Over the past few years, a continuous improvement has been observed in the confocal detection of human and bacterial cells, facilitated by the application of specific, fluorochrome-tagged antibodies. These assays, when used together, offer both the tools for precise measurement and the adaptability to fit any specific experimental requirement.Mycobacterial infections, including the debilitating tuberculosis, are a major global health problem. Tuberculosis's prevention and treatment are complicated by the inadequate protection offered by the current vaccine and the growing presence of drug-resistant forms of the bacteria. Subsequently, a more thorough comprehension of the pathogenic strategies employed by mycobacteria and the corresponding immune responses of the host during infection in the complex in vivo environment is critically important. Infection models currently available, while instrumental in exploring mycobacterial pathogenesis, still suffer from limitations that necessitate the development of models that enhance them. This paper details recent progress in the murine model of Mycobacterium marinum infection, specifically the localized nature of the infection restricted to the mouse's tail. A key benefit of the M. marinum model lies in its capacity to replicate aspects of human tuberculosis, absent in the murine Mycobacterium tuberculosis model, including the formation of granulomas with central caseating necrosis, and the spontaneous development of a latent phase. Besides its non-lethal nature, the model permits longitudinal analysis of disease evolution in live animals. The methods outlined in this chapter pertain to the preparation of infected tissue samples, permitting detailed and quantitative evaluation of the immune response via flow cytometry, immunofluorescence microscopy, RT-qPCR, ELISA, and Western blot analyses. These methods also include analyses of bacterial load and localization within the tissue.Bacterial host cell penetration studies are often conducted using gentamicin protection assays, which are resource-intensive and present challenges due to their inherent experimental variability. An internally controlled method, designed for medium- to high-throughput applications, is presented in this chapter to assess the capacity of multiple Salmonella virulence factor mutant strains to attach to and invade host cells. Consortia of genetically-tagged isogenic bacterial strains, combined with a modified gentamicin protection assay, underpins a method with broad applicability to other pathogens. A flexible toolset within these protocols allows for precise quantification of host cell binding and invasive properties in different mutant types. Furthermore, the technique can be deployed for both infections of cultured host cells and in vivo animal models, generating a similar genetic response, which greatly simplifies the process of comparing findings across experimental systems.Bacterial sepsis, along with other systemic inflammatory responses, exhibits the hallmark of vascular dysfunction. az304 inhibitor Glycans and proteoglycans are the fundamental components of the glycocalyx, which forms a dense layer on the luminal surface of blood vessels. Among the glycocalyx's constituents are glycoproteins, arising from the endothelium, pericytes, intravascular leukocytes, and plasma. These create a vascular cell surface proteome dynamic in its nature, attuned to specific tissues, and acutely responsive to fluctuations in vascular equilibrium, blood infections, and inflammatory reactions. We describe a detailed experimental protocol for the chemical labeling and measurement of the vascular cell surface proteome in mouse models of bacteremia, employing a time-dependent and organ-specific approach. This method not only helps to identify markers of vascular activation but also gives a molecular framework for understanding the contribution of vascular dysfunction to the organ pathology of systemic inflammation.Inflammasomes, substantial multiprotein complexes, predominantly form within innate immune cells following the recognition of microbial or sterile provocations. Infection triggers the activation of inflammasomes, a critical pro-inflammatory event, prompting numerous pathogens to develop specialized strategies to avoid or suppress this process. One of the infectious agents, group A Streptococcus (GAS), is responsible for a broad assortment of diseases with differing levels of severity. A primary driver of inflammasome activation from GAS is the pore-forming protein streptolysin O (SLO), among a variety of other secreted virulence factors. We detail a protocol for the dependable assessment of inflammasome activation in murine bone marrow-derived macrophages (BMDMs) infected with Group A Streptococcus (GAS), encompassing procedures for BMDM generation and bacterial cultivation. A simple modification to this protocol enables its use with a broad range of bacterial pathogens, or, with particular focus on human macrophages.Antibody binding to bacterial surfaces is integral to the immune response, and the protein-protein interaction's binding affinity is a key characteristic. Predicting the function of an antibody within a physiological system, considering the