Host Pathogen responses of chicken cells in vitro and in vivo to a diverse population of Campylobacter strains

The epithelial lining of the lung is often the first point of interaction between the host and inhaled pathogens, allergens and medications. Epithelial cells are therefore the main focus of studies which aim to shed light on host-pathogen interactions, to dissect the mechanisms of local host immunity and study toxicology. If these studies are not to be conducted exclusively in vivo, it is imperative that in vitro models are developed with a high in vitro-in vivo correlation. We describe here a co-culture bilayer model of the bovine alveolus, designed to overcome some of the limitations encountered with mono-culture and live animal models. Our system includes bovine pulmonary arterial endothelial cells (BPAECs) seeded onto a permeable membrane in 24 well Transwell format. The BPAECs are overlaid with immortalised bovine alveolar type II epithelial cells and the bilayer cultured at air-liquid interface for 14 days before use; in our case to study host-mycobacterial interactions. Characterisation of novel cell lines and the bilayer model have provided compelling evidence that immortalised bovine alveolar type II cells are an authentic substitute for primary alveolar type II cells and their culture as a bilayer in conjunction with BPAECs provides a physiologically relevant in vitro model of the bovine alveolus. The bilayer model may be used to study dynamic intracellular and extracellular host-pathogen interactions, using proteomics, genomics, live cell imaging, in-cell ELISA and confocal microscopy. The model presented in this article enables other researchers to establish an in vitro model of the bovine alveolus that is easy to set up, malleable and serves as a comparable alternative to in vivo models, whilst allowing study of early host-pathogen interactions, currently not feasible in vivo. The model therefore achieves one of the 3Rs objectives in that it replaces the use of animals in research of bovine respiratory diseases.

Download Full-text

Illuminating Macrophage Contributions to Host-Pathogen Interactions In Vivo: the Power of Zebrafish

Infection and Immunity ◽

10.1128/iai.00906-19 ◽

2020 ◽

Vol 88 (7) ◽

Cited By ~ 1

Author(s):

Emily E. Rosowski

Keyword(s):

Cell Culture ◽

Innate Immunity ◽

Imaging Techniques ◽

Innate Immune ◽

Host Pathogen Interactions ◽

Culture Studies ◽

Larval Zebrafish ◽

Host Pathogen

ABSTRACT Macrophages are a key cell type in innate immunity. Years of in vitro cell culture studies have unraveled myriad macrophage pathways that combat pathogens and demonstrated how pathogen effectors subvert these mechanisms. However, in vitro cell culture studies may not accurately reflect how macrophages fit into the context of an innate immune response in whole animals with multiple cell types and tissues. Larval zebrafish have emerged as an intermediate model of innate immunity and host-pathogen interactions to bridge the gap between cell culture studies and mammalian models. These organisms possess an innate immune system largely conserved with that of humans and allow state-of-the-art genetic and imaging techniques, all in the context of an intact organism. Using larval zebrafish, researchers are elucidating the function of macrophages in response to many different infections, including both bacterial and fungal pathogens. The goal of this review is to highlight studies in zebrafish that utilized live-imaging techniques to analyze macrophage activities in response to pathogens. Recent studies have explored the roles of specific pathways and mechanisms in macrophage killing ability, explored how pathogens subvert these responses, identified subsets of macrophages with differential microbicidal activities, and implicated macrophages as an intracellular niche for pathogen survival and trafficking. Research using this model continues to advance our understanding of how macrophages, and specific pathways inside these cells, fit into complex multicellular innate immune responses in vivo, providing important information on how pathogens evade these pathways and how we can exploit them for development of treatments against microbial infections.

