scholarly journals Penicillin G-Induced Chlamydial Stress Response in a Porcine Strain ofChlamydia pecorum

2016 ◽  
Vol 2016 ◽  
pp. 1-10 ◽  
Author(s):  
Cory Ann Leonard ◽  
Frederic Dewez ◽  
Nicole Borel
Keyword(s):  
Stress Response ◽  
De Novo ◽  
Protein Translation ◽  
Asymptomatic Infection ◽  
Host Cells ◽  
Penicillin G ◽  
Beta Lactam ◽  
Chlamydia Pecorum ◽  
Response Persistence

Chlamydia pecorumcauses asymptomatic infection and pathology in ruminants, pigs, and koalas. We characterized the antichlamydial effect of the beta lactam penicillin G onChlamydia pecorumstrain 1710S (porcine abortion isolate). Penicillin-exposed and mock-exposed infected host cells showed equivalent inclusions numbers. Penicillin-exposed inclusions contained aberrant bacterial forms and exhibited reduced infectivity, while mock-exposed inclusions contained normal bacterial forms and exhibited robust infectivity. Infectious bacteria production increased upon discontinuation of penicillin exposure, compared to continued exposure.Chlamydia-induced cell death occurred in mock-exposed controls; cell survival was improved in penicillin-exposed infected groups. Similar results were obtained both in the presence and in the absence of the eukaryotic protein translation inhibitor cycloheximide and at different times of initiation of penicillin exposure. These data demonstrate that penicillin G induces the chlamydial stress response (persistence) and is not bactericidal, for this chlamydial species/strainin vitro, regardless of host cellde novoprotein synthesis.

scholarly journals Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in COVID-19 patients and healthy individuals

2021 ◽  
pp. eabd6990
Author(s):  
Sang Il Kim ◽  
Jinsung Noh ◽  
Sujeong Kim ◽  
Younggeun Choi ◽  
Duck Kyun Yoo ◽  
...  
Keyword(s):  
B Cells ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
De Novo ◽  
Binding Domain ◽  
Host Cells ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Healthy Individuals

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 out of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were comprised of immunoglobulin heavy variable (IGHV)3-53 or IGHV3-66 and immunoglobulin heavy joining (IGHJ)6 genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different immunoglobulin heavy variable chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in six out of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

scholarly journals Francisella tularensis ΔpyrF Mutants Show that Replication in Nonmacrophages Is Sufficient for Pathogenesis In Vivo

2010 ◽  
Vol 78 (6) ◽  
pp. 2607-2619 ◽  
Author(s):  
Joseph Horzempa ◽  
Dawn M. O'Dee ◽  
Robert M. Q. Shanks ◽  
Gerard J. Nau
Keyword(s):  
Francisella Tularensis ◽  
De Novo ◽  
Human Fibroblasts ◽  
Host Cells ◽  
Hek 293 ◽  
293 Cells ◽  
Animal Infection ◽  
Schu S4

ABSTRACT The pathogenesis of Francisella tularensis has been associated with this bacterium's ability to replicate within macrophages. F. tularensis can also invade and replicate in a variety of nonphagocytic host cells, including lung and kidney epithelial cells and hepatocytes. As uracil biosynthesis is a central metabolic pathway usually necessary for pathogens, we characterized ΔpyrF mutants of both F. tularensis LVS and Schu S4 to investigate the role of these mutants in intracellular growth. As expected, these mutant strains were deficient in de novo pyrimidine biosynthesis and were resistant to 5-fluoroorotic acid, which is converted to a toxic product by functional PyrF. The F. tularensis ΔpyrF mutants could not replicate in primary human macrophages. The inability to replicate in macrophages suggested that the F. tularensis ΔpyrF strains would be attenuated in animal infection models. Surprisingly, these mutants retained virulence during infection of chicken embryos and in the murine model of pneumonic tularemia. We hypothesized that the F. tularensis ΔpyrF strains may replicate in cells other than macrophages to account for their virulence. In support of this, F. tularensis ΔpyrF mutants replicated in HEK-293 cells and normal human fibroblasts in vitro. Moreover, immunofluorescence microscopy showed abundant staining of wild-type and mutant bacteria in nonmacrophage cells in the lungs of infected mice. These findings indicate that replication in nonmacrophages contributes to the pathogenesis of F. tularensis.