presence of competing protein interactions, hinges on initial determination of its affinity for its antigen. Antibody-antigen interaction strength is frequently measured using separated protein molecules. Understanding antibody-antigen interactions becomes more profound by examining binding on a bacterial surface, which abounds with diverse antigens, potentially containing multiple copies of the target antigen, and in a context reflecting its native structure. A flow cytometry assay is described in this chapter, enabling the measurement and calculation of cell surface binding affinity or avidity for both monoclonal and polyclonal antibody preparations.Regulated cell death, including the release of neutrophil extracellular traps (NETs), plays a significant role in combating bacterial infections. Generally, the release of RCD and NETs' dynamic processes is evaluated independently through methods which are either non-specific or time-consuming. This description details a high-throughput flow cytometry method analyzing neutrophil reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) release, validated by visual live-cell imaging after ex vivo exposure to bacteria. This approach enables a rigorous quantification and detailed monitoring of the dynamic neutrophil effector response toward bacterial infection.Phagocytosis's relevance extends across a multitude of research domains, often being quantified as a practical measure of function. Despite the importance of accurate quantification, significant challenges arise, creating difficulties for researchers to conduct robust studies. Numerous methods exist for evaluating phagocytic activity, but the importance of the experimental design, including the planned analysis, is frequently underemphasized. Experimental parameters, including reaction volume, time, and phagocyte-prey concentrations, frequently exert a considerable influence on the final outcome. The choice of detection method, incorporating different fluorescent or colorimetric labels for prey and phagocytes, is equally important. For enhanced phagocytosis quantification, utilizing dose-response curve principles within the experimental design and analytical processes increases accuracy and consistency across diverse experiments and systems. The prey, initially fluorescently double-stained, may then be opsonized, and finally introduced to phagocytes at a range of quantities. Data collection using flow cytometry takes place after the incubation process. Phagocyte assessment, incorporating both population-based and single-cell-level measurements, allows for the separation of adhesion and internalization. The following experimental protocol describes the phagocytic uptake of opsonized Streptococcus pyogenes by THP-1 cells. Existing phagocytosis assays are compatible with this approach, yielding consistently reproducible results and high sensitivity.Cultivation techniques pose a significant barrier to comprehending the impact of anaerobic bacteria on the human host. Nonetheless, a dramatic rise in research during the last ten years has showcased the significance of these disregarded pathogens. Within this chapter, we present a survey of how neutrophils and monocytes respond, in terms of surface and intracellular inflammation markers, to the Gram-positive anaerobic cocci (GPAC) species Peptoniphilus (P.) harei.The characterization of individual proteins is possible due to the single-molecule capabilities of mass photometry (MP). The Refeyn OneMP tool is used in this detailed procedure for the examination of molecular complexes, focusing on M53, a plasminogen-binding group A streptococcal M-like protein (PAM), and human plasminogen. The methodology detailed in this document validated a binding stoichiometry of 11 for the M53-plasminogen complex. To ascertain the oligomerization state, homogeneity, purity, and approximate molecular weights of each protein, MP was applied.The intricate and fluctuating protein-protein interactions between host cells and pathogens drive crucial steps in pathogen adherence to, penetration of, and colonization within the host, alongside the circumvention of the host's immune defenses. Specialized virulence factors and effector proteins, characteristic of bacterial interactions, predominantly target and interact with essential host proteins. Employing a proteomics approach centered on mass spectrometry, I'm presenting a method to identify host-pathogen interactions. This entails affinity purification to isolate the proteins, followed by cross-linking mass spectrometry and structural modeling to visualize their interaction interfaces.During bacterial infection, host receptors are significantly influenced by the actions of bacterial virulence factors. In conclusion, the study of interactions between host receptors and bacterial virulence factors not only exposes the molecular mechanisms of bacterial infection, but also furnishes a blueprint for devising novel therapeutic and preventative strategies. High-resolution quantitative mass spectrometry, in combination with an APEX2-based live cell proximity labeling strategy, is used to characterize the substrates of the Helicobacter pylori HtrA protease on the membrane of human stomach epithelial cells.