Download Full-text

Efficacy of Humanized Exposures of Cefiderocol (S-649266) against a Diverse Population of Gram-Negative Bacteria in a Murine Thigh Infection Model

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01022-17 ◽

2017 ◽

Vol 61 (11) ◽

Cited By ~ 35

Author(s):

Marguerite L. Monogue ◽

Masakatsu Tsuji ◽

Yoshinori Yamano ◽

Roger Echols ◽

David P. Nicolau

Keyword(s):

Antibacterial Activity ◽

Multidrug Resistant ◽

Infection Model ◽

Gram Negative Bacteria ◽

Diverse Population ◽

Gram Negative ◽

Content Type ◽

Log Reduction

ABSTRACT Cefiderocol (S-649266) is a novel siderophore cephalosporin with potent in vitro activity against clinically encountered multidrug-resistant (MDR) Gram-negative isolates; however, its spectrum of antibacterial activity against these difficult-to-treat isolates remains to be fully explored in vivo. Here, we evaluated the efficacy of cefiderocol humanized exposures in a neutropenic murine thigh model to support a suitable MIC breakpoint. Furthermore, we compared cefiderocol's efficacy with humanized exposures of meropenem and cefepime against a subset of these phenotypically diverse isolates. Ninety-five Gram-negative isolates were studied. Efficacy was determined as the change in log10 CFU at 24 h compared with 0-h controls. Bacterial stasis or ≥1 log reduction in 67 isolates with MICs of ≤4 μg/ml was noted in 77, 88, and 85% of Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas aeruginosa, respectively. For isolates with MICs of ≥8 μg/ml, bacterial stasis or ≥1 log10 reduction was observed in only 2 of 28 (8 Enterobacteriaceae, 19 A. baumannii, and 1 P. aeruginosa) strains. Against highly resistant meropenem and cefepime organisms, cefiderocol maintained its in vivo efficacy. Overall, humanized exposures of cefiderocol produced similar reductions in bacterial density for organisms with MICs of ≤4 μg/ml, whereas isolates with MICs of ≥8 μg/ml generally displayed bacterial growth in the presence of the compound. Data derived in the current study will assist with the delineation of MIC susceptibility breakpoints for cefiderocol against these important nosocomial Gram-negative pathogens; however, additional clinical data are required to substantiate these observations.

Download Full-text

The Development of Ovine Gastric and Intestinal Organoids for Studying Ruminant Host-Pathogen Interactions

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.733811 ◽

2021 ◽

Vol 11 ◽

Author(s):

David Smith ◽

Daniel R. G. Price ◽

Alison Burrells ◽

Marc N. Faber ◽

Katie A. Hildersley ◽

...

Keyword(s):

Animal Health ◽

Bacterial Pathogen ◽

Serial Passage ◽

Apical Surface ◽

Transcript Profile ◽

Intestinal Organoids ◽

Host Pathogen ◽

Gi Infections

Gastrointestinal (GI) infections in sheep have significant implications for animal health, welfare and productivity, as well as being a source of zoonotic pathogens. Interactions between pathogens and epithelial cells at the mucosal surface play a key role in determining the outcome of GI infections; however, the inaccessibility of the GI tract in vivo significantly limits the ability to study such interactions in detail. We therefore developed ovine epithelial organoids representing physiologically important gastric and intestinal sites of infection, specifically the abomasum (analogous to the stomach in monogastrics) and ileum. We show that both abomasal and ileal organoids form self-organising three-dimensional structures with a single epithelial layer and a central lumen that are stable in culture over serial passage. We performed RNA-seq analysis on abomasal and ileal tissue from multiple animals and on organoids across multiple passages and show the transcript profile of both abomasal and ileal organoids cultured under identical conditions are reflective of the tissue from which they were derived and that the transcript profile in organoids is stable over at least five serial passages. In addition, we demonstrate that the organoids can be successfully cryopreserved and resuscitated, allowing long-term storage of organoid lines, thereby reducing the number of animals required as a source of tissue. We also report the first published observations of a helminth infecting gastric and intestinal organoids by challenge with the sheep parasitic nematode Teladorsagia circumcincta, demonstrating the utility of these organoids for pathogen co-culture experiments. Finally, the polarity in the abomasal and ileal organoids can be inverted to make the apical surface directly accessible to pathogens or their products, here shown by infection of apical-out organoids with the zoonotic enteric bacterial pathogen Salmonella enterica serovar Typhimurium. In summary, we report a simple and reliable in vitro culture system for generation and maintenance of small ruminant intestinal and gastric organoids. In line with 3Rs principals, use of such organoids will reduce and replace animals in host-pathogen research.

