scholarly journals Oropharyngeal Carriage of hpl-Containing Haemophilus haemolyticus Predicts Lower Prevalence and Density of NTHi Colonisation in Healthy Adults

Pathogens ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 577
Author(s):  
Brianna Atto ◽  
Dale Kunde ◽  
David A. Gell ◽  
Stephen Tristram
Keyword(s):  
Healthy Adults ◽  
Host Cells ◽  
Respiratory Pathogen ◽  
Lower Prevalence ◽  
Nontypeable Haemophilus Influenzae ◽  
Upper Airways ◽  
Reading Frame ◽  
Clinically Significant ◽  
Oropharyngeal Carriage

Nontypeable Haemophilus influenzae (NTHi) is a major respiratory pathogen that initiates infection by colonising the upper airways. Strategies that interfere with this interaction may therefore have a clinically significant impact on the ability of NTHi to cause disease. We have previously shown that strains of the commensal bacterium Haemophilus haemolyticus (Hh) that produce a novel haem-binding protein, haemophilin, can prevent NTHi growth and interactions with host cells in vitro. We hypothesized that natural pharyngeal carriage of Hh strains with the hpl open reading frame (Hh-hpl+) would be associated with a lower prevalence and/or density of NTHi colonisation in healthy individuals. Oropharyngeal swabs were collected from 257 healthy adults in Australia between 2018 and 2019. Real-time PCR was used to quantitatively compare the oropharyngeal carriage load of NTHi and Hh populations with the Hh-hpl+ or Hh-hpl− genotype. The likelihood of acquiring/maintaining NTHi colonisation status over a two- to six-month period was assessed in individuals that carried either Hh-hpl− (n = 25) or Hh-hpl+ (n = 25). Compared to carriage of Hh-hpl− strains, adult (18–65 years) and elderly (>65 years) participants that were colonised with Hh-hpl+ were 2.43 or 2.67 times less likely to carry NTHi in their oropharynx, respectively. Colonisation with high densities of Hh-hpl+ correlated with a low NTHi carriage load and a 2.63 times lower likelihood of acquiring/maintaining NTHi colonisation status between visits. Together with supporting in vitro studies, these results encourage further investigation into the potential use of Hh-hpl+ as a respiratory probiotic candidate for the prevention of NTHi infection.

scholarly journals Characterization of a Chlamydia psittaciDNA Binding Protein (EUO) Synthesized during the Early and Middle Phases of the Developmental Cycle

1998 ◽  
Vol 66 (3) ◽  
pp. 1167-1173 ◽  
Author(s):  
Li Zhang ◽  
Annemarie L. Douglas ◽  
Thomas P. Hatch
Keyword(s):  
Promoter Region ◽  
Chlamydia Psittaci ◽  
Host Cells ◽  
Upstream Open Reading Frame ◽  
Developmental Cycle ◽  
Binding Property ◽  
Logarithmic Growth Phase ◽  
Reading Frame

ABSTRACT The EUO gene (for early upstream open reading frame) ofChlamydia psittaci was previously found to be transcribed better at 1 than at 24 h postinfection. We found that the EUO gene encodes a minor protein that is expressed within 1 h of infection of host cells with C. psittaci 6BC but that protein quantity peaks during the logarithmic growth phase of reticulate bodies (RBs), declines late in the infection (after 20 h) when RBs reorganize into elementary bodies (EBs), and is absent in infectious EBs. EUO protein lacks homology to known proteins but does contain a putative helix-turn-helix motif. We found that recombinant EUO binds to DNA in vitro with a relatively broad specificity. Using the bp −200 to +67 promoter region of the cysteine-rich envelope protein (crp) operon as a model, we show that EUO protein preferentially binds to AT-rich sequences and protects crpDNA from DNase I from approximately bp −60 to −9. We also found that native EUO protein in extracts of RBs binds to the promoter region of the crp operon, demonstrating that the DNA binding property of EUO protein is not an artifact of recombinant methods. Although EUO protein appears to bind to the crp operon with high affinity in vitro (Kd of about 15 nM), it is not known whether the protein binds the crp DNA in vivo.

