Derivative of Scorpion Neurotoxin BeM9 Is Selective for Insect Voltage-Gated Sodium Channels

Scorpion α-toxins are small proteins inhibiting the inactivation of voltage-gated sodium channels. They can selectively act on either mammalian (mammal toxins) or insect channels (insect toxins), or affect both types of channels (α-like toxins). Currently no model has been proposed that fully explains the dependence of selectivity upon amino acid sequence, but some patterns have already been established. Thus, most mammal toxins have an aspartic acid residue in position 8, which is involved in the formation of the nest motif, but it is still not clear whether this residue interacts directly with channels. The objective of our study was to obtain a derivative of the α-like toxin BeM9 with the replacement of lysine in position 8 by glutamate (K8E), changing the charge, but excluding the formation of the nest motif. In addition, we replaced the tyrosine in position 17 with glycine (Y17G), which is characteristic of mammal toxins. Surprisingly, the double-mutant derivative BeM9EG lost its activity on mammalian channels, becoming an insect toxin. To explain these changes, we constructed models of BeM9 and BeM9EG complexes with channels, and also performed molecular dynamics of isolated toxins. Analysis of intermolecular contacts in the complexes did not explain the reason for the selectivity change. Nevertheless, the structure of intramolecular contacts and data on molecular mobility indicate an important role of residues K8 and Y17 in stabilizing a certain conformation of BeM9 loops. We assume that the replacement of these residues allosterically affects the efficiency of toxin binding to channels.

AzuCR RNA modulates carbon metabolism as a dual-function RNA in Escherichia coli

Bacteria have evolved small RNAs (sRNAs) to regulate numerous biological processes and stress responses. While sRNAs generally are considered to be “noncoding”, a few have been found to also encode a small protein. Here we describe one such dual-function RNA that modulates carbon utilization in Escherichia coli. The 164 nucleotide RNA was previously shown to encode a 28 amino acid protein (denoted AzuC). We discovered the membrane-associated AzuC protein interacts with GlpD, the aerobic glycerol-3-phosphate dehydrogenase, leading to increased GlpD activity. Overexpression of the RNA encoding AzuC results in a growth defect in glycerol and galactose medium. The defect in galactose medium was still observed for a stop codon mutant derivative, suggesting a potential regulatory role for the RNA. Consistent with this observation, we found that cadA and galE are repressed by base pairing with the RNA (denoted AzuCR). Interestingly, translation of AzuC interferes with the observed repression of cadA and galE by AzuCR and base pairing interferes with AzuC translation, demonstrating that the translation and base pairing functions compete.

Proteome remodeling in the Mycobacterium tuberculosis PknG knockout: molecular evidence for the role of this kinase in cell envelope biogenesis and hypoxia response.

Mycobacterium tuberculosis, the ethiological agent of tuberculosis, is among the deadliest human pathogens. One of M. tuberculosis pathogenic hallmarks is its ability to persist in a dormant state in the host for long periods, reinitiating the infectious cycle when favorable environmental conditions are found. Thus, it is not surprising that this pathogen has developed different mechanisms to withstand the stressful conditions found in the host. In particular, the Ser/Thr protein kinase PknG has gained special relevance since it regulates nitrogen metabolism and facilitates bacterial survival inside macrophages. Nevertheless, the molecular mechanisms underlying these effects are far from being elucidated. To further investigate these issues, we performed quantitative proteomics analyses of protein extracts from M. tuberculosis H37Rv and a mutant derivative lacking pknG. Our results showed that in the absence of PknG the mycobacterial proteome was remodeled since 5.7% of the proteins encoded by M. tuberculosis presented significant changes in its relative abundance when compared to the wild-type strain. The main biological processes affected by pknG deletion were the biosynthesis of cell envelope components and the response to hypoxic conditions. As many as 13 DosR-regulated proteins were underrepresented in the pknG deletion mutant, including the distinctive Hrp-1, which was found to be 12-fold decreased according to Parallel Reaction Monitoring experiments. Altogether, the results presented here allow us to postulate that PknG regulation of bacterial adaptation to stress conditions might be an important mechanism underlying its reported effect on intracellular bacterial survival.

