Study Of Host-pathogen Interactions Research Articles

A chronic Pseudomonas aeruginosa mouse lung infection modeling the pathophysiology and inflammation of human cystic fibrosis.

Host-pathogen interaction studies of Pseudomonas aeruginosa cystic fibrosis (CF) lung infections have been hampered by limitations of mouse infection models. Here we combine synthetic CF sputum medium (SCFM) agar beads and Scnn1b -Tg transgenic mice to model the mucus obstructed airways and complex inflammatory characteristic of the human cystic fibrosis lung environment. In this model, which we name SCFM-Tg-mice, we use SCFM to cause changes in bacterial gene expression consistent with sputum collected from people with CF and the Scnn1b-Tg mice produce excessive airway mucus like people with CF. We show that SCMF-Tg-mice infected with P. aeruginosa have defects in lung function and increased inflammation that is consistent with human CF lung infections. This model can be adapted for other bacterial species and can be used to test hypotheses about bacterial pathogenesis and potential treatments in a CF human-like system.

A nisin-inducible chromosomal gene expression system based on ICE Tn5253 of Streptococcus pneumoniae, transferable among streptococci and enterococci

The present work reports the development and validation of a chromosomal expression system in Streptococcus pneumoniae which permits gene expression under the control of Lactococcus lactis lantibiotic nisin. The system is based on the integrative and conjugative element (ICE) Tn5253 of S. pneumoniae capable of site-specific chromosomal integration and conjugal transfer to a variety of bacterial species. We constructed an insertion vector that integrates in Tn5251, an ICE contained in Tn5253, which carries the tetracycline resistance tet(M) gene. The vector contains the nisRK regulatory system operon, the L. lactis nisin inducible promoter PnisA upstream of a multiple cloning site for target DNA insertion, and is flanked by two DNA regions of Tn5251 which drive homologous recombination in ICE Tn5253. For system evaluation, the emm6.1::ha1 fusion gene was cloned and integrated into the chromosome of the Tn5253-carrying pneumococcal strain FR24 by transformation. This gene encodes a fusion protein containing the signal peptide, the 122 N-terminal and the 140 C-terminal aa of the Streptococcus pyogenes M6 surface protein joined to the HA1 subunit of the influenza virus A hemagglutinin. Quantitative RT-PCR analysis carried out on total RNA purified from nisin treated and untreated cultures showed an increase in emm6.1::ha1 transcript copy number with growing nisin concentration. The expression of M6-HA1 protein was detected by Western blot and quantified by Dot blot, while Flow cytometry analysis confirmed the presence on the pneumococcal surface. Recombinant ICE Tn5253::[nisRK]-[emm6.1::ha1] containing the nisin-inducible expression system was successfully transferred by conjugation in different streptococcal species including Streptococcus gordonii, S. pyogenes, Streptococcus agalactiae and Enterococcus faecalis. As for S. pneumoniae, the emm6.1::ha1 transcript copy number and the amount of M6-HA1 protein produced correlated with the nisin concentration used for induction in all investigated bacterial hosts. We demonstrated that this host-vector expression system is stably integrated as a single copy within the bacterial chromosome, is transferable to both transformable and non transformable bacterial species, and allows fine tuning of protein expression modulated by nisin concentration. These characteristics make our system suitable for a wide range of applications including complementation assays, physiological studies, host-pathogen interaction studies.

Morphological and immunological characterization of primary cultured chicken caecal epithelial cells.

Cell cultures are models in biological and medical research to understand physiological and pathological processes. Cell lines are not always available depending on cell type and required species. In addition, the immortalization process often affects cell biology. Primary cells generally maintain a greater degree of similarity in short-term culture to the cells in tissue. Goal of this study was to verify the suitability of chicken primary epithelial caecal cells (PECCs) for in vitro investigations of host‒pathogen interactions. Epithelial nature of PECCs was confirmed by detection of tight and adherens junctions and cobblestone-like cell morphology. Sialic acids distribution was similar to that in caecal cyrosections. To understand the capacity of PECCs to respond to microbial challenges, the Toll-like receptors (TLRs) repertoire was determined. Exposure of PECCs to polyinosinic-polycytidylic acid (poly(I:C)) or lipopolysaccharide (LPS) led to upregulation of type I and III interferon (IFN) as well as interleukin (IL-) 1β, IL-6 and IL-8 mRNA expression. Overall, the PECCs showed properties of polarized epithelial cells. The presence of TLRs, their differential expression, as well as pattern recognition receptor dependent immune responses enable PECCs to act as suitable in vitro model for host‒pathogen interaction studies, which are difficult to conduct under in vivo conditions.

