scholarly journals The Central Role and Possible Mechanisms of Bacterial DNAs in Sepsis Development

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Zhenxing Cheng ◽  
Simon T. Abrams ◽  
James Austin ◽  
Julien Toh ◽  
Susan Siyu Wang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Cytokine Production ◽  
Severe Disease ◽  
Host Cells ◽  
Signal Pathways ◽  
Dna Sensors ◽  
Bacterial Dna ◽  
Viral Dnas ◽  
Novel Therapeutic Strategies ◽  
Cellular Compartments

The pathological roles of bacterial DNA have been documented many decades ago. Bacterial DNAs are different from mammalian DNAs; the latter are heavily methylated. Mammalian cells have sensors such as TLR-9 to sense the DNAs with nonmethylated CpGs and distinguish them from host DNAs with methylated CpGs. Further investigation has identified many other types of DNA sensors distributed in a variety of cellular compartments. These sensors not only sense foreign DNAs, including bacterial and viral DNAs, but also sense damaged DNAs from the host cells. The major downstream signalling pathways includeTLR-9-MyD88-IKKa-IRF-7/NF-κB pathways to increase IFN/proinflammatory cytokine production, STING-TBK1-IRF3 pathway to increase IFN-beta, and AIM2-ASC-caspas-1 pathway to release IL-1beta. The major outcome is to activate host immune response by inducing cytokine production. In this review, we focus on the roles and potential mechanisms of DNA sensors and downstream pathways in sepsis. Although bacterial DNAs play important roles in sepsis development, bacterial DNAs alone are unable to cause severe disease nor lead to death. Priming animals with bacterial DNAs facilitate other pathological factors, such as LPS and other virulent factors, to induce severe disease and lethality. We also discuss compartmental distribution of DNA sensors and pathological significance as well as the transport of extracellular DNAs into cells. Understanding the roles of DNA sensors and signal pathways will pave the way for novel therapeutic strategies in many diseases, particularly in sepsis.

scholarly journals Isolation of reconstructed functional ribonucleoprotein complexes of Machupo virus

2021 ◽  
Author(s):  
Jesse D. Pyle ◽  
Sean P. J. Whelan
Keyword(s):  
Rna Polymerase ◽  
Mammalian Cells ◽  
Severe Disease ◽  
Host Cells ◽  
Genomic Rna ◽  
Replication Machinery ◽  
Rna Dependent Rna Polymerase ◽  
Genomic Rnas ◽  
Ribonucleoprotein Complexes ◽  
Machupo Virus

Arenaviruses initiate infection by delivering a transcriptionally-competent ribonucleoprotein (RNP) complex into the cytosol of host cells. The arenavirus RNP consists of the large (L) RNA-dependent RNA polymerase (RdRP) bound to a nucleoprotein (NP)-encapsidated genomic RNA (vRNA) template. During transcription and replication, L must transiently displace RNA-bound NP to allow for template access into the RdRP active site. Concomitant with RNA replication new subunits of NP must be added to the nascent complementary RNAs (cRNA) as they emerge from the product exit channel of L. Interactions between L and NP thus play a central role in arenavirus gene expression. We developed an approach to purify recombinant functional RNPs from mammalian cells in culture using a synthetic vRNA, affinity-tagged L and NP. Negative-stain electron microscopy of purified RNPs revealed they adopt diverse and flexible structures, like RNPs of other Bunyavirales members. Monodisperse L-NP and trimeric ring-like NP complexes were also obtained in excess of flexible RNPs, suggesting that these heterodimeric structures self-assemble in the absence of suitable RNA templates. This work allows for further biochemical analysis of the interaction between arenavirus L and NP proteins and provides a framework for future high-resolution structural analyses of this replication-associated complex. IMPORTANCE Arenaviruses are rodent-borne pathogens that can cause severe disease in humans. All arenaviruses begin the infection cycle with delivery of the virus replication machinery into the cytoplasm of the host cell. This machinery consists of an RNA-dependent RNA polymerase – which copies the viral genome segments and synthesizes all four viral mRNAs – bound to the two nucleoprotein-encapsidated genomic RNAs. How this complex assembles remains a mystery. Our findings provide direct evidence for the formation of diverse intracellular arenavirus replication complexes using purification strategies for the polymerase, nucleoprotein, and genomic RNA of Machupo virus, which causes Bolivian hemorrhagic fever in humans. We demonstrate that the polymerase and nucleoprotein assemble into higher-order structures within cells, providing a model for the molecular events of arenavirus RNA synthesis. These findings provide a framework for probing the architectures and functions of the arenavirus replication machinery, and thus advancing antiviral strategies targeting this essential complex.

