Elucidation of the Gemcitabine Transporters of Escherichia coli K-12 and Gamma-Proteobacteria Linked to Gemcitabine-Related Chemoresistance.

Gemcitabine (2',2'-difluoro-2'-deoxycytidine), a widely used anticancer drug, is considered a gold standard in treating aggressive pancreatic cancers. Gamma-proteobacteria that colonize the pancreatic tumors contribute to chemoresistance against gemcitabine by metabolizing the drug to a less active and deaminated form. The gemcitabine transporters of these bacteria are unknown to date. Furthermore, there is no complete knowledge of the gemcitabine transporters in Escherichia coli or any other related proteobacteria. In this study, we investigate the complement of gemcitabine transporters in E. coli K-12 and two common chemoresistance-related bacteria (Klebsiella pneumoniae and Citrobacter freundii). We found that E. coli K-12 has two high-affinity gemcitabine transporters with distinct specificity properties, namely, NupC and NupG, whereas the gemcitabine transporters of C. freundii and K. pneumoniae include the NupC and NupG orthologs, functionally indistinguishable from their counterparts, and, in K. pneumoniae, one additional NupC variant, designated KpNupC2. All these bacterial transporters have a higher affinity for gemcitabine than their human counterparts. The highest affinity (KM 2.5-3.0 µΜ) is exhibited by NupGs of the bacteria-specific nucleoside-H+ symporter (NHS) family followed by NupCs (KM 10-13 µΜ) of the concentrative nucleoside transporter (CNT) family, 15-100 times higher than the affinities reported for the human gemcitabine transporter hENT1/SLC29A1, which is primarily associated with gemcitabine uptake in the pancreatic adenocarcinoma cells. Our results offer a basis for further insight into the role of specific bacteria in drug availability within tumors and for understanding the structure-function differences of bacterial and human drug transporters.

Functional analysis of Escherichia coli K12 toxin-antitoxin systems as novel drug targets using a network biology approach.

Bacterial resistance to various drugs and antibiotics has become a significant issue in the fight against infectious diseases. Due to the presence of diverse toxin-antitoxin (TA) systems, bacteria undergo adaptive metabolic alterations and can tolerate the effects of drugs and antibiotics. Bacterial TA systems are unique and can be therapeutic targets for developing new antimicrobial agents, owing to their ability to influence bacterial fate. With this background, our study aims to identify novel drug targets against Escherichia coli K12 MG1655 antitoxin using homology modelling approach. In this study, the protein-protein interaction network of 87 E. coli K12 MG1655 TA systems identified through literature mining was screened for the identification of hub proteins. The model evaluation, assessment, and homology modelling of the hub proteins were evaluated. Furthermore, computer-aided mathematical models of selected phytochemicals have been tested against the identified hub proteins. The TA system was functionally enriched in regulation of cell growth, negative regulation of cell growth, regulation of mRNA stability, mRNA catabolic process and RNA phosphodiester bond hydrolysis. RelE, RelB, MazE, MazF, MqsR, MqsA, and YoeB were identified as hub proteins. The robustness and superior quality of the RelB and MazE modelled structure were discovered by model evaluation, quality assessment criteria, and homology modelling of hub proteins. Clorobiocin was found to be a strong inhibitor by docking these modelled structures. Clorobiocin could be utilized as an antibacterial agent against multidrug resistant E. coli which may inactivate antitoxins and cause programmed cell death.

Inactivation of Escherichia coli K12 on raw almonds using supercritical carbon dioxide and thyme oil.

Raw almonds could be contaminated by pathogens, but the current pasteurization practice using propylene oxide in the U.S. has flammability and carcinogenicity concerns. Supercritical carbon dioxide (scCO2) is a water-free technology and is a solvent of essential oils that are effective antimicrobial preservatives. The objective of this study was to investigate the possibility of combining scCO2 and thyme oil (TO) to reduce Escherichia coli K12 inoculated on raw almonds. Raw almonds inoculated with ∼6 log CFU/g E. coli K12 were batch-treated with scCO2 alone or the combination of presoaking in pure TO followed by scCO2 treatments at different combinations of temperature, pressure, and duration. Compared to scCO2 alone treatments, the combination of TO and scCO2 treatments significantly improved the disinfection effectiveness. Temperature had the most significant effect on the log reduction. At 70 °C, the log reduction by the combination treatment was over 4-log CFU/g and the maximum reduction was 5.16 log CFU/g. The findings suggest that the combination of TO and scCO2 may be a potential water-free technology to meet the requirement of over 4-log reduction of target microorganism for almond and other tree nut products.

