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Cytotoxicity studies determining hemolytic properties of antimicrobial peptides or other drugs are an important step in the development of novel therapeutics for clinical use. Hemolysis is an affordable, accessible, and rapid method for initial assessment of cellular toxicity for all drugs under development. However, variability in species of red blood cells and protocols used may result in significant differences in results. AMPs generally possess higher selectivity for bacterial cells but can have toxicity against host cells at high concentrations. Knowing the hemolytic activity of the peptides we are developing contributes to our understanding of their potential toxicity. Computational approaches for predicting hemolytic activity of AMPs exist and were tested head-to-head with our experimental results. Starting with an observation of high hemolytic activity of LL-37 peptide against human red blood cells that were collected in EDTA, we explored alternative approaches to develop a more robust, accurate and simple hemolysis assay using defibrinated human blood. We found significant differences between the sensitivity of defibrinated red blood cells and EDTA treated red blood cells. Accurately determining the hemolytic activity using human red blood cells will allow for a more robust calculation of the therapeutic index of our potential antimicrobial compounds, a critical measure in their pre-clinical development. We introduce a standardized, more accurate protocol for assessing hemolytic activity using defibrinated human red blood cells. This approach, facilitated by the increased commercial availability of de-identified human blood and defibrination methods, offers a robust tool for evaluating toxicity of emerging drug compounds, especially AMPs. [Display omitted] [ABSTRACT FROM AUTHOR]

Development of new therapeutics against Select Agents such as Francisella is critical preparation in the event of bioterrorism. Testing FDA-approved drugs for this purpose may yield new activities unrelated to their intended purpose and may hasten the discovery of new therapeutics. A library of 420 FDA-approved drugs was screened for antibiofilm activity against a model organism for human tularemia, Francisella (F.) novicida, excluding drugs that significantly inhibited growth. The initial screen was based on the 2-component system (TCS) dependent biofilm effect, thus, the QseC dependence of maprotiline anti-biofilm action was demonstrated. By comparing their FDA-approved uses, chemical structures, and other properties of active drugs, toremifene and polycyclic antidepressants maprotiline and chlorpromazine were identified as being highly active against F. novicida biofilm formation. Further down-selection excluded toremifene for its membrane active activity and chlorpromazine for its high antimicrobial activity. The mode of action of maprotiline against F. novicida was sought. It was demonstrated that maprotiline was able to significantly down-regulate the expression of the virulence factor IglC, encoded on the Francisella Pathogenicity Island (FPI), suggesting that maprotiline is exerting an effect on bacterial virulence. Further studies showed that maprotiline significantly rescued F. novicida infected wax worm larvae. In vivo studies demonstrated that maprotiline treatment could prolong time to disease onset and survival in F. novicida infected mice. These results suggest that an FDA-approved drug such as maprotiline has the potential to combat Francisella infection as an antivirulence agent, and may have utility in combination with antibiotics. [ABSTRACT FROM AUTHOR]

Our understanding of the virulence and pathogenesis of Francisella spp. has significantly advanced in recent years, including a new understanding that this organism can form biofilms. What is known so far about Francisella spp. biofilms is summarized here and future research questions are suggested. The molecular basis of biofilm production has begun to be studied, especially the role of extracellular carbohydrates and capsule, quorum sensing and two-component signaling systems. Further work has explored the contribution of amoebae, pili, outer-membrane vesicles, chitinases, and small molecules such as c-di-GMP to Francisella spp. biofilm formation. A role for Francisella spp. biofilm in feeding mosquito larvae has been suggested. As no strong role in virulence has been found yet, Francisella spp. biofilm formation is most likely a key mechanism for environmental survival and persistence. The significance and importance of Francisella spp.'s biofilm phenotype as a critical aspect of its microbial physiology is being developed. Areas for further studies include the potential role of Francisella spp. biofilms in the infection of mammalian hosts and virulence regulation. [ABSTRACT FROM AUTHOR]

