scholarly journals The AmiC/NlpD pathway dominates peptidoglycan breakdown in Neisseria meningitidisand affects cell separation, NOD1 agonist production, and infection

2021 ◽  
Author(s):  
Jia Mun Chan ◽  
Kathleen T Hackett ◽  
Katelynn L Woodhams ◽  
Ryan E Schaub ◽  
Joseph P Dillard
Keyword(s):  
Cell Separation ◽  
Innate Immune ◽  
Reporter Cell ◽  
Flag Epitope ◽  
Human Whole Blood ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein ◽  
Low Levels ◽  
Optical Reconstruction

The human-restricted pathogen Neisseria meningitidis, which is best known for causing invasive meningococcal disease, has a nonpathogenic lifestyle as an asymptomatic colonizer of the human naso- and oropharyngeal space. N. meningitidis releases small peptidoglycan (PG) fragments during growth. It was demonstrated previously that N. meningitidis releases low levels of tripeptide PG monomer, which is an inflammatory molecule recognized by the human intracellular innate immune receptor NOD1. In this present study, we demonstrated that N. meningitidis released more PG-derived peptides compared to PG monomers. Using a reporter cell line overexpressing human NOD1, we showed that N. meningitidis activates NOD1 using PG-derived peptides. Generation of such peptides required the presence of the periplasmic N- acetylmuramyl-L-alanine amidase AmiC, and the outer membrane lipoprotein, NlpD. AmiC and NlpD were found to function in cell separation, and mutation of either amiC or nlpD resulted in large clumps of unseparated N. meningitidis cells instead of the characteristic diplococci. Using stochastic optical reconstruction microscopy, we demonstrated that FLAG epitope-tagged NlpD localized to the septum, while similarly-tagged AmiC was found at the septum in some diplococci but distributed around the cell in most cases. In a human whole blood infection assay, an nlpD mutant was severely attenuated and showed particular sensitivity to complement. Thus, in N. meningitidis the cell separation proteins AmiC and NlpD are necessary for NOD1 stimulation and for survival during infection of human blood.

Stochastic Optical Reconstruction Microscopy Optimization for Investigating Innate Immune Response

2012 ◽  
Vol 18 (S2) ◽  
pp. 158-159
Author(s):  
Q.B. Smith ◽  
J.S. Aaron ◽  
B.D. Carson ◽  
J.A. Timlin
Keyword(s):  
Immune Response ◽  
Innate Immune Response ◽  
Innate Immune ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Optical Reconstruction

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.

scholarly journals Nanoscopic cell-wall architecture of an immunogenic ligand in Candida albicans during antifungal drug treatment

2016 ◽  
Vol 27 (6) ◽  
pp. 1002-1014 ◽  
Author(s):  
Jia Lin ◽  
Michael J. Wester ◽  
Matthew S. Graus ◽  
Keith A. Lidke ◽  
Aaron K. Neumann
Keyword(s):  
Cell Wall ◽  
Candida Albicans ◽  
Cell Walls ◽  
Innate Immune ◽  
Immune Recognition ◽  
Cell Wall Polysaccharide ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Before And After ◽  
Antifungal Chemotherapy ◽  
Optical Reconstruction

The cell wall of Candida albicans is composed largely of polysaccharides. Here we focus on β-glucan, an immunogenic cell-wall polysaccharide whose surface exposure is often restricted, or “masked,” from immune recognition by Dectin-1 on dendritic cells (DCs) and other innate immune cells. Previous research suggested that the physical presentation geometry of β-glucan might determine whether it can be recognized by Dectin-1. We used direct stochastic optical reconstruction microscopy to explore the fine structure of β-glucan exposed on C. albicans cell walls before and after treatment with the antimycotic drug caspofungin, which alters glucan exposure. Most surface-accessible glucan on C. albicans yeast and hyphae is limited to isolated Dectin-1–binding sites. Caspofungin-induced unmasking caused approximately fourfold to sevenfold increase in total glucan exposure, accompanied by increased phagocytosis efficiency of DCs for unmasked yeasts. Nanoscopic imaging of caspofungin-unmasked C. albicans cell walls revealed that the increase in glucan exposure is due to increased density of glucan exposures and increased multiglucan exposure sizes. These findings reveal that glucan exhibits significant nanostructure, which is a previously unknown physical component of the host– Candida interaction that might change during antifungal chemotherapy and affect innate immune activation.