Urinary tract infections: pathogenesis, host susceptibility and emerging therapeutics.

Urinary tract infections (UTIs), which include any infection of the urethra, bladder or kidneys, account for an estimated 400 million infections and billions of dollars in health-care spending per year. The most common bacterium implicated in UTI is uropathogenic Escherichia coli, but diverse pathogens including Klebsiella, Enterococcus, Pseudomonas, Staphylococcus and even yeast such as Candida species can also cause UTIs. UTIs occur in both women and men and in both healthy and immunocompromised patients. However, certain patient factors predispose to disease: for example, female sex, history of prior UTI, or the presence of a urinary catheter or other urinary tract abnormality. The current clinical paradigm for the treatment of UTIs involves the use of antibiotics. Unfortunately, the efficacy of this approach is dwindling as the prevalence of antimicrobial resistance rises among UTI isolates, and the immense quantity of antibiotics prescribed annually for these infections contributes to the emergence of resistant pathogens. Therefore, there is an urgent need for new antibiotics and non-antibiotic treatment and prevention strategies. In this Review, we discuss how recent studies of bacterial pathogenesis, recurrence, persistence, host-pathogen interactions and host susceptibility factors have elucidated new and promising targets for the treatment and prevention of UTIs.

Integrating Large-Scale Protein Structure Prediction into Human Genetics Research.

The last five years have seen impressive progress in deep learning models applied to protein research. Most notably, sequence-based structure predictions have seen transformative gains in the form of AlphaFold2 and related approaches. Millions of missense protein variants in the human population lack annotations, and these computational methods are a valuable means to prioritize variants for further analysis. Here, we review the recent progress in deep learning models applied to the prediction of protein structure and protein variants, with particular emphasis on their implications for human genetics and health. Improved prediction of protein structures facilitates annotations of the impact of variants on protein stability, protein-protein interaction interfaces, and small-molecule binding pockets. Moreover, it contributes to the study of host-pathogen interactions and the characterization of protein function. As genome sequencing in large cohorts becomes increasingly prevalent, we believe that better integration of state-of-the-art protein informatics technologies into human genetics research is of paramount importance.

Establishment and identification of the head kidney cell line of yellowfin seabream (Acanthopagrus latus) and its application in a virus susceptibility study

The yellowfin seabream (Acanthopagrus latus) is a crucial marine resource owing to its economic significance. Acanthopagrus latus aquaculture faces numerous challenges from viral diseases, but a robust in-vitro research model to understand and address these threats is lacking. Therefore, we developed a novel A. latus cell line from head kidney cells called ALHK1. This study details the development, characterisation, and viral susceptibility properties of ALHK cells. This cell line primarily comprises fibroblast-like cells and has robust proliferative capacity when cultured at 28 °C in Leibovitz's L-15 medium supplemented with 10–20% foetal bovine serum. It exhibited remarkable stability after more than 60 consecutive passages and validation through cryopreservation techniques. The specificity of the ALHK cell line's origin from A. latus was confirmed via polymerase chain reaction (PCR) amplification of the cytochrome B gene, and a chromosomal karyotype analysis revealed a diploid count of 48 (2n = 48). Furthermore, the lipofection-mediated transfection efficiency using the pEGFP-N3 plasmid was high, at nearly 40%, suggesting that ALHK cells could be used for studies involving exogenous gene manipulation. In addition, ALHK cells displayed heightened sensitivity to the large mouth bass virus (LMBV), substantiated through observations of cytopathic effects, quantitative real-time PCR, and viral titration assays. Finally, the response of ALHK cells to LMBV infection resulted in differentially expressed antiviral genes associated with innate immunity. In conclusion, the ALHK cell line is a dependable in-vitro platform for elucidating the mechanisms of viral diseases in yellowfin seabream. Moreover, this cell line could be valuable for immunology, vaccine development, and host-pathogen interaction studies.