LptB-LptF coupling mediates the closure of the substrate-binding cavity in the LptB2 FGC transporter through a rigid-body mechanism to extract LPS.

Lipopolysaccharides (LPS) are essential envelope components in many Gram-negative bacteria and provide intrinsic resistance to antibiotics. LPS molecules are synthesized in the inner membrane and then transported to the cell surface by the LPS transport (Lpt) machinery. In this system, the ATP-binding cassette (ABC) transporter LptB2 FGC extracts LPS from the inner membrane and places it onto a periplasmic protein bridge through a poorly understood mechanism. Here, we show that residue E86 of LptB is essential for coupling the function of this ATPase to that of its partners LptFG, specifically at the step where ATP binding drives the closure of the LptB dimer and the collapse of the LPS-binding cavity in LptFG that moves LPS to the Lpt periplasmic bridge. We also show that defects caused by changing residue E86 are suppressed by mutations altering either LPS structure or transmembrane helices in LptG. Furthermore, these suppressors also fix defects in the coupling helix of LptF, but not of LptG. Together, these results support a transport mechanism in which the ATP-driven movements of LptB and those of the substrate-binding cavity in LptFG are bi-directionally coordinated through the rigid-body coupling, with LptF's coupling helix being important in coordinating cavity collapse with LptB dimerization.

Reconstruction of transcriptional regulatory networks of Fis and H-NS in Escherichia coli from genome-wide data analysis.

Fis (Factor for inversion stimulation) and H-NS (Histone-like nucleoid-structuring protein) are two well-known nucleoid-associated proteins (NAPs) in proteobacteria, which play crucial roles in genome organization and transcriptional regulation. We performed RNA-sequencing to identify genes regulated by these NAPs. Study reveals that Fis and H-NS affect expression of 462 and 88 genes respectively in Escherichia coli at mid-exponential growth phase. By integrating available ChIP-seq data, we identified direct and indirect regulons of Fis and H-NS proteins. Functional analysis reveals that Fis controls expression of genes involved in translation, oxidative phosphorylation, sugar metabolism and transport, amino acid metabolism, bacteriocin transport, cell division, two-component system, biofilm formation, pilus organization and lipopolysaccharide biosynthesis pathways. However, H-NS represses expression of genes in cell adhesion, recombination, biofilm formation and lipopolysaccharide biosynthesis pathways under mid-exponential growth condition. The current regulatory networks thus provide a global glimpse of coordinated regulatory roles for these two important NAPs.

MhpA Is a Hydroxylase Catalyzing the Initial Reaction of 3-(3-Hydroxyphenyl)Propionate Catabolism in Escherichia coli K-12.

Escherichia coli K-12 and some other strains have been reported to be capable of utilizing 3-(3-hydroxyphenyl)propionate (3HPP), one of the phenylpropanoids from lignin. Although other enzymes involved in 3HPP catabolism and their corresponding genes from its degraders have been identified, 3HPP 2-hydroxylase, catalyzing the first step of its catabolism, has yet to be functionally identified at biochemical and genetic levels. In this study, we investigated the function and characteristics of MhpA from E. coli strain K-12 (MhpAK-12). Gene deletion and complementation showed that mhpA was vital for its growth on 3HPP, but the mhpA deletion strain was still able to grow on 3-(2,3-dihydroxyphenyl)propionate (DHPP), the hydroxylation product transformed from 3HPP by MhpAK-12 MhpAK-12 was overexpressed and purified, and it was likely a polymer and tightly bound with an approximately equal number of moles of FAD. Using NADH or NADPH as a cofactor, purified MhpAK-12 catalyzed the conversion of 3HPP to DHPP at a similar efficiency. The conversion from 3HPP to DHPP by purified MhpAK-12 was confirmed using high-performance liquid chromatography and liquid chromatography-mass spectrometry. Bioinformatics analysis indicated that MhpAK-12 and its putative homologues belonged to taxa that were phylogenetically distant from functionally identified FAD-containing monooxygenases (hydroxylases). Interestingly, MhpAK-12 has approximately an extra 150 residues at its C terminus in comparison to its close homologues, but its truncated versions MhpAK-12400 and MhpAK-12480 (with 154 and 74 residues deleted from the C terminus, respectively) both lost their activities. Thus, MhpAK-12 has been confirmed to be a 3HPP 2-hydroxylase catalyzing the conversion of 3HPP to DHPP, the initial reaction of 3HPP degradation.IMPORTANCE Phenylpropionate and its hydroxylated derivatives resulted from lignin degradation ubiquitously exist on the Earth. A number of bacterial strains have the ability to grow on 3HPP, one of the above derivatives. The hydroxylation was thought to be the initial and vital step for its aerobic catabolism via the meta pathway. The significance of our research is the functional identification and characterization of the purified 3HPP 2-hydroxylase MhpA from Escherichia coli K-12 at biochemical and genetic levels, since this enzyme has not previously been expressed from its encoding gene, purified, and characterized in any bacteria. It will not only fill a gap in our understanding of 3HPP 2-hydroxylase and its corresponding gene for the critical step in microbial 3HPP catabolism but also provide another example of the diversity of microbial degradation of plant-derived phenylpropionate and its hydroxylated derivatives.

