Cell Wall Synthesis Research Articles

Durlobactam, a Diazabicyclooctane β-Lactamase Inhibitor, Inhibits BlaC and Peptidoglycan Transpeptidases of Mycobacterium tuberculosis.

Peptidoglycan synthesis is an underutilized drug target in Mycobacterium tuberculosis (Mtb). Diazabicyclooctanes (DBOs) are a class of broad-spectrum β-lactamase inhibitors that also inhibit certain peptidoglycan transpeptidases that are important in mycobacterial cell wall synthesis. We evaluated the DBO durlobactam as an inhibitor of BlaC, the Mtb β-lactamase, and multiple Mtb peptidoglycan transpeptidases (PonA1, LdtMt1, LdtMt2, LdtMt3, and LdtMt5). Timed electrospray ionization mass spectrometry (ESI-MS) captured acyl-enzyme complexes with BlaC and all transpeptidases except LdtMt5. Inhibition kinetics demonstrated durlobactam was a potent and efficient DBO inhibitor of BlaC (KIapp 9.2 ± 0.9 μM, k2/K 5600 ± 560 M-1 s-1) and similar to clavulanate (KIapp 3.3 ± 0.6 μM, k2/K 8400 ± 840 M-1 s-1); however, durlobactam had a lower turnover number (tn = kcat/kinact) than clavulanate (1 and 8, respectively). KIapp values with durlobactam and clavulanate were similar for peptidoglycan transpeptidases, but ESI-MS captured durlobactam complexes at more time points. Molecular docking and simulation demonstrated several productive interactions of durlobactam in the active sites of BlaC, PonA1, and LdtMt2. Antibiotic susceptibility testing was conducted on 11 Mtb isolates with amoxicillin, ceftriaxone, meropenem, imipenem, clavulanate, and durlobactam. Durlobactam had a minimum inhibitory concentration (MIC) range of 0.5-16 μg/mL, similar to the ranges for meropenem (1-32 μg/mL) and imipenem (0.5-64 μg/mL). In β-lactam + durlobactam combinations (1:1 mass/volume), MICs were lowered 4- to 64-fold for all isolates except one with meropenem-durlobactam. This work supports further exploration of novel β-lactamase inhibitors that target BlaC and Mtb peptidoglycan transpeptidases.

Physiological and molecular mechanisms of ZnO quantum dots mitigating cadmium stress in Salvia miltiorrhiza

This study delved into the physiological and molecular mechanisms underlying the mitigation of cadmium (Cd) stress in the model medicinal plant Salvia miltiorrhiza through the application of ZnO quantum dots (ZnO QDs, 3.84 nm). A pot experiment was conducted, wherein S. miltiorrhiza was subjected to Cd stress for six weeks with foliar application of 100 mg/L ZnO QDs. Physiological analyses demonstrated that compared to Cd stress alone, ZnO QDs improved biomass, reduced Cd accumulation, increased the content of photosynthetic pigments (chlorophyll and carotenoids), and enhanced the levels of essential nutrient elements (Ca, Mn, and Cu) under Cd stress. Furthermore, ZnO QDs significantly lowered Cd-induced reactive oxygen species (ROS) content, including H2O2, O2−, and MDA, while enhancing the activity of antioxidant enzymes (SOD, POD, APX, and GSH-PX). Additionally, ZnO QDs promoted the biosynthesis of primary and secondary metabolites, such as total protein, soluble sugars, terpenoids, and phenols, thereby mitigating Cd stress in S. miltiorrhiza. At the molecular level, ZnO QDs were found to activate the expression of stress signal transduction-related genes, subsequently regulating the expression of downstream target genes associated with metal transport, cell wall synthesis, and secondary metabolite synthesis via transcription factors. This activation mechanism contributed to enhancing Cd tolerance in S. miltiorrhiza. In summary, these findings shed light on the mechanisms underlying the mitigation of Cd stress by ZnO QDs, offering a potential nanomaterial-based strategy for enhancing Cd tolerance in medicinal plants.