Laboratory Management of Mammalian Hosts for Ixodes scapularis -Host-Pathogen Interaction Studies.

Due to their hematophagous life cycle, hard-bodied ticks including the genus Ixodes are a potential vector for numerous pathogenic organisms including bacteria, protozoa, viruses, and infectious prions. The natural geographic range of several hard tick species, includig Ixodes scapularis, has expanded over recent decades. Consequently, there is an ongoing need to maintain, feed, and propagate ticks for host-pathogen interaction studies to better understand and mitigate their impact on human and animal health. Artificial membrane feeding of hard ticks has advanced in recent years, has study design advantages, and should be used, when possible, to reduce animal use, but it also has several limitations that require the continued use of mammalian hosts including mice, guinea pigs, and rabbits. In this overview, we discuss the best management practices for these relevant species with respect to biosafety, health, and optimal host comfort when used in studies that depend on tick feeding. The capsule-jacket method is preferred over the ear sock-E-collar method of tick feeding on rabbit hosts because of better host health, comfort, and increased study versatility.

Direct Salmonella injection into enteroid cells allows the study of host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Intestinal epithelial cells (IECs) play pivotal roles in nutrient uptake and in the protection against gut microorganisms. However, certain enteric pathogens, such as Salmonella enterica serovar Typhimurium (S. Tm), can invade IECs by employing flagella and type III secretion systems (T3SSs) with cognate effector proteins and exploit IECs as a replicative niche. Detection of flagella or T3SS proteins by IECs results in rapid host cell responses, i.e., the activation of inflammasomes. Here, we introduce a single-cell manipulation technology based on fluidic force microscopy (FluidFM) that enables direct bacteria delivery into the cytosol of single IECs within a murine enteroid monolayer. This approach allows to specifically study pathogen-host cell interactions in the cytosol uncoupled from preceding events such as docking, initiation of uptake, or vacuole escape. Consistent with current understanding, we show using a live-cell inflammasome reporter that exposure of the IEC cytosol to S. Tm induces NAIP/NLRC4 inflammasomes via its known ligands flagellin and T3SS rod and needle. Injected S. Tm mutants devoid of these invasion-relevant ligands were able to grow in the cytosol of IECs despite the absence of T3SS functions, suggesting that, in the absence of NAIP/NLRC4 inflammasome activation and the ensuing cell death, no effector-mediated host cell manipulation is required to render the epithelial cytosol growth-permissive for S. Tm. Overall, the experimental system to introduce S. Tm into single enteroid cells enables investigations into the molecular basis governing host-pathogen interactions in the cytosol with high spatiotemporal resolution.

Cell-Free Culture Supernatant of Lactobacillus acidophilus AG01 and Bifidobacterium animalis subsp. lactis AG02 Reduces the Pathogenicity of NetB-Positive Clostridium perfringens in a Chicken Intestinal Epithelial Cell Line.

The worldwide reduction in the use of antibiotics in animal feed is fueling the need for alternatives for the prevention and control of poultry intestinal diseases such as necrotic enteritis (NE), which is caused by Clostridium perfringens. This is the first report on the use of an intestinal epithelial chicken cell line (CHIC-8E11) to study the pathogenic traits of C. perfringens and to investigate the mode of action of cell-free supernatants (CFS) from probiotic Lactobacillus acidophilus AG01 and Bifidobacterium animalis subsp. lactis AG02 in reducing the pathogenicity of C. perfringens. The cell adhesion, permeability and cytotoxicity were assessed under challenge with four C. perfringens strains isolated from broiler NE episodes of differing geographical origin (CP1-UK; CP10-Sweden; 25037-CP01 and CP22-USA). All the C. perfringens strains could adhere to the CHIC-8E11 cells, with varying affinity (0.05-0.48% adhesion across the strains). The CFS from one out of two strains (CP22) increased the cell permeability (+4.5-fold vs. the control, p < 0.01), as measured by the fluorescein isothiocyanate-dextran (FD4) content, with NetB toxin implicated in this effect. The CFS from all the strains was cytotoxic against the CHIC-8E11 cells in a dose- and strain-dependent manner (cytotoxicity 23-62% across the strains when dosed at 50 µL/mL, as assessed by the MTT cell viability assay). Pre-treatment of the cells with CFS from B. animalis subsp. lactis AG02 but not L. acidophilus AG01 reduced the cell adhesion of three out of four C. perfringens strains (by 77-85% vs. the control, p < 0.001) and reduced the negative effect of two NetB-positive strains on the cell permeability. The CFS of both probiotics alleviated the cytotoxicity of all the C. perfringens strains, which was dependent on the dose. The results confirm the suitability of the CHIC-8E11 cell line for the study of host-pathogen cell interactions in the context of NE caused by C. perfringens and reveal a beneficial mode of action of B. animalis subsp. lactis AG02 in reducing C. perfringens cell adhesion and, together with L. acidophilus AG01, in reducing C. perfringens cytotoxicity.