scholarly journals Dynamic localisation of DamX regulates bacterial filamentation and division during UPEC dispersal from host cells

2021 ◽  
Author(s):  
Bill Soderstrom ◽  
Daniel O. Daley ◽  
Iain G. Duggin
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Stress Response ◽  
Epithelial Cells ◽  
Membrane Fusion ◽  
Host Cells ◽  
Division Rings ◽  
Bladder Cells ◽  
Rod Cell

Uropathogenic Escherichia coli (UPEC) cells can grow into highly filamentous forms during infection of bladder epithelial cells, but this process is poorly understood. Herein we found that some UPEC filaments released from infected bladder cells in vitro grew very rapidly and by more than 100 μm before initiating division, whereas others did not survive, suggesting that filamentation is a stress response that promotes dispersal. The DamX bifunctional division protein, which is essential for UPEC filamentation, was initially non-localized but then assembled at multiple division sites in the filaments prior to division. DamX rings maintained consistent thickness during constriction and remained at the septum until after membrane fusion was completed, like in rod cell division. Our findings suggest a mechanism involving regulated dissipation of DamX, leading to division arrest and filamentation, followed by its reassembly into division rings to promote UPEC dispersal and survival during infection.

scholarly journals Isoalantolactone Enhances the Antimicrobial Activity of Penicillin G against Staphylococcus aureus by Inactivating β-Lactamase during Protein Translation

Pathogens ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 161 ◽  
Author(s):  
Yonglin Zhou ◽  
Yan Guo ◽  
Zhongmei Wen ◽  
Xinxin Ci ◽  
Lining Xia ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Inhibitory Concentration ◽  
Synergistic Effects ◽  
Protein Translation ◽  
Multidrug Resistant ◽  
Cd Spectroscopy ◽  
Penicillin G ◽  
Pcr Assay ◽  
Mechanism Of Inhibition

β-Lactamase-positive Staphylococcus aureus is one of the most prevalent multidrug-resistant pathogens worldwide and is associated with increasing threats to clinical therapeutics and public health. Here, we showed that isoalantolactone (IAL), in combination with penicillin G, exhibited significant synergism against 21 β-lactamase-positive S. aureus strains (including methicillin resistant S. aureus). An enzyme inhibition assay, a checkerboard minimum inhibitory concentration (MIC) assay, a growth curve assay, a time-killing assay, a RT-PCR assay and Circular Dichroism (CD) spectroscopy were performed on different β-lactamases or β-lactamase-positive S. aureus strains, in vitro, to confirm the mechanism of inhibition of β-lactamase and the synergistic effects of the combination of penicillin G and IAL. All the fractional inhibitory concentration (FIC) indices of penicillin G, in combination with IAL, against β-lactamase-positive S. aureus, were less than 0.5, and ranged from 0.10 ± 0.02 to 0.38 ± 0.17. The survival rate of S. aureus-infected mice increased significantly from 35.29% to 88.24% within 144 h following multiple compound therapy approaches. Unlike sulbactam, IAL inactivated β-lactamase during protein translation, and the therapeutic effect of combination therapy with IAL and penicillin G was equivalent to that of sulbactam with penicillin G. Collectively, our results indicated that IAL is a promising and leading drug that can be used to restore the antibacterial effect of β-lactam antibiotics such as penicillin G and to address the inevitable infection caused by β-lactamase-positive S. aureus.

scholarly journals Carbonic Anhydrase Is Essential for Streptococcus pneumoniae Growth in Environmental Ambient Air

