more potent than HopA1pss. In the resistance-compromised ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) null-mutant, transcriptomic changes associated with HopA1pss-elicited ETI are still induced and carry resemblance to PAMP-triggered immunity (PTI) signatures. Enrichment of HopA1pss interactome identifies proteins with regulatory roles in post-transcriptional and translational processes. With our demonstration here that both HopA1 suppress reporter-gene translations in vitro imply that the above effector-associations with plant target carry inhibitory consequences. Overall, with our results here we unravel possible virulence role(s) of HopA1 in suppressing PTI and provide newer insights into its detection in resistant plants.

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