Download Full-text

In vivo host-pathogen interaction as revealed by global proteomic profiling of zebrafish larvae

10.1101/140509 ◽

2017 ◽

Author(s):

Francisco Díaz-Pascual ◽

Javiera Ortíz-Severín ◽

Macarena A. Varas ◽

Miguel L. Allende ◽

Francisco P. Chávez

Keyword(s):

Microbial Pathogens ◽

Cytokine Signaling ◽

Proteomic Profiling ◽

Zebrafish Larvae ◽

Host Pathogen Interaction ◽

Host Pathogen ◽

Associated Proteins ◽

Static Immersion

AbstractThe outcome of a host-pathogen interaction is determined by the conditions of the host, the pathogen, and the environment. Although numerous proteomic studies of in vitro-grown microbial pathogens have been performed, in vivo proteomic approaches are still rare. In addition, increasing evidence supports that in vitro studies inadequately reflect in vivo conditions. Choosing the proper host is essential to detect the expression of proteins from the pathogen in vivo. Numerous studies have demonstrated the suitability of zebrafish (Danio rerio) embryos as a model to in vivo studies of Pseudomonas aeruginosa infection. In most zebrafish-pathogen studies, infection is achieved by microinjection of bacteria into the larvae. However, few reports using static immersion of bacterial pathogens have been published. In this study we infected 3 days post-fertilization (DPF) zebrafish larvae with P. aeruginosa PAO1 by immersion and injection and tracked the in vivo immune response by the zebrafish. Additionally, by using non-isotopic (Q-exactive) metaproteomics we simultaneously evaluated the proteomic response of the pathogen (P. aeruginosa PAO1) and the host (zebrafish). We found some zebrafish metabolic pathways, such as hypoxia response via HIF activation pathway, exclusively enriched in the larvae exposed by static immersion. In contrast, we found that inflammation mediated by chemokine and cytokine signaling pathways was exclusively enriched in the larvae exposed by injection, while the integrin signaling pathway and angiogenesis were solely enriched in the larvae exposed by immersion. We also found important virulence factors from P. aeruginosa that were enriched only after exposure by injection, such as the Type-III secretion system and flagella-associated proteins. On the other hand, P. aeruginosa proteins involved in processes like biofilm formation, cellular responses to antibiotic and starvation were enriched exclusively after an exposure by immersion.We demonstrated the suitability of zebrafish embryos as a model for in vivo host-pathogen based proteomic studies in P. aeruginosa. Our global proteomic profiling identifies novel molecular signatures that give systematic insight into zebrafish-Pseudomonas interaction.

Download Full-text

Host Airway Proteins Interact with Staphylococcus aureus during Early Pneumonia

Infection and Immunity ◽

10.1128/iai.01301-07 ◽

2008 ◽

Vol 76 (3) ◽

pp. 888-898 ◽

Cited By ~ 17

Author(s):

Christy L. Ventura ◽

Roger Higdon ◽

Eugene Kolker ◽

Shawn J. Skerrett ◽

Craig E. Rubens

Keyword(s):