scholarly journals Dual RNA-seq of Nontypeable Haemophilus influenzae and Host Cell Transcriptomes Reveals Novel Insights into Host-Pathogen Cross Talk

mBio ◽  
2015 ◽  
Vol 6 (6) ◽  
Author(s):  
Buket Baddal ◽  
Alessandro Muzzi ◽  
Stefano Censini ◽  
Raffaele A. Calogero ◽  
Giulia Torricelli ◽  
...  
Keyword(s):  
Haemophilus Influenzae ◽  
Cross Talk ◽  
Induced Defense ◽  
Bronchial Epithelium ◽  
Host Cells ◽  
Central Metabolism ◽  
Nontypeable Haemophilus Influenzae ◽  
Human Respiratory Tract ◽  
Content Type

ABSTRACTThe ability to adhere and adapt to the human respiratory tract mucosa plays a pivotal role in the pathogenic lifestyle of nontypeableHaemophilus influenzae(NTHi). However, the temporal events associated with a successful colonization have not been fully characterized. In this study, by reconstituting the ciliated human bronchial epitheliumin vitro, we monitored the global transcriptional changes in NTHi and infected mucosal epithelium simultaneously for up to 72 h by dual RNA sequencing. The initial stage of colonization was characterized by the binding of NTHi to ciliated cells. Temporal profiling of host mRNA signatures revealed significant dysregulation of the target cell cytoskeleton elicited by bacterial infection, with a profound effect on the intermediate filament network and junctional complexes. In response to environmental stimuli of the host epithelium, NTHi downregulated its central metabolism and increased the expression of transporters, indicating a change in the metabolic regime due to the availability of host substrates. Concurrently, the oxidative environment generated by infected cells instigated bacterial expression of stress-induced defense mechanisms, including the transport of exogenous glutathione and activation of the toxin-antitoxin system. The results of this analysis were validated by those of confocal microscopy, Western blotting, Bio-plex, and real-time quantitative reverse transcription-PCR (qRT-PCR). Notably, as part of our screening for novel signatures of infection, we identified a global profile of noncoding transcripts that are candidate small RNAs (sRNAs) regulated during human host infection inHaemophilusspecies. Our data, by providing a robust and comprehensive representation of the cross talk between the host and invading pathogen, provides important insights into NTHi pathogenesis and the development of efficacious preventive strategies.IMPORTANCESimultaneous monitoring of infection-linked transcriptome alterations in an invading pathogen and its target host cells represents a key strategy for identifying regulatory responses that drive pathogenesis. In this study, we report the progressive events of NTHi colonization in a highly differentiated model of ciliated bronchial epithelium. Genome-wide transcriptome maps of NTHi during infection provided mechanistic insights into bacterial adaptive responses to the host niche, with modulation of the central metabolism as an important signature of the evolving milieu. Our data indicate that infected epithelia respond by substantial alteration of the cytoskeletal network and cytokine repertoire, revealing a dynamic cross talk that is responsible for the onset of inflammation. This work significantly enhances our understanding of the means by which NTHi promotes infection on human mucosae and reveals novel strategies exploited by this important pathogen to cause invasive disease.

scholarly journals Impairment in inflammasome signaling by the chronic Pseudomonas aeruginosa isolates from cystic fibrosis patients results in an increase in inflammatory response

2021 ◽  
Vol 12 (3) ◽  
Author(s):  
Melissa S. Phuong ◽  
Rafael E. Hernandez ◽  
Daniel J. Wolter ◽  
Lucas R. Hoffman ◽  
Subash Sad
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Cell Death ◽  
Inflammatory Cytokines ◽  
Immune Cell ◽  
Host Cells ◽  
Respiratory Pathogen ◽  
Cell Death Pathways ◽  
The Relationship

