Molecular Microbiology最新文献

Apicomplexan parasites are aetiological agents of numerous diseases in humans and livestock. Functional genomics studies in these parasites enable the identification of biological mechanisms and protein functions that can be targeted for therapeutic intervention. Recent improvements in forward genetics and whole-genome screens utilising CRISPR/Cas technology have revolutionised the functional analysis of genes during Apicomplexan infection of host cells. Here, we highlight key discoveries from CRISPR/Cas9 screens in Apicomplexa or their infected host cells and discuss remaining challenges to maximise this technology that may help answer fundamental questions about parasite-host interactions.

Transmission electron microscopy (TEM) has been essential to study virus-cell interactions. The architecture of viral replication factories, the principles of virus assembly and the components of virus egress pathways are known thanks to the contribution of TEM methods. Specially, when studying viruses in cells, methodologies for labeling proteins and other macromolecules are important tools to correlate morphology with function. In this review, we present the most widely used labeling method for TEM, immunogold, together with a lesser known technique, metal-tagging transmission electron microscopy (METTEM) and how they can contribute to study viral infections. Immunogold uses the power of antibodies and electron dense, colloidal gold particles while METTEM uses metallothionein (MT), a metal-binding protein as a clonable tag. MT molecules build gold nano-clusters inside cells when these are incubated with gold salts. We describe the necessary controls to confirm that signals are specific, the advantages and limitations of both methods, and show some examples of immunogold and METTEM of cells infected with viruses.

Since its inception in the 1930s, transmission electron microscopy (TEM) has been a powerful method to explore the cellular structure of parasites. TEM usually requires samples of <100 nm thick and with protozoans being larger than 1 μm, their study requires resin embedding and ultrathin sectioning. During the past decade, several new methods have been developed to improve, facilitate, and speed up the structural characterisation of biological samples, offering new imaging modalities for the study of protozoans. In particular, scanning transmission electron microscopy (STEM) can be used to observe sample sections as thick as 1 μm thus becoming an alternative to conventional TEM. STEM can also be performed under cryogenic conditions in combination with cryo-electron tomography providing access to the study of thicker samples in their native hydrated states in 3D. This method, called cryo-scanning transmission electron tomography (cryo-STET), was first developed in 2014. This review presents the basic concepts and benefits of STEM methods and provides examples to illustrate the potential for new insights into the structure and ultrastructure of protozoans.

Invasive candidiasis caused by non-albicans species has been on the rise, with Candida glabrata emerging as the second most common etiological agent. Candida glabrata possesses an intrinsically lower susceptibility to azoles and an alarming propensity to rapidly develop high-level azole resistance during treatment. In this study, we have developed an efficient piggyBac (PB) transposon-mediated mutagenesis system in C. glabrata to conduct genome-wide genetic screens and applied it to profile genes that contribute to azole resistance. When challenged with the antifungal drug fluconazole, PB insertion into 270 genes led to significant resistance. A large subset of these genes has a role in the mitochondria, including almost all genes encoding the subunits of the F₁F₀ ATPase complex. We show that deleting ATP3 or ATP22 results in increased azole resistance but does not affect susceptibility to polyenes and echinocandins. The increased azole resistance is due to increased expression of PDR1 that encodes a transcription factor known to promote drug efflux pump expression. Deleting PDR1 in the atp3Δ or atp22Δ mutant resulted in hypersensitivity to fluconazole. Our results shed light on the mechanisms contributing to azole resistance in C. glabrata. This PB transposon-mediated mutagenesis system can significantly facilitate future genome-wide genetic screens.

Microbial cells must continually adapt their physiology in the face of changing environmental conditions. Archaea living in extreme conditions, such as saturated salinity, represent important examples of such resilience. The model salt-loving organism Haloferax volcanii exhibits remarkable plasticity in its morphology, biofilm formation, and motility in response to variations in nutrients and cell density. However, the mechanisms regulating these lifestyle transitions remain unclear. In prior research, we showed that the transcriptional regulator, TrmB, maintains the rod shape in the related species Halobacterium salinarum by activating the expression of enzyme-coding genes in the gluconeogenesis metabolic pathway. In Hbt. salinarum, TrmB-dependent production of glucose moieties is required for cell surface glycoprotein biogenesis. Here, we use a combination of genetics and quantitative phenotyping assays to demonstrate that TrmB is essential for growth under gluconeogenic conditions in Hfx. volcanii. The ∆trmB strain rapidly accumulated suppressor mutations in a gene encoding a novel transcriptional regulator, which we name trmB suppressor, or TbsP (a.k.a. "tablespoon"). TbsP is required for adhesion to abiotic surfaces (i.e., biofilm formation) and maintains wild-type cell morphology and motility. We use functional genomics and promoter fusion assays to characterize the regulons controlled by each of TrmB and TbsP, including joint regulation of the glucose-dependent transcription of gapII, which encodes an important gluconeogenic enzyme. We conclude that TrmB and TbsP coregulate gluconeogenesis, with downstream impacts on lifestyle transitions in response to nutrients in Hfx. volcanii.

Positive-sense single-stranded RNA viruses significantly reshape intracellular membranes to generate viral replication organelles that form a controlled niche in which nucleic acids, enzymes, and cofactors accumulate to assure an efficient replication of the viral genome. In recent years, advancements in electron microscopy (EM) techniques have enabled imaging of these viral factories in a near-native state providing significantly higher molecular details that have led to progress in our general understanding of virus biology. In this review, we describe the contribution of the cutting-edge EM approaches to the current knowledge of replication organelles biogenesis, structure, and functions.

Bacterial secretion systems, such as the type 3, 4, and 6 are multiprotein nanomachines expressed at the surface of pathogens with Gram-negative like envelopes. They are known to be crucial for virulence and to translocate bacteria-encoded effector proteins into host cells to manipulate cellular functions. This facilitates either pathogen attachment or invasion of the targeted cell. Effector proteins also promote evasion of host immune recognition. Imaging by cryo-electron microscopy in combination with structure determination has become a powerful approach to understand how these nanomachines work. Still, questions on their assembly, the precise secretion mechanisms, and their direct involvement in pathogenicity remain unsolved. Here, we present an overview of the recent developments in in situ cryo-electron microscopy. We discuss its potential for the investigation of the role of bacterial secretion systems during the host-bacterial crosstalk at the molecular level. These in situ studies open new perspectives for our understanding of secretion system structure and function.

Super-resolution fluorescence microscopy technologies developed over the past two decades have pushed the resolution limit for fluorescently labeled molecules into the nanometer range. These technologies have the potential to study bacterial structures, for example, macromolecular assemblies such as secretion systems, with single-molecule resolution on a millisecond time scale. Here we review recent applications of super-resolution fluorescence microscopy with a focus on bacterial secretion systems. We also describe MINFLUX fluorescence nanoscopy, a relatively new technique that promises to one day produce molecular movies of molecular machines in action.