A rapid anti-Helicobacter pylori biofilm drug screening biosensor based on AlpB outer membrane protein and colloidal gold/nanoporous gold framework.

Inhibition or disruption of biofilms has been recognized as an important means to eradicate Helicobacter pylori (H. pylori) infection. However, a fast and efficient drug screening method against H. pylori biofilms has not yet been established. Therefore, AlpB, an important outer membrane protein in H. pylori biofilm formation, was selected as a biological recognition element to screen anti-biofilm drugs in this study. A novel AlpB/colloidal gold (CG)/nanoporous gold (NPG)/Nafion-reduced graphene oxide (rGO)/glassy carbon electrode (GCE) biosensor was constructed based on the heterologous expression of AlpB. The prepared AlpB-based biosensor not only successfully identified six anti-biofilm drugs, but also evaluated the sensitivity and action intensity of different anti-biofilm drugs binding to AlpB by interaction kinetics analysis. The sensitivity order of AlpB to the six anti-biofilm drugs was: allicin > erythromycin > SCC > curcumin > rifampicin > NAC and the action intensity of the six anti-biofilm drugs on AlpB was: rifampicin > NAC > allicin > erythromycin > SCC > curcumin. In addition, molecular docking results showed that the six anti-biofilm drugs might exert their anti-biofilm effects by spontaneously binding to the conserved region of AlpB protein. This study provided a rapid screening platform and a unified data processing method for potential anti-biofilm drug development.

BtuB-Dependent Infection of the T5-like Yersinia Phage ϕR2-01.

Yersinia enterocolitica is a food-borne Gram-negative pathogen responsible for several gastrointestinal disorders. Host-specific lytic bacteriophages have been increasingly used recently as an alternative or complementary treatment to combat bacterial infections, especially when antibiotics fail. Here, we describe the proteogenomic characterization and host receptor identification of the siphovirus vB_YenS_ϕR2-01 (in short, ϕR2-01) that infects strains of several Yersinia enterocolitica serotypes. The ϕR2-01 genome contains 154 predicted genes, 117 of which encode products that are homologous to those of Escherichia bacteriophage T5. The ϕR2-01 and T5 genomes are largely syntenic, with the major differences residing in areas encoding hypothetical ϕR2-01 proteins. Label-free mass-spectrometry-based proteomics confirmed the expression of 90 of the ϕR2-01 genes, with 88 of these being either phage particle structural or phage-particle-associated proteins. In vitro transposon-based host mutagenesis and ϕR2-01 adsorption experiments identified the outer membrane vitamin B12 receptor BtuB as the host receptor. This study provides a proteogenomic characterization of a T5-type bacteriophage and identifies specific Y. enterocolitica strains sensitive to infection with possible future applications of ϕR2-01 as a food biocontrol or phage therapy agent.

Therapeutic Effects of Inhibitor of ompA Expression against Carbapenem-Resistant Acinetobacter baumannii Strains.

The widespread of carbapenem-resistant Acinetobacter baumannii (CRAB) is of great concern in clinical settings worldwide. It is urgent to develop new therapeutic agents against this pathogen. This study aimed to evaluate the therapeutic potentials of compound 62520, which has been previously identified as an inhibitor of the ompA promoter activity of A. baumannii, against CRAB isolates, both in vitro and in vivo. Compound 62520 was found to inhibit the ompA expression and biofilm formation in A. baumannii ATCC 17978 at sub-inhibitory concentrations in a dose-dependent manner. These inhibitory properties were also observed in clinical CRAB isolates belonging to sequence type (ST) 191. Additionally, compound 62520 exhibited a bacteriostatic activity against clinical clonal complex (CC) 208 CRAB isolates, including ST191, and ESKAPE pathogens. This bacteriostatic activity was not different between STs of CRAB isolates. Bacterial clearance was observed in mice infected with bioimaging A. baumannii strain 24 h after treatment with compound 62520. Compound 62520 was shown to significantly increase the survival rates of both immunocompetent and neutropenic mice infected with A. baumannii ATCC 17978. This compound also increased the survival rates of mice infected with clinical CRAB isolate. These results suggest that compound 62520 is a promising scaffold to develop a novel therapeutic agent against CRAB infections.

Screening of small molecules attenuating biofilm formation of Acinetobacter baumannii by inhibition of ompA promoter activity.