Background: We report the sequencing, assembly and analysis of the genome of the Komodo dragon (Varanus komodoensis), the largest extant lizard, with a focus on antimicrobial host-defense peptides. The Komodo dragon diet includes carrion, and a complex milieu of bacteria, including potentially pathogenic strains, has been detected in the saliva of wild dragons. They appear to be unaffected, suggesting that dragons have robust defenses against infection. While little information is available regarding the molecular biology of reptile immunity, it is believed that innate immunity, which employs antimicrobial host-defense peptides including defensins and cathelicidins, plays a more prominent role in reptile immunity than it does in mammals.. Results: High molecular weight genomic DNA was extracted from Komodo dragon blood cells. Subsequent sequencing and assembly of the genome from the collected DNA yielded a genome size of 1.6 Gb with 45x coverage, and the identification of 17,213 predicted genes. Through further analyses of the genome, we identified genes and gene-clusters corresponding to antimicrobial host-defense peptide genes. Multiple β-defensin-related gene clusters were identified, as well as a cluster of potential Komodo dragon ovodefensin genes located in close proximity to a cluster of Komodo dragon β-defensin genes. In addition to these defensins, multiple cathelicidin-like genes were also identified in the genome. Overall, 66 β-defensin genes, six ovodefensin genes and three cathelicidin genes were identified in the Komodo dragon genome. Conclusions: Genes with important roles in host-defense and innate immunity were identified in this newly sequenced Komodo dragon genome, suggesting that these organisms have a robust innate immune system. Specifically, multiple Komodo antimicrobial peptide genes were identified. Importantly, many of the antimicrobial peptide genes were found in gene clusters. We found that these innate immunity genes are conserved among reptiles, and the organization is similar to that seen in other avian and reptilian species. Having the genome of this important squamate will allow researchers to learn more about reptilian gene families and will be a valuable resource for researchers studying the evolution and biology of the endangered Komodo dragon. [ABSTRACT FROM AUTHOR]

Alligator mississippiensis (American alligator), a member of order Crocodilia, lives in bacteria-laden environments but is not often known to succumb to bacterial infections. Their serum has been shown to have antibacterial activity beyond that of human serum, and it is believed that this activity is partially due to cationic antimicrobial peptides (CAMPs). CAMPs are produced by many organisms as part of the innate immune system. CAMPs are attractive possible therapies against multi-drug resistant bacteria, such as those found in biofilm-infected war wounds, because they seldom cause genetic resistance in bacteria and are effective against antibiotic resistant bacteria. In this work, we identified, synthesized, and characterized a cathelicidin and two shorter fragments from the American alligator. We discovered the cathelicidin using Basic Local Alignment Search Tool (BLAST) alignment and by comparing A. mississippiensis expressed sequence tags (ESTs) with propeptide cathelicidins of other reptiles. We analyzed the structure using bioinformatics tools and circular dichroism and predicted that the full-length cathelicidin peptide has a mixed structure, with an N-terminal α-helix and a center Pro hinge. In minimal inhibitory concentration (MIC) assays, it was determined that the cathelicidin and the two shorter fragments have strong activity against multiple Gram-negative bacteria, including clinical isolates of multi-drug resistant (MDR) Acinetobacter baumannii and carbapenem-resistant Klebsiella pneumoniae. Using the ethidium bromide uptake assay, it was found that these peptides permeabilize the bacterial membrane and are less sensitive to salt inhibition than many other known CAMPs. The alligator cathelicidin peptides were not hemolytic against sheep red blood cells at 300 μg/ml and were not significantly cytotoxic against A549 human lung epithelial cells after 24 h exposure in 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays. These alligator cathelicidin peptides have activity similar to other CAMPs from reptiles such as NA-CATH. It is possible that the alligator cathelicidins play an important role in the innate immune response of A. mississippiensis , similar to LL-37 in humans. In addition, due to their activities against MDR bacteria and lack of cytotoxicity, the AM-CATH peptides could be an attractive platform for further development as a potential therapeutic. [ABSTRACT FROM AUTHOR]