Seed treatment with prodigiosin controls damping-off of cucumber caused by Pythium ultimum

Ethanol extract of cell mass of Serratia marcescens strain N4-5, when applied as a treatment to cucumber seed, has been shown to provide control of the oomycete soil-borne plant pathogen Pythium ultimum equivalent to that provided by a seed-treatment chemical pesticide in some soils. Two dominant compounds in this extract, prodigiosin and the serratamolide serrawetin W1, were identified based on mass and collision induced dissociation mass fragmentation spectra. An additional four compounds with M+H+ masses (487, 541, 543, and 571) consistent with serratamolides reported in the literature were also detected. Several other compounds with M+H+ masses of 488, 536, 684, 834, 906, and 908 m/z were detected in this ethanol extract inconsistently over multiple liquid chromatography coupled with tandem mass spectrometry (LC/MS–MS) runs. A purified preparation of prodigiosin provided control of damping-off of cucumber caused by P. ultimum when applied as a seed treatment while ethanol extract of cell mass of strain Tn246, a transposon-mutant-derivative of strain N4-5, did not. Strain Tn246 contained a mini-Tn5 Km insertion in a prodigiosin biosynthetic gene and was deficient in production of prodigiosin. All other compounds detected in N4-5 extract were detected in the Tn246 extract. This is the first report demonstrating that prodigiosin can control a plant disease. Other compounds in ethanol extract of strain N4-5 may contribute to disease control.

Strain and Growth Conditions may Regulate Resistance of Listeria monocytogenes Biofilms to Benzalkonium Chloride

Listeria monocytogenes is one of the main foodborne pathogens. The formation of biofilms by L. monocytogenes contributes to its resistance to disinfectants, which represents a serious risk for food production plants. The aim of this study was to compare the effect of sub-inhibitory concentrations of benzalkonium chloride (BAC) (1.25 or 2.5 mg/L) on biofilm production and on biofilm reduction after exposure to an inhibitory concentration of BAC (1280 mg/L) in two isogenic L. monocytogenes strains: the BAC-sensitive wild-type strain S2-1 and its BAC-resistant mutant derivative S2BAC, which presented a multidrug resistance phenotype. The biofilm-forming ability of the strains under different BAC concentrations was evaluated by the resazurin method using polystyrene microplates. The biofilm reduction after BAC exposure was evaluated by using stainless steel coupons (SSCs). When the resazurin method was used, S2BAC produced significantly more biofilm in the presence of a sub-inhibitory concentration of BAC compared to that in the culture medium without BAC (p < 0.05). When the SSC method was used, the presence of sub-inhibitory concentrations of BAC resulted in a higher resistance of the biofilm for S2BAC compared to that in the culture medium without BAC (p < 0.05). This was not observed with the sensitive S2-1 strain. These results suggest that biofilm behavior depends on the strain and sub-inhibitory concentrations of disinfectants and may explain the ability of certain isolates to persist in niches of food processing plants.

Increases in Hydrophilicity and Charge on the Polar Face of Alyteserin 1c Helix Change its Selectivity towards Gram-Positive Bacteria