Regulatory role of CsqR (YihW) in transcription of the genes for catabolism of the anionic sugar sulfoquinovose (SQ) in Escherichia coli K-12.

The binding sites of YihW, an uncharacterized DeoR-family transcription factor (TF) of Escherichia coli K-12, were identified using Genomic SELEX screening at two closely located sites, one inside the spacer between the bidirectional transcription units comprising the yihUTS operon and the yihV gene, and another one upstream of the yihW gene itself. Recently the YihUTS and YihV proteins were identified as catalysing the catabolism of sulfoquinovose (SQ), a hydrolysis product of sulfoquinovosyl diacylglycerol (SQDG) derived from plants and other photosynthetic organisms. Gel shift assay in vitro and reporter assay in vivo indicated that YihW functions as a repressor for all three transcription units. De-repression of the yih operons was found to be under the control of SQ as inducer, but not of lactose, glucose or galactose. Furthermore, a mode of its cooperative DNA binding was suggested for YihW by atomic force microscopy. Hence, as a regulator of the catabolism of SQ, we renamed YihW as CsqR.

Dimethyl phthalate damaged the cell membrane of Escherichia coli K12.

Dimethyl phthalate (DMP), a phthalate ester (PAE), is a ubiquitous and organic pollutant. In this study, the toxicity of DMP to Escherichia coli K12 and its underlying mechanism were investigated. The results showed that DMP inhibited the growth of E. coli K12 and induced cell inactivation and/or death. DMP caused serious damage to the cell membrane of E. coli K12, and the damage increased with higher DMP concentrations. DMP exposure disrupted cell membranes, as evidenced by dose-dependent variations of cell structures, surface properties, and membrane compositions. Increases in the malondialdehyde (MDA) content indicated an increase in oxidative stress induced by DMP in E. coli K12. The activity of succinic dehydrogenase (SDH) was changed by DMP, which could affect energy metabolism in the membrane of E. coli K12. The expression levels of OmpA and OmpX were increased, and the expression levels of OmpF and OmpW were decreased, in E. coli K12 exposed to DMP. The toxicities of DMP to E. coli K12 could be ascribed to membrane disruption and oxidative stress-induced cell inactivation and/or death. The outcomes will shed new light on the assessment of the ecological effects of DMP.

Selenocompounds as Novel Antibacterial Agents and Bacterial Efflux Pump Inhibitors.

Bacterial multidrug resistance is becoming a growing problem for public health, due to the development and spreading of bacterial strains resistant to antimicrobials. In this study, the antibacterial and multidrug resistance reversing activity of a series of seleno-carbonyl compounds has been evaluated. The effects of eleven selenocompounds on bacterial growth were evaluated in Staphylococcus aureus, methicillin resistant S. aureus (MRSA), Enterococcus faecalis, Escherichia coli, and Chlamydia trachomatis D. The combination effect of compounds with antibiotics was examined by the minimum inhibitory concentration reduction assay. Their efflux pump (EP) inhibitory properties were assessed using real-time fluorimetry. Relative expressions of EP and quorum-sensing genes were studied by quantitative PCR. Results showed that a methylketone selenoester had remarkable antibacterial activity against Gram-positive bacteria and potentiated the activity of oxacillin in MRSA. Most of the selenocompounds showed significant anti-chlamydial effects. The selenoanhydride and the diselenodiester were active inhibitors of the AcrAB-TolC system. Based on these results it can be concluded that this group of selenocompounds can be attractive potential antibacterials and EP inhibitors. The discovery of new derivatives with a significant antibacterial activity as novel selenocompounds, is of high impact in the fight against resistant pathogens.