scholarly journals A polymeric approach toward resistance-resistant antimicrobial agent with dual-selective mechanisms of action

2021 ◽  
Vol 7 (5) ◽  
pp. eabc9917
Author(s):  
Silei Bai ◽  
Jianxue Wang ◽  
Kailing Yang ◽  
Cailing Zhou ◽  
Yangfan Xu ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Mechanisms Of Action ◽  
Model Systems ◽  
General Strategy ◽  
Antimicrobial Polymers ◽  
Bacterial Dna ◽  
Bacterial Membranes ◽  
Severe Infections ◽  
Multiple Drugs ◽  
Resistance Rates

Antibiotic resistance is now a major threat to human health, and one approach to combating this threat is to develop resistance-resistant antibiotics. Synthetic antimicrobial polymers are generally resistance resistant, having good activity with low resistance rates but usually with low therapeutic indices. Here, we report our solution to this problem by introducing dual-selective mechanisms of action to a short amidine-rich polymer, which can simultaneously disrupt bacterial membranes and bind to bacterial DNA. The oligoamidine shows unobservable resistance generation but high therapeutic indices against many bacterial types, such as ESKAPE strains and clinical isolates resistant to multiple drugs, including colistin. The oligomer exhibited excellent effectiveness in various model systems, killing extracellular or intracellular bacteria in the presence of mammalian cells, removing all bacteria from Caenorhabditis elegans, and rescuing mice with severe infections. This “dual mechanisms of action” approach may be a general strategy for future development of antimicrobial polymers.

scholarly journals Neurotrophin Receptor TrkC Is an Entry Receptor for Trypanosoma cruzi in Neural, Glial, and Epithelial Cells

2011 ◽  
Vol 79 (10) ◽  
pp. 4081-4087 ◽  
Author(s):  
Craig Weinkauf ◽  
Ryan Salvador ◽  
Mercio PereiraPerrin
Keyword(s):  
Trypanosoma Cruzi ◽  
Chagas Disease ◽  
Mammalian Cells ◽  
Chinese Hamster ◽  
Neuronal Cell ◽  
Host Cells ◽  
Neurotrophin Receptor ◽  
Content Type

ABSTRACTTrypanosoma cruzi, the agent of Chagas' disease, infects a variety of mammalian cells in a process that includes multiple cycles of intracellular division and differentiation starting with host receptor recognition by a parasite ligand(s). Earlier work in our laboratory showed that the neurotrophin-3 (NT-3) receptor TrkC is activated byT. cruzisurfacetrans-sialidase, also known as parasite-derived neurotrophic factor (PDNF). However, it has remained unclear whether TrkC is used byT. cruzito enter host cells. Here, we show that a neuronal cell line (PC12-NNR5) relatively resistant toT. cruzibecame highly susceptible to infection when overexpressing human TrkC but not human TrkB. Furthermore,trkCtransfection conferred an ∼3.0-fold intracellular growth advantage. Sialylation-deficient Chinese hamster ovarian (CHO) epithelial cell lines Lec1 and Lec2 also became much more permissive toT. cruziafter transfection with thetrkCgene. Additionally, NT-3 specifically blockedT. cruziinfection of the TrkC-NNR5 transfectants and of naturally permissive TrkC-bearing Schwann cells and astrocytes, as did recombinant PDNF. Two specific inhibitors of Trk autophosphorylation (K252a and AG879) and inhibitors of Trk-induced MAPK/Erk (U0126) and Akt kinase (LY294002) signaling, but not an inhibitor of insulin-like growth factor 1 receptor, abrogated TrkC-mediated cell invasion. Antibody to TrkC blockedT. cruziinfection of the TrkC-NNR5 transfectants and of cells that naturally express TrkC. The TrkC antibody also significantly and specifically reduced cutaneous infection in a mouse model of acute Chagas' disease. TrkC is ubiquitously expressed in the peripheral and central nervous systems, and in nonneural cells infected byT. cruzi, including cardiac and gastrointestinal muscle cells. Thus, TrkC is implicated as a functional PDNF receptor in cell entry, independently of sialic acid recognition, mediating broadT. cruziinfection bothin vitroandin vivo.