Comparison of broth microdilution and E-test for susceptibility of MRSA to vancomycin

Introduction: Healthcare-associated methicillin-resistant Staphylococcus aureus (MRSA) developed soon after the introduction of methicillin. These bacteria have shown resistance to multiple drugs and therefore vancomycin became the antibiotic of choice for treatment of MRSA infections. Vancomycin is a bactericidal antibiotic that acts by inhibiting bacterial cell wall synthesis. This study aimed to compare broth microdilution (BMD) and E-test in determining vancomycin minimum inhibitory concentration (MIC) against MRSA.Methods: A total of 30 clinical isolates of MRSA were acquired from Colombo South Teaching Hospital, Sri Lanka. These MRSA strains were identified by cefoxitin disk diffusion and the vancomycin MIC was determined through BMD and E-test. The Kruskal-Wallis test was applied to test if the samples originated from the same distribution with post hoc determination of the correlation between methods using the Mann-Whitney U test.Results: All 30 MRSA isolates were 100% vancomycin susceptible (≤2 μg/mL) irrespective of methodology, according to the Clinical and Laboratory Standards Institute (CLSI) established breakpoints. However, the E-test MIC values were 1 to 2 dilutions higher than those of BMD. A statistically significant difference between vancomycin MICs of BMD and E-test (p = <0.00001) was calculated which indicated a difference in accuracy.Conclusion: Due to cost, convenience, and the ability to detect vancomycin intermediate Staphylococcus aureus (VISA), exploring the possibility of using E-test as an alternative to BMD is worthwhile.

Modelling metabolic fluxes of tomato stems reveals that nitrogen shapes central metabolism for defence against Botrytis cinerea.

Among plant pathogens, the necrotrophic fungus Botrytis cinerea is one of the most prevalent, leading to severe crop damage. Studies related to its colonization of different plant species have reported variable host metabolic responses to infection. In tomato, high N availability leads to decreased susceptibility. Metabolic flux analysis can be used as an integrated method to better understand which metabolic adaptations lead to effective host defence and resistance. Here, we investigated the metabolic response of tomato infected by B. cinerea in symptomless stem tissues proximal to the lesions, with a reconstructed metabolic model constrained with a large and consistent metabolic dataset acquired under four different N supplies, throughout 7 days post inoculation (dpi). An overall comparison of 48 flux solution vectors of Botrytis- and mock-inoculated plants showed that fluxes were higher in Botrytis-inoculated plants, and the difference increased with a reduction in available N, accompanying an unexpected increase in radial growth. Despite higher fluxes, such as those involved in cell wall synthesis and other pathways, fluxes related to glycolysis, the TCA cycle, amino acids and protein synthesis were limited under very low N, which might explain the enhanced susceptibility. Limiting starch synthesis and enhancing fluxes towards redox and specialized metabolism also contributed to defence independent of N supply.

Structural and kinetic analysis of the monofunctional Staphylococcus aureus PBP1

Staphylococcus aureus, an ESKAPE pathogen, is a major clinical concern due to its pathogenicity and manifold antimicrobial resistance mechanisms. The commonly used β-lactam antibiotics target bacterial penicillin-binding proteins (PBPs) and inhibit crosslinking of peptidoglycan strands that comprise the bacterial cell wall mesh, initiating a cascade of effects leading to bacterial cell death. S. aureus PBP1 is involved in synthesis of the bacterial cell wall during division and its presence is essential for survival of both antibiotic susceptible and resistant S. aureus strains. Here, we present X-ray crystallographic data for S. aureus PBP1 in its apo form as well as acyl-enzyme structures with distinct classes of β-lactam antibiotics representing the penicillins, carbapenems, and cephalosporins, respectively: oxacillin, ertapenem and cephalexin. Our structural data suggest that the PBP1 active site is readily accessible for substrate, with little conformational change in key structural elements required for its covalent acylation of β-lactam inhibitors. Stopped-flow kinetic analysis and gel-based competition assays support the structural observations, with even the weakest performing β-lactams still having comparatively high acylation rates and affinities for PBP1. Our structural and kinetic analysis sheds insight into the ligand–PBP interactions that drive antibiotic efficacy against these historically useful antimicrobial targets and expands on current knowledge for future drug design and treatment of S. aureus infections.