A Multiscale Agent-Based Model for the Investigation of E. coli K12 Metabolic Response During Biofilm Formation.

Bacterial biofilm formation is an organized collective response to biochemical cues that enables bacterial colonies to persist and withstand environmental insults. We developed a multiscale agent-based model that characterizes the intracellular, extracellular, and cellular scale interactions that modulate Escherichia coli MG1655 biofilm formation. Each bacterium's intracellular response and cellular state were represented as an outcome of interactions with the environment and neighboring bacteria. In the intracellular model, environment-driven gene expression and metabolism were captured using statistical regression and Michaelis-Menten kinetics, respectively. In the cellular model, growth, death, and type IV pili- and flagella-dependent movement were based on the bacteria's intracellular state. We implemented the extracellular model as a three-dimensional diffusion model used to describe glucose, oxygen, and autoinducer 2 gradients within the biofilm and bulk fluid. We validated the model by comparing simulation results to empirical quantitative biofilm profiles, gene expression, and metabolic concentrations. Using the model, we characterized and compared the temporal metabolic and gene expression profiles of sessile versus planktonic bacterial populations during biofilm formation and investigated correlations between gene expression and biofilm-associated metabolites and cellular scale phenotypes. Based on our in silico studies, planktonic bacteria had higher metabolite concentrations in the glycolysis and citric acid cycle pathways, with higher gene expression levels in flagella and lipopolysaccharide-associated genes. Conversely, sessile bacteria had higher metabolite concentrations in the autoinducer 2 pathway, with type IV pili, autoinducer 2 export, and cellular respiration genes upregulated in comparison with planktonic bacteria. Having demonstrated results consistent with in vitro static culture biofilm systems, our model enables examination of molecular phenomena within biofilms that are experimentally inaccessible and provides a framework for future exploration of how hypothesized molecular mechanisms impact bulk community behavior.

Efficacy of UV-TiO2 photocatalysis technology for inactivation of Escherichia coli K12 on the surface of blueberries and a model agar matrix and the influence of surface characteristics.

Surface disinfection of fresh blueberries is an important food safety challenge due to the delicate texture and short shelf life of these small fruits. A newly designed water-assisted photocatalytic reactor was developed for disinfection of fruits with a delicate texture and complex surface characteristics. Efficacy of UV-TiO2 photocatalysis was evaluated in comparison with UV alone for inactivation of Escherichia coli K12 (as a surrogate for Escherichia coli O157:H7) inoculated onto the surface of the blueberry skin, calyx, and an experimentally prepared agar matrix that was used as a model matrix. Influence of surface characteristics such as surface hydrophobicity and surface free energy on bacterial adhesion were also investigated. The initial bacterial population on all surfaces was approximately 7.0 log CFU/g. UV-TiO2 photocatalysis (4.5 mW/cm2) for 30 s achieved comparatively higher bacterial reductions of 5.3 log and 4.6 log CFU/g on blueberry skin and agar matrix surfaces, respectively, than 4.5 log and 3.4 log CFU/g reductions for UV alone (6.0 mW/cm2). Total phenolic and total anthocyanin contents of fruits were significantly increased after both UV-TiO2 and UV treatments, compared with water washed control fruits. UV-TiO2 photocatalysis technology is a non-chemical and residue-free method with reduced water usage for surface disinfection of fresh blueberries.

Spontaneous mutations in the flhD operon generate motility heterogeneity in Escherichia coli biofilm.