Antimicrobial peptides are components of both vertebrate and invertebrate innate immune systems that are expressed in response to exposure to bacterial antigens. Naturally occurring antimicrobial peptides from evolutionarily ancient species have been extensively studied and are being developed as potential therapeutics against antibiotic resistant microorganisms. In this study, a putative Cimex lectularius (bedbug, CL) defensin is characterized for its effectiveness against human skin flora including Gram-negative and Gram-positive bacteria. The bedbug defensin (CL-defensin), belonging to family of insect defensins, is predicted to have a characteristic N-terminal loop, an α-helix, and an antiparallel β-sheet, which was supported by circular dichroism spectroscopy. The defensin was shown to be antimicrobial against Gram-positive bacteria commonly found on human skin ( Micrococcus luteus, Corynebacterium renale, Staphylococcus aureus and Staphylococcus epidermidis ); however, it was ineffective against common skin Gram-negative bacteria ( Pseudomonas aeruginosa and Acinetobacter baumannii ) under low-salt conditions. CL-defensin was also effective against M. luteus and C. renale in high-salt (MIC) conditions. Our studies indicate that CL-defensin functions by depolarization and pore-formation in the bacterial cytoplasmic membrane. [ABSTRACT FROM AUTHOR]

In many bacteria, the ability to modulate biofilm production relies on specific signaling molecules that are either self-produced or made by neighboring microbes within the ecological niche. We analyzed the potential interspecies signaling effect of the Burkholderia diffusible signal factor (BDSF) on Francisella novicida, a model organism for Francisella tularensis, and demonstrated that BDSF both inhibits the formation and causes the dispersion of Francisella biofilm. Specificity was demonstrated for the cis versus the trans form of BDSF. Using transcriptome sequencing, quantitative reverse transcription-PCR, and activity assays, we found that BDSF altered the expression of many F. novicida genes, including genes involved in biofilm formation, such as chitinases. Using a chitinase inhibitor, the antibiofilm activity of BDSF was also shown to be chitinase dependent. In addition, BDSF caused an increase in RelA expression and increased levels of (p)ppGpp, leading to decreased biofilm production. These results support our observation that exposure of F. novicida to BDSF causes biofilm dispersal. Furthermore, BDSF upregulated the genes involved in iron acquisition (figABCD), increasing siderophore production. Thus, this study provides evidence for a potential role and mechanism of diffusible signal factor (DSF) signaling in the genus Francisella and suggests the possibility of interspecies signaling between Francisella and other bacteria. Overall, this study suggests that in response to the interspecies DSF signal, F. novicida can alter its gene expression and regulate its biofilm formation. [ABSTRACT FROM AUTHOR]

In many bacteria, the ability to modulate biofilm production relies on specific signaling molecules that are either self-produced or made by neighboring microbes within the ecological niche. We analyzed the potential interspecies signaling effect of the Burkholderia diffusible signal factor (BDSF) on Francisella novicida, a model organism for Francisella tularensis, and demonstrated that BDSF both inhibits the formation and causes the dispersion of Francisella biofilm. Specificity was demonstrated for the cis versus the trans form of BDSF. Using transcriptome sequencing, quantitative reverse transcription-PCR, and activity assays, we found that BDSF altered the expression of many F. novicida genes, including genes involved in biofilm formation, such as chitinases. Using a chitinase inhibitor, the antibiofilm activity of BDSF was also shown to be chitinase dependent. In addition, BDSF caused an increase in RelA expression and increased levels of (p)ppGpp, leading to decreased biofilm production. These results support our observation that exposure of F. novicida to BDSF causes biofilm dispersal. Furthermore, BDSF upregulated the genes involved in iron acquisition (figABCD), increasing siderophore production. Thus, this study provides evidence for a potential role and mechanism of diffusible signal factor (DSF) signaling in the genus Francisella and suggests the possibility of interspecies signaling between Francisella and other bacteria. Overall, this study suggests that in response to the interspecies DSF signal, F. novicida can alter its gene expression and regulate its biofilm formation. [ABSTRACT FROM AUTHOR]