Anti-virulence therapeutic strategies are promising alternatives against drug-resistant pathogens. Outer membrane protein A (OmpA) plays a versatile role in the pathogenesis and antimicrobial resistance of Acinetobacter baumannii. Therefore, OmpA is an innovative target for anti-virulence therapy against A. baumannii. This study aimed to develop a high-throughput screening (HTS) system to discover small molecules inhibiting the ompA promoter activity of A. baumannii and screen chemical compounds using the bacterial growth-based HTS system. The ompA promoter and open reading frame of nptI fusion plasmids that controlled the expression of nptI encoding resistance to kanamycin by the ompA promoter were constructed and then transformed into A. baumannii ATCC 17978. This reporter strain was applied to screen small molecules inhibiting the ompA promoter activity in a chemical library. Of the 7,520 chemical compounds, 15 exhibited ≥ 70% growth inhibition of the report strain cultured in media containing kanamycin. Three compounds inhibited the expression of ompA and OmpA in the outer membrane of A. baumannii ATCC 17978, which subsequently reduced biofilm formation. In conclusion, our reporter strain is useful for large-scale screening of small molecules inhibiting the ompA expression in A. baumannii. Hit compounds identified by the HTS system are promising scaffolds to develop novel therapeutics against A. baumannii.

Enhanced Sensitivity of Salmonella to Antimicrobial Blue Light Caused by Inactivating rfaC Gene Involved in Lipopolysaccharide Biosynthesis.

Salmonella is a global foodborne pathogen that causes human diseases ranging from mild gastroenteritis to severe systemic infections. Recently, antimicrobial blue light (aBL) showed effective bactericidal activity against a variety of bacteria (e.g., Salmonella) with varying efficiency. However, the antimicrobial mechanism of aBL has not been fully elucidated. Our previous report showed that the outer membrane (OM) is a key target of aBL. The major component of the OM, lipopolysaccharide (LPS), may play a role in aBL bactericidal effect. Therefore, the influence of LPS truncation on the sensitivity of Salmonella Typhimurium SL1344 to aBL was investigated for the first time. First, the rfaC gene in the SL1344 strain likely involved in linking lipid A to the core region of LPS was inactivated and the influence on LPS structure was verified in the mutant strain SL1344ΔrfaC. SL1344ΔrfaC showed a significant increase in sensitivity to aBL, and the bactericidal efficiency exceeded 8 log CFU at an aBL dose of 383 J/cm2, while that of its parental SL1344 strain approached 4 log CFU. To discover the possible mechanism of higher sensitivity, the permeability of OM was determined. Compared to SL1344, SL1344ΔrfaC showed 2.7-fold higher permeability of the OM at 20 J/cm2, this may explain the higher vulnerability of the OM to aBL. Furthermore, the fatty acid profile was analyzed to reveal the detailed changes in the OM and inner membrane of the mutant. Results showed that the membrane lipids of SL1344ΔrfaC were markedly different to SL1344, indicating that change in fatty acid profile might mediate the enhancement of OM permeability and the increased sensitivity to aBL in SL1344ΔrfaC. Hence, we concluded that disruption of rfaC in Salmonella Typhimurium led to the formation of truncated LPS and thus enhanced the permeability of the OM, which contributed to the increased sensitivity to aBL.

Embelin-loaded chitosan gold nanoparticles interact synergistically with ciprofloxacin by inhibiting efflux pumps in multidrug-resistant Pseudomonas aeruginosa and Escherichia coli.

A global upsurge in emergence and spread of antibiotic resistance (ABR) in bacterial populations is a serious threat for human health. Unfortunately, ABR is no longer confined to nosocomial environments and is frequently reported from community microbes as well. The ABR is resulting in shrinking potent antibiotics pool and thus necessitating novel and alternative therapies and therapeutics. Current investigation was aimed to assess the synergistic potential of a synthesized, phytomolecule-loaded, polysaccharide-stabilized metallic nanoparticles (NPs) against Pseudomonas aeruginosa (PA) and Escherichia coli (EC) isolated from river waters. ABR profiling of these strains characterized them as multidrug resistant (MDR). Synthesized embelin (Emb, isolated from Embelia tsjeriam-cottam)-loaded, chitosan-gold (Emb-Chi-Au) NPs were assessed for their potential synergistic activity with ciprofloxacin (CIP) via checker-board assay and time-kill curve analysis. The NPs reduced the minimal inhibitory concentration (MIC) of CIP by 16- and 4-fold against MDR PA (PA-r) and EC (EC-r) strains, respectively. Fractional inhibitory concentration (FIC) indices with ≤0.5 values confirmed the synergy between the Emb-Chi-Au NPs and CIP, which was further confirmed at ½ MICs in both PA-r and EC-r via time-kill curve analysis. In order to decipher the mode of action, efflux pump inhibitory effects of Emb-Chi-Au NPs were evaluated in terms of the increase in the EtBr mediated fluorescence in control versus NP-treated MDR strains. Molecular docking based in silico simulations were used to predict the interactions between Emb and the active sites of the efflux pump related proteins in PA-r (MexA, MexB and OprM) and EC-r (AcrA, AcrB and TolC), which revealed the probable bond formation between Emb and respective amino acid residues.

