Innate Immune Mechanisms to Protect Against Infection at the Human Decidual-Placental Interface.

During pregnancy, the placenta forms the anatomical barrier between the mother and developing fetus. Infectious agents can potentially breach the placental barrier resulting in pathogenic transmission from mother to fetus. Innate immune responses, orchestrated by maternal and fetal cells at the decidual-placental interface, are the first line of defense to avoid vertical transmission. Here, we outline the anatomy of the human placenta and uterine lining, the decidua, and discuss the potential capacity of pathogen pattern recognition and other host defense strategies present in the innate immune cells at the placental-decidual interface. We consider major congenital infections that access the placenta from hematogenous or decidual route. Finally, we highlight the challenges in studying human placental responses to pathogens and vertical transmission using current experimental models and identify gaps in knowledge that need to be addressed. We further propose novel experimental strategies to address such limitations

Role of the capsule locus in the virulence of Bordetella pertussis

Ph.DDOCTOR OF PHILOSOPH

Comparative Heterochromatin Profiling Reveals Conserved and Unique Epigenome Signatures Linked to Adaptation and Development of Malaria Parasites.

Heterochromatin-dependent gene silencing is central to the adaptation and survival of Plasmodium falciparum malaria parasites, allowing clonally variant gene expression during blood infection in humans. By assessing genome-wide heterochromatin protein 1 (HP1) occupancy, we present a comprehensive analysis of heterochromatin landscapes across different Plasmodium species, strains, and life cycle stages. Common targets of epigenetic silencing include fast-evolving multi-gene families encoding surface antigens and a small set of conserved HP1-associated genes with regulatory potential. Many P. falciparum heterochromatic genes are marked in a strain-specific manner, increasing the parasite's adaptive capacity. Whereas heterochromatin is strictly maintained during mitotic proliferation of asexual blood stage parasites, substantial heterochromatin reorganization occurs in differentiating gametocytes and appears crucial for the activation of key gametocyte-specific genes and adaptation of erythrocyte remodeling machinery. Collectively, these findings provide a catalog of heterochromatic genes and reveal conserved and specialized features of epigenetic control across the genus Plasmodium

COVID-19 progression and convalescence in common variable immunodeficiency patients shows incomplete adaptive responses and persistent inflammasome activation

Patients with common variable immunodeficiency (CVID), the most prevalent symptomatic primary immunodeficiency, are characterized by hypogammaglobulinemia, poorly protective vaccine titers and increased susceptibility to infections. New pathogens such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19), might constitute a particular threat to these immunocompromised patients since many of them experience a slower recovery and do not achieve full response to SARS-CoV-2 vaccines. To define the molecular basis of the altered immune responses caused by SARS-CoV-2 infection in CVID patients, we generated longitudinal single-cell datasets of peripheral blood immune cells along viral infection and recovery. We sampled the same individuals before, during and after SARS-CoV-2 infection to model their specific immune response dynamics while removing donor variability. We observed that COVID-19 CVID patients show defective canonical NF-κB pathway activation and dysregulated expression of BCR-related genes in naïve B cells, as well as enhanced cytotoxic activity but incomplete cytokine response in NK and T cells. Moreover, monocytes from COVID-19 CVID patients show persistent activation of several inflammasome-related genes, including the pyrin and NLRC4 inflammasomes. Our results shed light on the molecular basis of the prolonged clinical manifestations observed in these immunodeficient patients upon SARS-CoV-2 infection, which might illuminate the development of tailored treatments for COVID-19 CVID patients.We thank the CERCA Program/Generalitat de Catalunya and the Josep Carreras Foundation for institutional support. This publication is part of the Human Cell Atlas: www.humancellatlas.org/publications. This study was funded by ”la Caixa” Foundation under the grant agreement LCF/PR/HR22/52420002, Spanish Ministry of Science and Innovation (grant number PID2020-117212RB-I00/AEI/10.13038/501100011033) (E.B.), by the Wellcome Trust Grant 206194 and 108413/A/15/D (R.V.-T.), Instituto de Salud Carlos III (ISCIII), Ref. AC18/00057, associated with i-PAD project (ERARE European Union program) (E.B.), and the Chan Zuckerberg Initiative (grant 2020-216799) (R.V.-T. and E.B.). This publication has also been supported by the Unstoppable campaign of the Josep Carreras Leukaemia Foundation. We are indebted to the donors for participating in this research.N