Chitosan‐Stabilized PtAu Nanoparticles with Multienzyme‐Like Activity for Mixed Bacteria Infection Wound Healing and Insights into Its Antibacterial Mechanism

Traditional antibacterial agents are often observed to be ineffective because bacteria evolved to strains with greater antibiotic resistance. Here, vigorous chitosan‐stabilized PtAu nanoparticles (CSPA) with multienzyme‐like activity are successfully fabricated, which serve an effective artificial nanozyme to enhance antibacterial activity for mixed bacterial infection wound treatment. Ultrasmall size CSPA exhibits excellent hydrophilicity and biocompatibility, possesses strong oxidase‐ and peroxidase‐like activity generating a substantial amount of ROS (, 1O2, ·OH) to cause oxidative damage to bacteria, also demonstrates nicotinamide adenine dinucleotide dehydrogenase‐like activity disrupting the bacterial respiratory chains, and subsequently impedes adenosine triphosphate production. CSPA exhibits favorable broad‐spectrum antibacterial activity at very low concentrations, prevents bacterial resistance, and completely inhibits bacterial biofilm formation. Antibacterial Mechanism of CSPA by the transcriptomics is further revealed that CSPA can induce bacterial oxidative stress, hinder bacterial energy metabolism, and disrupt the synthesis and function of bacterial cell walls and cell membranes. In vivo, CSPA inhibits the mixed bacterial population at the wound site and promotes wound healing in rats. This study introduces a novel antibacterial approach, providing important insight into the antibacterial mechanism of CSPA nanozymes and promoting the advancement of nanocatalytic materials in biomedical applications.

Targeting MurB from Helicobacter pylori: insights from virtual screening, molecular docking and molecular dynamics simulation

ABSTRACT Helicobacter pylori is a gram-negative, helical-shaped bacteria found in the mucus lining of the stomach of humans responsible for causing sores or an ulcer. H. pylori is becoming a multi-drug resistant bacterium, which in turn demands the need for development of alternative treatment strategies for the proper management of diseases. The peptidoglycan biosynthesis pathway is critical for the synthesis of cell walls and for the survival of H. pylori. There are several crucial enzymes in this biosynthesis pathway which can act as significant drug targets against H. pylori. One of these important enzymes is UDP-N-acetylenolpyruvoylglucosamine reductase (MurB), which catalyses an important committed step of cell wall biosynthesis. In the present study, we performed a high throughput virtual screening using the Enamine HTSC library against the MurB enzyme. The molecules were also screened for druglike properties by ADME screening, toxicity, and tumorigenic nature, followed by MD simulations. We found that Z102621114_1 and Z228235240_1 ligand molecules can act as potential inhibitors of MurB protein. Our results suggest, these molecules may be able to meet the demand for novel antimicrobials to treat H. pylori medication resistance. The subsequent experimental investigations can increase their potential as a novel agents against H. pylori infections. Research highlights Helicobacter pylori infects 50% of the Global population. Helicobacter pylori infection is associated with gastric cancer. MurB is a key drug target protein as it is responsible for cell wall synthesis. Z102621114_1 and Z228235240_1 were identified as potential inhibitors of MurB of Helicobacter pylori.

Antibiotic's target site affects the potentiation of Lactiplantibacillus plantarum inhibition and inactivation by electroporation.

