Microbial Cell最新文献

Microbial biofilms can cause chronic infection. In the clinical setting, the biofilm-related infections usually persist and reoccur; the main reason is the increased antibiotic resistance of biofilms. Traditional antibiotic therapy is not effective and might increase the threat of antibiotic resistance to public health. Therefore, it is urgent to study the tolerance and resistance mechanism of biofilms to antibiotics and find effective therapies for biofilm-related infections. The tolerance mechanism and host reaction of biofilm to antibiotics are reviewed, and bacterial biofilm related diseases formed by human pathogens are discussed thoroughly. The review also explored the role of biofilms in the development of bacterial resistance mechanisms and proposed therapeutic intervention strategies for biofilm related diseases.

Salmonella enterica subsp. enterica sv. Typhimurium str. LT2 (hereafter S. Typhimurium) synthesizes adenosylcobalamin (AdoCbl, CoB₁₂) de novo only under anoxic conditions, but it can assemble the lower ligand loop (a.k.a. the nucleotide loop) and can form the unique C-Co bond present in CoB₁₂ in the presence or absence of molecular oxygen. During studies of nucleotide loop assembly in S. Typhimurium, we noticed that the growth of this bacterium could be arrested by the lower ligand nucleobase, namely 5,6-dimethylbenzimidazole (DMB). Here we report in vitro and in vivo evidence that shows that the structural similarity of DMB to the isoalloxazine moiety of flavin cofactors causes its deleterious effect on cell growth. We studied DMB inhibition of the housekeeping flavin dehydrogenase (Fre) and three flavoenzymes that initiate the catabolism of tricarballylate, succinate or D-alanine in S. Typhimurium. Notably, while growth with tricarballylate was inhibited by 5-methyl-benzimidazole (5-Me-Bza) and DMB, growth with succinate or glycerol was arrested by DMB but not by 5-Me-Bza. Neither unsubstituted benzimidazole nor adenine inhibited growth of S. Typhimurium at DMB inhibitory concentrations. Whole genome sequencing analysis of spontaneous mutant strains that grew in the presence of inhibitory concentrations of DMB identified mutations effecting the cycA (encodes D-Ala/D-Ser transporter) and dctA (encodes dicarboxylate transporter) genes and in the coding sequence of the tricarballylate transporter (TcuC), suggesting that increased uptake of substrates relieved DMB inhibition. We discuss two possible mechanisms of inhibition by DMB.

In yeast, Elongator-dependent tRNA modifications are regulated by the Kti11•Kti13 dimer and hijacked for cell killing by zymocin, a tRNase ribotoxin. Kti11 (alias Dph3) also controls modification of elongation factor 2 (EF2) with diphthamide, the target for lethal ADP-ribosylation by diphtheria toxin (DT). Diphthamide formation on EF2 involves four biosynthetic steps encoded by the DPH1-DPH7 network and an ill-defined KTI13 function. On further examining the latter gene in yeast, we found that kti13Δ null-mutants maintain unmodified EF2 able to escape ADP-ribosylation by DT and to survive EF2 inhibition by sordarin, a diphthamide-dependent antifungal. Consistently, mass spectrometry shows kti13Δ cells are blocked in proper formation of amino-carboxyl-propyl-EF2, the first diphthamide pathway intermediate. Thus, apart from their common function in tRNA modification, both Kti11/Dph3 and Kti13 share roles in the initiation step of EF2 modification. We suggest an alias KTI13/DPH8 nomenclature indicating dual-functionality analogous to KTI11/DPH3.

