Aromatic Hydrazide Compounds that Inhibit the Growth of Mycobacterium Tuberculosis

Aims: To demonstrate the efficacy of aromatic hydrazide compounds to inhibit growth of Mycobacterium tuberculosis. Study Design: To synthesize tuberculostats and test their antibacterial activity in-vitro. Place and Duration of Study: University of Nebraska, Durham Science Center, 6001 Dodge Street, Omaha NE 68182, and Texas A&M Health Science Center, Department of Microbial Pathogenesis and Immunology, 8447 State Hwy 47, Medical Research and Education Building, Room #3012, Bryan, TX 7780. From March 2019 to October 2019. Methodology: Hydrazide functional groups were formed by covalently bonding hydrazine onto a carbonyl carbon that is a substituent of a single aromatic ring. Microwave excitation was utilized for synthesis, followed by evaluation of antibacterial activity. These compounds were placed into tissue culture media at various concentrations and then Mycobacterium tuberculosis bacteria was added, in order to determine the level of growth inhibition. Growth inhibition of the bacteria was measured as a function of compound concentration versus growth inhibition. Results: Compounds A, B, C, and D carry hydrazide groups as a substituent to a single aromatic ring. All four compounds show zero violations of Rule of 5, indicating favorable drug-likeness. All four compounds showed greater than 50% growth inhibition of bacteria at concentrations below 50 micrograms per milliliter. Growth inhibition was measured by colony forming units and luminescence. Polar surface area, Log P, molecular volume, and other molecular properties were determined for these four compounds. Conclusion: These four hydrazide compounds induced substantial inhibition of bacterial growth. Microwave excitation for the synthesis of hydrazide compounds is effective. These compounds have favorable drug-likeness properties and are highly effective inhibiting the growth of Mycobacterium tuberculosis

Four Compounds Suppressing Growth of Mycobacterium tuberculosis

Aims: To demonstrate the efficacy of several small molecular weight compounds having hydrazide groups, for inhibiting the growth of Mycobacterium tuberculosis. To show these same compounds have favorable drug-likeness properties. Study Design: To synthesize tuberculostats and test their antibacterial activity in-vitro. Place and Duration of Study: University of Nebraska, Durham Science Center, 6001 Dodge Street, Omaha NE 68182, and Texas A&M Health Science Center, Department of Microbial Pathogenesis and Immunology, 8447 State Hwy 47, Medical Research and Education Building, Room #3012, Bryan, TX 7780. From January 2015 to June 2015. Methodology: Hydrazide groups were formed by covalently bonding hydrazine onto small molecules having a single aromatic ring by utilizing microwave excitation and evaluating for antibacterial activity. These compounds were placed into tissue culture media at various concentrations and then tuberculosis bacteria were added to determine the level of growth inhibition. Growth inhibition of the bacteria was measured as a function of compound concentration for assessment and comparison. Results: Compounds A, B, C, and D carry hydrazide groups with various substituents that are bonded to a single aromatic ring. All four compounds show zero violations of Rule of 5, indicating favorable absorption and membrane permeation. All four compounds showed greater than 85% growth inhibition of bacteria at concentrations below 50 micrograms per milliliter, while assayed by colony forming units and luminescence. Values of Log BB suggests compounds A and C will have greater penetration into the central nervous system than isoniazid. Conclusion: These four hydrazide compounds induced substantial inhibition of bacterial growth. Microwave excitation for the synthesis of hydrazide compounds is effective. These compounds have favorable drug-likeness properties and are highly effective inhibiting growth of Mycobacterium tuberculosis

Novel Tuberculostatic Agents Suitable for Treatment of Mycobacterium tuberculosis Infections of the Central Nervous System

Aims: To demonstrate the efficacy of five small molecule compounds for inhibiting the growth of Mycobacterium tuberculosis. To present evidence that these compounds will penetrate into the central nervous system. Study Design: Five small molecule compounds bearing a hydrazide group were synthesized utilizing microwave excitation. These compounds were then placed into tissue culture with Mycobacterium tuberculosis at various concentrations for evaluation of bacterial growth inhibition. Place and Duration of Study: The compounds to be tested were prepared at the University of Nebraska Chemistry Department August 2013. The evaluation of antibacterial activity was determined at the Texas A&M Health Science Center during October to December of 2013. Methodology: Applying microwave excitation for generation of hydrazide groups within the structure of small molecule carboxylic acids, five agents were prepared for evaluation of bacterial growth inhibition. These agents were dissolved into tissue culture media at various concentrations. Having various levels of tuberculostatic agents, then tuberculosis bacteria were added to determine level of growth inhibition. Growth inhibition of the bacteria was achieved and measured by compound concentration for comparison and evaluation. Results: Five compounds having a hydrazide functional group greatly inhibited the growth of Mycobacterium tuberculosis. All five agents had molecular weight less than 215 grams/mole and polar surface area of less than 70 Angstroms2. Values of Log P ranged from -0.226 to 0.998. Values of Log BB (Log [Cbrain/Cblood]) ranged from -0.711 to - 0.525, with a range in central nervous system penetration Cbrain/Cblood of 0.195 to 0.299. All compounds showed zero violations of the Rule of 5. Substantial inhibition of bacterial growth was observed at concentrations as low as 30 micrograms/mL, as measured by optical density and colony forming units. Conclusion: These five hydrazide compounds substantially decreased the proliferation of tuberculosis bacteria at concentrations as low as 30 micrograms/mL. In addition, their physicochemical properties are shown to allow high levels of penetration into the central nervous system

