The phosphoproteome of choroid plexus epithelial cells following infection with Neisseria meningitidis

The Gram-negative bacterium Neisseria meningitidis, which causes meningitis in humans, has been demonstrated to manipulate or alter host signalling pathways during infection of the central nervous system (CNS). However, these complex signalling networks are not completely understood. We investigate the phosphoproteome of an in vitro model of the blood-cerebrospinal fluid barrier (BCSFB) based on human epithelial choroid plexus (CP) papilloma (HIBCPP) cells during infection with the N. meningitidis serogroup B strain MC58 in presence and absence of the bacterial capsule. Interestingly, our data demonstrates a stronger impact on the phosphoproteome of the cells by the capsule-deficient mutant of MC58. Using enrichment analyses, potential pathways, molecular processes, biological processes, cellular components and kinases were determined to be regulated as a consequence of N. meningitidis infection of the BCSFB. Our data highlight a variety of protein regulations that are altered during infection of CP epithelial cells with N. meningitidis, with the regulation of several pathways and molecular events only being detected after infection with the capsule-deficient mutant. Mass spectrometry proteomics data are available via ProteomeXchange with identifier PXD038560

Manipulation von Wirtszellmechanismen während der Infektion durch Gram-positive und Gram-negative Erreger in einem humanen Modell der Blut-Liquor-Schranke

Infektionen durch Gram-positive und Gram-negative Bakterien können zur Entwicklung der lebensbedrohlichen Erkrankung Meningitis führen. Um eine Meningitis zu verursachen, müssen die Krankheitserreger die Schutzbarrieren des Gehirns überwinden. Eine dieser Barrieren, die das zentrale Nervensystem (ZNS) schützt, ist die Blut-Liquor-Schranke (BLS), deren morphologisches Korrelat die Epithelzellen des Plexus choroideus (PC) sind. Für den Gram-positiven Erreger Streptococcus suis (S. suis) und die Gram-negativen Erreger Haemophilus influenzae (H. influenzae) und Neisseria meningitidis (N. meningitidis) wurde die BLS als mögliche Eintrittspforte in das ZNS diskutiert. In vitro Studien, unter Verwendung eines Modells des BLS, das durch humane Papillomzellen des PC (HIBCPP-Zellen) dargestellt wird, haben eine polare Infektion dieser Erreger von der basolateralen Seite der Zellen gezeigt. Es ist bekannt, dass bakterielle Erreger die Signaltransduktionswege des Wirts während der Infektion zu ihrem eigenen Vorteil manipulieren können. Dazu gehören Endozytosemechanismen wie die Dynamin-vermittelte Endozytose, um die Aufnahme der Krankheitserreger in die Wirtszellen zu erleichtern. Während von den untersuchten Bakterien die S. suis Stämme und die N. meningitidis Kapselmutante eine Abhängigkeit von diesem Mechanismus für eine optimale Infektion der HIBCPP-Zellen aufwiesen, konnte für die N. meningitidis Wildtyp Stämme und die Varianten von H. influenzae keine signifikante Abhängigkeit von der Dynamin-vermittelten Endozytose bei Infektion der HIBCPP-Zellen beobachtet werden. Weitere, von Bakterien häufig manipulierte, Signalkaskaden sind die extrazellulären signalregulierten Kinasen 1 und 2 (Erk1/2) und p38 Mitogen-aktivierten Proteinkinasen (MAPK). Die Pathogene S. suis und H. influenzae konnten HIBCPP-Zellen, unabhängig von der Kapselexpression, nicht effizient infizieren, wenn diese MAPK inhibiert wurden. In ähnlicher Weise wurde eine Abhängigkeit von beiden Signalwegen für die N. meningitidis Wildtyp Stämme beobachtet. Jedoch konnte die N. meningitidis Kapselmutante die HIBCPP-Zellen unabhängig von der Aktivität beider MAPK infizieren. Aus diesem Grund wurde eine Transkriptomanalyse der HIBCPP-Zellen nach Infektion mit dem N. meningitidis Wildtyp Stamm und der Kapselmutante durchgeführt, um die zugrunde liegenden Prozesse während der Infektion der HIBCPP-Zellen weiter zu charakterisieren. Die Ergebnisse deuten auf eine Rolle des PC bei der Entzündungsreaktion hin und heben Unterschiede in der Stärke der Auswirkungen auf die Genexpression nach Infektion mit den N. meningitidis Stämmen in Gegenwart oder Abwesenheit der Bakterienkapsel hervor. Darüber hinaus ergab eine Analyse des Phosphoproteoms nach Infektion von HIBCPP-Zellen mit N. meningitidis eine Vielzahl von regulierten Signalwegen und Proteinen, mit deren Hilfe die genauen Infektionsmechanismen der BLS durch N. meningitidis weiter untersucht werden könne