Enhanced levels of acetic acid reduce the activity of yeast strains employed for industrial fermentation-based applications. Therefore, unraveling the genetic factors underlying the regulation of the tolerance and sensitivity of yeast towards acetic acid is imperative for optimising various industrial processes. In this communication, we have attempted to decipher the acetic acid stress response of the previously reported acetic acid-sensitive histone mutants. Revalidation using spot-test assays and growth curves revealed that five of these mutants, viz., H3K18Q, H3S28A, H3K42Q, H3Q68A, and H3F104A, are most sensitive towards the tested acetic acid concentrations. These mutants demonstrated enhanced acetic acid stress response as evidenced by the increased expression levels of AIF1, reactive oxygen species (ROS) generation, chromatin fragmentation, and aggregated actin cytoskeleton. Additionally, the mutants exhibited active cell wall damage response upon acetic acid treatment, as demonstrated by increased Slt2-phosphorylation and expression of cell wall integrity genes. Interestingly, the mutants demonstrated increased sensitivity to cell wall stress-causing agents. Finally, screening of histone H3 N-terminal tail truncation mutants revealed that the tail truncations exhibit general sensitivity to acetic acid stress. Some of these N-terminal tail truncation mutants viz., H3 [del 1-24], H3 [del 1-28], H3 [del 9-24], and H3 [del 25-36] are also sensitive to cell wall stress agents such as Congo red and caffeine suggesting that their enhanced acetic acid sensitivity may be due to cell wall stress induced by acetic acid.

Metallothionein (MT), which is a small metal-binding protein with cysteine-rich motifs, functions in the detoxification of heavy metals in a variety of organisms. Even though previous studies suggest that MT is involved in the tolerance mechanisms against nitrosative stress induced by toxic levels of nitric oxide (NO) in mammalian cells, the physiological functions of MT in relation to NO have not been fully understood. In this study, we analyzed the functions of MT in nitrosative stress tolerance in the yeast Saccharomyces cerevisiae. Our phenotypic analyses showed that deletion or overexpression of the MT-encoding gene, CUP1, led to higher sensitivity or tolerance to nitrosative stress in S. cerevisiae cells, respectively. We further examined whether the yeast MT Cup1 in the cell-free lysate scavenges NO. These results showed that the cell-free lysate containing a higher level of Cup1 degraded NO more efficiently. On the other hand, the transcription level of CUP1 was not affected by nitrosative stress treatment. Our findings suggest that the yeast MT Cup1 contributes to nitrosative stress tolerance, possibly as a constitutive rather than an inducible defense mechanism.

Caspase 3 activation is a hallmark of cell death and there is a strong correlation between elevated protease activity and evolving pathology in neurodegenerative disease, such as amyotrophic lateral sclerosis (ALS). At the cellular level, ALS is characterized by protein aggregates and inclusions, comprising the RNA binding protein TDP-43, which are hypothesized to trigger pathogenic activation of caspase 3. However, a growing body of evidence indicates this protease is essential for ensuring cell viability during growth, differentiation and adaptation to stress. Here, we explored whether caspase 3 acts to disperse toxic protein aggregates, a proteostasis activity first ascribed to the distantly related yeast metacaspase ScMCA1. We demonstrate that human caspase 3 can functionally substitute for the ScMCA1 and limit protein aggregation in yeast, including TDP-43 inclusions. Proteomic analysis revealed that disrupting caspase 3 in the same yeast substitution model resulted in detrimental TDP-43/mitochondrial protein associations. Similarly, suppression of caspase 3, in either murine or human skeletal muscle cells, led to accumulation of TDP-43 aggregates and impaired mitochondrial function. These results suggest that caspase 3 is not inherently pathogenic, but may act as a compensatory proteostasis factor, to limit TDP-43 protein inclusions and protect organelle function in aggregation related degenerative disease.

Staphylococcus aureus is a major human pathogen that utilises many surface-associated and secreted proteins to form biofilms and cause disease. However, our understanding of these processes is limited by challenges of using fluorescent protein reporters in their native environment, because they must be exported and fold correctly to become fluorescent. Here, we demonstrate the feasibility of using the monomeric superfolder GFP (msfGFP) exported from S. aureus. By fusing msfGFP to signal peptides for the Secretory (Sec) and Twin Arginine Translocation (Tat) pathways, the two major secretion pathways in S. aureus, we quantified msfGFP fluorescence in bacterial cultures and cell-free supernatant from the cultures. When fused to a Tat signal peptide, we detected msfGFP fluorescence inside but not outside bacterial cells, indicating a failure to export msfGFP. However, when fused to a Sec signal peptide, msfGFP fluorescence was present outside cells, indicating successful export of the msfGFP in the unfolded state, followed by extracellular folding and maturation to the photoactive state. We applied this strategy to study coagulase (Coa), a secreted protein and a major contributor to the formation of a fibrin network in S. aureus biofilms that protects bacteria from the host immune system and increases attachment to host surfaces. We confirmed that a genomically integrated C-terminal fusion of Coa to msfGFP does not impair the activity of Coa or its localisation within the biofilm matrix. Our findings demonstrate that msfGFP is a good candidate fluorescent reporter to consider when studying proteins secreted by the Sec pathway in S. aureus.