scholarly journals SpyAD, a Moonlighting Protein of Group A Streptococcus Contributing to Bacterial Division and Host Cell Adhesion

2014 ◽  
Vol 82 (7) ◽  
pp. 2890-2901 ◽  
Author(s):  
Marilena Gallotta ◽  
Giovanni Gancitano ◽  
Giampiero Pietrocola ◽  
Marirosa Mora ◽  
Alfredo Pezzicoli ◽  
...  
Keyword(s):  
Cell Division ◽  
Mammalian Cells ◽  
Group A Streptococcus ◽  
Host Cells ◽  
Knockout Mutant ◽  
Content Type ◽  
Moonlighting Protein ◽  
Group A

ABSTRACTGroup A streptococcus (GAS) is a human pathogen causing a wide repertoire of mild and severe diseases for which no vaccine is yet available. We recently reported the identification of three protein antigens that in combination conferred wide protection against GAS infection in mice. Here we focused our attention on the characterization of one of these three antigens, Spy0269, a highly conserved, surface-exposed, and immunogenic protein of unknown function. Deletion of thespy0269gene in a GAS M1 isolate resulted in very long bacterial chains, which is indicative of an impaired capacity of the knockout mutant to properly divide. Confocal microscopy and immunoprecipitation experiments demonstrated that the protein was mainly localized at the cell septum and could interactin vitrowith the cell division protein FtsZ, leading us to hypothesize that Spy0269 is a member of the GAS divisome machinery. Predicted structural domains and sequence homologies with known streptococcal adhesins suggested that this antigen could also play a role in mediating GAS interaction with host cells. This hypothesis was confirmed by showing that recombinant Spy0269 could bind to mammalian epithelial cellsin vitroand thatLactococcus lactisexpressing Spy0269 on its cell surface could adhere to mammalian cellsin vitroand to mice nasal mucosain vivo. On the basis of these data, we believe that Spy0269 is involved both in bacterial cell division and in adhesion to host cells and we propose to rename this multifunctional moonlighting protein as SpyAD (StreptococcuspyogenesAdhesion andDivision protein).

scholarly journals Heterologously Expressed Staphylococcus aureusFibronectin-Binding Proteins Are Sufficient for Invasion of Host Cells

2000 ◽  
Vol 68 (12) ◽  
pp. 6871-6878 ◽  
Author(s):  
Bhanu Sinha ◽  
Patrice Francois ◽  
Yok-Ai Que ◽  
Muzaffar Hussain ◽  
Christine Heilmann ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Binding Proteins ◽  
Surface Protein ◽  
Latex Agglutination ◽  
Surface Proteins ◽  
Host Cells ◽  
Gram Positive ◽  
Fibronectin Receptor ◽  
293 Cells ◽  
Accessible Surface