Virulence Factors of Meningitis-Causing Bacteria: Enabling Brain Entry across the Blood–Brain Barrier

Infections of the central nervous system (CNS) are still a major cause of morbidity and mortality worldwide. Traversal of the barriers protecting the brain by pathogens is a prerequisite for the development of meningitis. Bacteria have developed a variety of different strategies to cross these barriers and reach the CNS. To this end, they use a variety of different virulence factors that enable them to attach to and traverse these barriers. These virulence factors mediate adhesion to and invasion into host cells, intracellular survival, induction of host cell signaling and inflammatory response, and affect barrier function. While some of these mechanisms differ, others are shared by multiple pathogens. Further understanding of these processes, with special emphasis on the difference between the blood–brain barrier and the blood–cerebrospinal fluid barrier, as well as virulence factors used by the pathogens, is still needed

Capsule-dependent impact of MAPK signalling on host cell invasion and immune response during infection of the choroid plexus epithelium by Neisseria meningitidis

Background!#!The Gram-negative bacterium Neisseria meningitidis (Nm) can cause meningitis in humans, but the host signalling pathways manipulated by Nm during central nervous system (CNS) entry are not completely understood.!##!Methods!#!We investigate the role of the mitogen-activated protein kinases (MAPK) Erk1/2 and p38 in an in vitro model of the blood-cerebrospinal fluid barrier (BCSFB) based on human epithelial choroid plexus (CP) papilloma (HIBCPP) cells during infection with Nm serogroup B (NmB) and serogroup C (NmC) strains. A transcriptome analysis of HIBCPP cells following infection with Nm by massive analysis of cDNA ends (MACE) was done to further characterize the cellular response to infection of the barrier.!##!Results!#!Interestingly, whereas NmB and NmC wild type strains required active Erk1/2 and p38 pathways for infection, invasion by capsule-deficient mutants was independent of Erk1/2 and, in case of the NmB strain, of p38 activity. The transcriptome analysis of HIBCPP cells following infection with Nm demonstrated specific regulation of genes involved in the immune response dependent on Erk1/2 signalling. Gene ontology (GO) analysis confirmed loss of MAPK signalling after Erk1/2 inhibition and revealed an additional reduction of cellular responses including NFκB and JAK-STAT signalling. Interestingly, GO terms related to TNF signalling and production of IL6 were lost specifically following Erk1/2 inhibition during infection with wild type Nm, which correlated with the reduced infection rates by the wild type in absence of Erk1/2 signalling.!##!Conclusion!#!Our data point towards a role of MAPK signalling during infection of the CP epithelium by Nm, which is strongly influenced by capsule expression, and affects infection rates as well as the host cell response

Functional characterisation of filamentous actin probe expression in neuronal cells

<div><p>Genetically encoded filamentous actin probes, Lifeact, Utrophin and F-tractin, are used as tools to label the actin cytoskeleton. Recent evidence in several different cell types indicates that these probes can cause changes in filamentous actin dynamics, altering cell morphology and function. Although these probes are commonly used to visualise actin dynamics in neurons, their effects on axonal and dendritic morphology has not been systematically characterised. In this study, we quantitatively analysed the effect of Lifeact, Utrophin and F-tractin on neuronal morphogenesis in primary hippocampal neurons. Our data show that the expression of actin-tracking probes significantly impacts on axonal and dendrite growth these neurons. Lifeact-GFP expression, under the control of a pBABE promoter, caused a significant decrease in total axon length, while another Lifeact-GFP expression, under the control of a CAG promoter, decreased the length and complexity of dendritic trees. Utr261-EGFP resulted in increased dendritic branching but Utr230-EGFP only accumulated in cell soma, without labelling any neurites. Lifeact-7-mEGFP and F-tractin-EGFP in a pEGFP-C1 vector, under the control of a CMV promoter, caused only minor changes in neuronal morphology as detected by Sholl analysis. The results of this study demonstrate the effects that filamentous actin tracking probes can have on the axonal and dendritic compartments of neuronal cells and emphasise the care that must be taken when interpreting data from experiments using these probes.</p></div

Summary of quantitative morphological analysis.