Evidence for a role of the polysaccharide capsule transport proteins in pertussis pathogenesis

Polysaccharide (PS) capsules are important virulence determinants for many bacterial pathogens. Bordetella pertussis, the agent of whooping cough, produces a surface associated microcapsule but its role in pertussis pathogenesis remained unknown. Here we showed that the B. pertussis capsule locus is expressed in vivo in murine lungs and that absence of the membrane-associated protein KpsT, involved in the transport of the PS polymers across the envelope, but not the surface-exposed PS capsule itself, affects drastically B. pertussis colonization efficacy in mice. Microarray analysis revealed that absence of KpsT in B. pertussis resulted in global down-regulation of gene expression including key virulence genes regulated by BvgA/S, the master two-component system. Using a BvgS phase-locked mutant, we demonstrated a functional link between KpsT and BvgA/S-mediated signal transduction. Whereas pull-down assays do not support physical interaction between BvgS sensor and any of the capsule locus encoded proteins, absence of KpsT impaired BvgS oligomerization, necessary for BvgS function. Furthermore, complementation studies indicated that instead of KpsT alone, the entire PS capsule transport machinery spanning the cell envelope likely plays a role in BvgS-mediated signal transduction. Our work thus provides the first experimental evidence of a role for a virulence-repressed gene in pertussis pathogenesis.Published versio

Comparative Heterochromatin Profiling Reveals Conserved and Unique Epigenome Signatures Linked to Adaptation and Development of Malaria Parasites

Contains fulltext : 190233.pdf (publisher's version ) (Open Access

Rapid activation of distinct members of multigene families in Plasmodium spp

© 2020, The Author(s). The genomes of Plasmodium spp. encode a number of different multigene families that are thought to play a critical role for survival. However, with the exception of the P. falciparum var genes, very little is known about the biological roles of any of the other multigene families. Using the recently developed Selection Linked Integration method, we have been able to activate the expression of a single member of a multigene family of our choice in Plasmodium spp. from its endogenous promoter. We demonstrate the usefulness of this approach by activating the expression of a unique var, rifin and stevor in P. falciparum as well as yir in P. yoelii. Characterization of the selected parasites reveals differences between the different families in terms of mutual exclusive control, co-regulation, and host adaptation. Our results further support the application of the approach for the study of multigene families in Plasmodium and other organisms

Selective expression of variant surface antigens enables Plasmodium falciparum to evade immune clearance in vivo

AbstractPlasmodium falciparum has developed extensive mechanisms to evade host immune clearance. Currently, most of our understanding is based on in vitro studies of individual parasite variant surface antigens and how this relates to the processes in vivo is not well-understood. Here, we have used a humanized mouse model to identify parasite factors important for in vivo growth. We show that upregulation of the specific PfEMP1, VAR2CSA, provides the parasite with protection from macrophage phagocytosis and clearance in the humanized mice. Furthermore, parasites adapted to thrive in the humanized mice show reduced NK cell-mediated killing through interaction with the immune inhibitory receptor, LILRB1. Taken together, these findings reveal new insights into the molecular and cellular mechanisms that the parasite utilizes to coordinate immune escape in vivo. Identification and targeting of these specific parasite variant surface antigens crucial for immune evasion provides a unique approach for therapy.</jats:p

Construction of Δ<i>kpsT</i>, Δ<i>kpsE</i> and Δ<i>vipC B. pertussis</i> mutants.

