Defense Mechanism Of Bacteria Research Articles

Sub-MIC antibiotics increased the fitness cost of CRISPR-Cas in Acinetobacter baumannii.

The escalating prevalence of bacterial resistance, particularly multidrug-resistant bacteria like Acinetobacter baumannii, has become a significant global public health concern. The CRISPR-Cas system, a crucial defense mechanism in bacteria against foreign genetic elements, provides a competitive advantage. Type I-Fb and Type I-Fa are two subtypes of CRISPR-Cas systems that were found in A. baumannii, and the I-Fb CRISPR-Cas system regulates antibiotic resistance in A. baumannii. However, it is noteworthy that a majority of clinical isolates of A. baumannii lack or have incomplete CRISPR-Cas systems and most of them are multidrug-resistant. In light of this, our study aimed to examine the impact of antibiotic pressure on the fitness cost of the I-Fb CRISPR-Cas system in A. baumannii. In the study, we conducted in vitro competition experiments to investigate the influence of sub-minimum inhibitory concentration (sub-MIC) on the CRISPR-Cas systems' fitness cost in A. baumannii. We found that the fitness cost of the CRISPR-Cas system was increased under sub-MIC conditions. The expression of CRISPR-Cas-related genes was decreased, while the conjugation frequency was increased in AB43 under sub-MIC conditions. Through metabolomic analysis, we identified that sub-MIC conditions primarily affected energy metabolism pathways. In particular, we observed increased carbon metabolism, nitrogen metabolism, and intracellular ATP. Notably, the CRISPR-Cas system demonstrated resistance to the efflux pump-mediated resistance. Furthermore, the expression of efflux pump-related genes was increased under sub-MIC conditions. Our findings suggest that the I-Fb CRISPR-Cas system confers a significant competitive advantage in A. baumanni. However, under sub-MIC conditions, its function and the ability to inhibit the energy required for efflux pumps are reduced, resulting in an increased fitness cost and loss of competitive advantage.

Why Phage Can't be the End of Superbug on its Own?

The emergence of ultra-resistant superbug due to the widespread use and even abuse of antibiotics has caused people to start dying from common wound infections. It has become a global consensus to crack the superbug problem as soon as possible. In this battle, a virus called "phage" discovered a century ago has become a research hotspot again because of its characteristic of engulfing bacteria. Especially, the emergence of "phage display" technology has made the targeted evolution of viruses possible, and phage has become a potential weapon against superbug. By reviewing the discovery history of phage, dissecting the relationship between phage and bacteria as well as the molecular mechanism of interaction, this paper found that phage and bacteria are not in a simple parasitic relationship with each other, and that bacteria will evolve various active and passive defense mechanisms against phage infection, while phage will further evolve their responses against the various defense mechanisms of bacteria. In this invasion-defense-counter-defense arms race, phage and bacteria interact with each other and coexist in co-evolution. Phage not only indirectly affects human health by influencing the pathogenicity of bacteria, but also directly affects human health by interacting with human immune cells. The specificity and replication variability of phage, the large number of phage-bacteria, the complexity of phage-bacteria interactions and their unknown evolutionary mechanisms, and the insecurity of phage entry into the human body also contribute to the fact that the efficacy of phagotherapy, which has a century-long history, has always been uncertain and not reliable for large-scale application. Therefore, this study concludes that phage cannot defeat superbug alone, providing a new way of thinking about how humans can finally overcome the superbug problem.

Polystyrene nanoparticles induce biofilm formation in Pseudomonas aeruginosa

In recent years, micro/nanoplastics have garnered widespread attention due to their ecological risks. In this study, we investigated the effects of polystyrene nanoparticles (PS-NPs) of different sizes on the growth and biofilm formation of Pseudomonas aeruginosa PAO1. The results demonstrated that exposure to certain concentrations of PS-NPs significantly promoted bacterial biofilm formation. Meanwhile, we comprehensively revealed its mechanism whereby PS-NPs induced oxidative stress and altered bacterial membrane permeability by contacting or penetrating bacterial membranes. To counteract the stimulation by PS-NPs and reduce their toxicity, bacteria enhanced biofilm formation by upregulating the expression of biofilm-related genes, increasing EPS and virulence factors secretion, and enhancing bacterial motility through the participation of the quorum sensing (QS) system. Additionally, we also found that exposure to PS-NPs enhanced bacterial antibiotic resistance, posing a challenge to antimicrobial therapy. Our study reveals the toxic effects of nanoplastics and the defense mechanisms of bacteria, which has important implications for the risk assessment and management of environmental nanoplastics.