Initial in vivo testing of TPO-receptor agonist eltrombopag in osteosarcoma patient-derived xenograft models by the pediatric preclinical testing consortium.

Eltrombopag is a small molecule, thrombopoietin receptor agonist approved for the treatment of patients with aplastic anemia and chronic immune thrombocytopenia. It is also a polyvalent cation chelator and inhibits leukemia cell proliferation via reduction of intracellular iron. The in vivo efficacy of eltrombopag was tested against a panel of six Pediatric Preclinical Testing Consortium osteosarcoma xenografts at doses of 5 mg/kg/day (moderate dose) and 50 mg/kg/day (high dose). Eltrombopag, at moderate doses, failed to significantly improve event-free survival (EFS) in 6/6 models. At high doses, eltrombopag significantly prolonged EFS in 2/2 models, though the effect size was small. All models tested demonstrated progressive disease. While eltrombopag did not meaningfully inhibit osteosarcoma growth, it also did not stimulate tumor growth, suggesting it may be safely investigated as a supportive care agent to enhance platelet recovery post chemotherapy.

Design, synthesis and drug resistance reversal potential of novel curcumin mimics Van D: Synergy potential of curcumin mimics.

Being crucial part of plant-based novel discovery of drug from natural resources, a study was done to explore the antibacterial potential of curcumin mimics in combination with antibiotics against multidrug resistant isolates of Pseudomonas aeruginosa. The best candidate Van D, a curcumin mimics reduced the MIC of tetracycline (TET) up to 16 folds against multidrug resistant clinical isolates. VanD further inhibited the efflux pumps as evident by ethidium bromide efflux and by in-silico docking studies. In another experiment, it was also found that Van D inhibits biofilm synthesis. This derivative kills the KG-P2, an isolate of P. aeruginosa in a time dependent manner, the post-antibiotic effect (PAE) of tetracycline was extended as well as mutant prevention concentration (MPC) of TET was also decreased. In Swiss albino mice, Van D reduced the proinflammatory cytokines concentration. In acute oral toxicity study, this derivative was well tolerated and found to be safe up to 1000 mg/kg dose. To the best of our knowledge, this is the first report on curcumin mimics as synergistic agent via inhibition of efflux pump.

Roles of the EnvZ/OmpR Two-Component System and Porins in Iron Acquisition in Escherichia coli.

Escherichia coli secretes high-affinity Fe3+ chelators to solubilize and transport chelated Fe3+ via specific outer membrane receptors. In microaerobic and anaerobic growth environments, where the reduced Fe2+ form is predominant, ferrous transport systems fulfill the bacterial need for iron. Expression of genes coding for iron metabolism is controlled by Fur, which when bound to Fe2+ acts as a repressor. Work carried out here shows that the constitutively activated EnvZ/OmpR two-component system, which normally controls expression of the ompC and ompF porin genes, dramatically increases the intracellular pool of accessible iron, as determined by whole-cell electron paramagnetic resonance spectroscopy, by inducing the OmpC/FeoB-mediated ferrous transport pathway. Elevated levels of intracellular iron in turn activated Fur, which inhibited the ferric transport pathway but not the ferrous transport pathway. The data show that the positive effect of constitutively activated EnvZ/OmpR on feoB expression is sufficient to overcome the negative effect of activated Fur on feoB In a tonB mutant, which lacks functional ferric transport systems, deletion of ompR severely impairs growth on rich medium not supplemented with iron, while the simultaneous deletion of ompC and ompF is not viable. These data, together with the observation of derepression of the Fur regulon in an OmpC mutant, show that the porins play an important role in iron homeostasis. The work presented here also resolves a long-standing paradoxical observation of the effect of certain mutant envZ alleles on iron regulon.IMPORTANCE The work presented here solved a long-standing paradox of the negative effects of certain missense alleles of envZ, which codes for kinase of the EnvZ/OmpR two-component system, on the expression of ferric uptake genes. The data revealed that the constitutive envZ alleles activate the Feo- and OmpC-mediated ferrous uptake pathway to flood the cytoplasm with accessible ferrous iron. This activates the ferric uptake regulator, Fur, which inhibits ferric uptake system but cannot inhibit the feo operon due to the positive effect of activated EnvZ/OmpR. The data also revealed the importance of porins in iron homeostasis.