AbstractPseudomonas aeruginosa is a common respiratory pathogen in cystic fibrosis (CF) patients which undergoes adaptations during chronic infection towards reduced virulence, which can facilitate bacterial evasion of killing by host cells. However, inflammatory cytokines are often found to be elevated in CF patients, and it is unknown how chronic P. aeruginosa infection can be paradoxically associated with both diminished virulence in vitro and increased inflammation and disease progression. Thus, we investigated the relationship between the stimulation of inflammatory cell death pathways by CF P. aeruginosa respiratory isolates and the expression of key inflammatory cytokines. We show that early respiratory isolates of P. aeruginosa from CF patients potently induce inflammasome signaling, cell death, and expression of IL-1β by macrophages, yet little expression of other inflammatory cytokines (TNF, IL-6 and IL-8). In contrast, chronic P. aeruginosa isolates induce relatively poor macrophage inflammasome signaling, cell death, and IL-1β expression but paradoxically excessive production of TNF, IL-6 and IL-8 compared to early P. aeruginosa isolates. Using various mutants of P. aeruginosa, we show that the premature cell death of macrophages caused by virulent bacteria compromises their ability to express cytokines. Contrary to the belief that chronic P. aeruginosa isolates are less pathogenic, we reveal that infections with chronic P. aeruginosa isolates result in increased cytokine induction due to their failure to induce immune cell death, which results in a relatively intense inflammation compared with early isolates.

scholarly journals Human metapneumovirus induces autophagy through JNK and MEK/ERK pathway

2019 ◽  
Author(s):  
Pan Zhang ◽  
Suhua Chen ◽  
Hui Yang ◽  
Bin Tan ◽  
Yao Zhao
Keyword(s):  
Airway Epithelial Cells ◽  
Human Metapneumovirus ◽  
Host Cells ◽  
Respiratory Pathogen ◽  
Erk Pathway ◽  
Airway Epithelial ◽  
Host Interaction ◽  
Hmpv Infection ◽  
Lc3 Puncta

Abstract Background: Human metapneumovirus (hMPV) is a ubiquitous respiratory pathogen, especially in infants and young children. Virus-host interaction affects viral replication and host immune responses. Autophagy plays an important role in virus-host interaction. Airway epithelial cells serve as the first line in host to against respiratory virus infection. However, it is still unknown whether autophagy is activated in human metapneumovirus (hMPV) infected host cells. Methods: In this study, we demonstrated the occurrence of autophagy through some autophagic features including the conversion of GFP-RFP-LC3 plasmid, RFP-LC3 puncta and expression of autophagic-related gene. The pathways involved in autophagy were detected by Western Blot and verified by specific pathway inhibitors. The relationship between the replication of hMPV and autophagy was tested by Rapamycin, 3-MA, siRNA-LC3 and pathway inhibitors. We also used BALB/c mice to verified the autophagy and pathways in hMPV infection. Results: We found that the GFP-RFP-LC3 plasmid, RFP-LC3 puncta and expression of ATG5, ATG7, Becllin1, LC3 were increased in hMPV group in vitro . JNK and MEK/ERK signaling pathway are activated in hMPV induced-autophagy and we confirmed the two pathways with specific inhibitors SP600125 and PD98059. Furthermore, we found that rapamycin suppressed hMPV infection significantly, but reversed when treated with autophagy inhibitor 3-MA, siRNA-LC3 and pathway inhibitors. hMPV infected mice also induce autophagy through JNK and MEK/ERK pathways. Conclusion: Taken together, our results show strong evidence that autophagy involved in hMPV infection through JNK, MEK/ERK pathway which plays an antiviral role in the process of hMPV infection.

scholarly journals Recent Progress in Mycoplasma pneumoniae Infection

2015 ◽  
Vol 4 (2) ◽  
pp. 31-34
Author(s):  
Anqiao Zhong
Keyword(s):  
Mycoplasma Pneumoniae ◽  
Clinical Symptoms ◽  
Host Cells ◽  
Respiratory Pathogen ◽  
Culture Methods ◽  
Serological Tests ◽  
Sensitivity Tests ◽  
Host Cell Surface ◽  
Mycoplasma Pneumoniae Infection

