Microbial Cell最新文献

Budding yeast Saccharomyces cerevisiae is widely used as a model organism to study the biogenesis and architecture of organellar membranes, which can be visualized by transmission electron microscopy (TEM). Preparation of yeast cells for TEM can be quite challenging and time-consuming. Here, we describe an optimized protocol for conventional fixation of yeast cells with potassium permanganate combined with cell wall permeabilization with sodium metaperiodate and embedding in Epon. We have replaced time-consuming incubation steps by short treatments with microwaves and developed a microwave-assisted permanganate fixation and Epon embedding protocol that reduces the time required for sample preparation to one working day. We expect that these protocols will be useful for routine analysis of membrane ultrastructure in yeast.

Staphylococcus aureus, a versatile human pathogen, poses a significant challenge in healthcare settings due to its ability to develop antibiotic resistance and form robust biofilms. Understanding the intricate mechanisms underlying the antibiotic resistance is crucial for effective infection treatment and control. This comprehensive review delves into the multifaceted roles of efflux pumps in S. aureus, with a focus on their contribution to antibiotic resistance and biofilm formation. Efflux pumps, integral components of the bacterial cell membrane, are responsible for expelling a wide range of toxic substances, including antibiotics, from bacterial cells. By actively extruding antibiotics, these pumps reduce intracellular drug concentrations, rendering antibiotics less effective. Moreover, efflux pumps have emerged as significant contributors to both antibiotic resistance and biofilm formation in S. aureus. Biofilms, structured communities of bacterial cells embedded in a protective matrix, enable S. aureus to adhere to surfaces, evade host immune responses, and resist antibiotic therapy. Efflux pumps play a pivotal role in the development and maintenance of S. aureus biofilms. However, the interplay between efflux pumps, antibiotic resistance and biofilm formation remains unexplored in S. aureus. This review aims to elucidate the complex relationship between efflux pumps, antibiotic resistance and biofilm formation in S. aureus with the aim to aid in the development of potential therapeutic targets for combating S. aureus infections, especially those associated with biofilms. The insights provided herein may contribute to the advancement of novel strategies to overcome antibiotic resistance and disrupt biofilm formation in this clinically significant pathogen.

Concurrent infections with two or more pathogens with analogous tropism, such as RSV and SARS-CoV-2, may antagonize or facilitate each other, modulating disease outcome. Clinically, discrepancies in the severity of symptoms have been reported in children with RSV/SARS-CoV-2 co-infection. Herein, we propose an in vitro co-infection model to assess how RSV/SARS-CoV-2 co-infection alters cellular homeostasis. To this end, A549-hACE2 expressing cells were either infected with RSV or SARS-CoV-2 alone or co-infected with both viruses. Viral replication was assessed at 72 hours post infection by droplet digital PCR, immunofluorescence, and transmission electron microscopy. Anti-viral/receptor/autophagy gene expression was evaluated by RT-qPCR and confirmed by secretome analyses and intracellular protein production. RSV/SARS-CoV-2 co-infection in A549-hACE2 cells was characterized by: 1) an increase in the replication rate of RSV compared to single infection; 2) an increase in one of the RSV host receptors, ICAM1; 3) an upregulation in the expression/secretion of pro-inflammatory genes; 4) a rise in the number and length of cellular conduits; and 5) augmented autophagosomes formation and/or alteration of the autophagy pathway. These findings suggest that RSV/SARS-CoV-2 co-infection model displays a unique and specific viral and molecular fingerprint and shed light on the viral dynamics during viral infection pathogenesis. This in vitro co-infection model may represent a potential attractive cost-effective approach to mimic both viral dynamics and host cellular responses, providing in future readily measurable targets predictive of co-infection progression.