Antibiotic resistance represents a growing global threat, and thus the motivation to develop novel and combined methods of bacterial inactivation is increasing. Electroporation is a technique in which electric pulses of sufficient strength are applied to permeabilize cells, including bacteria. Combining antibacterials with electroporation is a promising strategy to potentiate their bactericidal and bacteriostatic effectiveness. This approach has already proved useful for increasing bacterial inactivation, yet most studies so far have mainly focused on the maximal achievable effects, and less on the underlying mechanisms. We recently demonstrated that in the Gram-negative (G-) bacterium Escherichia coli, electroporation potentiates antibacterials targeting the peptidoglycan wall more than those with intracellular targets. However, in Gram-positive (G+) bacteria, the wall is directly accessible from the outside, and thus the dependence of potentiation on the antibacterial's target may be rather different. Here, we compare the inactivation and growth inhibition of the G+ bacterium Lactiplantibacillus plantarum for two antibiotics with different modes of action: ampicillin (inhibits cell-wall synthesis) and tetracycline (inhibits intracellular protein synthesis). We used antibiotic concentrations ranging from 0 to 30 × MIC (minimum inhibitory concentration that we predetermined for each antibiotic), a single 1-ms electric pulse with an amplitude from 0 to 20 kV/cm, and post-pulse pre-dilution incubation of 24 h or 1 h. Electroporation increased the inhibition and inactivation efficiency of both antibiotics, but this was more pronounced for tetracycline, with statistical significance mostly limited to 24-h incubation. In general, both inhibition and inactivation grew stronger with increasing antibiotic concentration and electric field amplitude. Our results indicate that electroporation potentiates inactivation of G+ bacteria to a larger extent for antibiotics that inhibit intracellular processes and require transport into the cytoplasm, and to a smaller extent for antibiotics that inhibit cell-wall synthesis. This is the inverse of the relation observed in G- bacteria, and can be explained by the difference in the envelope structure: in G- bacteria the outer membrane must be breached for wall-inhibiting antibiotics to access their target, whereas in G+ bacteria the wall is inherently accessible from the outside and permeabilization does not affect this access.

Genome-wide profiling of Hfq-bound RNAs reveals the iron-responsive small RNA RusT in Caulobacter crescentus.

The alphaproteobacterium Caulobacter crescentus thrives in oligotrophic environments and is able to optimally exploit minimal resources by entertaining an intricate network of gene expression control mechanisms. Numerous transcriptional activators and repressors have been reported to contribute to these processes, but only few studies have focused on regulation at the post-transcriptional level in C. crescentus. Small RNAs (sRNAs) are a prominent class of regulators of bacterial gene expression, and most sRNAs characterized today engage in direct base-pairing interactions to modulate the translation and/or stability of target mRNAs. In many cases, the ubiquitous RNA chaperone, Hfq, contributes to the establishment of RNA-RNA interactions. Although the deletion of the hfq gene is associated with a severe loss of fitness in C. crescentus, the RNA ligands of the chaperone have remained largely unexplored. Here we report on the identification of coding and non-coding transcripts associated with Hfq in C. crescentus and demonstrate Hfq-dependent post-transcriptional regulation in this organism. We show that the Hfq-bound sRNA RusT is transcriptionally controlled by the NtrYX two-component system and induced in response to iron starvation. By combining RusT pulse expression with whole-genome transcriptome analysis, we determine 16 candidate target transcripts that are deregulated, many of which encode outer membrane transporters. We hence suggest RusT to support remodeling of the C. crescentus cell surface when iron supplies are limited.IMPORTANCEThe conserved RNA-binding protein Hfq contributes significantly to the adaptation of bacteria to different environmental conditions. Hfq not only stabilizes associated sRNAs but also promotes inter-molecular base-pairing interactions with target transcripts. Hfq plays a pivotal role for growth and survival, controlling central metabolism and cell wall synthesis in the oligotroph Caulobacter crescentus. However, direct evidence for Hfq-dependent post-transcriptional regulation and potential oligotrophy in C. crescentus has been lacking. Here, we identified sRNAs and mRNAs associated with Hfq in vivo, and demonstrated the requirement of Hfq for sRNA-mediated regulation, particularly of outer membrane transporters in C. crescentus.

Reciprocal regulation of enterococcal cephalosporin resistance by products of the autoregulated yvcJ-glmR-yvcL operon enhances fitness during cephalosporin exposure.