Long-term exposure of polytetrafluoroethylene-nanoplastics on the nitrogen removal and extracellular polymeric substances in sequencing batch reactor

The impact of polytetrafluoroethylene-nanoplastics (PTFE-NPs) on biological sewage disposal was delved, containing nitrogen remotion, microbiological activity and composition of extracellular polymer (EPS). The addition of PTFE-NPs reduced the removal efficiencies of chemical oxygen demand (COD) and ammonia nitrogen (NH4+-N) by 3.43 % and 2.35 %, respectively. In comparison with no PTFE-NPs, the specific oxygen uptake rate (SOUR), specific ammonia oxidation rate (SAOR), specific nitrite oxidation rate (SNOR) and specific nitrate reduction rate (SNRR) decreased by 65.26 %, 65.24 %, 41.77 % and 54.56 %, respectively. The PTFE-NPs inhibited the activities of nitrobacteria and denitrobacteria. It was worth noting that, nitrite oxidized bacterium was more resistant to adverse environments than ammonia oxidizing bacterium. Compared with no PTFE-NPs, the reactive oxygen species (ROS) content and lactate dehydrogenase (LDH) grew by 130 % and 50 % under PTFE-NPs pressure. The appearance of PTFE-NPs affected the normal function of microorganisms by inducing endocellular oxidative stress and destroying the completeness of the cytomembrane. The protein (PN) and polysaccharide (PS) levels in loosely bound EPS (LB-EPS) and tightly bound EPS (TB -EPS) increased by 4.96, 0.70, 3.07 and 0.71 mg g−1 VSS, under PTFE-NPs. Meanwhile, the PN/PS ratios of LB-EPS and TB -EPS increased from 6.18 and 6.41–11.04 and 9.29, respectively. The LB-EPS might provide sufficient binding sites for PTFE-NPs adsorption due to its loose and porous structure. The defense mechanism of bacteria against PTFE-NPs was mainly the PN in loosely bound EPS. Moreover, the functional groups referred to the complexation of EPS with PTFE-NPs were mainly related to N-H, CO, and C-N in proteins and O-H in polysaccharides.

Genome Editing Approaches with CRISPR/Cas9 for Cancer Treatment: Critical Appraisal of Preclinical and Clinical Utility, Challenges, and Future Research.

The increasing burden on human malignant diseases became a major concern for healthcare practitioners, that must deal with tumor relapse and the inability to efficiently treat metastasis, in addition to side effects. Throughout the decades, many therapeutic strategies have been employed to improve the clinical outcomes of cancer patients and great efforts have been made to develop more efficient and targeted medicines. The malignant cell is characterized by genetic and epigenetic modifications, therefore targeting those specific drivers of carcinogenesis is highly desirable. Among the genome editing technologies, CRISPR/Cas9 stood as a promising candidate for cancer treatment alternatives, due to its low complexity design. First described as a defense mechanism of bacteria against invading foreign DNA, later it was shown that CRISPR components can be engineered to target specific DNA sequences in a test tube, a discovery that was awarded later with the Nobel Prize in chemistry for its rapid expansion as a reliable genome editing tool in many fields of research, including medicine. The present paper aims of describing CRISPR/Cas9 potential targets for malignant disorders, and the approaches used for achieving this goal. Aside from preclinical studies, we also present the clinical trials that use CRISPR-based technology for therapeutic purposes of cancer. Finally, a summary of the presented studies adds a more focused view of the therapeutic value CRISPR/Cas9 holds and the associated shortcomings.

Comparative proteomic analysis reveals metabolic variability of probiotic Enterococcus durans during aerobic and anaerobic cultivation

The variation in the bioavailability of oxygen constitutes the environmental conditions found by bacteria in their passage through the host gastro-intestinal tract. Given the importance of oxygen in the defense mechanism of bacteria, it is important to understand how bacteria respond to this stress at a metabolic level. The probiotic strain Enterococcus durans LAB18S was cultivated under aerobic and anaerobic conditions using prebiotic oligosaccharides as carbon source. The whole cell proteome and secretome were analyzed through label-free quantitative proteomics approach. The results showed that the LAB18S isolate when grown with fructo-oligosacchrides (FOS) showed a higher number of differentially expressed proteins compared to samples with galacto-oligosaccharides (GOS) or glucose. Clinically important enzymes for the treatment of cancer, L-asparaginase and arginine deiminase, were overexpressed when the isolate was cultured in FOS. In addition, the absence of oxygen induced the strain to produce proteins related to cell multiplication, cell wall integrity and resistance, and H2O2 detoxification. This study showed that E. durans LAB18S growing on FOS was stimulated to produce clinically important biomolecules, including proteins that have been investigated as potential antineoplastic agents.Significance: The probiotic strain E. durans LAB18S produce clinically relevant enzymes for the treatment of cancer when cultivated in symbiosis with fructo-oligosacchrides (FOS). In addition, proteins associated with cellular multiplication, cell wall integrity and resistance, and H2O2 detoxification were induced under anaerobic growth. These characteristics could be relevant to support maintenance of intestinal health.