<p>Summary of quantitative morphological analysis.</p

Effects of filamentous actin tracking probes on the morphology of primary mouse hippocampal neurons [Lifeact-GFP(2) and Lifeact-EGFP].

<p>Representative images of neurons transfected with Lifeact-GFP(2) (A-D) or Lifeact-EGFP (E-H). (A, E) expressed probes; (B, F) axonal marker Tau1; (C, G) pan-neuronal β3-tubulin; (D, H) merged images. Scale bar = 50μm.</p

Quantitative analysis of axonal morphology at DIV3 after transfection with EGFP, Lifeact-GFP(1), Lifeact-GFP(2), Lifeact-EGFP, Lifeact-7-mEGFP, Utr261-EGFP and F-tractin-EGFP expressing constructs.

<p>Neurons transfected with Lifeact-GFP(1) show decreased total axon length (A), decreased primary axon total length (B) and decreased total length of primary axon branches (D), compared to EGFP control. Neurons transfected with Lifeact-EGFP and Utr261-EGFP show also show decreased primary axon total length (B). Neurons transfected with Lifeact-GFP(2), Lifeact-7-mEGFP and F-tractin-EGFP did not show any significant changes in axonal morphology when compared to EGFP control (A-F). Between 17 and 47 cells, collected from at least 3 biological replicates, were analysed per construct. Error bars represent standard error of the mean. Significance was determined by Kruskal-Wallis Test (non-parametric one-way ANOVA) and Dunn's multiple corrections test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p

Sholl analysis of dendritic complexity of mouse hippocampal neurons transfected with filamentous actin tracking probes.

<p><b>Dendritic complexity was</b> measured as number of intersections per shell as a function of distance from the soma. A Neurons transfected with Lifeact-GFP(1) displayno significant changes in dendritic compliexity compared to EGFP control. Lifeact-GFP(2) transfected neuron consistently had the lowest number of intersections per shell from 0–60 μm from the soma. Lifeact-EGFP transfected neurons showed an increase in dendritic complexity from 0–20 μm and a decrease from 20–60 μm compared to EGFP control. Lifeact-7-mEGFP transfected neurons showed an increase in dendritic complexity from 0–30 μm and a significant decrease from 30–40 μm. Utr261-EGFP also showed an increase in dendritic complexity from 0–30 μm. F-tractin-EGFP transfection resulted in increased dendritic complexity at 0–20 μm from the cell soma. Between 17 and 47 cells, collected from at least 3 biological replicates, were analysed per construct. Error bars represent standard error of the mean. Significance was determined by two-way ANOVA with Tukey's test for multiple corrections. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. Significant difference between EGFP and each actin tracking probe is indicated by the following symbols: Lifeact-GFP(2) = #, Lifeact-EGFP = §, Lifeact-7-mEGFP = £, Utr-261-EGFP = ⁺, F-tractin-EGFP = ‡.</p

Quantitative analysis of dendritic morphology at DIV3 after transfection with EGFP, Lifeact-GFP(1), Lifeact-GFP(2), Lifeact-EGFP, Lifeact-7-mEGFP, Utr261-EGFP and F-tractin-EGFP expressing constructs.

<p>Compared to EGFP control, neurons transfected with Lifeact-GFP(2) showed significant decreases in the total length of dendritic trees (B), the mean length of primary dendritic shaft (C), the mean length of primary dendritic branches (E) and the mean length of dendritic trees (H). Neurons transfected with Utr261-EGFP showed an increase in the number of primary dendritic branches (D) and mean number of primary dendritic branches per dendritic tree per cell (G), compared to EGFP control. Neurons transfected with Lifeact-GFP(1), Lifeact-EGFP, Lifeact-7-mEGFP and F-tractin-EGFP did not show any significant changes in dendritic morphology when compared to EGFP control (A-H) Between 17 and 47 cells, collected from at least 3 biological replicates, were analysed per construct. Error bars represent standard error of the mean. Significance was determined by Kruskal-Wallis Test (non-parametric one-way ANOVA) and Dunn's multiple corrections test. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.</p