Bacterial Infections and Mycoses Commons^™

Soluble Antimicrobial Peptide (Amp) Screening To Rationally Design Amp-Hydrogels That Selectively Prevent Biofilm Formation, Matthias Recktenwald, Muskanjot Kaur, Mohammed M. Benmassaoud, Aryanna Copling, Tulika Khanna, Michael Curry, Denise Cortes, Gilbert Fleischer, Valerie J. Carabetta, Sebastián L. Vega

Rowan-Virtua Research Day

Staphylococcus aureus is an opportunistic pathogen that lives on surfaces and skin and can cause serious infections once inside the body. While antibiotics effectively kill bacteria, there are a growing number of infections with antibiotic-resistant strains. Antimicrobial peptides (AMPs) are part of the innate immune system and can eliminate pathogens including bacteria, fungi, and viruses, and are a promising alternative to antibiotics. Although studies have reported that AMP-functionalized hydrogels can prevent bacterial adhesion and biofilm formation, these materials generally consist of one AMP at an arbitrary concentration, and AMP dosing and the combined effects of multiple AMPs are not well …

Broad-Spectrum Activity Of Membranolytic Cationic Macrocyclic Peptides Against Multi-Drug Resistant Bacteria And Fungi, Sandeep Lohan, Anastasia G. Konshina, Rakesh K. Tiwari, Roman G. Efremov, Innokentiy Maslennikov, Keykavous Parang

Pharmacy Faculty Articles and Research

The emergence of multidrug-resistant (MDR) strains causes severe problems in the treatment of microbial infections owing to limited treatment options. Antimicrobial peptides (AMPs) are drawing considerable attention as promising antibiotic alternative candidates to combat MDR bacterial and fungal infections. Herein, we present a series of small amphiphilic membrane-active cyclic peptides composed, in part, of various nongenetically encoded hydrophilic and hydrophobic amino acids. Notably, lead cyclic peptides 3b and 4b showed broad-spectrum activity against drug-resistant Gram-positive (MIC = 1.5–6.2 µg/mL) and Gram-negative (MIC = 12.5–25 µg/mL) bacteria, and fungi (MIC = 3.1–12.5 µg/mL). Furthermore, lead peptides displayed substantial antibiofilm action comparable …

Managing Stress: A Study Of Stress Response Mechanisms In Mycobacteria, Augusto C. Hunt Serracin

Biology Dissertations

Mycobacteria encompass many pathogenic species known to cause severe disease in humans. A well-known example is Mycobacterium tuberculosis (Mtb), the causative agent of the lung disease tuberculosis, which kills millions of humans worldwide yearly. Pathogenic mycobacteria like Mtb are challenging to treat because of their innate ability to adapt to environmental stress. Their unique cell physiology and conserved stress responses allow them to combat biological insults, regulate growth, and regulate genes involved in stress; all these responses increase tolerance to antibiotics. The current therapies to treat mycobacterial infections are lengthy and, at times, unsuccessful, partly due to antibiotic tolerance. A …

Additive Effects Of Cyclic Peptide [R4w4] When Added Alongside Azithromycin And Rifampicin Against Mycobacterium Avium Infection, Melissa Kelley, Kayvan Sasaninia, Arbi Abnousian, Ali Badaoui, James Owens, Abrianna Beever, Nala Kachour, Rakesh Kumar Tiwari, Vishwanath Venketaraman

Pharmacy Faculty Articles and Research

Mycobacterium avium (M. avium), a type of nontuberculous mycobacteria (NTM), poses a risk for pulmonary infections and disseminated infections in immunocompromised individuals. Conventional treatment consists of a 12-month regimen of the first-line antibiotics rifampicin and azithromycin. However, the treatment duration and low antibiotic tolerability present challenges in the treatment of M. avium infection. Furthermore, the emergence of multidrug-resistant mycobacterium strains prompts a need for novel treatments against M. avium infection. This study aims to test the efficacy of a novel antimicrobial peptide, cyclic [R4W4], alongside the first-line antibiotics azithromycin and rifampicin in reducing M. avium survival. Colony-forming unit (CFU) …

Amphiphilic Cell-Penetrating Peptides Containing Natural And Unnatural Amino Acids As Drug Delivery Tools And Antimicrobial Agents, David Salehi

Pharmaceutical Sciences (MS) Theses

Cell-penetrating peptides containing arginine as positively charged residues and tryptophan or diphenylalanine as hydrophobic residues were synthesized. The synthesis was accomplished through the Fmoc solid-phase peptide synthesis in the presence of HBTU and DIPEA. The side-chain protected linear peptides were cleaved from the resin and cyclized in the presence of DIC and HOAt in the solution phase overnight. MALDI-TOF mass spectrometry was used to characterize the peptides.