ABSTRACT Staphylococcus aureus invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, critically depends on fibronectin bridging between S. aureusfibronectin-binding proteins (FnBPs) and the host fibronectin receptor integrin α5β1 (B. Sinha et al., Cell. Microbiol. 1:101–117, 1999). However, it is unknown whether this mechanism is sufficient for S. aureus invasion. To address this question, various S. aureus adhesins (FnBPA, FnBPB, and clumping factor [ClfA]) were expressed in Staphylococcus carnosus and Lactococcus lactis subsp.cremoris. Both noninvasive gram-positive microorganisms are genetically distinct from S. aureus, lack any knownS. aureus surface protein, and do not bind fibronectin. Transformants of S. carnosus and L. lactisharboring plasmids coding for various S. aureus surface proteins (FnBPA, FnBPB, and ClfA) functionally expressed adhesins (as determined by bacterial clumping in plasma, specific latex agglutination, Western ligand blotting, and binding to immobilized and soluble fibronectin). FnBPA or FnBPB but not of ClfA conferred invasiveness to S. carnosus and L. lactis. Invasion of 293 cells by transformants was comparable to that of strongly invasive S. aureus strain Cowan 1. Binding of soluble and immobilized fibronectin paralleled invasiveness, demonstrating that the amount of accessible surface FnBPs is rate limiting. Thus, S. aureus FnBPs confer invasiveness to noninvasive, apathogenic gram-positive cocci. Furthermore, FnBP-coated polystyrene beads were internalized by 293 cells, demonstrating that FnBPs are sufficient for invasion of host cells without the need for (S. aureus-specific) coreceptors.

scholarly journals Mammalian cell invasion and intracellular trafficking by Trypanosoma cruzi infective forms

2005 ◽  
Vol 77 (1) ◽  
pp. 77-94 ◽  
Author(s):  
Renato A. Mortara ◽  
Walter K. Andreoli ◽  
Noemi N. Taniwaki ◽  
Adriana B. Fernandes ◽  
Claudio V. da Silva ◽  
...  
Keyword(s):  
Trypanosoma Cruzi ◽  
Host Cell ◽  
Mammalian Cells ◽  
Parasitophorous Vacuole ◽  
Host Cells ◽  
Mammalian Host ◽  
Target Cells ◽  
Sylvatic Cycle ◽  
Final Destination ◽  
Different Strains

Trypanosoma cruzi, the etiological agent of Chagas’ disease, occurs as different strains or isolates that may be grouped in two major phylogenetic lineages: T. cruzi I, associated with the sylvatic cycle and T. cruzi II, linked to the human disease. In the mammalian host the parasite has to invade cells and many studies implicated the flagellated trypomastigotes in this process. Several parasite surface components and some of host cell receptors with which they interact have been identified. Our work focused on how amastigotes, usually found growing in the cytoplasm, can invade mammalian cells with infectivities comparable to that of trypomastigotes. We found differences in cellular responses induced by amastigotes and trypomastigotes regarding cytoskeletal components and actin-rich projections. Extracellularly generated amastigotes of T. cruzi I strains may display greater infectivity than metacyclic trypomastigotes towards cultured cell lines as well as target cells that have modified expression of different classes of cellular components. Cultured host cells harboring the bacterium Coxiella burnetii allowed us to gain new insights into the trafficking properties of the different infective forms of T. cruzi, disclosing unexpected requirements for the parasite to transit between the parasitophorous vacuole to its final destination in the host cell cytoplasm.

scholarly journals Analysis of Virus Population Profiles within Pigs Infected with Virulent Classical Swine Fever Viruses: Evidence for Bottlenecks in Transmission but Absence of Tissue-Specific Virus Variants

2020 ◽  
Vol 94 (19) ◽  
Author(s):  
Camille Melissa Johnston ◽  
Ulrik Fahnøe ◽  
Louise Lohse ◽  
Jens Bukh ◽  
Graham J. Belsham ◽  
...  
Keyword(s):  
Classical Swine Fever Virus ◽  
Population Change ◽  
Virulent Strain ◽  
Severe Disease ◽  
Clinical Signs ◽  
Classical Swine Fever ◽  
Fever Virus ◽  
Host Cells ◽  
Virus Population ◽  
Highly Virulent