Recently, resistance of pathogens towards conventional antibiotics has increased, representing a threat to public health globally. As part of the fight against this, studies on alternative antibiotics such as antimicrobial peptides have been performed, and it has been shown that their sequence and structure are closely related to their antimicrobial activity. Against this background, we here evaluated the antibacterial activity of two peptides developed by solid-phase synthesis, Alyteserin 1c (WT) and its mutant derivative (ΔM), which shows increased net charge and reduced hydrophobicity. These structural characteristics were modified as a result of amino acid substitutions on the polar face of the WT helix. The minimum inhibitory concentration (MIC) of both peptides was obtained in Gram-positive and Gram-negative bacteria. The results showed that the rational substitutions of the amino acids increased the activity in Gram-positive bacteria, especially against Staphylococcus aureus, for which the MIC was one-third of that for the WT analog. In contrast to the case for Gram-positive bacteria, these substitutions decreased activity against Gram-negative bacteria, especially in Escherichia coli, for which the MIC was eight-fold higher than that exhibited by the WT peptide. To understand this, models of the peptide behavior upon interacting with membranes of E. coli and S. aureus created using molecular dynamics were studied and it was determined that the helical stability of the peptide is indispensable for antimicrobial activity. The hydrogen bonds between the His20 of the peptides and the phospholipids of the membranes should modulate the selectivity associated with structural stability at the carboxy-terminal region of the peptides.

A mutation in the glycosyltransferase gene lafB causes daptomycin hypersusceptibility in Enterococcus faecium

Objectives To verify dissemination of daptomycin-non-susceptible Enterococcus faecium in a hospital where daptomycin was not in use and to understand the evolutionary pathways connecting daptomycin hypersusceptibility to non-susceptibility. Methods Clonality of 26 E. faecium was assessed by PFGE and the STs of these isolates were determined. The most daptomycin-susceptible isolate was evolved in vitro by stepwise daptomycin selection, generating isolates for genome comparisons. Results The spread of a high-risk daptomycin-non-susceptible VRE clone was detected, as was the occurrence of an unusual daptomycin-hypersusceptible strain (HBSJRP18). To determine the basis for daptomycin hypersusceptibility, we evolved HBSJRP18 in vitro and identified candidate genetic alterations potentially related to daptomycin susceptibility. Both lafB, encoding glycosyltransferase, which is putatively involved in lipoteichoic acid (LTA) biosynthesis, and dak, encoding a dihydroxyacetone kinase likely involved in fatty acid metabolism, were mutated in multiple independent experiments. Trans-complementation showed that the lafB polymorphism naturally occurring in HBSJRP18 caused its daptomycin hypersusceptibility. Fourier-transform infrared spectroscopy identified differences between the extracted LTA spectra from the hypersusceptible isolate and its revertant, as well as other non-susceptible variants, supporting a role for LafB in E. faecium LTA biosynthesis. Zeta potential difference was detected in one evolved dak mutant derivative. While much more susceptible to daptomycin, HBSJRP18 showed enhanced growth in the presence of piperacillin, suggesting that this, or another cell wall-targeting antibiotic, may have selected for the daptomycin-hypersusceptible phenotype. Conclusions Our findings provide new information on the basis for daptomycin susceptibility in E. faecium, with implications for limiting the development and spread of daptomycin resistance.

Cezomycin Is Activated by CalC to Its Ester Form for Further Biosynthesis Steps in the Production of Calcimycin inStreptomyces chartreusisNRRL 3882

