Emerging methods in cellular microbiology

In an era where new viruses can emerge suddenly and anti-microbial resistance is now widespread, a full understanding of the pathogen and host factors that contribute to disease pathology and spread remains a critically important goal. Methods and technologies to study the cell biology of infection are changing rapidly. The interdisciplinary requirements to interrogate host-pathogen interactions are ever more complex and indispensable to identify the cellular and immune factors that lead to the resolution of infection. The ability of a pathogen to replicate in the host likewise needs a thorough knowledge of the pathogen determinants required to cause disease. In this special issue, Cellular Microbiology highlights some of the most cutting-edge approaches and techniques to study pathogen biology and infection.

Light based and electron microscopic imaging has long played an important role in defining the intracellular mechanisms of infection. Such methods can now be quickly adapted through the design of fluorescent reporters and utilised for high throughout applications. Cortese and Laketa (2021) describe recent advances in high-throughput microscopy and electron microscopy and explain how these were applied during the SARS-CoV-2 outbreak (Cortese & Laketa, 2021). For example, the development of a mNeonGreen reporter microscopy-based virus neutralisation assay outperformed standard plaque assays in sensitivity and time in the critical early stages of the COVID-19 pandemic (Muruato et al., 2020). Cryo-electron microscopy was instrumental in rapidly defining the structure of the SARS-CoV-2 spike protein in complex with its cellular receptor, angiotensin convertase enzyme 2 (ACE2), directly informing an understanding of neutralising antibodies (Cortese & Laketa, 2021). In bacterial pathogens, Dufrêne, Viljoen, Mignolet, and Mathelié-Guinlet (2021) report on the utility of atomic force microscopy (AFM) to provide super-resolution imaging and nanomechanical measurements of bacterial cell surfaces and receptor-ligand interactions. This is providing new insight into the fine structure and biophysical function of adhesins and also informing vaccine and drug development (Dufrêne et al., 2021).

The subcellular host cell compartment occupied by intracellular pathogens is highly specialised to support pathogen replication. Given their role in sampling of extracellular fluid, macropinosomes are hijacked by diverse pathogens to establish entry into the intracellular environment. However, macropinosomes are also highly dynamic organelles with a range of cellular functions. Chang, Enninga, and Stévenin (2021) describe imaging-based and proteomic methods for the tracking and characterisation of these important organelles as they are modified as a niche for invasion and/or replication by bacteria such as Shigella, Salmonella, Brucella and Chlamydia (Chang et al., 2021). Similarly, Simeone, Sayes, Lawarée, and Brosch (2021) explore the intracellular niche of Mycobacteria, in particular the bacterial factors that allow phagosomal escape of Mycobacterium tuberculosis into the cell cytosol (Simeone et al., 2021). Multiple techniques used in parallel, such as labelling with galectin-3 and ubiquitin to identify damaged phagosomal membranes, as well as FRET-based reporter and cytofluorometric approaches have provided new insight into phagosomal rupture by M. tuberculosis.

Cell lines and primary cells remain important tools in the study of host-pathogen interactions. However, the development of new models such as human stem cell models and microfluidic ‘organ on chip’ technologies can offer an additional and more physiological perspective. Pellegrino and Gutierrez (2021) report on the use of stem cell-derived models engineered into three-dimensional microenvironments as a novel means to study the biology of infection, with and without other cell types in cellular co-culture (Pellegrino & Gutierrez, 2021). These so-called ‘organoids’ are particularly helpful for pre-clinical studies where no small animal model of infection exists and can be adapted to incorporate genome editing tools. Another three-dimensional stem cell approach is ‘organ on-a-chip’ technology which utilises microfluidics and biomaterials to mimic the interface between the pathogen and host tissue during infection. Feaugas and Sauvonnet (2021) provide a timely update on the advantages of using organ on-a-chip and the different elements to consider as well as examples of how it has re-defined our knowledge of host pathogen interactions (Feaugas & Sauvonnet, 2021). For example, organ-on-a-chip has provided insight into how mechanical forces in the gut influence Shigella invasion (Grassart et al., 2019).

High throughput screening is an integral part of drug development as well as large scale genomic editing approaches. Subhash and Sundaramurthy describe screening platforms that take the host environment into account (Subhash & Sundaramurthy, 2021). This allows the identification of potential host directed therapeutics which may be used as adjunct therapies against chronic intractable infections like tuberculosis, particularly where the pathogen is intracellular. Imaging is a desirable output for many high throughput screening approaches for a number of reasons. An automated image analysis program is discussed by Fisch et al. (2021), HRMAn (Host Response to Microbe Analysis), that uses machine learning and artificial intelligence to assess infection by pathogens in an unbiased manner. Parameters include pathogen growth, pathogen killing and activation of host cell signalling. When incorporated into a functional screen, HRMAn provides unprecedented capacity for high throughput and high-content analysis (Fisch et al., 2021).

In summary, the techniques presented in this special issue of Cellular Microbiology provide inspiration and direction to many investigators studying bacterial, viral and parasite infections. The state-of-the-art approaches explained here will allow transformational research questions to be addressed and deepen our knowledge of host-pathogen interactions that could lead to new disease interventions. We anticipate that further issues of Cellular Microbiology will be dedicated to emerging technologies in order to highlight the most important methodological developments in our field.