Phage-Encoded Cationic Antimicrobial Peptide Required for Lysis

Abstract: Most phages of Gram-negative bacteria hosts encode spanins for disruption of the outer membrane, which is the last step in host lysis. However, bioinformatic analysis indicates that ∼15% of these phages lack a spanin gene, suggesting they have an alternate way of disrupting the outer membrane (OM). Here, we show that the T7-like coliphage phiKT causes an explosive cell lysis associated with spanin activity despite not encoding spanins. A putative lysis cassette cloned from the phiKT late gene region includes the hypothetical novel gene 28 located between the holin and endolysin genes and supports inducible lysis in Escherichia coli K-12. Moreover, induction of an isogenic construct lacking gene 28 resulted in divalent cation-stabilized spherical cells rather than lysis, implicating gp28 in OM disruption. Additionally, gp28 was shown to complement the lysis defect of a spanin-null λ lysogen. Gene 28 encodes a 56-amino acid cationic protein with predicted amphipathic helical structure and is membrane-associated after lysis. Urea and KCl washes did not release gp28 from the particulate, suggesting a strong hydrophobic membrane interaction. Fluorescence microscopy supports membrane localization of the gp28 protein before lysis. The protein gp28 is similar in size, charge, predicted fold, and membrane association to the human cathelicidin antimicrobial peptide LL-37. Synthesized gp28 behaved similarly to LL-37 in standard assays mixing peptide and cells to measure bactericidal and inhibitory effects. Taken together, these results indicate that phiKT gp28 is a phage-encoded cationic antimicrobial peptide that disrupts bacterial outer membranes during host lysis and, thus, establishes a new class of phage lysis proteins, the disruptins. Importance: We provide evidence that phiKT produces an antimicrobial peptide for outer membrane disruption during lysis. This protein, designated a disruptin, is a new paradigm for phage lysis and has no similarities to other known lysis genes. Although many mechanisms have been proposed for the function of antimicrobial peptides, there is no consensus on the molecular basis of membrane disruption. Additionally, there is no established genetic system to support such studies. Therefore, the phiKT disruptin may represent the first genetically tractable antimicrobial peptide, facilitating mechanistic analyses

Efficacy of cathelicidin-mimetic antimicrobial peptoids against staphylococcus aureus

Staphylococcus aureus is one of the most common pathogens associated with infection in wounds. The current standard of care uses a combination of disinfection and drainage followed by conventional antibiotics such as methicillin. Methicillin and vancomycin resistance has rendered these treatments ineffective, often causing the reemergence of infection. This study examines the use of antimicrobial peptoids (sequence-specific poly-N-substituted glycines) designed to mimic naturally occurring cationic, amphipathic host defense peptides, as an alternative to conventional antibiotics. These peptoids also show efficient and fast (<30 min) killing of methicillin-susceptible S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA) at low micromolar concentrations without having apparent cytotoxic side effects in vivo. Additionally, these novel peptoids show excellent efficacy against biofilm formation and detachment for both MSSA and MRSA. In comparison, conventional antibiotics were unable to detach or prevent formation of biofilms. One cationic 12mer, Peptoid 1, shows great promise, as it could prevent formation of and detach biofilms at concentrations as low as 1.6 μM. The use of a bioluminescent S. aureus murine incision wound model demonstrated clearance of infection in peptoid-treated mice within 8 days, conveying another advantage these peptoids have over conventional antibiotics. These results provide clear evidence of the potential for antimicrobial peptoids for the treatment of S. aureus wound infections. IMPORTANCE Staphylococcus aureus resistance is a consistent problem with a large impact on the health care system. Infections with resistant S. aureus can cause serious adverse effects and can result in death. These antimicrobial peptoids show efficient killing of bacteria both as a biofilm and as free bacteria, often doing so in less than 30 min. As such, these antimicrobials have the potential to alleviate the burden that Staphylococcus infections have on the health care system and cause better outcomes for infected patients

Relating gene expression data on two-component systems to functional annotations in Escherichia coli