Enterococci are commensal members of the gastrointestinal tract and also major nosocomial pathogens. They possess both intrinsic and acquired resistance to many antibiotics, including intrinsic resistance to cephalosporins that target bacterial cell wall synthesis. These antimicrobial resistance traits make enterococcal infections challenging to treat. Moreover, prior therapy with antibiotics, including broad-spectrum cephalosporins, promotes enterococcal proliferation in the gut, resulting in dissemination to other sites of the body and subsequent infection. As a result, a better understanding of mechanisms of cephalosporin resistance is needed to enable development of new therapies to treat or prevent enterococcal infections. We previously reported that flow of metabolites through the peptidoglycan biosynthesis pathway is one determinant of enterococcal cephalosporin resistance. One factor that has been implicated in regulating flow of metabolites into cell wall biosynthesis pathways of other Gram-positive bacteria is GlmR. In enterococci, GlmR is encoded as the middle gene of a predicted 3-gene operon along with YvcJ and YvcL, whose functions are poorly understood. Here we use genetics and biochemistry to investigate the function of the enterococcal yvcJ-glmR-yvcL gene cluster. Our results reveal that YvcL is a DNA-binding protein that regulates expression of the yvcJ-glmR-yvcL operon in response to cell wall stress. YvcJ and GlmR bind UDP-GlcNAc and reciprocally regulate cephalosporin resistance in E. faecalis, and binding of UDP-GlcNAc by YvcJ appears essential for its activity. Reciprocal regulation by YvcJ/GlmR is essential for fitness during exposure to cephalosporin stress. Additionally, our results indicate that enterococcal GlmR likely acts by a different mechanism than the previously studied GlmR of Bacillus subtilis, suggesting that the YvcJ/GlmR regulatory module has evolved unique targets in different species of bacteria.

Effects of simulated microgravity on current generation of electrochemically active bacteria: Insights from case-control study using random positioning machine

Electrochemically active bacteria (EAB) exhibit promising prospects for space exploration and life support systems. However, the effects of the space environment on EAB are unclear. In this study, the effects of simulated microgravity on the current generation of mixed-culture EAB were illustrated, and the underlying mechanism was elucidated. The results demonstrated that the electrochemical activity of mixed-culture EAB was enhanced, which was mainly due to the enrichment of Geobacter and the increase in EAB biomass. Additionally, the genes and proteins of the biofilm changed obviously under simulated microgravity conditions, including: I) genes related to signal transfer, II) genes related to cell wall synthesis, and III) genes related to riboflavin synthesis. This study first revealed the enrichment in EAB abundance, the increase in EAB biomass, and the promotion of current generation under simulated microgravity.

Synergistic inhibition mechanism of quinazolinone and piperacillin on penicillin-binding protein 2a: a promising approach for combating methicillin-resistant Staphylococcus aureus

The production of penicillin-binding protein 2a (PBP2a), a cell wall synthesis protein, is primarily responsible for the high-level resistance observed in methicillin-resistant Staphylococcus aureus (MRSA). PBP2a exhibits a significantly reduced affinity for most β-lactam antibiotics owing to its tightly closed active site. Quinazolinones (QNE), a novel class of non-β-lactam antibiotics, could initiate the allosteric regulation of PBP2a, resulting in the opening of the initially closed active pocket. Based on our previous study, we have a basic understanding of the dual-site inhibitor ceftaroline (CFT) induced allosteric regulation of PBP2a. However, there are still limitations in the knowledge of how combining medicines, QNE and piperacillin (PIP), induce the allosteric response of PBP2a and inhibit its function. Herein, molecular dynamics (MD) simulations were performed to elucidate the intricate mechanisms underlying the combination mode of QNE and PIP. Our study successfully captured the opening process of the active pocket upon the binding of the QNE at the allosteric site, which alters the signaling pathways with a favorable transmission to the active site. Subsequent docking experiments with different conformational states of the active pocket indicated that all three inhibitors, PIP, QNE, and CFT, exhibited higher docking scores and more favorable docking poses to the open active pocket. These findings reveal the implied mechanism of QNE-mediated allostery underlying combination therapy and provide novel insights into developing innovative therapeutic modalities against MRSA. Communicated by Ramaswamy H. Sarma

Drought decreases cotton fiber strength by altering sucrose flow route.