Defining the physiological role of a gene product relies on interpreting phenotypes caused by the lack, or alteration, of the respective gene product. Mutations in critical genes often lead to easily recognized phenotypes that can include changes in cellular growth, metabolism, structure etc. However, mutations in many important genes may fail to generate an obvious defect unless additional perturbations are caused by medium or genetic background. The latter scenario is exemplified by RidA proteins. In vitro RidA proteins deaminate numerous imine/enamines, including those generated by serine/threonine dehydratase IlvA (EC:4.3.1.19) from serine or threonine - 2-aminoacrylate (2AA) and 2-aminocrotonate (2AC), respectively. Despite this demonstrable biochemical activity, a lack of RidA has little to no effect on growth of E. coli or S. enterica without the application of additional metabolic perturbation. A cellular role of RidA is to prevent accumulation of 2AA which, if allowed to persist, can irreversibly damage pyridoxal 5'-phosphate (PLP)-dependent enzymes, causing global metabolic stress. Because the phenotypes caused by a lack of RidA are dependent on the unique structure of each metabolic network, the link between RidA function and 2AA stress is difficult to demonstrate in some organisms. The current study used coculture experiments to exacerbate differences in growth caused by the lack of RidA in S. enterica and E. coli. Results described here solidify the established role of RidA in removing 2AA, while also presenting evidence for a role of RidA in enhancing flux towards isoleucine biosynthesis in E. coli. Overall, these data emphasize that metabolic networks can generate distinct responses to perturbation, even when the individual components are conserved.

Alcohol-associated liver disease is highly prevalent worldwide, with alcohol-associated hepatitis as a severe form characterized by substantial morbidity, mortality, and economic burden. Gut bacterial dysbiosis has been linked to progression of alcohol-associated hepatitis. Fecal cytolysin secreted by the pathobiont Enterococcus faecalis (E. faecalis) is associated with increased mortality in patients with alcohol-associated hepatitis. Although gelatinase is considered a virulence factor in E. faecalis, its prevalence and impact on alcohol-associated hepatitis patient outcomes remains unclear. In this study, 20 out of 65 (30.8%) patients with alcohol-associated hepatitis tested positive for gelatinase in their stool. There were no significant differences in 30-day and 90-day mortality between gelatinase-positive and gelatinase-negative patients (p=0.97 and p=0.48, respectively). Fecal gelatinase had a low discriminative ability for 30-day mortality (area under the curve [AUC] 0.50 vs fibrosis-4 Index (FIB-4) 0.75) and 90-day mortality compared with other established liver disease markers (AUC 0.57 vs FIB-4 0.79 or 'age, serum bilirubin, INR, and serum creatinine' (ABIC) score 0.78). Furthermore, fecal gelatinase was not an important feature for 30-day or 90-day mortality per random forest analysis. Finally, gelatinase-positive patients with alcohol-associated hepatitis did not exhibit more severe liver disease compared with gelatinase-negative patients. In conclusion, fecal gelatinase does not predict mortality or disease severity in patients with alcohol-associated hepatitis from our cohort.

Proteins are the principal macromolecular constituent of proliferating cells, and protein synthesis is viewed as a primary metric of cell growth. While there are celebrated examples of proteins whose levels are periodic in the cell cycle (e.g., cyclins), the concentration of most proteins was not thought to change in the cell cycle, but some recent results challenge this notion. The 'bulk' protein is the focus of this article, specifically the rate of its synthesis, in the budding yeast Saccharomyces cerevisiae.

The alarmone (p)ppGpp serves as the signalling molecule for the bacterial universal stringent response and plays a crucial role in bacterial virulence, persistence, and stress adaptation. Consequently, there is a significant focus on developing new drugs that target and modulate the levels of (p)ppGpp as a potential strategy for controlling bacterial infections. However, despite the availability of various methods for detecting (p)ppGpp, a simple and straightforward detection method is needed. In this study, we demonstrated that malachite green, a well-established compound used for phosphate detection, can directly detect (p)ppGpp and its analogues esp., pGpp. By utilizing malachite green, we identified three new inhibitors of the hydrolase activity of SpoT, one of the two RelA-SpoT homolog (RSH) proteins responsible for making and hydrolyzing (p)ppGpp in Escherichia coli. These findings highlight the convenience and practicality of malachite green, which can be widely employed in high-throughput studies to investigate (pp)pGpp in vitro and discover novel regulators of RSH proteins.