In addition tomembrane disruption, the cathelicidin antimicrobial peptide (AMP) LL-37 translocates through the bacterial inner membrane to target intracellular molecules. The present study aims to identify an alternate mechanism and a cytoplasmic target of LL-37 in Francisella. LL-37 binding proteins from Francisella novicida U112 bacterial lysates were precipitated by using biotinylated LL-37 (B-LL-37) and NeutrAvidin-agarose beads. Bound proteins were identified by LC-MS/MS, validated and characterized by bead pull-down assays and differential scanning fluorimetry (DSF). The cationic AMP (CAMP) LL- 37 was able to interact with Francisella cytoplasmic acyl carrier protein (AcpP; FTN1340/FTT1376). Further study confirmed that LL-37 peptide could bind to AcpP and that the sheep cathelicidin SMAP-29 (Sheep Myeloid Antimicrobial Peptide 29) further increased LL-37 binding to AcpP, suggesting a synergistic effect of SMAP-29 on the binding. LL-37 could also bind to both AcpP of Escherichia coli and Bacillus anthracis, implying amechanism of broad action of LL-37-AcpP binding. Overexpression of the acpP gene in F. novicida led to an increase in LL-37 susceptibility. LL-37 binding to AcpP changed the fatty acid composition profiles. Taken together, we identified a novel cytoplasmic target of LL-37 in Francisella, suggesting a mechanism of action of this peptide beyond membrane permeabilization. Our findings highlight a novel mechanism of antimicrobial activity of this peptide and document a previously unexplored target of α-helical CAMPs. [ABSTRACT FROM AUTHOR]

Burkholderia thailandensis is a Gram-negative soil bacterium used as a model organism for B. pseudomallei, the causative agent of melioidosis and an organism classified category B priority pathogen and a Tier 1 select agent for its potential use as a biological weapon. Burkholderia species are reportedly “highly resistant” to antimicrobial agents, including cyclic peptide antibiotics, due to multiple resistance systems, a hypothesis we decided to test using antimicrobial (host defense) peptides. In this study, a number of cationic antimicrobial peptides (CAMPs) were tested in vitro against B. thailandensis for both antimicrobial activity and inhibition of biofilm formation. Here, we report that the Chinese cobra (Naja atra) cathelicidin NA-CATH was significantly antimicrobial against B. thailandensis. Additional cathelicidins, including the human cathelicidin LL-37, a sheep cathelicidin SMAP-29, and some smaller ATRA peptide derivatives of NA-CATH were also effective. The D-enantiomer of one small peptide (ATRA-1A) was found to be antimicrobial as well, with EC50 in the range of the L-enantiomer. Our results also demonstrate that human alpha-defensins (HNP-1 & -2) and a short beta-defensin-derived peptide (Peptide 4 of hBD-3) were not bactericidal against B. thailandensis. We also found that the cathelicidin peptides, including LL-37, NA-CATH, and SMAP-29, possessed significant ability to prevent biofilm formation of B. thailandensis. Additionally, we show that LL-37 and its D-enantiomer D-LL-37 can disperse pre-formed biofilms. These results demonstrate that although B. thailandensis is highly resistant to many antibiotics, cyclic peptide antibiotics such as polymyxin B, and defensing peptides, some antimicrobial peptides including the elapid snake cathelicidin NA-CATH exert significant antimicrobial and antibiofilm activity towards B. thailandensis. [ABSTRACT FROM AUTHOR]