The potential mechanisms by which drought restricts cotton fiber cell wall synthesis and fiber strength formation are still not fully understood. Herein, drought experiments were conducted using two upland cotton cultivars of Dexiamian 1 (drought-tolerant) and Yuzaomian 9110 (drought-sensitive). Results showed that drought notably reduced sucrose efflux from cottonseed coats to fibers by down-regulating the expression of GhSWEET10 and GhSWEET15 in outer cottonseed coats, leading to promoted sucrose accumulation in cottonseed coats but decreased sucrose accumulation in fibers. Within cotton fibers, drought restricted the hydrolysis from sucrose to UDPG by suppressing sucrose synthase activity, and drought favored the conversion of UDPG to β-1, 3-glucan rather than cellulose by up-regulating GhCALS5. Hence, cellulose content was reduced, which was the main reason for the decreased fiber strength under drought. Moreover, drought promoted lignin synthesis by up-regulating the expression of Gh4CL4, GhPAL9, GhCCR5, GhCAD11, and GhOMT6, which partly offset the negative influence of reduced cellulose content on fiber strength. Compared with Yuzaomian 9110, the drought-tolerance of Dexiamian 1 was evidenced in the following ways: (1) slighter blocked sucrose flow from seedcoat to fiber, (2) less β-1, 3-glucan accumulation, and (3) more lignin biosynthesis under drought. Overall, this study provides new insights into the mechanism of drought impacting cotton fiber strength formation.

Exploring Leptospira interrogans FDAARGOS_203: Insights into AMR and Anti-Phage Defense.

Leptospira, which are known to be important disease-causing agents transmitted between animals and humans, result in significant illness and, in some cases, significant death in human populations. This purpose of this study was to examine the genomic structure of Leptospira interrogans serovar Copenhageni strain FDAARGOS_203 to identify the specific genetic factors that contribute to antimicrobial resistance (AMR) and defense against phages. The genome, consisting of two contigs totaling 4,630,574 base pairs, underwent thorough examination for protein-coding sequences, transfer RNA genes, and ribosomal RNA genes. A total of twenty-two antibiotic resistance genes that specifically target essential cellular processes such as cell wall synthesis, DNA replication, and protein synthesis have been identified. Significant among these were gidB, gdpD, and ggsA, each involved in separate aspects of antibiotic resistance. In addition, the investigation explored the defense mechanisms of bacteriophages, revealing the presence of defense islands that contain a range of anti-phage systems, including RM_Type_IV, PrrC, Borvo, CAS_Class1-Subtype-IC, and CAS_Class1-Subtype-IB. This comprehensive genomic analysis enhances our understanding of the molecular mechanisms that determine Leptospira's ability to adapt to various environments. The identified genetic factors linked to AMR and defense against phages not only enhance our scientific comprehension, but also provide a basis for focused interventions to reduce the impact of leptospirosis.

Mechanisms Underlying the Differential Sensitivity to Mesotrione in Sweet Corn

Mesotrione is a widely used post-emergence herbicide for maize. The toxicity of mesotrione to maize (especially sweet corn) has been widely reported, and some sweet corn varieties are highly sensitive to mesotrione, which affects subsequent plant growth periods. However, the molecular mechanisms responsible for the differences in susceptibility to mesotrione are not known. By comparing changes in the transcriptome of mesotrione-tolerant line 301 and mesotrione-sensitive line 276 after mesotrione treatment, we found that the genes coding light-harvesting chlorophyll protein complex were induced in 301, and the genes coding loosening cell walls were overrepresented in 276. The net photosynthetic rate, maximum photochemical efficiency of leaf PSII, photochemical quenching of chlorophyll fluorescence, and the electron transport rate were significantly higher in 301 than in 276 after mesotrione treatment, and these effects became more severe as time passed. In addition, oxidative balance was also affected by mesotrione. Compared with 301, SOD, POD, and CAT activities were significantly reduced with longer exposure time in 276. The results suggested that sweet corn can mitigate herbicide mesotrione toxicity by improving photosynthesis, ROS scavenging, and cell wall synthesis.