The cytotoxicity of the synthesized peptides was determined in CCRF-CEM (human, lymphoblast peripheral blood), and HEK-293 (human, embryonic epithelial kidney healthy) cells using the MTS assay. A concentration of 10 µM was found …

Cyclic Peptide [R4w4] In Improving The Ability Of First-Line Antibiotics To Inhibit Mycobacterium Tuberculosis Inside In Vitro Human Granulomas, Joshua Hernandez, David Ashley, Ruoqiong Cao, Rachel Abrahem, Timothy Nguyen, Kimberly To, Aram Yegiazaryan, Ajayi Akinwale David, Rakesh Kumar Tiwari, Vishwanath Venketaraman

Pharmacy Faculty Articles and Research

Tuberculosis (TB) is currently one of the leading causes of global mortality. Medical non-compliance due to the length of the treatment and antibiotic side effects has led to the emergence of multidrug-resistant (MDR) strains of Mycobacterium tuberculosis (M. tb) that are difficult to treat. A current therapeutic strategy attempting to circumvent this issue aims to enhance drug delivery to reduce the duration of the antibiotic regimen or dosage of first-line antibiotics. One such agent that may help is cyclic peptide [R₄W₄], as it has been shown to have antibacterial properties (in combination with tetracycline) …

Determining The Antibacterial Activity And Mode Of Action Of Tirandamycin, Hailey Bouchard

CMC Senior Theses

Tirandamycin is a small molecule natural product that has been isolated from various species of marine and terrestrial Streptomyces. The natural product has shown antibacterial activity against an array of Gram-positive and Gram-negative bacteria, showing promise as a pharmaceutical drug. Tirandamycin has 14 known derivatives, many of which have been created synthetically. Some of its derivatives are particularly potent against the high-risk bacteria vancomycin-resistant Enterococcus faecium, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus pneumoniae and Escherichia coli. However, the antibacterial potency of these derivatives has not been tested systematically leading to the possibility of discovering more potent …

Iiv-6 Inhibits Nf-Kappab Responses In Drosophila, Cara C. West, Florentina Rus, Ying Chen, Anni Kleino, Monique Gangloff, Don B. Gammon, Neal S. Silverman

Neal Silverman

The host immune response and virus-encoded immune evasion proteins pose constant, mutual selective pressure on each other. Virally encoded immune evasion proteins also indicate which host pathways must be inhibited to allow for viral replication. Here, we show that IIV-6 is capable of inhibiting the two Drosophila NF-kappaB signaling pathways, Imd and Toll. Antimicrobial peptide (AMP) gene induction downstream of either pathway is suppressed when cells infected with IIV-6 are also stimulated with Toll or Imd ligands. We find that cleavage of both Imd and Relish, as well as Relish nuclear translocation, three key points in Imd signal transduction, occur …

Design, Synthesis, And Evaluation Of Amphiphilic Cyclic And Linear Peptides Composed Of Hydrophobic And Positively-Charged Amino Acids As Antibacterial Agents, Neda Riahifard, Saghar Mozaffari, Taibah Aldakhil, Francisco Nunez, Qamar Alshammari, Saud Alshammari, Jason Yamaki, Keykavous Parang, Rakesh Kumar Tiwari

Pharmacy Faculty Articles and Research

Antimicrobial peptides (AMPs) contain amphipathic structures and are derived from natural resources. AMPs have been found to be effective in treating the infections caused by antibiotic-resistant bacteria (ARB), and thus, are potential lead compounds against ARB. AMPs’ physicochemical properties, such as cationic nature, amphiphilicity, and their size, will provide the opportunity to interact with membrane bilayers leading to damage and death of microorganisms. Herein, AMP analogs of [R₄W₄] were designed and synthesized by changing the hydrophobicity and cationic nature of the lead compound with other amino acids to provide insights into a structure-activity relationship against selected …