Abstract: Post translational modifications are required for proteins to be fully functional. The three step process, prenylation, leads to farnesylation or geranylgeranylation, which increase the hydrophobicity of the prenylated protein for efficient anchoring into plasma membranes and/or organellar membranes. Prenylated proteins function in a number of signaling and regulatory pathways that are responsible for basic cell operations. Well characterized prenylated proteins include Ras, Rac and Rho. Recently, pathogenic prokaryotic proteins, such as SifA and AnkB, have been shown to be prenylated by eukaryotic host cell machinery, but their functions remain elusive. The identification of other bacterial proteins undergoing this type of host-directed post-translational modification shows promise in elucidating host–pathogen interactions to develop new therapeutics. This review incorporates new advances in the study of protein prenylation into a broader aspect of biology with a focus on host–pathogen interaction. [Copyright &y& Elsevier]

Background: Chronic, infected wounds typically contain multiple genera of bacteria, including Staphylococcus aureus, many of which are strong biofilm formers. Bacterial biofilms are thought to be a direct impediment to wound healing. New therapies that focus on a biofilm approach may improve the recovery and healing rate for infected wounds. In this study, cathelicidins and related short, synthetic peptides were tested for their antimicrobial effectiveness as well as their ability to inhibit the ability of S. aureus to form biofilms. Results: The helical human cathelicidin LL-37 was tested against S. aureus, and was found to exhibit effective antimicrobial, anti-attachment as well as anti-biofilm activity at concentrations in the low üg/ml range. The effect of peptide chirality and associated protease-resistance was explored through the use of an all-D amino acid peptide, D-LL-37, and in turn compared to scrambled LL-37. Helical cathelicidins have been identified in other animals such as the Chinese cobra, Naja atra (NA-CATH). We previously identified an 11-residue imperfectly repeated pattern (ATRA motif) within the sequence of NA-CATH. A series of short peptides (ATRA-1, -2, -1A), as well as a synthetic peptide, NA-CATH:ATRA1-ATRA1, were designed to explore the significance of the conserved residues within the ATRA motif for anti-microbial activity. The CD spectrum of NA-CATH and NA-CATH:ATRA1-ATRA1 revealed the structural properties of these peptides and suggested that helicity may factor into their anti-microbial and antibiofilm activities. Conclusions: The NA-CATH:ATRA1-ATRA1 peptide inhibits the production of biofilm by S. aureus in the presence of salt, exhibiting anti-biofilm activity at lower peptide concentrations than NA-CATH, LL-37 and D-LL-37; and demonstrates low cytoxicity against host cells but does not affect bacterial attachment. The peptides utilized in this anti-biofilm approach may provide templates for a new group of anti-microbials and potential future topical therapeutics for treating chronic wound infections. [ABSTRACT FROM AUTHOR]

Abstract: Francisella infects the lungs causing pneumonic tularemia. Focusing on the lung’s host defense, we have examined antimicrobial peptides as part of the innate immune response to Francisella infection. Interest in antimicrobial peptides, such as the cathelicidins, has grown due their potential therapeutic applications and the increasing problem of bacterial resistance to commonly used antibiotics. Only one human cathelicidin, LL-37, has been characterized. Helical cathelicidins have also been discovered in snakes including the Chinese King Cobra, Naja atra (NA-CATH). Four synthetic 11-residue peptides (ATRA-1, -2, -1A and -1P) containing variations of a repeated motif within NA-CATH were designed. We hypothesized that these smaller synthetic peptides could have excellent antimicrobial effectiveness with shorter length (and less cost), making them strong potential candidates for development into broad-spectrum antimicrobial compounds. We tested the susceptibility of F. novicida to four ATRA peptides, LL-37, and NA-CATH. Two of the ATRA peptides had high antimicrobial activity (μM), while the two proline-containing ATRA peptides had low activity. The ATRA peptides did not show significant hemolytic activity even at high peptide concentration, indicating low cytotoxicity against host cells. NA-CATH killed Francisella bacteria more quickly than LL-37. However, LL-37 was the most effective peptide against F. novicida (EC50=50nM). LL-37 mRNA was induced in A549 cells by Francisella infection. We recently demonstrated that F. novicida forms in vitro biofilms. LL-37 inhibited F. novicida biofilm formation at sub-antimicrobial concentrations. Understanding the properties of these peptides, and their endogenous expression in the lung could lead to potential future therapeutic interventions for this lung infection. [Copyright &y& Elsevier]