Mechanistic insights on the antibacterial action of the kyotorphin peptide derivatives revealed by in vitro studies and Galleria mellonella proteomic analysis

ObjectivesThe selected kyotorphin derivatives were tested to improve their antimicrobial and antibiofilm activity. The antimicrobial screening of the KTP derivatives were ascertained in the representative strains of bacteria, including Streptococcus pneumoniae, Streptococcus pyogenes, Escherichia coli and Pseudomonas aeruginosa. MethodsKyotorphin derivatives, KTP-NH2, KTP–NH2–DL, IbKTP, IbKTP-NH2, MetKTP-DL, MetKTP-LD, were designed and synthesized to improve lipophilicity and resistance to enzymatic degradation. Peptides were synthesized by standard solution or solid-phase peptide synthesis and purified using RP-HPLC, which resulted in >95 % purity, and were fully characterized by mass spectrometry and 1H NMR. The minimum inhibitory concentrations (MIC) determined for bacterial strains were between 20 and 419 μM. The direct effect of IbKTP-NH2 on bacterial cells was imaged using scanning electron microscopy. The absence of toxicity, high survival after infection and an increase in the hemocytes count was evaluated by injections of derivatives in Galleria mellonella larvae. Proteomics analyses of G. mellonella hemolymph were performed to investigate the underlying mechanism of antibacterial activity of IbKTP-NH2 at MIC. ResultsIbKTP-NH2 induces morphological changes in bacterial cell, many differentially expressed proteins involved in DNA replication, synthesis of cell wall, and virulence were up-regulated after the treatment of G. mellonella with IbKTP-NH2. ConclusionWe suggest that this derivative, in addition to its physical activity on the bacterial membranes, can elicit a cellular and humoral immune response, therefore, it could be considered for biomedical applications.

Analysis of the Growth Response of Cassava Plants (Manihot Esculenta) to Variability in Water Availability

In recent decades, there has been a very pronounced climate change on earth.  Reduced rainfall and the occurrence of long droughts are direct impacts that can trigger other problems in the agricultural sector such as crop failure and weakening food security.  This will also have an impact on the process of growth and agricultural production in the Lampung Province area, especially on cassava plants. Water often limits the growth and development of cultivated plants. The response of plants to lack of water can be seen in their metabolic activity, morphology, growth rate, or productivity. Cell growth is the function of plants that are most sensitive to water shortages.  Lack of water will affect cell turgor so that it will reduce cell development, protein synthesis, and cell wall synthesis. The cassava clones used in this study were Garuda Clones with 5 treatment levels, namely the first treatment (P1) 0L, the second treatment (P2) 0.5L, the third treatment (P3) 1L, the fourth treatment (P4) 1.5L, the fifth treatment (P5) 2L. The result of this study is that there is a cassava growth response to water availability in each treatment except for the variable number of shoots. In the variable plant height, significant response variations are obtained, and it is known that the availability of water needed is directly proportional to the response to plant height growth that also increases.