Engineered P450 BM3 and cpADH5 coupled cascade reaction for ß-oxo fatty acid methyl ester production in whole cells.

Hydroxy- or ketone- functionalized fatty acid methyl esters (FAMEs) are important compounds for production of pharmaceuticals, vitamins, cosmetics or dietary supplements. Biocatalysis through enzymatic cascades has drawn attention to the efficient, sustainable, and greener synthetic processes. Furthermore, whole cell catalysts offer important advantages such as cofactor regeneration by cell metabolism, omission of protein purification steps and increased enzyme stability. Here, we report the first whole cell catalysis employing an engineered P450 BM3 variant and cpADH5 coupled cascade reaction for the biosynthesis of hydroxy- and keto-FAMEs. Firstly, P450 BM3 was engineered through the KnowVolution approach yielding P450 BM3 variant YE_M1_2, (R47S/Y51W/T235S/N239R/I401 M) which exhibited boosted performance toward methyl hexanoate. The initial oxidation rate of YE_M1_2 toward methyl hexanoate was determined to be 23-fold higher than the wild type enzyme and a 1.5-fold increase in methyl 3-hydroxyhexanoate production was obtained (YE_M1_2; 2.75 mM and WT; 1.8 mM). Subsequently, the whole cell catalyst for the synthesis of methyl 3-hydroxyhexanoate and methyl 3-oxohexanoate was constructed by combining the engineered P450 BM3 and cpADH5 variants in an artificial operon. A 2.06 mM total product formation was achieved by the whole cell catalyst including co-expressed channel protein, FhuA and co-solvent addition. Moreover, the generated whole cell biocatalyst also accepted methyl valerate, methyl heptanoate as well as methyl octanoate as substrates and yielded ω-1 ketones as the main product.

The Effect of Environmental Stresses on lipL32 Gene Expression in Pathogenic Leptospira spp. through Real-Time PCR.

Leptospirosis is a worldwide infectious and zoonotic disease. The incidence of this disease is high in temperate regions, especially in northern Iran. The aim of this study was to investigate the effects of temperature, pH, and Phyllanthus amarus plant extract on the lipL32 gene expression in pathogenic Leptospira spp. Fifty water samples were collected. Culture and PCR technique were used to isolate and identify the bacterium and the presence of the lipL32 gene. The samples were exposed to different temperatures and pH levels for one day and the Ph. amarus plant extract at different concentrations for one and seven days. RNA was extracted, and cDNA synthesis was performed for all the samples. All cDNAs were evaluated by the real-time PCR (SYBR green) technique. Out of the 50 samples, ten samples (20%), using PCR were determined to contain the pathogenic Leptospira. Fold change of the expression of the lipL32 gene associated with stresses was as follows: temperature stress of 40°C, 35°C, and 25°C reduced the lipL32 gene expression in all three isolates, especially in the isolates type 1. The pH stress, i.e., pH values equal to 8 or 9 reduced the gene expression in three types of isolates, and pH = 6 stress increases the lipL32 gene expression in the isolates of type 1. Ph. amarus plant extract stress reduced the mentioned gene expression only in isolates of type 2. Temperature and pH stresses could lead to differences in the expression level and cause the lipL32 gene expression decrease in three pathogenic isolates. The MIC results showed anti-leptospiral effect of Ph. amarus plant extract.

Anti-virulence activity of polyphenolic fraction isolated from Kombucha against Vibrio cholerae.