AbstractMycoplasma pneumoniae(Mp) is a definite respiratory pathogen affecting people of all ages. This organism is adsorbed on host cell surface through extreme adherent organelles, generating peroxide ions and possible exotoxins. Mp may directly invade host cells and cause latent infections. Induced immunoreactive injury is one of the main factors resulting in clinical symptoms of Mp infection. Polymerase chain reaction (PCR) and serological detection should be combined for diagnosis of Mp infection when nasopharyngeal and oropharyngeal specimens are simultaneously obtained and detected by PCR. The most reliable basis for diagnosis can be obtained with two serological tests in different courses of the disease. Culture methods also bear significance in diagnosing Mp infections andin vitrodrug sensitivity tests.

scholarly journals Vibrio vulnificus rtxE Is Important for Virulence, and Its Expression Is Induced by Exposure to Host Cells

2008 ◽  
Vol 76 (4) ◽  
pp. 1509-1517 ◽  
Author(s):  
Byung Cheol Lee ◽  
Jeong Hyun Lee ◽  
Myung Won Kim ◽  
Byoung Sik Kim ◽  
Man Hwan Oh ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Vibrio Vulnificus ◽  
Lethal Dose ◽  
Host Cells ◽  
Transcriptional Unit ◽  
Intestinal Epithelial ◽  
Wild Type ◽  
Reading Frame ◽  
Human Intestinal Epithelial Cells

ABSTRACT Numerous secreted virulence factors have been proposed to account for the fulminating and destructive nature of Vibrio vulnificus infections. A mutant of V. vulnificus that exhibited less cytotoxicity to INT-407 human intestinal epithelial cells was screened from a library of mutants constructed by random transposon mutagenesis. A transposon-tagging method was used to identify and clone an open reading frame encoding an RTX toxin secretion ATP binding protein, RtxE, from V. vulnificus. The deduced amino acid sequence of RtxE from V. vulnificus was 91% identical to that reported from Vibrio cholerae. Functions of the rtxE gene in virulence were assessed by constructing an isogenic mutant whose rtxE gene was inactivated by allelic exchanges and by evaluating the differences between its virulence phenotype and that of the wild type in vitro and in mice. The disruption of rtxE blocked secretion of RtxA to the cell exterior and resulted in a significant reduction in cytotoxic activity against epithelial cells in vitro. Also, the intraperitoneal 50% lethal dose of the rtxE mutant was 104 to 105 times higher than that of the parental wild type, indicating that RtxE is essential for the virulence of V. vulnificus. Furthermore, the present study demonstrated that the rtxBDE genes are transcribed as one transcriptional unit under the control of a single promoter, P rtxBDE . The activity of V. vulnificus P rtxBDE is induced by exposure to INT-407 cells, and the induction requires direct contact of the bacteria with the host cells.

scholarly journals Promoter Sequences of Varicella-Zoster Virus Glycoprotein I Targeted by Cellular Transactivating Factors Sp1 and USF Determine Virulence in Skin and T Cells in SCIDhu Mice In Vivo

2003 ◽  
Vol 77 (1) ◽  
pp. 489-498 ◽  
Author(s):  
Hideki Ito ◽  
Marvin H. Sommer ◽  
Leigh Zerboni ◽  
Hongying He ◽  
Dwayne Boucaud ◽  
...  
Keyword(s):  
T Cells ◽  
Varicella Zoster Virus ◽  
Host Cells ◽  
Upstream Sequence ◽  
Upstream Stimulatory Factor ◽  
Reading Frame ◽  
Varicella Zoster ◽  
Glycoprotein I