Colicins - A Sound Antimicrobial Approach For The Prevention Of Catheter-Associated Urinary Tract Infections, Sandra M. Roy

Doctoral Dissertations

The emergence and spread of antibiotic resistance has created one of the greatest challenges in fighting infectious disease. We address the rise of antibiotic-resistant pathogens by examining the evolutionary history of a class of resistance determinants, the SHV b-lactamases. We isolated the genes that encode the SHV beta-lactamases (bla_SHV genes) from clinical settings and from an environment essentially devoid of antibiotic use. Our data suggests that, counter to current dogma, the use of antibiotics in the clinic is not creating these resistance genes; genes for antibiotic resistance already exist in nature and our use of antibiotics in clinical …

Mucosal Fluid Glycoprotein Dmbt1 Suppresses Twitching Motility And Virulence Of The Opportunistic Pathogen Pseudomonas Aeruginosa, Jianfang Li, Matteo E. O. Metruccio, David J. Evans, Suzanne M. J. Fleiszig

Faculty Publications & Research of the TUC College of Pharmacy

It is generally thought that mucosal fluids protect underlying epithelial surfaces against opportunistic infection via their antimicrobial activity. However, our published data show that human tear fluid can protect against the major opportunistic pathogen Pseudomonas aeruginosa independently of bacteriostatic activity. Here, we explored the mechanisms for tear protection, focusing on impacts of tear fluid on bacterial virulence factor expression. Results showed that tear fluid suppressed twitching motility, a type of surface-associated movement conferred by pili. Previously, we showed that twitching is critical for P. aeruginosa traversal of corneal epithelia, exit from epithelial cells after internalization, and corneal virulence. Inhibition …

Discovering A Novel Antifungal Target In Downstream Sterol Biosynthesis Using A Squalene Synthase Functional Motif, Kristin Brooke Linscott

Theses and Dissertations--Molecular and Cellular Biochemistry

The sterol biosynthetic pathway is essential for growth of all eukaryotic cells and the main target of antifungal agents. The emergence of resistance to these antifungals in an already ill patient population indicates a need to develop drugs that have a broad spectrum of activity among pathogenic fungi and have minimal patient toxicity. Squalene synthase is the first committed step in the sterol pathway and has been studied intensively for development of antifungal agents. While the overall architecture of this enzyme is identical throughout eukaryotes, it was shown that plant and animal genes cannot complement a squalene synthase knockout mutation …

Exploring The Mechanisms Of Action Of Antifungal Peptides Using Saccharomyces Cerevisiae., Michelle L. Mason

Biological Sciences Undergraduate Honors Theses

Candida albicans is a normal inhabitant of the skin and mucosal membranes of humans, however, in individuals with depressed immune systems or disrupted cutaneous flora, Candida can overgrow and cause serious infection. Candida infection is the fourth leading cause of nosocomial infection in the United States. These infections are often associated with longer hospital stays and higher mortality. Current drug therapies for this infection are largely ineffective due to the increased drug resistance of Candida species, and for some therapeutics, high levels of drug toxicity to humans. Histatin 5 is a naturally occurring salivary peptide that has strong antifungal properties. …

Identification Of A Human Monoclonal Antibody To Replace Equine Diphtheria Anti-Toxin For The Treatment Of Diphtheria, Leila M. Sevigny, Brian J. Booth, Kirk J. Rowley, Brett A. Leav, Peter S. Cheslock, Kerry A. Garrity, Susan Sloan, Gregory J. Babcock, William D. Thomas, Mark Klempner, Yang Wang

William D Thomas Jr

Diphtheria anti-toxin (DAT) has been used to treat Corynebacterium diphtheriae infection for over one hundred years. While the global incidence of diphtheria has declined in the 20th century, the disease remains endemic in many parts of the world and significant outbreaks still occur. Diphtheria anti-toxin is an equine polyclonal antibody with considerable side effects that is in critically short supply globally. A safer, more readily available alternative to DAT would be desirable. In the current study, we cloned human monoclonal antibodies (HuMabs) directly from antibody secreting cells of human volunteers immunized with Td vaccine. We isolated a diverse panel of …

The Use Of A Ditopic Gd(Iii) Paramagnetic Probe For Investigating Α-Bungarotoxin Surface Accessibility, Andrea Bernini, Ottavia Spiga, Vincenzo Venditti, Filippo Prischi, Mauro Botta, Gianluca Croce, Angela Pui-Ling Tong, Wing-Talk Wong, Neri Niccolai