Abstract: The ability of human beta-defensins hBD-1, hBD-2, and hBD-3 to exert direct in vitro antimicrobial effects was evaluated using Francisella tularensis Live Vaccine Strain (LVS) and Francisella novicida. While hBD-2 showed some antimicrobial activity in these assays, only hBD-3 demonstrated significant potency against Francisella. Francisella tularensis LVS infection induced elevated levels of hBD-2 mRNA in human airway epithelial (A549) cells, while having no significant impact on the levels of hBD-3 and only a moderate effect on the level of hBD-1 mRNA. Francisella infection avoided stimulating the production of the most potent anti-Francisella host peptide, hBD-3, in A549 cells, although hBD-3 is stimulated by other treatments. The differential induction of beta-defensins in Francisella infected lung epithelial cells suggests a complex dynamic in the expression of antimicrobial peptides and the innate immune response. [Copyright &y& Elsevier]

Background: Many pathogens, including Yersinia pestis, cannot be consistently and reliably cultured from blood. New approaches are needed to facilitate the detection of proteins, nucleic acid and microorganisms in whole blood samples to improve downstream assay performance. Detection of biomarkers in whole blood is difficult due to the presence of host proteins that obscure standard detection mechanisms. Nanotrap® particles are micron-sized hydrogel structures containing a dye molecule as the affinity bait and used to detect host biomarkers, viral nucleic acids and proteins as well as some bacterial markers. Nanotraps have been shown to bind and enrich a wide variety of biomarkers and viruses in clinically relevant matrices such as urine and plasma. Our objective was to characterize the binding ability of Nanotrap particle type CN3080 to Y. pestis bacteria, bacterial proteins and nucleic acids from whole human blood in order to potentially improve detection and diagnosis. Results: CN3080 Nanotraps bind tightly to Yersinia bacteria, even after washing, and we were able to visualize the co-localized Nanotraps and bacteria by electron microscopy. These magnetic hydrogel Nanotraps were able to bind Yersinia DNA, supporting the utility of Nanotraps for enhancing nucleic acid-based detection methods. Nanotraps were capable of increasing Y. pestis nucleic acid yield by fourfold from whole human blood compared to standard nucleic acid extraction. Interestingly, we found CN3080 Nanotraps to have a high affinity for multiple components of the Yersinia type III secretion system (T3SS), including chaperone proteins, Yop effector proteins and virulence factor protein LcrV (V). Using Nanotraps as a rapid upstream sample-prep tool, we were able to detect LcrV in human blood by western blotting with minimal blood interference in contrast to direct western blotting of blood samples in which LcrV was obscured. We were able to computationally model the interaction of LcrV with the CN3080 Nanotrap dye and found that it had a low delta-G, suggesting high affinity. Importantly, Nanotraps were also able to enhance detection of secreted Yersinia proteins by mass spectrometry. Conclusion: Upstream use of magnetic CN3080 Nanotrap particles may improve the downstream workflow though binding and enrichment of biomarkers and speed of processing. Utilization of Nanotrap particles can improve detection of Yersinia pestis proteins and nucleic acid from whole human blood and contribute to downstream assays and diagnostics including molecular methods such as sequencing and PCR and protein-based methods. [ABSTRACT FROM AUTHOR]