ABSTRACT Varicella-zoster virus (VZV) glycoprotein I is dispensable in cell culture but necessary for infection of human skin and T cells in SCIDhu mice in vivo. The gI promoter contains an activating upstream sequence that binds the cellular transactivators specificity factor 1 (Sp1) and upstream stimulatory factor (USF) and an open reading frame 29 (ORF29)-responsive element (29RE), which mediates enhancement by ORF29 DNA binding protein of immediate-early 62 (IE62)-induced transcription. Recombinants, rOKAgI-Sp1 and rOKAgI-USF, with two base pair substitutions in Sp1 or USF sites, replicated like rOKA in vitro, but infectivity of rOKAgI-Sp1 was significantly impaired in skin and T cells in vivo. A double mutant, rOKAgI-Sp1/USF, did not replicate in skin but yielded low titers of infectious virus in T cells. The repaired protein, rOKAgI:rep-Sp1/USF, was as infectious as rOKA. Thus, disrupting gI promoter sites for cellular transactivators altered VZV virulence in vivo, with variable consequences related to the cellular factor and the host cell type. Mutations in the 29RE of the gI promoter were made by substituting each of four 10-bp blocks in this region with a 10-bp sequence, GATAACTACA, that was predicted to interfere with enhancer effects of the ORF29 protein. One of these mutants, which was designated rOKAgI-29RE-3, had diminished replication in skin and T cells, indicating that ORF29 protein-mediated enhancement of gI expression contributes to VZV virulence. Mutations within promoters of viral genes that are nonessential in vitro should allow construction of recombinant herpesviruses that have altered virulence in specific host cells in vivo and may be useful for designing herpesviral gene therapy vectors and attenuated viral vaccines.

The Effect of Warfarin Sodium on the Duration of Platelet Aggregation

1967 ◽  
Vol 18 (03/04) ◽  
pp. 766-778 ◽  
Author(s):  
H. J Knieriem ◽  
A. B Chandler
Keyword(s):  
Platelet Count ◽  
Placebo Group ◽  
Platelet Aggregation ◽  
Prothrombin Time ◽  
Platelet Rich Plasma ◽  
Healthy Adults ◽  
Double Blind Study ◽  
Double Blind ◽  
Warfarin Sodium

SummaryThe effect of the administration of warfarin sodium (Coumadin®) on the duration of platelet aggregation in vitro was studied. Coumadin was given for 4 consecutive days to 10 healthy adults who were followed over a period of 9 days. The duration of adenosine diphosphate-induced platelet aggregation in platelet-rich plasma, the prothrombin time, and the platelet count of platelet-rich plasma were measured. Four other healthy adults received placebos and participated in a double-blind study with those receiving Coumadin.Although administration of Coumadin caused a prolongation of the prothrombin time to 2 or 21/2 times the normal value, a decrease in the duration of platelet aggregation was not observed. In most individuals who received Coumadin an increase in the duration of platelet aggregation occurred. The effect of Coumadin on platelet aggregation was not consistently related to the prothrombin time or to the platelet count. In the placebo group there was a distinct relation between the duration of platelet aggregation and the platelet count in platelet-rich plasma.The mean increase in the duration of platelet aggregation when compared to the control value before medication with Coumadin was 37.7%. In the placebo group there was a mean increase of 8.4%. The difference between the two groups is significant (p <0.001). Increased duration of platelet aggregation also occurred in two individuals who received Coumadin over a period of 10 and 16 days respectively.