2010 ◽  
Vol 192 (15) ◽  
pp. 4054-4062 ◽  
Author(s):  
Peter Burghout ◽  
Lorelei E. Cron ◽  
Henrik Gradstedt ◽  
Beatriz Quintero ◽  
Elles Simonetti ◽  
...  
Keyword(s):  
Streptococcus Pneumoniae ◽  
Carbonic Anhydrase ◽  
Unsaturated Fatty Acids ◽  
De Novo ◽  
Ambient Air ◽  
Host Cells ◽  
Ambient Conditions ◽  
Growth And Survival ◽  
Cellular Processes

ABSTRACT The respiratory tract pathogen Streptococcus pneumoniae needs to adapt to the different levels of carbon dioxide (CO2) it encounters during transmission, colonization, and infection. Since CO2 is important for various cellular processes, factors that allow optimal CO2 sequestering are likely to be important for pneumococcal growth and survival. In this study, we showed that the putative pneumococcal carbonic anhydrase (PCA) is essential for in vitro growth of S. pneumoniae under the CO2-poor conditions found in environmental ambient air. Enzymatic analysis showed that PCA catalyzes the reversible hydration of CO2 to bicarbonate (HCO3 −), an essential step to prevent the cellular release of CO2. The addition of unsaturated fatty acids (UFAs) reversed the CO2-dependent in vitro growth inhibition of S. pneumoniae strains lacking the pca gene (Δpca), indicating that PCA-mediated CO2 fixation is at least associated with HCO3 −-dependent de novo biosynthesis of UFAs. Besides being necessary for growth in environmental ambient conditions, PCA-mediated CO2 fixation pathways appear to be required for intracellular survival in host cells. This effect was especially pronounced during invasion of human brain microvascular endothelial cells (HBMEC) and uptake by murine J774 macrophage cells but not during interaction of S. pneumoniae with Detroit 562 pharyngeal epithelial cells. Finally, the highly conserved pca gene was found to be invariably present in both CO2-independent and naturally circulating CO2-dependent strains, suggesting a conserved essential role for PCA and PCA-mediated CO2 fixation pathways for pneumococcal growth and survival.

scholarly journals A nadA Mutation Confers Nicotinic Acid Auxotrophy in Pro-carcinogenic Intestinal Escherichia coli NC101

2021 ◽  
Vol 12 ◽  
Author(s):  
Lacey R. Lopez ◽  
Cassandra J. Barlogio ◽  
Christopher A. Broberg ◽  
Jeremy Wang ◽  
Janelle C. Arthur
Keyword(s):  
Escherichia Coli ◽  
Nicotinic Acid ◽  
De Novo ◽  
Host Cells ◽  
Gene Encoding ◽  
E Coli ◽  
Functional Studies ◽  
Bowel Diseases ◽  
Mechanistic Investigations

Inflammatory bowel diseases (IBDs) and inflammation-associated colorectal cancer (CRC) are linked to blooms of adherent-invasive Escherichia coli (AIEC) in the intestinal microbiota. AIEC are functionally defined by their ability to adhere/invade epithelial cells and survive/replicate within macrophages. Changes in micronutrient availability can alter AIEC physiology and interactions with host cells. Thus, culturing AIEC for mechanistic investigations often involves precise nutrient formulation. We observed that the pro-inflammatory and pro-carcinogenic AIEC strain NC101 failed to grow in minimal media (MM). We hypothesized that NC101 was unable to synthesize a vital micronutrient normally found in the host gut. Through nutrient supplementation studies, we identified that NC101 is a nicotinic acid (NA) auxotroph. NA auxotrophy was not observed in the other non-toxigenic E. coli or AIEC strains we tested. Sequencing revealed NC101 has a missense mutation in nadA, a gene encoding quinolinate synthase A that is important for de novo nicotinamide adenine dinucleotide (NAD) biosynthesis. Correcting the identified nadA point mutation restored NC101 prototrophy without impacting AIEC function, including motility and AIEC-defining survival in macrophages. Our findings, along with the generation of a prototrophic NC101 strain, will greatly enhance the ability to perform in vitro functional studies that are needed for mechanistic investigations on the role of intestinal E. coli in digestive disease.