The role of model organisms such as yeasts in life science research is crucial. Although the baker's yeast (Saccharomyces cerevisiae) is the most popular model among yeasts, the contribution of the fission yeasts (Schizosaccharomyces) to life science is also indisputable. Since both types of yeasts share several thousands of common orthologous genes with humans, they provide a simple research platform to investigate many fundamental molecular mechanisms and functions, thereby contributing to the understanding of the background of human diseases. In this review, we would like to highlight the many advantages of fission yeasts over budding yeasts. The usefulness of fission yeasts in virus research is shown as an example, presenting the most important research results related to the Human Immunodeficiency Virus Type 1 (HIV-1) Vpr protein. Besides, the potential role of fission yeasts in the study of prion biology is also discussed. Furthermore, we are keen to promote the uprising model yeast Schizosaccharomyces japonicus, which is a dimorphic species in the fission yeast genus. We propose the hyphal growth of S. japonicus as an unusual opportunity as a model to study the invadopodia of human cancer cells since the two seemingly different cell types can be compared along fundamental features. Here we also collect the latest laboratory protocols and bioinformatics tools for the fission yeasts to highlight the many possibilities available to the research community. In addition, we present several limiting factors that everyone should be aware of when working with yeast models.

The PD-1/PD-L1 pathway plays a pivotal role in T cell activity and is involved in the pathophysiology of Mycobacterium tuberculosis (MTB) infection. DNA methylation is a mechanism that modulates PD-L1 expression in cancer cells. However, its effect on PD-L1 expression in macrophages after MTB infection remains unknown. We prospectively enrolled patients with active tuberculosis (TB) and non-TB subjects. The expression of PD-L1 and methylation-related genes in peripheral blood mononuclear cells (PBMCs) were investigated and their correlation with disease severity and treatment outcomes were examined. PD-L1 promoter methylation status was evaluated using bisulfite sequencing. Immunohistochemistry (IHC) and immunofluorescence (IF) staining were used to visualize PD-L1- and TET-1-expressing cells in lung tissues from patients with TB and in macrophage cell lines with MTB-related stimulation. In total, 80 patients with active TB and 40 non-TB subjects were enrolled in the analysis. Patients with active TB had significantly higher expression of PD-L1, DNMT3b, TET1, TET2, and lower expression of DNMT1, compared to that in the non-TB subjects. The expression of PD-L1 and TET-1 was significantly associated with 1-month smear and culture non-conversion. IHC and IF staining demonstrated the co-localization of PD-L1- and TET-1-expressing macrophages in patients with pulmonary TB and in human macrophage cell lines after MTB-related stimulation. DNMT inhibition and TET-1 knockdown in human macrophages increased and decreased PD-L1 expression, respectively. Overall, PD-L1 expression is increased in patients with active TB and is correlated with treatment outcomes. DNA methylation is involved in modulating PD-L1 expression in human macrophages.

The ability of Candida albicans to switch its morphology from yeast to filaments, known as polymorphism, may bias the methods used in microbial quantification. Here, we compared the quantification methods [cell/mL, colony forming units (CFU)/mL, and the number of nuclei estimated by viability polymerase chain reaction (vPCR)] of three strains of C. albicans (one reference strain and two clinical isolates) grown as yeast, filaments, and biofilms. Metabolic activity (XTT assay) was also used for biofilms. Comparisons between the methods were evaluated by agreement analyses [Intraclass and Concordance Correlation Coefficients (ICC and CCC, respectively) and Bland-Altman Plot] and Pearson Correlation (α = 0.05). Principal Component Analysis (PCA) was employed to visualize the similarities and differences between the methods. Results demonstrated a lack of agreement between all methods irrespective of fungal morphology/growth, even when a strong correlation was observed. Bland-Altman plot also demonstrated proportional bias between all methods for all morphologies/growth, except between CFU/mL X vPCR for yeasts and biofilms. For all morphologies, the correlation between the methods were strong, but without linear relationship between them, except for yeast where vPCR showed weak correlation with cells/mL and CFU/mL. XTT moderately correlated with CFU/mL and vPCR and weakly correlated with cells/mL. For all morphologies/growth, PCA showed that CFU/mL was similar to cells/mL and vPCR was distinct from them, but for biofilms vPCR became more similar to CFU/mL and cells/mL while XTT was the most distinct method. As conclusions, our investigation demonstrated that CFU/mL underestimated cells/mL, while vPCR overestimated both cells/mL and CFU/mL, and that the methods had poor agreement and lack of linear relationship, irrespective of C. albicans morphology/growth.1.