<p>(<b>A</b>) <b>Schematic organization of </b><b><i>B</i></b><b>. </b><b><i>pertussis</i></b><b> capsule operon.</b> The capsule operon of <i>B. pertussis</i> BPSM strain regulated under the capsule promoter is as shown in panel <b>a</b>, black cross represents mutational insertion found in the locus. Black, hashed and open arrows represent genes involved in polysaccharide transport, polysaccharide modification/translocation and polysaccharide biosynthesis respectively. Adapted from GeneDB. Dotted triangle in panel <b>b</b> (Δ<i>kpsT</i>), <b>c</b> (Δ<i>kpsE</i>) and <b>d</b> (Δ<i>vipC</i>) indicate site of deletion that render each mutant non-capsulated. The DIG-labeled probe binding region (black rounded arrow), restriction sites and size of restriction-digested chromosomal DNA for Southern blot analysis are as shown. (<b>B</b>) <b>Southern blot analysis.</b> Restriction-digested chromosomal DNA from BPSM and Δ<i>kpsT</i>, Δ<i>kpsE</i> and Δ<i>vipC</i> were electrophoresed, transferred onto a nitrocellulose membrane and hybridized with the DIG-labeled probe (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0115243#pone-0115243-g001" target="_blank">Fig. 1A</a> for probe binding site). Panel <b>a</b>, <i>EcoRI</i>-restricted BPSM and Δ<i>kpsT</i> DNA yielded 2.7-kb and 3.2-kb respectively. Panel <b>b</b>, Hind<i>III</i>-Nco<i>I</i> restricted BPSM and Δ<i>kpsE</i> DNA yielded 4.8-kb and 3.9-kb respectively. Panel <b>c</b>, Hind<i>III</i>-Nco<i>I</i> restricted BPSM and Δ<i>vipC</i> DNA yielded 4.8-kb and 3.4-kb respectively. (<b>C</b>) <b>Transcription efficacy of downstream gene.</b> Total RNA was extracted from exponential SS liquid cultures of BPSM, KO<i>caps</i>, Δ<i>kpsT</i>, Δ<i>kpsE</i> and Δ<i>vipC</i> was reverse-transcribed followed by PCR amplification using primers specific to the endogenous control gene <i>recA</i>, and primers mapping to the respective downstream region of the deleted ORFs. The KO<i>caps</i> strain which was deleted for the entire capsule locus was used as a negative control. (<b>D</b>) <b>Growth kinetic profiles.</b> SS liquid medium was inoculated with BPSM (closed circles), Δ<i>kpsT</i> (open circles), Δ<i>kpsE</i> (closed triangles) and Δ<i>vipC</i> (open triangles) at initial OD_{600 nm} of 0.5 at time-point 0 hour. OD_{600 nm} was monitored for 52 hrs incubation at 37°C. The growth kinetics assay was performed twice independently for each strain and each culture condition. The data shown is representative of two independent experiments.</p

Phenotypic characterization of the Δ<i>kpsT</i>, Δ<i>kpsE</i> and Δ<i>vipC</i> mutants.

<p>(<b>A</b>) <b>Detection of the polysaccharide capsule at the bacterial surface.</b> Mouse polyclonal anti-<i>Salmonella typhi</i> Vi antigen immune serum was co-incubated with non-permeabilized BPSM, KO<i>caps</i>, Δ<i>kpsT</i>, Δ<i>kpsE</i> and Δ<i>vipC</i> bacteria strains as indicated in each flow cytometry plot. All bacteria strains were grown in Bvg^- phase. Anti-mouse FITC-conjugated IgG was used as secondary antibody. Isotype-matched controls are incubated with an anti-mouse antibody as shown in black histogram. The fluorescent cells were detected by flow cytometry, with 20,000 events counted for each sample. A representative experiment is shown from three independent experiments, with percentage of fluorescent cells indicated in each panel. (<b>B</b>) <b>Lung colonization profiles.</b> Balb/C mice were infected intranasally with 5×10⁵ CFU of <i>B. pertussis</i> BPSM, KO<i>caps</i>, Δ<i>kpsT</i>, Δ<i>kpsT</i>com, Δ<i>kpsE</i> and Δ<i>vipC</i> as indicated at each of graph plot. At the indicated time points, four infected mice per group were euthanized and their lungs were harvested, homogenized and plated on blood agar to determine the total number of CFU per lung. The results are expressed as the mean ± SEM of four mice per group. ** <i>p</i> value<0.01 and * <i>p</i> value<0.05 relative to BPSM. Results are representative of two independent experiments.</p