scholarly journals Cell-based and cell-free biocatalysis for the production of d-glucaric acid

2020 ◽  
Vol 13 (1) ◽  
Author(s):  
Lu-Zhou Chen ◽  
Si-Ling Huang ◽  
Jin Hou ◽  
Xue-Ping Guo ◽  
Feng-Shan Wang ◽  
...  
Keyword(s):  
Metabolic Pathways ◽  
De Novo ◽  
Value Added ◽  
Host Cells ◽  
Efficient Production ◽  
Glucaric Acid ◽  
A Value ◽  
Potential Applications ◽  
Increasing Demand

Abstractd-Glucaric acid (GA) is a value-added chemical produced from biomass, and has potential applications as a versatile platform chemical, food additive, metal sequestering agent, and therapeutic agent. Marketed GA is currently produced chemically, but increasing demand is driving the search for eco-friendlier and more efficient production approaches. Cell-based production of GA represents an alternative strategy for GA production. A series of synthetic pathways for GA have been ported into Escherichia coli, Saccharomyces cerevisiae and Pichia pastoris, respectively, and these engineered cells show the ability to synthesize GA de novo. Optimization of the GA metabolic pathways in host cells has leapt forward, and the titer and yield have increased rapidly. Meanwhile, cell-free multi-enzyme catalysis, in which the desired pathway is constructed in vitro from enzymes and cofactors involved in GA biosynthesis, has also realized efficient GA bioconversion. This review presents an overview of studies of the development of cell-based GA production, followed by a brief discussion of potential applications of biosensors that respond to GA in these biosynthesis routes.

scholarly journals Gene bb0318 Is Critical for the Oxidative Stress Response and Infectivity of Borrelia burgdorferi

2016 ◽  
Vol 84 (11) ◽  
pp. 3141-3151 ◽  
Author(s):  
Adrienne C. Showman ◽  
George Aranjuez ◽  
Philip P. Adams ◽  
Mollie W. Jewett
Keyword(s):  
Oxidative Stress ◽  
Stress Response ◽  
Borrelia Burgdorferi ◽  
Molecular Mechanisms ◽  
De Novo ◽  
Oxidative Stress Response ◽  
Growth Defect ◽  
Content Type

A greater understanding of the molecular mechanisms that Borrelia burgdorferi uses to survive during mammalian infection is critical for the development of novel diagnostic and therapeutic tools to improve the clinical management of Lyme disease. By use of an in vivo expression technology (IVET)-based approach to identify B. burgdorferi genes expressed in vivo , we discovered the bb0318 gene, which is thought to encode the ATPase component of a putative riboflavin ABC transport system. Riboflavin is a critical metabolite enabling all organisms to maintain redox homeostasis. B. burgdorferi appears to lack the metabolic capacity for de novo synthesis of riboflavin and so likely relies on scavenging riboflavin from the host environment. In this study, we sought to investigate the role of bb0318 in B. burgdorferi pathogenesis. No in vitro growth defect was observed for the Δ bb0318 clone. However, the mutant spirochetes displayed reduced levels of survival when exposed to exogenous hydrogen peroxide or murine macrophages. Spirochetes lacking bb0318 were found to have a 100-fold-higher 50% infectious dose than spirochetes containing bb0318 . In addition, at a high inoculum dose, bb0318 was found to be important for effective spirochete dissemination to deep tissues for as long as 3 weeks postinoculation and to be critical for B. burgdorferi infection of mouse hearts. Together, these data implicate bb0318 in the oxidative stress response of B. burgdorferi and indicate the contribution of bb0318 to B. burgdorferi mammalian infectivity.

scholarly journals Impact of the RNA Chaperone Hfq on the Fitness and Virulence Potential of Uropathogenic Escherichia coli