Novel Toxin-antitoxin System Xn-mazEF from Xenorhabdus nematophi-la: Identification, Characterization and Functional Exploration

2020 ◽  
Vol 16 ◽  
Author(s):  
Jogendra Singh Nim ◽  
Mohit Yadav ◽  
Lalit Kumar Gautam ◽  
Chaitali Ghosh ◽  
Shakti Sahi ◽  
...  
Keyword(s):  
Interaction Analysis ◽  
Functional Characterization ◽  
Host Cells ◽  
System Control ◽  
Xenorhabdus Nematophila ◽  
Functional Features ◽  
Dynamics Simulations ◽  
Species Specific ◽  
Rna Interaction

Background: Xenorhabdus nematophila maintains species-specific mutual interaction with nematodes of Steinernema genus. Type II Toxin Antitoxin (TA) systems, the mazEF TA system controls stress and programmed cell death in bacteria. Objective: This study elucidates the functional characterization of Xn-mazEF, a mazEF homolog in X. nematophila by computational and in vitro approaches. Methods: 3 D- structural models for Xn-MazE toxin and Xn-MazF antitoxin were generated, validated and characterized for protein - RNA interaction analysis. Further biological and cellular functions of Xn-MazF toxin were also predicted. Molecular dynamics simulations of 50ns for Xn-MazF toxin complexed with nucleic acid units (DU, RU, RC, and RU) were performed. The MazF toxin and complete MazEF operon were endogenously expressed and monitored for the killing of Escherichia coli host cells under arabinose induced tightly regulated system. Results: Upon induction, E. coli expressing toxin showed rapid killing within four hours and attained up to 65% growth inhibition, while the expression of the entire operon did not show significant killing. The observation suggests that the Xn-mazEF TA system control transcriptional regulation in X. nematophila and helps to manage stress or cause toxicity leading to programmed death of cells. Conclusion: The study provides insights into structural and functional features of novel toxin, XnMazF and provides an initial inference on control of X. nematophila growth regulated by TA systems.

ACIS, A Novel KepTide™, Binds to ACE-2 Receptor and Inhibits the Infection of SARS-CoV2 Virus in vitro in Primate Kidney Cells: Therapeutic Implications for COVID-19 (Preprint)

2020 ◽  
Author(s):  
Avik Sotira Scientific
Keyword(s):  
Social Life ◽  
Plaque Assay ◽  
Host Cells ◽  
Surface Glycoprotein ◽  
Crystallographic Structure ◽  
Therapeutic Implications ◽  
Biochemical Assays ◽  
Targeted Medicine

UNSTRUCTURED Coronavirus disease 2019 (COVID-19) is a severe acute respiratory syndrome (SARS) caused by a virus known as SARS-Coronavirus 2 (SARS-CoV2). Without a targeted-medicine, this disease has been causing a massive humanitarian crisis not only in terms of mortality, but also imposing a lasting damage to social life and economic progress of humankind. Therefore, an immediate therapeutic strategy needs to be intervened to mitigate this global crisis. Here, we report a novel KepTide™ (Knock-End Peptide) therapy that nullifies SARS-CoV2 infection. SARS-CoV2 employs its surface glycoprotein “spike” (S-glycoprotein) to interact with angiotensin converting enzyme-2 (ACE-2) receptor for its infection in host cells. Based on our in-silico-based homology modeling study validated with a recent X-ray crystallographic structure (PDB ID:6M0J), we have identified that a conserved motif of S-glycoprotein that intimately engages multiple hydrogen-bond (H-bond) interactions with ACE-2 enzyme. Accordingly, we designed a peptide, termed as ACIS (ACE-2 Inhibitory motif of Spike), that displayed significant affinity towards ACE-2 enzyme as confirmed by biochemical assays such as BLItz and fluorescence polarization assays. Interestingly, more than one biochemical modifications were adopted in ACIS in order to enhance the inhibitory action of ACIS and hence called as KEpTide™. Consequently, a monolayer invasion assay, plaque assay and dual immunofluorescence analysis further revealed that KEpTide™ efficiently mitigated the infection of SARS-CoV2 in vitro in VERO E6 cells. Finally, evaluating the relative abundance of ACIS in lungs and the potential side-effects in vivo in mice, our current study discovers a novel KepTide™ therapy that is safe, stable, and robust to attenuate the infection of SARS-CoV2 virus if administered intranasally. INTERNATIONAL REGISTERED REPORT RR2-https://doi.org/10.1101/2020.10.13.337584

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