BACKGROUND: Heterogeneity and niche adaptation in bacterial biofilm involve changes to the genetic makeup of the bacteria and gene expression control. We hypothesized that i) spontaneous mutations in the flhD operon can either increase or decrease motility and that ii) the resulting motility heterogeneity in the biofilm might lead to a long-term increase in biofilm biomass. RESULTS: We allowed the highly motile E. coli K-12 strain MC1000 to form seven- and fourteen-day old biofilm, from which we recovered reduced motility isolates at a substantially greater frequency (5.4 %) than from a similar experiment with planktonic bacteria (0.1 %). Biofilms formed exclusively by MC1000 degraded after 2 weeks. In contrast, biofilms initiated with a 1:1 ratio of MC1000 and its isogenic flhD::kn mutant remained intact at 4 weeks and the two strains remained in equilibrium for at least two weeks. These data imply that an 'optimal' biofilm may contain a mixture of motile and non-motile bacteria. Twenty-eight of the non-motile MC1000 isolates contained an IS1 element in proximity to the translational start of FlhD or within the open reading frames for FlhD or FlhC. Two isolates had an IS2 and one isolate had an IS5 in the open reading frame for FlhD. An additional three isolates contained deletions that included the RNA polymerase binding site, five isolates contained point mutations and small deletions in the open reading frame for FlhC. The locations of all these mutations are consistent with the lack of motility and further downstream within the flhD operon than previously published IS elements that increased motility. We believe that the location of the mutation within the flhD operon determines whether the effect on motility is positive or negative. To test the second part of our hypothesis where motility heterogeneity in a biofilm may lead to a long-term increase in biofilm biomass, we quantified biofilm biomass by MC1000, MC1000 flhD::kn, and mixtures of the two strains at ratios of 1:1, 10:1, and 1:10. After 3 weeks, biofilm of the mixed cultures contained up to five times more biomass than biofilm of each of the individual strains. CONCLUSION: Mutations in the flhD operon can exert positive or negative effects on motility, depending on the site of the mutation. We believe that this is a mechanism to generate motility heterogeneity within E. coli biofilm, which may help to maintain biofilm biomass over extended periods of time.

Comparative reevaluation of FASP and enhanced FASP methods by LC-MS/MS.

Filter-aided sample preparation is a proteomic technique for the preparation and on column proteolysis of proteins. Recently an enhanced FASP protocol was developed that uses deoxycholic acid (DCA) and that reportedly enhances trypsin proteolysis, resulting in increases cytosolic and membrane protein representation. FASP and eFASP were re-evaluated by ultra-high-performance liquid chromatography coupled to a quadrupole mass filter Orbitrap analyzer (Q Exactive). Although there was no difference in trypsin activity, 14,099 and 13,414 peptides, describing 1723 and 1793 protein groups, from Escherichia coli K12 were identified using FASP and eFASP, respectively. Characterization of the physicochemical properties of identified peptides showed no significant differences other than eFASP extracting slightly more basic peptides. At the protein level, both methods extracted essentially the same number of hydrophobic transmembrane helix-containing proteins as well as proteins associated with the cytoplasm or the cytoplasmic and outer membranes. By employing state-of-the-art LC-MS/MS shot gun proteomics, our results indicate that FASP and eFASP showed no significant differences at the protein level. However, because of the slight differences in selectivity at the physicochemical level of peptides, these methods can be seen to be somewhat complementary for analyses of complex peptide mixtures.

You can bring plankton to fecal indicator organisms, but you cannot make the plankton graze: particle contribution to E. coli and MS2 inactivation in surface waters.