2008 ◽  
Vol 76 (7) ◽  
pp. 3019-3026 ◽  
Author(s):  
Richard R. Kulesus ◽  
Karen Diaz-Perez ◽  
E. Susan Slechta ◽  
Danelle S. Eto ◽  
Matthew A. Mulvey
Keyword(s):  
Escherichia Coli ◽  
Stress Response ◽  
Sigma Factor ◽  
Posttranscriptional Regulation ◽  
Polymyxin B ◽  
Host Cells ◽  
Infection Model ◽  
Rna Chaperone ◽  
Envelope Stress

ABSTRACT Hfq is a bacterial RNA chaperone involved in the posttranscriptional regulation of many stress-inducible genes via small noncoding RNAs. Here, we show that Hfq is critical for the uropathogenic Escherichia coli (UPEC) isolate UTI89 to effectively colonize the bladder and kidneys in a murine urinary tract infection model system. The disruption of hfq did not affect bacterial adherence to or invasion of host cells but did limit the development of intracellular microcolonies by UTI89 within the terminally differentiated epithelial cells that line the lumen of the bladder. In vitro, the hfq mutant was significantly impaired in its abilities to handle the antibacterial cationic peptide polymyxin B and reactive nitrogen and oxygen radicals and to grow in acidic medium (pH 5.0). Relative to the wild-type strain, the hfq mutant also had a substantially reduced migration rate on motility agar and was less prone to form biofilms. Hfq activities are known to impact the regulation of both the stationary-phase sigma factor RpoS (σS) and the envelope stress response sigma factor RpoE (σE). Although we saw similarities among hfq, rpoS, and rpoE deletion mutants in our assays, the rpoE and hfq mutants were phenotypically the most alike. Cumulatively, our data indicate that Hfq likely affects UPEC virulence-related phenotypes primarily by modulating membrane homeostasis and envelope stress response pathways.

scholarly journals Population Genomic Molecular Epidemiological Study of Macrolide-Resistant Streptococcus pyogenes in Iceland, 1995 to 2016: Identification of a Large Clonal Population with a pbp2x Mutation Conferring Reduced In Vitro β-Lactam Susceptibility

2020 ◽  
Vol 58 (9) ◽  
Author(s):  
Sara B. Southon ◽  
Stephen B. Beres ◽  
Priyanka Kachroo ◽  
Matthew Ojeda Saavedra ◽  
Helga Erlendsdóttir ◽  
...  
Keyword(s):  
Streptococcus Pyogenes ◽  
Efflux Pump ◽  
Macrolide Resistance ◽  
Resistant Isolate ◽  
Single Amino Acid ◽  
Health Concern ◽  
Penicillin G ◽  
Reference Laboratory ◽  
Beta Lactam

ABSTRACT Resistance to macrolide antibiotics is a global concern in the treatment of Streptococcus pyogenes (group A Streptococcus [GAS]) infections. In Iceland, since the detection of the first macrolide-resistant isolate in 1998, three epidemic waves of macrolide-resistant GAS infections have occurred, with peaks in 1999, 2004, and 2008. We conducted whole-genome sequencing of all 1,575 available GAS macrolide-resistant clinical isolates of all infection types collected at the national reference laboratory in Reykjavik, Iceland, from 1998 to 2016. Among 1,515 erythromycin-resistant isolates, 90.3% were of only three emm types, emm4 (n = 713), emm6 (n = 324), and emm12 (n = 332), with each being predominant in a distinct epidemic peak. The antibiotic efflux pump genes, mef(A) and msr(D), were present on chimeric mobile genetic elements in 99.3% of the macrolide-resistant isolates of these emm types. Of note, in addition to macrolide resistance, virtually all emm12 isolates had a single amino acid substitution in penicillin-binding protein PBP2X that conferred a 2-fold increased penicillin G and ampicillin MIC among the isolates tested. We conclude that each of the three large epidemic peaks of macrolide-resistant GAS infections occurring in Iceland since 1998 was caused by the emergence and clonal expansion of progenitor strains, with macrolide resistance being conferred predominantly by inducible Mef(A) and Msr(D) drug efflux pumps. The occurrence of emm12 strains with macrolide resistance and decreased beta-lactam susceptibility was unexpected and is of public health concern.

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