The bacterial stringent response and its effector alarmone guanosine penta- or tetra - phosphates (p)ppGpp are vital for bacterial tolerance and survival of various stresses in environments (including antibiotics) and host cells (virulence). (p)ppGpp does so by binding to its numerous target proteins and reprograming bacterial transcriptome to tune down the synthesis of nucleotides and rRNA/tRNA, and up-regulate amino acid biosynthesis genes. Recent identification of more novel (p)ppGpp direct binding proteins in Escherichia coli and their deep studies have unveiled unprecedented details of how (p)ppGpp coordinates the nucleotide and amino acid metabolic pathways upon stringent response; however, the mechanistic link between nucleotide and amino acid metabolisms remains still incompletely understood. Here we propose the metabolite ribose 5'-phosphate as the key link between nucleotide and amino acid metabolisms and a working model integrating both the transcriptional and metabolic effects of (p)ppGpp on E. coli physiological adaptation during the stringent response.

Toll-like receptor 3 (TLR3) is an innate immune receptor that recognizes double-stranded RNA (dsRNA) and induces inflammation in immune and normal cells to initiate anti-microbial responses. TLR3 acts also as a death receptor only in cancer cells but not in their normal counterparts, making it an attractive target for cancer therapies. To date, all of the TLR3-activating dsRNAs used at preclinical or clinical stages have major drawbacks such as structural heterogeneity, toxicity, and lack of specificity and/or efficacy. We conducted the discovery process of a new family of TLR3 agonists that are chemically manufactured on solid-phase support and perfectly defined in terms of sequence and size. A stepwise discovery process was performed leading to the identification of TL-532, a 70 base pair dsRNA that is potent without transfection reagent and is highly specific for TLR3 without activating other innate nucleic sensors such as RIG-I/MDA5, TLR7, TLR8, and TLR9. TL-532 induces inflammation in murine RAW264.7 myeloid macrophages, in human NCI-H292 lung cancer cells, and it promotes immunogenic apoptosis in tumor cells in vitro and ex vivo without toxicity towards normal primary cells. In conclusion, we identified a novel TLR3 agonist called TL-532 that has promising anticancer properties.

Autophagy promotes or inhibits cell death depending on the environment and cell type. Our previous findings suggested that Atg1 is genetically involved in the regulation of Pmk1 MAPK in fission yeast. Here, we showed that Δatg1 displays lower levels of Pmk1 MAPK phosphorylation than did the wild-type (WT) cells upon treatment with a 1,3-β-D-glucan synthase inhibitor micafungin or CaCl₂, both of which activate Pmk1. Moreover, the overproduction of Atg1, but not that of the kinase inactivating Atg1^D193A activates Pmk1 without any extracellular stimuli, suggesting that Atg1 may promote Pmk1 MAPK signaling activation. Notably, the overproduction of Atg1 induces a toxic effect on the growth of WT cells and the deletion of Pmk1 failed to suppress the cell death induced by Atg1, indicating that the Atg1-mediated cell death requires additional mechanism(s) other than Pmk1 activation. Moreover, atg1 gene deletion induces tolerance to micafungin and CaCl₂, whereas pmk1 deletion induces severe sensitivities to these compounds. The Δatg1Δpmk1 double mutants display intermediate sensitivities to these compounds, showing that atg1 deletion partly suppressed growth inhibition induced by Δpmk1. Thus, Atg1 may act to promote cell death upon micafungin and CaCl₂ stimuli regardless of Pmk1 MAPK activity. Since micafungin and CaCl₂ are intracellular calcium inducers, our data reveal a novel role of the autophagy regulator Atg1 to induce cell death upon calcium overload independent of its role in Pmk1 MAPK activation.