Organisms that are associated with feces ("fecal indicator organisms") are monitored to assess the potential for fecal contamination of surface water bodies in the United States. However, the effect of the complex mixtures of chemicals and the natural microbial community within surface water ("particles") on fecal indicator organism persistence is not well characterized. We aimed to better understand how particles, including biological (e.g., potential grazers) and inert (e.g., minerals) types, affect the fecal indicator organisms Escherichia coli K-12 ("E. coli") and bacteriophage MS2 in surface waters. A gradient of particles captured by a 0.2-µm-pore-size filter ("large particles") was generated, and the additional particles and dissolved constituents that passed through the filter were deemed "small particles." We measured the ratio of MS2 and E. coli that survived over a 24-h incubation period for each condition (0%-1,000% large-particle concentration in raw water) and completed a linear regression that included large- and small-particle coefficients. Particles were characterized by quantifying plankton, total bacterial cells, and total solids. E. coli and MS2 persistence was not significantly affected by large particles, but small particles had an effect in most waters. Small particles in higher-salinity waters had the largest, negative effect on E. coli and MS2 survival ratios: Significant small-particle coefficients ranged from -1.7 to -5.5 day-1 in the marine waters and -0.89 to -3.2 day-1 in the fresh and estuarine waters. This work will inform remediation efforts for impaired surface water bodies.IMPORTANCEMany surface water bodies in the United States have organisms associated with fecal contamination that exceed regulatory standards and prevent safe recreation. The process to remediate impaired water bodies is complicated because these fecal indicator organisms are affected by the local environmental conditions. For example, the effect of particles in surface water on fecal indicator concentrations are difficult to quantify in a way that is comparable between studies and water bodies. We applied a method that overcomes this limitation to assess the effects of large particles, including natural plankton that could consume the seeded fecal indicator organisms. Even in environmental water samples with diverse communities of plankton present, no effect of large particles on fecal indicator concentrations was observed. These findings have implications for the interpretation and design of future studies, including that particle characterization of surface water may be necessary to assess the fate of fecal indicators.

CRISPR-spacer integration reporter plasmids reveal distinct genuine acquisition specificities among CRISPR-Cas I-E variants of Escherichia coli.

Prokaryotes immunize themselves against transmissible genetic elements by the integration (acquisition) in clustered regularly interspaced short palindromic repeats (CRISPR) loci of spacers homologous to invader nucleic acids, defined as protospacers. Following acquisition, mono-spacer CRISPR RNAs (termed crRNAs) guide CRISPR-associated (Cas) proteins to degrade (interference) protospacers flanked by an adjacent motif in extrachomosomal DNA. During acquisition, selection of spacer-precursors adjoining the protospacer motif and proper orientation of the integrated fragment with respect to the leader (sequence leading transcription of the flanking CRISPR array) grant efficient interference by at least some CRISPR-Cas systems. This adaptive stage of the CRISPR action is poorly characterized, mainly due to the lack of appropriate genetic strategies to address its study and, at least in Escherichia coli, the need of Cas overproduction for insertion detection. In this work, we describe the development and application in Escherichia coli strains of an interference-independent assay based on engineered selectable CRISPR-spacer integration reporter plasmids. By using this tool without the constraint of interference or cas overexpression, we confirmed fundamental aspects of this process such as the critical requirement of Cas1 and Cas2 and the identity of the CTT protospacer motif for the E. coli K12 system. In addition, we defined the CWT motif for a non-K12 CRISPR-Cas variant, and obtained data supporting the implication of the leader in spacer orientation, the preferred acquisition from plasmids harboring cas genes and the occurrence of a sequential cleavage at the insertion site by a ruler mechanism.

Laboratory strains of Escherichia coli K-12: things are seldom what they seem.

Escherichia coli K-12 was originally isolated 100 years ago and since then it has become an invaluable model organism and a cornerstone of molecular biology research. However, despite its pedigree, since its initial isolation E. coli K-12 has been repeatedly cultured, passaged and mutagenized, resulting in an organism that carries many genetic changes. To understand more about this important model organism, we have sequenced the genomes of two ancestral K-12 strains, WG1 and EMG2, considered to be the progenitors of many key laboratory strains. Our analysis confirms that these strains still carry genetic elements such as bacteriophage lambda (λ) and the F plasmid, but also indicates that they have undergone extensive laboratory-based evolution. Thus, scrutinizing the genomes of ancestral E. coli K-12 strains leads us to examine whether E. coli K-12 is a sufficiently robust model organism for 21st century microbiology.

Dimethyl phthalate inhibits the growth of Escherichia coli K-12 by regulating sugar transport and energy metabolism.

Dimethyl phthalate (DMP) is one of the most widely used plasticizers, and it is easily released into the environment, posing a threat to microbes. In this study, the impact of DMP on the uptake and metabolism of sugars in E. coli K-12 was assessed using proteomics, computational simulation analysis, transcriptome analysis, and sugar utilization experiments. DMP contamination inhibited the growth of E. coli K-12 and downregulated the expression of proteins in ATP-binding cassette (ABC) transporters and the phosphotransferase (PTS) system of E. coli K-12, which are primarily involved in the transmembrane transport of sugars. DMP formed a stable complex with sugar transporters and changed the rigidity and stability of the proteins. Furthermore, DMP treatment decreased the utilization of L-arabinose, glucose, D-xylose, and maltose. Moreover, carbon metabolism and oxidative phosphorylation were also downregulated by DMP. Our study shows that DMP reduces the uptake of sugars and ATP production and subsequently inhibits the growth of E. coli K-12.