Vincenzo Venditti

Protein surface accessibility is a critical parameter which drives all intermolecular interaction processes. In this respect a big deal of information has been derived by analyzing paramagnetic perturbation profiles obtained from NMR protein spectra, particularly in the case that the effects due to different soluble paramagnets can be compared. Here Gd2L7, a neutral ditopic paramagnetic NMR probe, has been characterized in terms of structure and relaxivity and its paramagnetic perturbations on α-bungarotoxin CαH signals in 1H–13C HSQC (heteronuclear single quantum coherence) spectra have been analyzed. Then, these signal attenuations have been compared with the ones previously obtained in the presence …

An Efficient Protocol For Incorporation Of An Unnatural Amino Acid In Perdeuterated Recombinant Proteins Using Glucose-Based Media, Vincenzo Venditti, Nicolas L. Fawzi, G. Marius Clore

Vincenzo Venditti

The in vivo incorporation of unnatural amino acids into proteins is a well-established technique requiring an orthogonal tRNA/aminoacyl-tRNA synthetase pair specific for the unnatural amino acid that is incorporated at a position encoded by a TAG amber codon. Although this technology provides unique opportunities to engineer protein structures, poor protein yields are usually obtained in deuterated media, hampering its application in the protein NMR field. Here, we describe a novel protocol for incorporating unnatural amino acids into fully deuterated proteins using glucose-based media (which are relevant to the production, for example, of amino acid-specific methyl-labeled proteins used in the study …

Inflammatory Proteins, Genetic Variation, And Environmental Influences On Health Care Associated Infection Development In Sepsis, Reba Antoinette Umberger

Theses and Dissertations (ETD)

The purpose of this study was to determine the impact of baseline systemic inflammation (pro‑inflammatory cytokine, anti‑inflammatory cytokine, and their ratio), genetic variability, and environment on the development of health care associated infections (HAI) among sepsis patients during their ICU stay (up to 28 days).

Methods: A prospective observation study was conducted at the Veterans Affairs Medical Center in the Medical Intensive Care Unit over an 18 month period. A total of 78 patients were enrolled within 72 hours of presenting to the ICU with sepsis. Patient were excluded if they were receiving immunosuppressants (chemotherapy or greater than one mg/kg …

Automated Sequence- And Stereo-Specific Assignment Of Methyl-Labeled Proteins By Paramagnetic Relaxation And Methyl–Methyl Nuclear Overhauser Enhancement Spectroscopy, Vincenzo Venditti, Nicolas L. Fawzi, G. Marius Clore

Vincenzo Venditti

Methyl-transverse relaxation optimized spectroscopy is rapidly becoming the preferred NMR technique for probing structure and dynamics of very large proteins up to ~1 MDa in molecular size. Data interpretation, however, necessitates assignment of methyl groups which still presents a very challenging and time-consuming process. Here we demonstrate that, in combination with a known 3D structure, paramagnetic relaxation enhancement (PRE), induced by nitroxide spin-labels incorporated at only a few surface-exposed engineered cysteines, provides fast, straightforward and robust access to methyl group resonance assignments, including stereoassignments for the methyl groups of leucine and valine. Neither prior assignments, including backbone assignments, for the …

A Structurally Driven Analysis Of Thiol Reactivity In Mammalian Albumins, Ottavia Spiga, Domenico Summa, Simone Cirri, Andrea Bernini, Vincenzo Venditti, Matteo De Chiara, Raffaella Priora, Simona Frosail, Antonios Margaritis, Danila Di Giuseppe, Paolo Di Simplicio, Neri Niccolai

Vincenzo Venditti

Understanding the structural basis of protein redox activity is still an open question. Hence, by using a structural genomics approach, different albumins have been chosen to correlate protein structural features with the corresponding reaction rates of thiol exchange between albumin and disulfide DTNB. Predicted structures of rat, porcine, and bovine albumins have been compared with the experimentally derived human albumin. High structural similarity among these four albumins can be observed, in spite of their markedly different reactivity with DTNB. Sequence alignments offered preliminary hints on the contributions of sequence-specific local environments modulating albumin reactivity. Molecular dynamics simulations performed on experimental …