ABSTRACT Classical swine fever virus (CSFV) contains a specific motif within the E2 glycoprotein that differs between strains of different virulence. In the highly virulent CSFV strain Koslov, this motif comprises residues S763/L764 in the polyprotein. However, L763/P764 represent the predominant alleles in published CSFV genomes. In this study, changes were introduced into the CSFV strain Koslov (here called vKos_SL) to generate modified CSFVs with substitutions at residues 763 and/or 764 (vKos_LL, vKos_SP, and vKos_LP). The properties of these mutant viruses, in comparison to those of vKos_SL, were determined in pigs. Each of the viruses was virulent and induced typical clinical signs of CSF, but the vKos_LP strain produced them significantly earlier. Full-length CSFV cDNA amplicons (12.3 kb) derived from sera of infected pigs were deep sequenced and cloned to reveal the individual haplotypes that contributed to the single-nucleotide polymorphism (SNP) profiles observed in the virus population. The SNP profiles for vKos_SL and vKos_LL displayed low-level heterogeneity across the entire genome, whereas vKos_SP and vKos_LP displayed limited diversity with a few high-frequency SNPs. This indicated that vKos_SL and vKos_LL exhibited a higher level of fitness in the host and more stability at the consensus level, whereas several consensus changes were observed in the vKos_SP and vKos_LP sequences, pointing to adaptation. For each virus, only a subset of the variants present within the virus inoculums were maintained in the infected pigs. No clear tissue-dependent quasispecies differentiation occurred within inoculated pigs; however, clear evidence for transmission bottlenecks to contact animals was observed, with subsequent loss of sequence diversity. IMPORTANCE The surface-exposed E2 protein of classical swine fever virus is required for its interaction with host cells. A short motif within this protein varies between strains of different virulence. The importance of two particular amino acid residues in determining the properties of a highly virulent strain of the virus has been analyzed. Each of the different viruses tested proved highly virulent, but one of them produced earlier, but not more severe, disease. By analyzing the virus genomes present within infected pigs, it was found that the viruses which replicated within inoculated animals were only a subset of those within the virus inoculum. Furthermore, following contact transmission, it was shown that a very restricted set of viruses had transferred between animals. There were no significant differences in the virus populations present in various tissues of the infected animals. These results indicate mechanisms of virus population change during transmission between animals.

scholarly journals Characterization, modelling and mitigation of gene expression burden in mammalian cells

2019 ◽  
Author(s):  
T Frei ◽  
F Cella ◽  
F Tedeschi ◽  
J Gutierrez ◽  
GB Stan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Genome Engineering ◽  
Host Cells ◽  
Genetic Circuits ◽  
Circuit Performance ◽  
Resource Requirements ◽  
Synthetic Circuit ◽  
Resource Aware

AbstractDespite recent advances in genome engineering, the design of genetic circuits in mammalian cells is still painstakingly slow and fraught with inexplicable failures. Here we demonstrate that competition for limited transcriptional and translational resources dynamically couples otherwise independent co-expressed exogenous genes, leading to diminished performance and contributing to the divergence between intended and actual function. We also show that the expression of endogenous genes is likewise impacted when genetic payloads are expressed in the host cells. Guided by a resource-aware mathematical model and our experimental finding that post-transcriptional regulators have a large capacity for resource redistribution, we identify and engineer natural and synthetic miRNA-based incoherent feedforward loop (iFFL) circuits that mitigate gene expression burden. The implementation of these circuits features the novel use of endogenous miRNAs as integral components of the engineered iFFL device, a versatile hybrid design that allows burden mitigation to be achieved across different cell-lines with minimal resource requirements. This study establishes the foundations for context-aware prediction and improvement of in vivo synthetic circuit performance, paving the way towards more rational synthetic construct design in mammalian cells.

scholarly journals Associations Between the Use of Renin–Angiotensin System Inhibitors and the Risks of Severe COVID-19 and Mortality in COVID-19 Patients With Hypertension: A Meta-Analysis of Observational Studies

2021 ◽  
Vol 8 ◽  
Author(s):  
Xiao-Ce Dai ◽  
Zhuo-Yu An ◽  
Zi-Yang Wang ◽  
Zi-Zhen Wang ◽  
Yi-Ren Wang
Keyword(s):  
Angiotensin Converting Enzyme ◽  
Observational Studies ◽  
Meta Analysis ◽  
Severe Disease ◽  
Host Cells ◽  
Cochrane Library ◽  
Converting Enzyme ◽  
Significant Difference ◽  
Duration Of Hospitalization ◽  
The Impact

Angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) share a target receptor with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The use of ACEIs/ARBs may cause angiotensin-converting enzyme 2 receptor upregulation, facilitating the entry of SARS-CoV-2 into host cells. There is concern that the use of ACEIs/ARBs could increase the risks of severe COVID-19 and mortality. The impact of discontinuing these drugs in patients with COVID-19 remains uncertain. We aimed to assess the association between the use of ACEIs/ARBs and the risks of mortality and severe disease in patients with COVID-19. A systematic search was performed in PubMed, EMBASE, Cochrane Library, and MedRxiv.org from December 1, 2019, to June 20, 2020. We also identified additional citations by manually searching the reference lists of eligible articles. Forty-two observational studies including 63,893 participants were included. We found that the use of ACEIs/ARBs was not significantly associated with a reduction in the relative risk of all-cause mortality [odds ratio (OR) = 0.87, 95% confidence interval (95% CI) = 0.75–1.00; I2 = 57%, p = 0.05]. We found no significant reduction in the risk of severe disease in the ACEI subgroup (OR = 0.95, 95% CI = 0.88–1.02, I2 = 50%, p = 0.18), the ARB subgroup (OR = 1.03, 95% CI = 0.94–1.13, I2 = 62%, p = 0.48), or the ACEI/ARB subgroup (OR = 0.83, 95% CI = 0.65–1.08, I2 = 67%, p = 0.16). Moreover, seven studies showed no significant difference in the duration of hospitalization between the two groups (mean difference = 0.33, 95% CI = −1.75 to 2.40, p = 0.76). In conclusion, the use of ACEIs/ARBs appears to not have a significant effect on mortality, disease severity, or duration of hospitalization in COVID-19 patients. On the basis of the findings of this meta-analysis, there is no support for the cessation of treatment with ACEIs or ARBs in patients with COVID-19.

scholarly journals PB2 Residue 271 Plays a Key Role in Enhanced Polymerase Activity of Influenza A Viruses in Mammalian Host Cells

2010 ◽  
Vol 84 (9) ◽  
pp. 4395-4406 ◽  
Author(s):  
Kendra A. Bussey ◽  
Tatiana L. Bousse ◽  
Emily A. Desmet ◽  
Baek Kim ◽  
Toru Takimoto
Keyword(s):  
Mammalian Cells ◽  
Influenza A ◽  
Virus Titer ◽  
Polymerase Activity ◽  
Influenza Viruses ◽  
Host Cells ◽  
Mammalian Host ◽  
Influenza A Viruses ◽  
H5n1 Influenza ◽  
Avian Viruses

ABSTRACT The direct infection of humans with highly pathogenic avian H5N1 influenza viruses has suggested viral mutation as one mechanism for the emergence of novel human influenza A viruses. Although the polymerase complex is known to be a key component in host adaptation, mutations that enhance the polymerase activity of avian viruses in mammalian hosts are not fully characterized. The genomic comparison of influenza A virus isolates has identified highly conserved residues in influenza proteins that are specific to either human or avian viruses, including 10 residues in PB2. We characterized the activity of avian polymerase complexes containing avian-to-human mutations at these conserved PB2 residues and found that, in addition to the E627K mutation, the PB2 mutation T271A enhances polymerase activity in human cells. We confirmed the effects of the T271A mutation using recombinant WSN viruses containing avian NP and polymerase genes with wild-type (WT) or mutant PB2. The 271A virus showed enhanced growth compared to that of the WT in mammalian cells in vitro. The 271A mutant did not increase viral pathogenicity significantly in mice compared to that of the 627K mutant, but it did enhance the lung virus titer. Also, cell infiltration was more evident in lungs of 271A-infected mice than in those of the WT. Interestingly, the avian-derived PB2 of the 2009 pandemic H1N1 influenza virus has 271A. The characterization of the polymerase activity of A/California/04/2009 (H1N1) and corresponding PB2 mutants indicates that the high polymerase activity of the pandemic strain in mammalian cells is, in part, dependent on 271A. Our results clearly indicate the contribution of PB2 amino acid 271 to enhanced polymerase activity and viral growth in mammalian hosts.

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