Identification of an N-terminal tag (580N) that improves the biosynthesis of fluorescent proteins in Francisella tularensis and other Gram-negative bacteria

Utilization of fluorescent proteins is widespread for the study of microbial pathogenesis and host-pathogen interactions. Here, we discovered that linkage of the 36 N-terminal amino acids of FTL_0580 (a hypothetical protein of Francisella tularensis) to fluorescent proteins increases the fluorescence emission of bacteria that express these recombinant fusions. This N-terminal peptide will be referred to as 580N. Western blotting revealed that the linkage of 580N to Emerald Green Fluorescent Protein (EmGFP) in F. tularensis markedly improved detection of this protein. We therefore hypothesized that transcripts containing 580N may be translated more efficiently than those lacking the coding sequence for this leader peptide. In support, expression of emGFPFt that had been codon-optimized for F. tularensis, yielded significantly enhanced fluorescence than its non-optimized counterpart. Furthermore, fusing emGFP with coding sequence for a small N-terminal peptide (Serine-Lysine-Isoleucine-Lysine), which had previously been shown to inhibit ribosomal stalling, produced robust fluorescence when expressed in F. tularensis. These findings support the interpretation that 580N enhances the translation efficiency of fluorescent proteins in F. tularensis. Interestingly, expression of non-optimized 580N-emGFP produced greater fluorescence intensity than any other construct. Structural predictions suggested that RNA secondary structure also may be influencing translation efficiency. When expressed in Escherichia coli and Klebsiella pneumoniae bacteria, 580N-emGFP produced increased green fluorescence compared to untagged emGFP (neither allele was codon optimized for these bacteria). In conclusion, fusing the coding sequence for the 580N leader peptide to recombinant genes might serve as an economical alternative to codon optimization for enhancing protein expression in bacteria.

Koch's curse: How models of extreme pathology bias studies of host-pathogen interactions.

Koch's curse: How models of extreme pathology bias studies of host-pathogen interactions.

Ds-Seq: An Integrated Pipeline for In Silico Small RNA Se-quence Analysis for Host-pathogen Interaction Studies

Plant-pathogen interactions activate molecular activities wherein the host defends the pathogen while the pathogen tries to suppress the plant response. Small RNAs (sRNAs) mediate major mechanisms, including post-transcriptional gene silencing, histone modification and DNA methylation by which plants respond to the presence of pathogens. Genome-wide profiling of host and pathogen sRNAs is therefore pivotal to uncovering the mechanisms underlying the host-pathogen interaction and mechanisms for host resistance. sRNA high throughput sequencing (HTS) data analysis often involves multiple stages/tools. Most necessary tools are accessible only through the command line, making it challenging for those without a high level of Unix/Linux skills. Furthermore, installation of some of these tools may become difficult due to dependencies and software version compatibility. We have developed an integrated open-source pipeline, Ds-Seq, for end-to-end in silico analysis of sRNA HTS data with improved reproducibility. The pipeline combines in-house scripts and public tools in a shell script, which can be invoked with a single command. The pipeline's usefulness has been demonstrated with testing on publicly available and published data from independent sRNA-seq datasets of host-pathogen interaction studies. Ds-Seq is available on GitHub, while a Docker image can be obtained from the Docker hub.Availability: Ds-Seq is freely available from the GitHub repository at https://github.com/CEPHAS-01/small-RNASeq.ngs and Docker hub with ID cephas/ds-seq (https://hub.docker.com/r/cephas/ds-seq).

Proximity Ligation Assay to Detect the Proximity Between Host Proteins and Viral Proteins of HIV-1.