The use of traditional foods and beverages or their bioactive compounds as anti-virulence agents is a new alternative method to overcome the increased global emergence of antimicrobial resistance in enteric pathogens. In the present study, we investigated the anti-virulence activity of a polyphenolic fraction previously isolated from Kombucha, a 14-day fermented beverage of sugared black tea, against Vibrio cholerae O1. The isolated fraction was mainly composed of the polyphenols catechin and isorhamnetin. The fraction, the individual polyphenols and the combination of the individual polyphenols significantly inhibited bacterial swarming motility and expression of flagellar regulatory genes motY and flaC, even at sub-inhibitory concentrations. The polyphenolic compounds also decreased bacterial protease secretion and mucin penetration in vitro. In vivo study revealed that the polyphenolic fraction significantly inhibited V. cholerae induced fluid accumulation in the rabbit ileal loop model and intestinal colonization in suckling mice model. Therefore, the anti-virulence activity of the Kombucha polyphenolic fraction involved inhibition of motility and protease secretion of V. cholerae, thus preventing bacterial penetration through the mucin layer as well as fluid accumulation and bacterial colonization in the intestinal epithelial cells. The overall results implied that Kombucha might be considered as a potential alternative source of anti-virulence polyphenols against V. cholerae. To the best of our knowledge, this is the first report on the anti-virulence activity of Kombucha, mostly attributed to its polyphenolic content.

Enhanced Astaxanthin Production in Escherichia coli via Morphology and Oxidative Stress Engineering.

Astaxanthin is a carotenoid of high commercial value because of its excellent antioxidative, anti-inflammatory, and anticancer properties. Here, we developed a novel strategy for improving the production of astaxanthin via morphology and oxidative stress engineering. First, we identified the morphology-/membrane- and oxidative stress-related genes, which should be knocked down, using the CRISPRi system. Deleting the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) generated longer and larger cells with higher reactive oxygen species (ROS) levels, thus enhancing the production of astaxanthin and decreasing cell growth. To not only improve cell growth but also obtain longer and larger cells with higher ROS levels, a complementary expression system using a temperature-sensitive plasmid was established. Complementarily expressing the morphology-/membrane-related genes (lpp and bamB) and the oxidative stress-related genes (uspE and yggE) further improved the production of astaxanthin to 11.92 mg/g dry cell weight in shake flask cultures.

A small-molecule inhibitor of BamA impervious to efflux and the outer membrane permeability barrier.

The development of new antimicrobial drugs is a priority to combat the increasing spread of multidrug-resistant bacteria. This development is especially problematic in gram-negative bacteria due to the outer membrane (OM) permeability barrier and multidrug efflux pumps. Therefore, we screened for compounds that target essential, nonredundant, surface-exposed processes in gram-negative bacteria. We identified a compound, MRL-494, that inhibits assembly of OM proteins (OMPs) by the ß-barrel assembly machine (BAM complex). The BAM complex contains one essential surface-exposed protein, BamA. We constructed a bamA mutagenesis library, screened for resistance to MRL-494, and identified the mutation bamAE470K BamAE470K restores OMP biogenesis in the presence of MRL-494. The mutant protein has both altered conformation and activity, suggesting it could either inhibit MRL-494 binding or allow BamA to function in the presence of MRL-494. By cellular thermal shift assay (CETSA), we determined that MRL-494 stabilizes BamA and BamAE470K from thermally induced aggregation, indicating direct or proximal binding to both BamA and BamAE470K Thus, it is the altered activity of BamAE470K responsible for resistance to MRL-494. Strikingly, MRL-494 possesses a second mechanism of action that kills gram-positive organisms. In microbes lacking an OM, MRL-494 lethally disrupts the cytoplasmic membrane. We suggest that the compound cannot disrupt the cytoplasmic membrane of gram-negative bacteria because it cannot penetrate the OM. Instead, MRL-494 inhibits OMP biogenesis from outside the OM by targeting BamA. The identification of a small molecule that inhibits OMP biogenesis at the cell surface represents a distinct class of antibacterial agents.

In vivo antibacterial activity of Zataria multiflora Boiss extract and its components, carvacrol, and thymol, against colistin-resistant Acinetobacter baumannii in a pneumonic BALB/c mouse model.