ABSTRACTCalcimycin, N-demethyl calcimycin, and cezomycin are polyether divalent cation ionophore secondary metabolites produced byStreptomyces chartreusis. A thorough understanding of the organization of their encoding genes, biosynthetic pathway(s), and cation specificities is vitally important for their efficient future production and therapeutic use. So far, this has been lacking, as has information concerning any biosynthetic relationships that may exist between calcimycin and cezomycin. In this study, we observed that when a Cal−(calB1mutant) derivative of a calcimycin-producing strain ofS. chartreusis(NRRL 3882) was grown on cezomycin, calcimycin production was restored. This suggested that calcimycin synthesis may have resulted from postsynthetic modification of cezomycin rather than from ade novoprocess through a novel and independent biosynthetic mechanism. Systematic screening of a number of Cal−S. chartreusismutants lacking the ability to convert cezomycin to calcimycin allowed the identification of a gene, provisionally namedcalC, which was involved in the conversion step. Molecular cloning and heterologous expression of the CalC protein along with its purification to homogeneity and negative-staining electron microscopy allowed the determination of its apparent molecular weight, oligomeric forms in solution, and activity. These experiments allowed us to confirm that the protein possessed ATP pyrophosphatase activity and was capable of ligating coenzyme A (CoA) with cezomycin but not 3-hydroxyanthranilic acid. The CalC protein's apparentKmandkcatfor cezomycin were observed to be 190 μM and 3.98 min−1, respectively, and it possessed the oligomeric form in solution. Our results unequivocally show that cezomycin is postsynthetically modified to calcimycin by the CalC protein through its activation of cezomycin to a CoA ester form.IMPORTANCECalcimycin is a secondary metabolite divalent cation-ionophore that has been studied in the context of human health. However, detail is lacking with respect to both calcimycin's biosynthesis and its biochemical/biophysical properties as well as information regarding its, and its analogues', divalent cation binding specificities and other activities. Such knowledge would be useful in understanding how calcimycin and related compounds may be effective in modifying the calcium channel ion flux and might be useful in influencing the homeostasis of magnesium and manganese ions for the cure or control of human and bacterial infectious diseases. The results presented here unequivocally show that CalC protein is essential for the production of calcimycin, which is essentially a derivative of cezomycin, and allow us to propose a biosynthetic mechanism for calcimycin's production.

OXA-244-Producing Escherichia coli Isolates, a Challenge for Clinical Microbiology Laboratories

ABSTRACT OXA-244 is a single-point-mutant derivative of OXA-48 displaying reduced carbapenemase activity. Here, we report the microbiological features of seven OXA-244-producing Escherichia coli isolates. Only one isolate grew on ChromID Carba Smart medium (bioMérieux), but six of the seven isolates grew on ChromID extended-spectrum-β-lactamase (ESBL) medium (bioMérieux), as they coproduced an ESBL and/or a plasmid-encoded cephalosporinase. The production of a carbapenemase was detected in 57.1%, 71.4%, 71.4%, and 100% of the E. coli isolates using the Carba NP test, the Rapidec Carba NP test (bioMérieux), a matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) hydrolysis assay (Bruker), and the OXA-48 K-SeT assay (Coris BioConcept), respectively. Our results indicate that OXA-244-producing E. coli isolates are difficult to detect, which may lead to their silent spread.

A Highly Arginolytic Streptococcus Species That Potently Antagonizes Streptococcus mutans

ABSTRACTThe ability of certain oral biofilm bacteria to moderate pH through arginine metabolism by the arginine deiminase system (ADS) is a deterrent to the development of dental caries. Here, we characterize a novelStreptococcusstrain, designated strain A12, isolated from supragingival dental plaque of a caries-free individual. A12 not only expressed the ADS pathway at high levels under a variety of conditions but also effectively inhibited growth and two intercellular signaling pathways of the dental caries pathogenStreptococcus mutans. A12 produced copious amounts of H2O2via the pyruvate oxidase enzyme that were sufficient to arrest the growth ofS. mutans. A12 also produced a protease similar to challisin (Sgc) ofStreptococcus gordoniithat was able to block the competence-stimulating peptide (CSP)–ComDE signaling system, which is essential for bacteriocin production byS. mutans. Wild-type A12, but not ansgcmutant derivative, could protect the sensitive indicator strainStreptococcus sanguinisSK150 from killing by the bacteriocins ofS. mutans. A12, but notS. gordonii, could also block the XIP (comX-inducingpeptide) signaling pathway, which is the proximal regulator of genetic competence inS. mutans, but Sgc was not required for this activity. The complete genome sequence of A12 was determined, and phylogenomic analyses compared A12 to streptococcal reference genomes. A12 was most similar toStreptococcus australisandStreptococcus parasanguinisbut sufficiently different that it may represent a new species. A12-like organisms may play crucial roles in the promotion of stable, health-associated oral biofilm communities by moderating plaque pH and interfering with the growth and virulence of caries pathogens.