The study of host-pathogen interaction often requires interrogating the protein-protein interactions and examining post-translational modifications of the proteins. Traditional protein detection strategies are limited in their sensitivity, specificity, and multiplexing capabilities. The Proximity Ligation Assay (PLA), a versatile and powerful molecular technique, can overcome these limitations. PLA blends the specificity of antibodies, two antibodies detecting two different epitopes on the same or two different proteins, with the amplification efficiency of a polymerase to allow highly specific and sensitive detection of low-abundant proteins, protein-protein interactions, or protein modifications. In this protocol, we describe the application of PLA to detect the proximity between HIV-1 Tat with one of its cellular partners, p65, in an infected host cell. The protocol could be applied to any other context with slight modifications. Of note, PLA can only confirm the physical proximity between two epitopes or proteins; however, the proximity need not necessarily allude to the functional interaction between the two proteins.

Trypanosoma cruzi STIB980: A TcI Strain for Drug Discovery and Reverse Genetics.

Since the first published genome sequence of Trypanosoma cruzi in 2005, there have been tremendous technological advances in genomics, reverse genetics, and assay development for this elusive pathogen. However, there is still an unmet need for new and better drugs to treat Chagas disease. Here, we introduce a T. cruzi assay strain that is useful for drug research and basic studies of host-pathogen interactions. T. cruzi STIB980 is a strain of discrete typing unit TcI that grows well in culture as axenic epimastigotes or intracellular amastigotes. We evaluated the optimal parameters for genetic transfection and constructed derivatives of T. cruzi STIB980 that express reporter genes for fluorescence- or bioluminescence-based drug efficacy testing, as well as a Cas9-expressing line for CRISPR/Cas9-mediated gene editing. The genome of T. cruzi STIB980 was sequenced by combining short-read Illumina with long-read Oxford Nanopore technologies. The latter served as the primary assembly and the former to correct mistakes. This resulted in a high-quality nuclear haplotype assembly of 28 Mb in 400 contigs, containing 10,043 open-reading frames with a median length of 1077 bp. We believe that T. cruzi STIB980 is a useful addition to the antichagasic toolbox and propose that it can serve as a DTU TcI reference strain for drug efficacy testing.

Use of single-cell technology to improve our understanding of the role of TLR2 in macrophage-Mycobacterium tuberculosis interaction.

The interaction between Mycobacterium tuberculosis, the agent of tuberculosis (TB), and its host cell, the macrophage, is multifaceted, dynamic, and involves multiple molecular partners. A better understanding of this interaction could help researchers manipulate the immune system in order to design host-targeted immunotherapies and/or develop a novel vaccine protecting better adults against TB. Jani and coworkers studied the role of the macrophage receptor TLR2 in the response to M. tuberculosis using single-cell technologies (C. Jani, S. L. Solomon, J. M. Peters, and S. C. Pringle, et al., mSystems, https://doi.org/10.1128/msystems.00052-23, 2023). This work addresses the multiple challenges associated with such studies and shows how informative single-cell analysis can be for the study of heterogeneous and complex host-pathogen interactions.

Extracellular vesicles biogenesis and uptake concepts: A comprehensive guide to studying host-pathogen communication.

The study of host-pathogen interactions has increased considerably in recent decades. This intercellular communication has been mediated by extracellular vesicles (EVs) that play an important role during the interaction. EVs are particles of lipid bilayer and described in different types of cells, eukaryotic or prokaryotic. Depending on their biogenesis they are described as exosomes (derived from multivesicular bodies) and microvesicles (derived from the plasma membrane). The EVs carry biomolecules, including nucleic acids, lipids, and proteins that can be released or internalized by other cells in different pathways (endocytosis, macropinocytosis, phagocytosis, or membrane fusion) in the process described as uptake. The balance between biogenesis and uptake of EVs could modify physiological and pathophysiological processes of the cell. This review is focusing on the dynamic roles of release and capture of EVs during host-pathogen interaction. We also do a critical analysis of methodologies for obtaining and analyzing EVs. Finally, we draw attention to critical points to be considered in EV biogenesis and uptake studies.