PBP1a glycosyltransferase and transpeptidase activities are both required for maintaining cell morphology and envelope integrity in Shewanella oneidensis

2020 ◽  
Vol 367 (3) ◽  
Author(s):  
Jianhua Yin ◽  
Ting Zhang ◽  
Jingxiao Cai ◽  
Jie Lou ◽  
Dan Cheng ◽  
...  
Keyword(s):  
Cell Morphology ◽  
Cell Envelope ◽  
Shewanella Oneidensis ◽  
Gram Negative Bacteria ◽  
Penicillin Binding Protein ◽  
Normal Morphology ◽  
E Coli ◽  
Outer Membrane Lipoprotein ◽  
Membrane Lipoprotein ◽  
Low Levels

ABSTRACT In rod-shaped Gram-negative bacteria, penicillin binding protein 1a (PBP1a) and 1b (PBP1b) form peptidoglycan-synthesizing complexes with the outer membrane lipoprotein LpoA and LpoB, respectively. Escherichia coli mutants lacking PBP1b/LpoB are sicker than those lacking PBP1a/LpoA. However, we previously found that mutants lacking PBP1a/LpoA but not PBP1b/LpoB are deleterious in Shewanella oneidensis. Here, we show that S. oneidensis PBP1a (SoPBP1a) contains conserved signature motifs with its E. coli counterpart, EcPBP1a. Although EcPBP1a play a less prominent role in E. coli, it is capable of substituting for the SoPBP1a in a manner dependent on SoLpoA. In S. oneidensis, expression of PBP1b is lower than PBP1a, and therefore the additional expression of SoPBP1b at low levels can functionally compensate for the absence of SoPBP1a. Importantly, S. oneidensis PBP1a variants lacking either glycosyltransferase (GTase) or transpeptidase (TPase) activity fail to maintain normal morphology and cell envelope integrity. Similarly, SoPBP1b variants also fail to compensate for the loss of SoPBP1a. Furthermore, overproduction of variants of SoPBP1a, but not SoPBP1b, has detrimental effects on cell morphology in S. oneidensis wild type cells. Overall, our results indicate that the combined enzymatic activities of SoPBP1a are essential for cell wall homeostasis.

scholarly journals Blinking statistics and molecular counting in direct stochastic reconstruction microscopy (dSTORM)

2021 ◽  
Author(s):  
Lekha Patel ◽  
David Williamson ◽  
Dylan M Owen ◽  
Edward A K Cohen
Keyword(s):  
Probability Distribution ◽  
Single Molecule ◽  
Immunological Synapse ◽  
Training Data ◽  
Supplementary Information ◽  
Cellular Structures ◽  
Exact Probability ◽  
Stochastic Optical Reconstruction Microscopy ◽  
Exact Probability Distribution ◽  
Optical Reconstruction

Abstract Motivation Many recent advancements in single-molecule localization microscopy exploit the stochastic photoswitching of fluorophores to reveal complex cellular structures beyond the classical diffraction limit. However, this same stochasticity makes counting the number of molecules to high precision extremely challenging, preventing key insight into the cellular structures and processes under observation. Results Modelling the photoswitching behaviour of a fluorophore as an unobserved continuous time Markov process transitioning between a single fluorescent and multiple dark states, and fully mitigating for missed blinks and false positives, we present a method for computing the exact probability distribution for the number of observed localizations from a single photoswitching fluorophore. This is then extended to provide the probability distribution for the number of localizations in a direct stochastic optical reconstruction microscopy experiment involving an arbitrary number of molecules. We demonstrate that when training data are available to estimate photoswitching rates, the unknown number of molecules can be accurately recovered from the posterior mode of the number of molecules given the number of localizations. Finally, we demonstrate the method on experimental data by quantifying the number of adapter protein linker for activation of T cells on the cell surface of the T-cell immunological synapse. Availability and implementation Software and data available at https://github.com/lp1611/mol_count_dstorm. Supplementary information Supplementary data are available at Bioinformatics online.

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