CRISPR/Cas9 Technology as an Emerging Tool for Targeting Amyotrophic Lateral Sclerosis (ALS).

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein-9 nuclease (Cas9) is a genome editing tool that has recently caught enormous attention due to its novelty, feasibility, and affordability. This system naturally functions as a defense mechanism in bacteria and has been repurposed as an RNA-guided DNA editing tool. Unlike zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), CRISPR/Cas9 takes advantage of an RNA-guided DNA endonuclease enzyme, Cas9, which is able to generate double-strand breaks (DSBs) at specific genomic locations. It triggers cellular endogenous DNA repair pathways, contributing to the generation of desired modifications in the genome. The ability of the system to precisely disrupt DNA sequences has opened up new avenues in our understanding of amyotrophic lateral sclerosis (ALS) pathogenesis and the development of new therapeutic approaches. In this review, we discuss the current knowledge of the principles and limitations of the CRISPR/Cas9 system, as well as strategies to improve these limitations. Furthermore, we summarize novel approaches of engaging the CRISPR/Cas9 system in establishing an adequate model of neurodegenerative disease and in the treatment of SOD1-linked forms of ALS. We also highlight possible applications of this system in the therapy of ALS, both the inherited type as well as ALS of sporadic origin.

Physiological and transcriptomic analyses reveal mechanistic insight into the adaption of marine Bacillus subtilis C01 to alumina nanoparticles.

An increasing number of studies have investigated the effects of nanoparticles (NPs) on microbial systems; however, few existing reports have focused on the defense mechanisms of bacteria against NPs. Whether secondary metabolism biosynthesis is a response to NP stress and contributes to the adaption of bacteria to NPs is unclear. Here, a significant induction in the surfactin production and biofilm formation were detected by adding Al2O3 NPs to the B. subtilis fermentation broth. Physiological analysis showed that Al2O3 NP stress could also affect the cell and colony morphogenesis and inhibit the motility and sporulation. Exogenously adding commercial surfactin restored the swarming motility. Additionally, a suite of toxicity assays analyzing membrane damage, cellular ROS generation, electron transport activity and membrane potential was used to determine the molecular mechanisms of toxicity of Al2O3 NPs. Furthermore, whole transcriptomic analysis was used to elucidate the mechanisms of B. subtilis adaption to Al2O3 NPs. These results revealed several mechanisms by which marine B. subtilis C01 adapt to Al2O3 NPs. Additionally, this study broadens the applications of nanomaterials and describes the important effects on secondary metabolism and multicellularity regulation by using Al2O3 NPs or other nano-products.

Crystal Structure of the Csm1 Subunit of the Csm Complex and Its Single-Stranded DNA-Specific Nuclease Activity

The CRISPR-Cas system is the RNA-guided immune defense mechanism in bacteria and archaea. Csm1 belongs to the Cas10 family, which is the common signature protein of the type III system. Csm1 is the largest subunit of the Csm interference complex in the type III-A subtype, which targets foreign DNA or RNA. Here, we report crystallographic and biochemical analyses of Thermococcus onnurineus Csm1, revealing a five-domain organization and single-stranded DNA (ssDNA)-specific nuclease activity associated with the N-terminal HD domain. This domain folds into permuted secondary structures in comparison with the HD domain of Cas3 and contains all the catalytically important residues. It exhibited both endo- and exonuclease activities in an Ni(2+) or Mn(2+)-dependent manner. The narrow width of the active-site cleft appears to restrict the substrate specificity to ssDNA and thus to prevent Csm1 from cleaving double-stranded chromosomal DNA. These data suggest that Csm1 may function in DNA interference by the Csm effector complex.

CrisprGE: a central hub of CRISPR/Cas-based genome editing.