Ovine placental explants: A new ex vivo model to study host‒pathogen interactions in reproductive pathogens

Reproductive failure is one of the main performance constraints in ruminant livestock. Transmissible agents such as Toxoplasma gondii and Neospora caninum are commonly involved in the occurrence of abortion in ruminants, but little is known about the mechanisms involved. While in vivo models are optimal for the study of abortion pathogenesis, they have a high economic cost and come with ethical concerns. Unfortunately, alternative in vitro models fail to replicate the complex in vivo placental structure. To overcome the limitations of currently available models, we developed an ex vivo model based on the cultivation of fresh and cryopreserved sheep placental explants, enabling the biobanking of tissues. Reproducible and simple markers of tissue integrity (histology, RNA concentrations), viability (resazurin reduction), and functionality (synthesis of steroid hormones) were also investigated, allowing a clear quality assessment of the model. This work shows that, similar to fresh explants, tissues cryopreserved in ethylene glycol using slow freezing rates maintain not only their structure and function but also their receptivity to T. gondii and N. caninum infection. In addition, the findings demonstrate that explant lifespan is mainly limited by the culture method, with protocols requiring improvements to extend it beyond 2 days. These findings suggest that cryopreserved tissues can be exploited to study the initial host‒pathogen interactions taking place in the placenta, thus deepening the knowledge of the specific mechanisms that trigger reproductive failure in sheep. Importantly, this work paves the way for the development of similar models in related species and contributes to the reduction of experimental animal use in the future.

Cell scientist to watch – Géraldine Laloux

ABSTRACT Géraldine Laloux is an Associate Professor of bacterial cell biology at the de Duve Institute, UCLouvain, Belgium. After studying host–pathogen interactions during her PhD at the University of Namur, Belgium, Géraldine focused more on the cell biology of bacteria when she joined the Jacobs-Wagner lab at Yale University, USA. In 2017, she then returned to Belgium to set up her lab at UCLouvain, where she works with predatory bacteria to study their unusual cell cycle mechanisms and underlying cellular organisation. We caught up with Géraldine over Zoom to find out more about the interesting lifestyle of these predatory bacteria, as well as her approaches to mentoring and starting a new lab.

Establishment and characterization of an SV40 immortalized chicken intestinal epithelial cell line

Primary chicken intestinal epithelial cells or 3D enteroids are a powerful tool to study the different biological mechanisms that occur in the chicken intestine. Unfortunately, they are not ideal for large-scale screening or long-term studies due to their short lifespan. Moreover, they require expensive culture media, coatings, or the usage of live embryos for each isolation. The aim of this study was to establish and characterize an immortalized chicken intestinal epithelial cell line to help the study of host-pathogen interactions in poultry. This cell line was established by transducing into primary chicken enterocytes the SV40 large-T antigen through a lentiviral vector. The transduced cells grew without changes up to 40 passages maintaining, after a differentiation phase of 48 h with epidermal growth factor, the biological properties of mature enterocytes such as alkaline phosphatase activity and tight junction formation. Immortalized enterocytes were able to generate a cytokine response during an inflammatory challenge, and showed to be susceptible to Eimeria tenella sporozoites invasion and generate a proper immune response to parasitic and lipopolysaccharide (Escherichia coli) stimulation. This immortalized cell line could be a cost-effective and easy-to-maintain model for all the public health, food safety, or research and pharmaceutical laboratories that study host-pathogen interactions, foodborne pathogens, and food or feed science in vitro.

Co-infection of the respiratory epithelium, scene of complex functional interactions between viral, bacterial, and human neuraminidases.

The activity of sialic acids, known to play critical roles in biology and many pathological processes, is finely regulated by a class of enzymes called sialidases, also known as neuraminidases. These are present in mammals and many other biological systems, such as viruses and bacteria. This review focuses on the very particular situation of co-infections of the respiratory epithelium, the scene of complex functional interactions between viral, bacterial, and human neuraminidases. This intrinsically multidisciplinary topic combining structural biology, biochemistry, physiology, and the study of host-pathogen interactions, opens up exciting research perspectives that could lead to a better understanding of the mechanisms underlying virus-bacteria co-infections and their contribution to the aggravation of respiratory pathology, notably in the context of pre-existing pathological contexts. Strategies that mimic or inhibit the activity of the neuraminidases could constitute interesting treatment options for viral and bacterial infections.