Cell Surface最新文献

Infections resulting from Mycobacterium abscessus are increasing in prevalence worldwide, with the greatest risk posed to patients with underlying respiratory conditions. Treatment for infections is difficult due to wide ranging intrinsic antimicrobial resistance, which is compounded by the existence of a range of subspecies within the M. abscessus complex, each with varying additional antimicrobial resistance profiles. Previously, the use of β-lactam/β-lactamase inhibitors within a combination therapy has been proposed as an effective treatment option for pulmonary M. abscessus infections. Here, we assess the in vitro efficacy of two non-β-lactam based inhibitors, relebactam and avibactam, as agents against M. abscessus with their respective partner drugs imipenem and ceftazidime, as well as in triplicate combinations with additional β-lactam antibiotics against the M. abscessus complex. We have shown that the commercially available ratio of imipenem to relebactam is the appropriate ratio for bactericidal activity against M. abscessus, whereas the ratio between ceftazidime and avibactam is redundant, due to inactivity of ceftazidime to inhibit the bacteria. We have identified that the use of imipenem and meropenem alongside either relebactam or avibactam yield low minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) for each M. abscessus subspecies, which are within the therapeutically achievable concentration ranges within the epithelial lining fluid of the lungs. We propose the implementation of imipenem with relebactam in place of stand-alone imipenem into the current treatment regime, alongside meropenem, as a future front-line treatment option for M. abscessus complex infections.

A rapidly increasing body of literature suggests that many biological processes are driven by phase separation within polymer mixtures. Liquid-liquid phase separation can lead to the formation of membrane-less organelles, which are thought to play a wide variety of roles in cell metabolism, gene regulation or signaling. One of the characteristics of these systems is that they are poised at phase transition boundaries, which makes them perfectly suited to elicit robust cellular responses to often very small changes in the cell’s “environment”. Recent observations suggest that, also in the semi-solid environment of plant cell walls, phase separation not only plays a role in wall patterning, hydration and stress relaxation during growth, but also may provide a driving force for cell wall expansion. In this context, pectins, the major polyanionic polysaccharides in the walls of growing cells, appear to play a critical role. Here, we will discuss (i) our current understanding of the structure–function relationship of pectins, (ii) in vivo evidence that pectin modification can drive critical phase transitions in the cell wall, (iii) how such phase transitions may drive cell wall expansion in addition to turgor pressure and (iv) the periodic cellular processes that may control phase transitions underlying cell wall assembly and expansion.

Rhizospheres are microecological zones at the interface of roots and soils. Interactions between bacteria and roots are critical for maintaining plant and soil health but are difficult to study because of constraints inherent in working with underground systems. We have developed an in-situ rhizosphere imaging system based on transparent soils and molecular probes that can be imaged using confocal microscopy. We observed spatial patterning of polysaccharides along roots and on cells deposited into the rhizosphere and also co-localised fluorescently tagged soil bacteria. These studies provide insight into the complex glycan landscape of rhizospheres and suggest a means by which root / rhizobacteria interactions can be non-disruptively studied.

Bacterial infections are the most eminent public health challenge of the 21st century. The primary step leading to infection is bacterial adhesion to the surface of host cells or medical devices, which is mediated by a multitude of molecular interactions. At the interface of life sciences and physics, last years advances in atomic force microscopy (AFM)-based force spectroscopy techniques have made possible to measure the forces driving bacteria-cell and bacteria-materials interactions on a single molecule/cell basis (single molecule/cell force spectroscopy).

Among the bacteria-(bio)materials surface interactions, the life-threatening infections associated to medical devices involving Staphylococcus aureus and Escherichia coli are the most eminent. On the other hand, Pseudomonas aeruginosa binding to the pulmonary and urinary tract or the Helicobacter pylori binding to the gastric mucosa, are classical examples of bacteria-host cell interactions that end in serious infections.

As we approach the end of the antibiotic era, acquisition of a deeper knowledge of the fundamental forces involved in bacteria – host cells/(bio)materials surface adhesion is crucial for the identification of new ligand-binding events and its assessment as novel targets for alternative anti-infective therapies.

This article aims to highlight the potential of AFM-based force spectroscopy for new targeted therapies development against bacterial infections in which adhesion plays a pivotal role and does not aim to be an extensive overview on the AFM technical capabilities and theory of single molecule force spectroscopy.

Carbon catabolite repression enables fungi to utilize the most favourable carbon source in the environment, and is mediated by a key regulator, CreA, in most fungi. CreA-mediated regulation has mainly been studied at high monosaccharide concentrations, an uncommon situation in most natural biotopes. In nature, many fungi rely on plant biomass as their major carbon source by producing enzymes to degrade plant cell wall polysaccharides into metabolizable sugars. To determine the role of CreA when fungi grow in more natural conditions and in particular with respect to degradation and conversion of plant cell walls, we compared transcriptomes of a creA deletion and reference strain of the ascomycete Aspergillus niger during growth on sugar beet pulp and wheat bran. Transcriptomics, extracellular sugar concentrations and growth profiling of A. niger on a variety of carbon sources, revealed that also under conditions with low concentrations of free monosaccharides, CreA has a major effect on gene expression in a strong time and substrate composition dependent manner. In addition, we compared the CreA regulon from five fungi during their growth on crude plant biomass or cellulose. It showed that CreA commonly regulated genes related to carbon metabolism, sugar transport and plant cell wall degrading enzymes across different species. We therefore conclude that CreA has a crucial role for fungi also in adapting to low sugar concentrations as occurring in their natural biotopes, which is supported by the presence of CreA orthologs in nearly all fungi.