BACKGROUND: Acinetobacter baumannii has emerged as a major cause of nosocomial infections. Various resistance mechanisms of A. baumannii against antibiotics have transformed it into a successful nosocomial pathogen. Because of the limited number of available antibiotics, we used a medicinal plant with an antibacterial effect. Zataria multiflora Boiss (ZMB) extract and its components were used for the treatment of pneumonic mice infected with A. baumannii. The biological effects of this extract and the regulation of the outer membrane protein A (ompA) gene were used in a mouse model. METHODS: A pneumonic mouse model was prepared using clinical and standard strains (1.5 × 108 colony-forming units/mL) of A. baumannii. BALB/c mice groups were treated with a ZMB extract, carvacrol, thymol, and sensitive antibiotics. The lung tissues of the treated mice were cultured for 5 days and each day, bacterial clearance and the ompA gene expression were assessed by quantitative real-time polymerase chain reaction. RESULTS: In the lung tissue culture of pneumonic mice infected with standard or clinical isolate, no colony was detected when treated with the ZMB extract after 2 and 3 days (P < 0.01), respectively. In the carvacrol-treated group, bacterial clearance was seen at day 4 and day 5 (P < 0.05). Bacterial clearance was seen 5 days after treatment with thymol and imipenem and 6 days after ampicillin/sulbactam treatment. The regulation of ompA gene was significantly decreased in this order: ZMB extract, carvacrol, thymol, imipenem, and ampicillin/sulbactam. DISCUSSION: The ZMB extract had a potent bactericidal effect against A. baumannii that could downregulate the ompA gene. ZBM extract and carvacrol could be novel therapeutic agents for antibiotic-resistant A. baumannii.

Evaluation of the promiscuous component of several bacterial export pumps TolC as a biomarker for toxic pollutants in feedstuffs.

A significant risk to the food chain is the presence of noxious pollutants in the feeds of animals whose products are used in human nutrition. Consequently, analytical methods and biosensors have been developed to detect these types of contaminates in feeds. Here we have evaluated whether the expression of TolC, a promiscuous component of several ATP-dependent efflux pumps in E. coli, up-regulated in response to chemical stress, could be a useful biomarker for this aim. Changes in TolC expression in response to toxic compounds, with different abilities to induce DNA damage, were determined using two E. coli strains with (DH5α) and without (BL21(DE3)) inactivating mutation in RecA gene. Deoxycholic acid and potassium dichromate up-regulated TolC in both strains. In contrast, cisplatin-induced TolC up-regulation was abolished in the absence of a functional RecA. When the effect of several insecticides, herbicides, antibiotics and common soil pollutants on TolC expression was analyzed, a relationship between toxicity and their ability to up-regulate TolC was observed. However, this was not a general event because the insecticide α-cipermetrin induced a reduction in cell viability, which was not accompanied by TolC up-regulation. In contrast, the soil pollutant benzene was able to stimulate TolC expression at non-toxic concentrations. When this test was used to analyze aqueous extracts from different feedstuffs, up-regulation of TolC was found in the absence of cell toxicity and was even accompanied by enhanced cell viability. In conclusion, TolC expression is partly dependent on the integrity of the RecA/LexaA system. Although toxic compounds up-regulate TolC in a dose-dependent manner, this response is also activated by non-toxic agents. Thus, owing to its poor specificity regardless of its sensitivity, the use of TolC up-regulation in E. coli to detect the presence of toxic pollutants in conventional and unconventional sources of nutrients for ruminant feeding requires supplementary biomarkers.

Heterodimeric Rifampicin-Tobramycin conjugates break intrinsic resistance of Pseudomonas aeruginosa to doxycycline and chloramphenicol in vitro and in a Galleria mellonella in vivo model.

Intrinsic resistance in Pseudomonas aeruginosa, defined by chromosomally encoded low outer membrane permeability and constitutively over-expressed efflux pumps, is a major reason why the pathogen is refractory to many antibiotics. Herein, we report that heterodimeric rifampicin-tobramycin conjugates break this intrinsic resistance and sensitize multidrug and extensively drug-resistant P. aeruginosa to doxycycline and chloramphenicol in vitro and in vivo. Tetracyclines and chloramphenicol are model compounds for bacteriostatic effects, but when combined with rifampicin-tobramycin adjuvants, their effects became bactericidal at sub MIC levels. Potentiation of tetracyclines correlates with the SAR of this class of drugs and is consistent with outer membrane permeabilization and efflux pump inhibition. Overall, this strategy finds new uses for old drugs and presents an avenue to expand the therapeutic utility of legacy antibiotics to recalcitrant pathogens such as P. aeruginosa.