<p>Abstract</p> <p>Background</p> <p>Obtaining physiological insights from microarray experiments requires computational techniques that relate gene expression data to functional information. Traditionally, this has been done in two consecutive steps. The first step identifies important genes through clustering or statistical techniques, while the second step assigns biological functions to the identified groups. Recently, techniques have been developed that identify such relationships in a single step.</p> <p>Results</p> <p>We have developed an algorithm that relates patterns of gene expression in a set of microarray experiments to functional groups in one step. Our only assumption is that patterns co-occur frequently. The effectiveness of the algorithm is demonstrated as part of a study of regulation by two-component systems in <it>Escherichia coli</it>. The significance of the relationships between expression data and functional annotations is evaluated based on density histograms that are constructed using product similarity among expression vectors. We present a biological analysis of three of the resulting functional groups of proteins, develop hypotheses for further biological studies, and test one of these hypotheses experimentally. A comparison with other algorithms and a different data set is presented.</p> <p>Conclusion</p> <p>Our new algorithm is able to find interesting and biologically meaningful relationships, not found by other algorithms, in previously analyzed data sets. Scaling of the algorithm to large data sets can be achieved based on a theoretical model.</p

Phenotypic and Genotypic Effects of FlhC Mediated Gene Regulation in Escherichia Coli O157:H7

Escherichia coli (E.coli) 0157:H7, a pathogen belonging to the enterohemorrhagic group of E.coli, has long been a concern to human health. The pathogen causes a myriad of symptoms in humans, ranging from diarrhea and malaise to renal failure. Human infection with the spread of the pathogen is mainly attributed to consumption of contaminated food material such as meat. Decontamination of meat via sprays have to date been the most commonly practiced method to reduce contamination, which now has little relevance in the face of developing resistance by the pathogen. In the following study we investigated FlhC mediated gene regulation in E. coli 0157:H7 on the surface of meat, in an attempt to recognize FlhC regulated targets, which may ultimately serve as targets for the development of novel decontaminating sprays. Microarray experiments were conducted to compare gene expression levels between a parental E. coli 0157:H7 strain and its isogenic flhC mutant, both grown on meat. Putative FlhC targets were then grouped based on their function. Realtime PCR experiment was done to confirm the regulation. Additionally, experiments were done to investigate the phenotypic effects of the regulation. To test the effect of FlhC on biofilm formation, an ATP based assay was first developed in E.coli K-12, which has been detailed in the following dissertation. This assay was used to quantify biofilm biomass in E. coli 0157. Microarray experiments revealed 287 genes as being down regulated by FlhC. These genes belonged to functions relating to cell division, metabolism, biofilm formation and pathogenicity. Real-time PCR confirmed the regulation of 87% of the tested genes. An additional 13 genes were tested with real-time PCR. These belonged to the same functional groups, but were either not spotted on the microarray chips or had missing data points and were hence not included in the analysis. All 13 of these genes appeared to be regulated by FlhC. The phenotypic experiments performed elucidated that the FlhC mutants divided to 20 times higher cell densities, formed five times more biofilm biomass and were twice as pathogenic in a chicken embryo lethality assay, when compared to the parental strain. The following dissertation also reports the development of a combination assay for the quantification of biofilm that takes advantage of the previously mentioned ATP assay and the PhenotypeMicroarray TM (PM) system. The assay was developed using the parental E. coli strain AJW678 and later applied to its isogenic flhD mutant to elaborate on the differences in nutritional requirements between the two strains during biofilm formation. Metabolic modeling and statistical testing was also applied to the data obtained. This assay will be used in the future to study biofilm formation by the parental strain E. coli 0157:H7 strain and its isogenic FlhC mutants on single carbon sources, hence identifying potential metabolites which differentially support biofilm formation in the parental and the mutant strain

Legionella pneumophila p45 element influences host cell entry and sensitivity to sodium.

Legionella pneumophila are environmental bacteria found ubiquitously in both natural and man-made water reservoirs, sometimes as constituents of biofilm communities, but mostly intracellularly within protozoal hosts. In the event that Legionella become aerosolized in water droplets and inhaled by humans, they can cause a potentially fatal form of pneumonia called Legionnaires' disease. Strains of L. pneumophila have highly plastic genomes that harbor numerous inter- and intra-genomic elements, enhancing their ability to live under diverse environmental conditions. One such mobile genomic element, p45 carries ~45 kbp of genes, including the Lvh (Legionella Vir homolog) type IVa secretion system. This element was evaluated for its contribution to L. pneumophila environmental resilience and virulence-related characteristics by comparing clinically isolated strain Philadelphia-1 that carries p45, Lp01 that lacks p45, and Lp01 with p45 reintroduced, Lp01+p45. We found that the p45 element impacts host cell entry and resistance to sodium, both virulence-related characteristics in Legionella species