Whole genome sequencing identifies MprF mutations in a genetically diverse population of daptomycin non-susceptible Staphylococcus aureus in Australia

Daptomycin is one of the few last-line antimicrobials available for the treatment of multidrug-resistant Staphylococcus aureus infections. An increasing number of daptomycin non-susceptible S. aureus infections has been reported worldwide, including Australia. Resistance to daptomycin is multifactorial and involves chromosomal mutations in genes encoding proteins involved in cell membrane and cell wall synthesis. In this study, we performed broth microdilution (BMD) to determine the daptomycin minimum inhibitory concentration (MIC) of 66 clinical isolates of S. aureus previously reported as daptomycin non-susceptible by the VITEKⓇ 2. We used whole genome sequencing to characterise the isolates and screened the genomes for mutations associated with daptomycin non-susceptibility. Only 56 of the 66 isolates had a daptomycin MIC >1 mg/L by BMD. Although the 66 isolates were polyclonal, ST22 was the predominant sequence type and one-third of the isolates were multidrug resistant. Daptomycin non-susceptibility was primarily associated with MprF mutations-at least one MprF mutation was identified in the 66 isolates. Twelve previously reported MprF mutations associated with daptomycin non-susceptibility were identified in 83% of the isolates. Novel MprF mutations identified included P314A, P314F, P314T, S337T, L341V, F349del, and T423R. Daptomycin non-susceptible S. aureus causing infections in Australia are polyclonal and harbour MprF mutation(s). The identification of multidrug-resistant daptomycin non-susceptible S. aureus is a public health concern.

Minimalist Tetrazine N-Acetyl Muramic Acid Probes for Rapid and Efficient Labeling of Commensal and Pathogenic Peptidoglycans in Living Bacterial Culture and During Macrophage Invasion.

N-Acetyl muramic acid (NAM) probes containing alkyne or azide groups are commonly used to investigate aspects of cell wall synthesis because of their small size and ability to incorporate into bacterial peptidoglycan (PG). However, copper-catalyzed alkyne-azide cycloaddition (CuAAC) reactions are not compatible with live cells, and strain-promoted alkyne-azide cycloaddition (SPAAC) reaction rates are modest and, therefore, not as desirable for tracking the temporal alterations of bacterial cell growth, remodeling, and division. Alternatively, the tetrazine-trans-cyclooctene ligation (Tz-TCO), which is the fastest known bioorthogonal reaction and not cytotoxic, allows for rapid live-cell labeling of PG at biologically relevant time scales and concentrations. Previous work to increase reaction kinetics on the PG surface by using tetrazine probes was limited because of low incorporation of the probe. Described here are new approaches to construct a minimalist tetrazine (Tz)-NAM probe utilizing recent advancements in asymmetric tetrazine synthesis. This minimalist Tz-NAM probe was successfully incorporated into pathogenic and commensal bacterial PG where fixed and rapid live-cell, no-wash labeling was successful in both free bacterial cultures and in coculture with human macrophages. Overall, this probe allows for expeditious labeling of bacterial PG, thereby making it an exceptional tool for monitoring PG biosynthesis for the development of new antibiotic screens. The versatility and selectivity of this probe will allow for real-time interrogation of the interactions of bacterial pathogens in a human host and will serve a broader utility for studying glycans in multiple complex biological systems.

Vegetative cell wall protein OsGP1 regulates cell wall mediated soda saline-alkali stress in rice.

Plant growth and development are inhibited by the high levels of ions and pH due to soda saline-alkali soil, and the cell wall serves as a crucial barrier against external stresses in plant cells. Proteins in the cell wall play important roles in plant cell growth, morphogenesis, pathogen infection and environmental response. In the current study, the full-length coding sequence of the vegetative cell wall protein gene OsGP1 was characterized from Lj11 (Oryza sativa longjing11), it contained 660 bp nucleotides encoding 219 amino acids. Protein-protein interaction network analysis revealed possible interaction between CESA1, TUBB8, and OsJ_01535 proteins, which are related to plant growth and cell wall synthesis. OsGP1 was found to be localized in the cell membrane and cell wall. Furthermore, overexpression of OsGP1 leads to increase in plant height and fresh weight, showing enhanced resistance to saline-alkali stress. The ROS (reactive oxygen species) scavengers were regulated by OsGP1 protein, peroxidase and superoxide dismutase activities were significantly higher, while malondialdehyde was lower in the overexpression line under stress. These results suggest that OsGP1 improves saline-alkali stress tolerance of rice possibly through cell wall-mediated intracellular environmental homeostasis.