Staphylococcus Aureus ◽

Shotgun Proteomics ◽

Community Acquired Pneumonia ◽

Host Proteins ◽

Host Pathogen Interactions ◽

Complement Components ◽

Host Pathogen ◽

Hospital Acquired

ABSTRACT Staphylococcus aureus is a major cause of hospital-acquired pneumonia and is emerging as an important etiological agent of community-acquired pneumonia. Little is known about the specific host-pathogen interactions that occur when S. aureus first enters the airway. A shotgun proteomics approach was utilized to identify the airway proteins associated with S. aureus during the first 6 h of infection. Host proteins eluted from bacteria recovered from the airways of mice 30 min or 6 h following intranasal inoculation under anesthesia were subjected to liquid chromatography and tandem mass spectrometry. A total of 513 host proteins were associated with S. aureus 30 min and/or 6 h postinoculation. A majority of the identified proteins were host cytosolic proteins, suggesting that S. aureus was rapidly internalized by phagocytes in the airway and that significant host cell lysis occurred during early infection. In addition, extracellular matrix and secreted proteins, including fibronectin, antimicrobial peptides, and complement components, were associated with S. aureus at both time points. The interaction of 12 host proteins shown to bind to S. aureus in vitro was demonstrated in vivo for the first time. The association of hemoglobin, which is thought to be the primary staphylococcal iron source during infection, with S. aureus in the airway was validated by immunoblotting. Thus, we used our recently developed S. aureus pneumonia model and shotgun proteomics to validate previous in vitro findings and to identify nearly 500 other proteins that interact with S. aureus in vivo. The data presented here provide novel insights into the host-pathogen interactions that occur when S. aureus enters the airway.

Download Full-text

Models of intestinal infection by Salmonella enterica: introduction of a new neonate mouse model

F1000Research ◽

10.12688/f1000research.8468.1 ◽

2016 ◽

Vol 5 ◽

pp. 1498 ◽

Cited By ~ 4

Author(s):

Marc Schulte ◽

Michael Hensel

Keyword(s):

Animal Model ◽

Small Intestine ◽

Salmonella Enterica ◽

Intestinal Inflammation ◽

Foodborne Pathogen ◽

Small Animal ◽

Host Pathogen Interaction ◽

Host Pathogen

Salmonella entericaserovar Typhimurium is a foodborne pathogen causing inflammatory disease in the intestine following diarrhea and is responsible for thousands of deaths worldwide. Manyin vitroinvestigations using cell culture models are available, but these do not represent the real natural environment present in the intestine of infected hosts. Severalin vivoanimal models have been used to study the host-pathogen interaction and to unravel the immune responses and cellular processes occurring during infection. An animal model forSalmonella-induced intestinal inflammation relies on the pretreatment of mice with streptomycin. This model is of great importance but still shows limitations to investigate the host-pathogen interaction in the small intestinein vivo. Here, we review the use of mouse models forSalmonellainfections and focus on a new small animal model using 1-day-old neonate mice. The neonate model enables researchers to observe infection of both the small and large intestine, thereby offering perspectives for new experimental approaches, as well as to analyze theSalmonella-enterocyte interaction in the small intestinein vivo.

Download Full-text

Identification of Natural Target Proteins Indicates Functions of a Serralysin-Type Metalloprotease, PrtA, in Anti-Immune Mechanisms

Applied and Environmental Microbiology ◽

10.1128/aem.02271-08 ◽

2009 ◽

Vol 75 (10) ◽

pp. 3120-3126 ◽

Cited By ~ 26

Author(s):

Gabriella Felfï¿½ldi ◽

Judit Marokhï¿½zi ◽

Miklï¿½s Kï¿½pirï¿½ ◽

Istvï¿½n Venekei

Keyword(s):

Immune Defense ◽

Innate Immune ◽

Coagulation Cascade ◽

Sequence Information ◽

Specific Substrate ◽

Immune Recognition ◽

Host Pathogen ◽

Substrate Proteins

ABSTRACT Serralysins are generally thought to function as pathogenicity factors of bacteria, but so far no hard evidence of this (e.g., specific substrate proteins that are sensitive to the cleavage by these proteases) has been found. We have looked for substrate proteins to a serralysin-type proteinase, PrtA, in a natural host-pathogen molecular interaction system involving Manduca sexta and Photorhabdus luminescens. The exposure in vitro of hemolymph to PrtA digestion resulted in selective cleavage of 16 proteins, provisionally termed PAT (PrtA target) proteins. We could obtain sequence information for nine of these PrtA sensitive proteins, and by searching databases, we could identify six of them. Each has immune-related function involving every aspect of the immune defense: β-1,3 glucan recognition protein 2 (immune recognition), hemocyte aggregation inhibitor protein (HAIP), serine proteinase homolog 3, six serpin-1 variants, including serpin-1I (immune signaling and regulation), and scolexins A and B (coagulation cascade effector function). The functions of the identified PrtA substrate proteins shed new light on a possible participation of a serralysin in the virulence mechanism of a pathogen. Provided these proteins are targets of PrtA in vivo, this might represent, among others, a complex suppressive role on the innate immune response via interference with both the recognition and the elimination of the pathogen during the first, infective stage of the host-pathogen interaction. Our results also raise the possibility that the natural substrate proteins of serralysins of vertebrate pathogens might be found among the components of the innate immune system.