Application of QSAR Approach to Assess the Effects of Organic Pollutants on Bacterial Virulence Factors.

The release of a wide variety of persistent chemical contaminants into wastewater has become a growing concern due to their potential health and environmental risks. While the toxic effects of these pollutants on aquatic organisms have been extensively studied, their impact on microbial pathogens and their virulence mechanisms remains largely unexplored. This research paper focuses on the identification and prioritization of chemical pollutants that increase bacterial pathogenicity, which is a public health concern. In order to predict how chemical compounds, such as pesticides and pharmaceuticals, would affect the virulence mechanisms of three bacterial strains (Escherichia coli K12, Pseudomonas aeruginosa H103, and Salmonella enterica serovar. Typhimurium), this study has developed quantitative structure-activity relationship (QSAR) models. The use of analysis of variance (ANOVA) functions assists in developing QSAR models based on the chemical structure of the compounds, to predict their effect on the growth and swarming behavior of the bacterial strains. The results showed an uncertainty in the created model, and that increases in virulence factors, including growth and motility of bacteria, after exposure to the studied compounds are possible to be predicted. These results could be more accurate if the interactions between groups of functions are included. For that, to make an accurate and universal model, it is essential to incorporate a larger number of compounds of similar and different structures.

Effects of mannose on pathogenesis of Acanthamoeba castellanii.

Acanthamoeba spp. are single-celled protozoan organisms that are widely distributed in the environment. In this study, to understand functional roles of a mannose-binding protein (MBP), Acanthamoeba castellanii was treated with methyl-alpha-D-mannopyranoside (mannose), and adhesion and cytotoxicity of the amoeba were analyzed. In addition, to understand the association of MBP for amoeba phagocytosis, phagocytosis assay was analyzed using non-pathogenic bacterium, Escherichia coli K12. Amoebae treated with mannose for 20 cycles exhibited larger vacuoles occupying the most area of the amoebic cytoplasm in comparison with the control group amoebae and glucose-treated amoebae. Mannose-selected amoebae exhibited lower levels of binding to Chinese hamster ovary (CHO) cells. Exogenous mannose inhibited >50% inhibition of amoebae (control group) binding to CHO cells. Moreover, exogenous mannose inhibited amoebae (i.e., man-treated) binding to CHO cells by <15%. Mannose-selected amoebae exhibited significantly decreased cytotoxicity to CHO cells compared with the control group amoebae, 25.1% vs 92.1%. In phagocytic assay, mannose-selected amoebae exhibited significant decreases in bacterial uptake in comparison with the control group, 0.019% vs 0.03% (P<0.05). Taken together, it is suggested that mannose-selected A. castellanii trophozoites should be severely damaged and do not well interact with a target cell via a lectin of MBP.

Does Silver in Different Forms Affect Bacterial Susceptibility and Resistance? A Mechanistic Perspective.

The exceptional increase in antibiotic resistance in past decades motivated the scientific community to use silver as a potential antibacterial agent. However, due to its unknown antibacterial mechanism and the pattern of bacterial resistance to silver species, it has not been revolutionized in the health sector. This study deciphers mechanistic aspects of silver species, i.e., ions and lysozyme-coated silver nanoparticles (L-Ag NPs), against E. coli K12 through RNA sequencing analysis. The obtained results support the reservoir nature of nanoparticles for the controlled release of silver ions into bacteria. This study differentiates between the antibacterial mechanism of silver species by discussing the pathway of their entry in bacteria, sequence of events inside cells, and response of bacteria to overcome silver stress. Controlled release of ions from L-Ag NPs not only reduces bacterial growth but also reduces the likelihood of resistance development. Conversely, direct exposure of silver ions, leads to rapid activation of the bacterial defense system leading to development of resistance against silver ions, like the well-known antibiotic resistance problem. These findings provide valuable insight on the mechanism of silver resistance and antibacterial strategies deployed by E. coli K12, which could be a potential target for the generation of aim-based and effective nanoantibiotics.