Mycobacterium tuberculosis (Mtb) is an intracellular human pathogen that has evolved to survive in a nutrient limited environment within the host for decades. Accordingly, Mtb has developed strategies to acquire scarce nutrients and the mycobacterial transporter systems provide an important route for the import of key energy sources. However, the physiological role of the Mtb transporters and their substrate preference(s) are poorly characterised. Previous studies have established that the Mtb UspC solute-binding domain recognises amino- and phosphorylated-sugars, indicating that the mycobacterial UspABC transporter plays a key role in the import of peptidoglycan precursors. Herein, we have used a wide array of approaches to investigate the role of UspABC in Mycobacterium smegmatis by analysis of mutant strains that either lack the solute binding domain: ΔuspC or the entire transport complex: ΔuspABC. Analysis of mycobacterial transcripts shows that the uspABC system is functionally expressed in mycobacteria as a contiguous reading frame. Topology mapping confirms an N_in-C_in orientation of the UspAB integral membrane spanning domains. Phenotypic microarray profiling of commercially available sugars suggests, unexpectedly, that the uspC and ΔuspABC mutants had different carbon utilisation profiles and that neither strain utilised glucose-1-phosphate. Furthermore, proteomics analysis showed an alteration in the abundance of proteins involved in sugar and lipid metabolism, crucial for cell envelope synthesis, and we propose that UspABC has an important role in determining the interplay between these pathways.

The emergence of drug-resistant strains of Mycobacterium tuberculosis (Mtb) ensures that drug discovery efforts remain at the forefront of TB research. There are multiple different experimental approaches that can be employed in the discovery of anti-TB agents. Notably, inhibitors of MmpL3 are numerous and structurally diverse in Mtb and have been discovered through the generation of spontaneous resistant mutants and subsequent whole genome sequencing studies. However, this approach is not always reliable and can lead to incorrect target assignment and requires orthogonal confirmatory approaches. In fact, many of these inhibitors have also been shown to act as multi-target agents, with secondary targets in Mtb, as well as in other non-MmpL3-containing pathogens. Herein, we have investigated further the cellular targets of the MmpL3-inhibitor BM212 and a number of BM212 analogues. To determine the alternative targets of BM212, which may have been masked by MmpL3 mutations, we have applied a combination of chemo-proteomic profiling using bead-immobilised BM212 derivatives and protein extracts, along with whole-cell and biochemical assays. The study identified EthR2 (Rv0078) as a protein that binds BM212 analogues. We further demonstrated binding of BM212 to EthR2 through an in vitro tryptophan fluorescence assay, which showed significant quenching of tryptophan fluorescence upon addition of BM212. Our studies have demonstrated the value of revisiting drugs with ambiguous targets, such as MmpL3, in an attempt to find alternative targets and the study of off-target effects to understand more precisely target engagement of new hits emerging from drug screening campaigns.

Invasive fungal infections remain highly problematic for human health. Collectively, they account for more than 1 million deaths a year in addition to more than 100 million mucosal infections and 1 billion skin infections. To be able to make progress it is important to understand the pathobiology of fungal interactions with the immune system. Here, we highlight new advancements pointing out the pivotal role of fungal cell wall components (β-glucan, mannan, galactosaminogalactan and melanin) in modulating host immunity and discuss how these open new opportunities for the development of immunomodulatory strategies to combat deadly fungal infectious diseases.

The impact of fungal infections on humans is a serious public health issue that has received much less attention than bacterial infection and treatment, despite ever-increasing incidence exacerbated by an increased incidence of immunocompromised individuals in the population. Candida species, in particular, cause some of the most prevalent hospital-related fungal infections. Fungal infections are also detrimental to the well-being of grazing livestock, with milk production in dairy cows, and body and coat condition adversely affected by fungal infections. Fungal cell walls are essential for viability, morphogenesis and pathogenesis: numerous anti-fungal drugs rely on targeting either the cell wall or cell membrane, but the pipeline of available bioactives is limited. There is a clear and unmet need to identify novel targets and develop new classes of anti-fungal agents. This mini review focuses on fungal cell wall structure, composition and biosynthesis in Candida spp., including C. auris. In addition, an overview of current advances in the development of cell wall targeted therapies is considered.

Understanding the molecular mechanisms governing antifungal resistance is crucial for identifying new cellular targets for developing new antifungal therapeutics. In this study, we performed a transposon-mediated genome-wide genetic screen in haploid Candida albicans to identify mutants resistant to caspofungin, the first member of the echinocandin class of antifungal drugs. A mutant exhibiting the highest resistance possessed a transposon insertion that inactivates GPI7, a gene encoding the mannose-ethanolamine phosphotransferase. Deleting GPI7 in diploid C. albicans caused similar caspofungin resistance. gpi7Δ/Δ cells showed significantly elevated cell wall chitin content and enhanced phosphorylation of Mkc1, a core component of the PKC-MAPK cell-wall integrity pathway. Deleting MKC1 suppressed the chitin elevation and caspofungin resistance of gpi7Δ/Δ cells, but overexpressing the dominant inactive form of RHO1, an upstream activator of PKC-MAPK signaling, did not. Transcriptome analysis uncovered 406 differentially expressed genes in gpi7Δ/Δ cells, many related to cell wall construction. Our results suggest that GPI7 deletion impairs cell wall integrity, which triggers the cell-wall salvage mechanism via the PKC-MAPK pathway independently of Rho1, resulting in the compensatory chitin synthesis to confer caspofungin resistance.