The B12 receptor BtuB alters the membrane integrity of Caulobacter crescentus.

Vitamin B12 is one of the most complex biomolecules in nature. Since few organisms can synthesize B12de novo, most bacteria utilize highly sensitive and specialized transporters to scavenge B12 and its precursors. In Gram-negative bacteria, BtuB is the outer membrane TonB-dependent receptor for B12. In the fresh water bacterium Caulobacter crescentus, btuB is among the most highly expressed genes. In this study, we characterized the function of BtuB in C. crescentus and unveiled a potential new function of this receptor involved in cellular fitness. Under standard minimal or rich growth conditions, we found that supplements of vitamin B12 to cultures of C. crescentus provided no significant advantage in growth rate. Using a B12 methionine auxotroph, we showed that BtuB in C. crescentus is capable of transporting B12 at low pico-molar range. A btuB knockout strain displayed higher sensitivity to detergents and to changes in osmotic pressure compared to the wild-type. Electron micrographs of this knockout strain revealed a morphology defect. The sensitivity observed in the btuB knockout strain was not due to changes in membrane permeability or altered S-layer levels. Our results demonstrate that btuB deletion mutants exhibit increased susceptibility to membrane stressors, suggesting a potential role of this receptor in membrane homeostasis. Because we only tested BtuB's function under laboratory conditions, we cannot eliminate the possibility that BtuB also plays a key role as a B12 scavenger in C. crescentus when growing in its highly variable and nutrient-limited natural environment.

Identification of the heme acquisition system in Vibrio vulnificus M2799.

Vibrio vulnificus, the causative agent of serious, often fatal, infections in humans, requires iron for its pathogenesis. As such, it obtains iron via both vulnibactin and heme-mediated iron-uptake systems. In this study, we identified the heme acquisition system in V. vulnificus M2799. The nucleotide sequences of the genes encoding heme receptors HupA and HvtA and the ATP-binding cassette (ABC) transport system proteins HupB, HupC, and HupD were determined, and then used in the construction of deletion mutants developed from a Δics strain, which could not synthesize vulnibactin. Growth experiments using these mutants indicated that HupA and HvtA are major and minor heme receptors, respectively. The expressions of two proteins were analyzed by the quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). Furthermore, complementation analyses confirmed that the HupBCD proteins are the only ABC transport system shared by both the HupA and HvtA receptors. This is the first genetic evidence that the HupBCD proteins are essential for heme acquisition by V. vulnificus. Further investigation showed that hupA, hvtA, and hupBCD are regulated by Fur. The qRT-PCR analysis of the heme receptor genes revealed that HupR, a LysR-family positive transcriptional activator, upregulates the expression of hupA, but not hvtA. In addition, ptrB was co-transcribed with hvtA, and PtrB had no influence on growth in low-iron CM9 medium supplemented with hemin, hemoglobin, or cytochrome C.

Relationship between Adaptive Changing of Lysophosphatidylethanolamine Content in the Bacterial Envelope and Ampicillin Sensitivity of Yersinia pseudotuberculosis.

The low permeability of porin channels is the possible reason for Gram-negative bacterial resistance to antibiotics. The adaptive accumulation of lysophosphatidylethanolamine (LPE) in Yersinia pseudotuberculosis induces conformational changes of OmpF porin that may hinder the transport of antibiotics through this channel. The present study was aimed to test whether the changes in LPE content affect the resistance of bacteria to ampicillin. The addition of glucose to the culture medium was shown to simultaneously increase the level of LPE and minimum inhibitory concentration (MIC) for ampicillin of Y. pseudotuberculosis cells 6- and 2-fold, respectively. However, the coadministration of glucose and polyphenol extract from buckwheat husks reduced the content of LPE 2-fold and restored MIC to the control value. Thus, PBEH can be used as antibiotic adjuvant to improve an antibiotic's ability to cross the outer membrane. The present work demonstrated: (i) the role of adaptive changes in the lipid composition of Y. pseudotuberculosis in the development of antibiotic resistance, and (ii) the promising use of PBEH in combination therapy to increase the susceptibility of Gram-negative bacteria to the conventional ß-lactam antibiotics, probably attenuating in vivo a previously demonstrated effect of LPE on the conformation and function of the OmpF channel.