CRISPR system is a powerful defense mechanism in bacteria and archaea to provide immunity against viruses. Recently, this process found a new application in intended targeting of the genomes. CRISPR-mediated genome editing is performed by two main components namely single guide RNA and Cas9 protein. Despite the enormous data generated in this area, there is a dearth of high throughput resource. Therefore, we have developed CrisprGE, a central hub of CRISPR/Cas-based genome editing. Presently, this database holds a total of 4680 entries of 223 unique genes from 32 model and other organisms. It encompasses information about the organism, gene, target gene sequences, genetic modification, modifications length, genome editing efficiency, cell line, assay, etc. This depository is developed using the open source LAMP (Linux Apache MYSQL PHP) server. User-friendly browsing, searching facility is integrated for easy data retrieval. It also includes useful tools like BLAST CrisprGE, BLAST NTdb and CRISPR Mapper. Considering potential utilities of CRISPR in the vast area of biology and therapeutics, we foresee this platform as an assistance to accelerate research in the burgeoning field of genome engineering.Database URL: http://crdd.osdd.net/servers/crisprge/.

Efficient Gene Disruption in Diverse Strains of Toxoplasma gondii Using CRISPR/CAS9

ABSTRACTToxoplasma gondii has become a model for studying the phylum Apicomplexa, in part due to the availability of excellent genetic tools. Although reverse genetic tools are available in a few widely utilized laboratory strains, they rely on special genetic backgrounds that are not easily implemented in natural isolates. Recent progress in modifying CRISPR (clustered regularly interspaced short palindromic repeats), a system of DNA recognition used as a defense mechanism in bacteria and archaea, has led to extremely efficient gene disruption in a variety of organisms. Here we utilized a CRISPR/CAS9-based system with single guide RNAs to disrupt genes in T. gondii. CRISPR/CAS9 provided an extremely efficient system for targeted gene disruption and for site-specific insertion of selectable markers through homologous recombination. CRISPR/CAS9 also facilitated site-specific insertion in the absence of homology, thus increasing the utility of this approach over existing technology. We then tested whether CRISPR/CAS9 would enable efficient transformation of a natural isolate. Using CRISPR/CAS9, we were able to rapidly generate both rop18 knockouts and complemented lines in the type I GT1 strain, which has been used for forward genetic crosses but which remains refractory to reverse genetic approaches. Assessment of their phenotypes in vivo revealed that ROP18 contributed a greater proportion to acute pathogenesis in GT1 than in the laboratory type I RH strain. Thus, CRISPR/CAS9 extends reverse genetic techniques to diverse isolates of T. gondii, allowing exploration of a much wider spectrum of biological diversity.

Global Metabolic Response of Enterococcus faecalis to Oxygen

Oxygen and oxidative stress have become relevant components in clarifying the mechanism that weakens bacterial cells in parallel to the mode of action of bactericidal antibiotics. Given the importance of oxidative stress in the overall defense mechanism of bacteria and their apparent role in the antimicrobial mode of action, it is important to understand how bacteria respond to this stress at a metabolic level. The aim of this study was to determine the impact of oxygen on the metabolism of the facultative anaerobe Enterococcus faecalis using continuous culture, metabolomics, and (13)C enrichment of metabolic intermediates. When E. faecalis was rapidly transitioned from anaerobic to aerobic growth, cellular metabolism was directed toward intracellular glutathione production and glycolysis was upregulated 2-fold, which increased the supply of critical metabolite precursors (e.g., glycine and glutamate) for sulfur metabolism and glutathione biosynthesis as well as reducing power for cellular respiration in the presence of hemin. The ultimate metabolic response of E. faecalis to an aerobic environment was the upregulation of fatty acid metabolism and benzoate degradation, which was linked to important changes in the bacterial membrane composition as evidenced by changes in membrane fatty acid composition and the reduction of membrane-associated demethylmenaquinone. These key metabolic pathways associated with the response of E. faecalis to oxygen may represent potential new targets to increase the susceptibility of this bacterium to bactericidal drugs.

PH‐Dependent Coordination of AgI Ions by Histidine: Experiment, Theory, and a Model for SilE

Artificial implants and biomaterials lack the natural defense system of our body and, thus, have to be protected from bacterial adhesion and biofilm formation. In addition to the increasing number of implanted objects, the resistance of bacteria is also an important problem. Silver ions are well-known for their antimicrobial properties, yet not a lot is known about their mode of action. Silver is expected to interact on many levels, thus the development of silver resistance is very difficult. Nevertheless, some bacteria are able to resist silver, even at higher concentrations. One such defense mechanism of bacteria against heavy-metal intoxication includes an efflux system. SilE, a periplasmic silver-binding protein that is involved in this defense mechanism, has been shown to possess numerous histidine functions, which strongly bind to silver atoms, as demonstrated by ourselves previously. Herein, we address the question of how histidine binds to silver ions as a function of pH value. This property is important because the local proton concentration in cells varies. Thus, we solved the crystal structures of histidine-silver complexes at different pH values and also investigated the influence of the amino-acid configuration. These results were completed by DFT calculations on the binding strength and packing effects and led to the development of a model for the mode of action of SilE.