Download Full-text

Acinetobacter baumannii: An Ancient Commensal with Weapons of a Pathogen

Pathogens ◽

10.3390/pathogens10040387 ◽

2021 ◽

Vol 10 (4) ◽

pp. 387

Author(s):

Meysam Sarshar ◽

Payam Behzadi ◽

Daniela Scribano ◽

Anna Teresa Palamara ◽

Cecilia Ambrosi

Keyword(s):

Acinetobacter Baumannii ◽

Virulence Factors ◽

Current Data ◽

Comparative Genomic ◽

Host Immune System ◽

Host Pathogen Interactions ◽

In Vivo Models ◽

Host Pathogen

Acinetobacter baumannii is regarded as a life-threatening pathogen associated with community-acquired and nosocomial infections, mainly pneumonia. The rise in the number of A. baumannii antibiotic-resistant strains reduces effective therapies and increases mortality. Bacterial comparative genomic studies have unraveled the innate and acquired virulence factors of A. baumannii. These virulence factors are involved in antibiotic resistance, environmental persistence, host-pathogen interactions, and immune evasion. Studies on host–pathogen interactions revealed that A. baumannii evolved different mechanisms to adhere to in order to invade host respiratory cells as well as evade the host immune system. In this review, we discuss current data on A. baumannii genetic features and virulence factors. An emphasis is given to the players in host–pathogen interaction in the respiratory tract. In addition, we report recent investigations into host defense systems using in vitro and in vivo models, providing new insights into the innate immune response to A. baumannii infections. Increasing our knowledge of A. baumannii pathogenesis may help the development of novel therapeutic strategies based on anti-adhesive, anti-virulence, and anti-cell to cell signaling pathways drugs.

Download Full-text

P2RX7 at the Host-Pathogen Interface of Infectious Diseases

Microbiology and Molecular Biology Reviews ◽

10.1128/mmbr.00055-20 ◽

2021 ◽

Vol 85 (1) ◽

Author(s):

Alexandra Y. Soare ◽

Tracey L. Freeman ◽

Alice K. Min ◽

Hagerah S. Malik ◽

Elizabeth O. Osota ◽

...

Keyword(s):

Current Knowledge ◽

Inflammatory Diseases ◽

Critical Role ◽

Extracellular Nucleotides ◽

Host Pathogen ◽

Infected Cells ◽

Molecular Patterns

SUMMARY The P2X7 receptor (P2RX7) is an important molecule that functions as a danger sensor, detecting extracellular nucleotides from injured cells and thus signaling an inflammatory program to nearby cells. It is expressed in immune cells and plays important roles in pathogen surveillance and cell-mediated responses to infectious organisms. There is an abundance of literature on the role of P2RX7 in inflammatory diseases and the role of these receptors in host-pathogen interactions. Here, we describe the current knowledge of the role of P2RX7 in the host response to a variety of pathogens, including viruses, bacteria, fungi, protozoa, and helminths. We describe in vitro and in vivo evidence for the critical role these receptors play in mediating and modulating immune responses. Our observations indicate a role for P2X7 signaling in sensing damage-associated molecular patterns released by nearby infected cells to facilitate immunopathology or protection. In this review, we describe how P2RX7 signaling can play critical roles in numerous cells types in response to a diverse array of pathogens in mediating pathogenesis and immunity to infectious agents.

Download Full-text