Venezuelan equine encephalitis virus (VEEV) is a neurotropic virus that causes significant disease in both humans and equines. Here we characterized the impact of VEEV on signaling pathways regulating cell death in human primary astrocytes. VEEV productively infected primary astrocytes and caused an upregulation of early growth response 1 (EGR1) gene expression at 9 and 18 h post infection. EGR1 induction was dependent on extracellular signal-regulated kinase1/2 (ERK1/2) and protein kinase R (PKR)-like endoplasmic reticulum kinase (PERK), but not on p38 mitogen activated protein kinase (MAPK) or phosphoinositide 3-kinase (PI3K) signaling. Knockdown of EGR1 significantly reduced VEEV-induced apoptosis and impacted viral replication. Knockdown of ERK1/2 or PERK significantly reduced EGR1 gene expression, dramatically reduced viral replication, and increased cell survival as well as rescued cells from VEEV-induced apoptosis. These data indicate that EGR1 activation and subsequent cell death are regulated through ERK and PERK pathways in VEEV infected primary astrocytes. [ABSTRACT FROM AUTHOR]

Francisella tularensis is the cause of the zoonotic disease tularemia. In Sweden and Scandinavia, epidemiological studies have implicated mosquitoes as a vector. Prior research has demonstrated the presence of Francisella DNA in infected mosquitoes but has not shown definitive transmission of tularemia from a mosquito to a mammalian host. We hypothesized that antimicrobial peptides, an important component of the innate immune system of higher organisms, may play a role in mosquito host-defense to Francisella . We established that Francisella sp. are susceptible to two cecropin antimicrobial peptides derived from the mosquito Aedes albopictus as well as Culex pipiens . We also demonstrated induced expression of Aedes albopictus antimicrobial peptide genes by Francisella infection C6/36 mosquito cell line. We demonstrate that mosquito antimicrobial peptides act against Francisella by disrupting the cellular membrane of the bacteria. Thus, it is possible that antimicrobial peptides may play a role in the inability of mosquitoes to establish an effective natural transmission of tularemia. [ABSTRACT FROM AUTHOR]

Background: Our group has developed a new process for isolating and identifying novel cationic antimicrobial peptides from small amounts of biological samples. Previously, we identified several active antimicrobial peptides from 100 µl of plasma from Alligator mississippiensis. These peptides were found to have in vitro antimicrobial activity against Pseudomonas aeruginosa and Staphylococcus aureus. In this work, we further characterize three of the novel peptides discovered using this process: Apo5, Apo6, and A1P. Results: We examined the activity of these peptides against multi-drug resistant strains and clinical isolates of common human pathogens. We investigated their structural characteristics using circular dichroism and tested for membrane disruption and DNA binding. These peptides were found to have strong in vitro activity against multi-drug resistant and clinically isolated strains of S. aureus, Escherichia coli, P. aeruginosa, and Acinetobacter baumannii. Apo5 and Apo6, peptides derived from alligator apolipoprotein C-1, depolarized the bacterial membrane. A1P, a peptide from the serpin proteinase inhibitor, did not permeabilize membranes. Performing circular dichroism analysis, Apo5 and Apo6 were found to be predominantly helical in SDS and TFE buffer, while A1P has significantly different structures in phosphate buffer, SDS, and TFE. None of these peptides were found to be hemolytic to sheep red blood cells or significantly cytotoxic up to 100 µg/ml after 24 h exposure. Conclusions: Overall, we suggest that Apo5 and Apo6 have a different mode of action than A1P, and that all three peptides make promising candidates for the treatment of drug-resistant bacteria, such as A. baumannii. [ABSTRACT FROM AUTHOR]

Background: The Institute of Theoretical and Experimental Biophysics in Moscow recently developed a new nanoaerosol generator. This study evaluated this novel technology, which has the potential to enhance therapeutic delivery, with the goal of using the generator to treat pulmonary Francisella tularensis subsp. novicida (F. novicida) infections in BALB/c mice. Results: First, the analysis of quantum dots distribution in cryosections of murine lungs demonstrated that nanoaerosols penetrate the alveoli and spread more homogenously in the lungs than upon intranasal delivery. Second, the generator was used to aerosolize the antibiotic levofloxacin to determine the effectiveness of nanoaerosolized levofloxacin as treatment against F. novicida. The generator was capable of delivering a sufficient dose of nanoaerosolized liposome-encapsulated levofloxacin to rescue mice against 100LD50 of F. novicida. Conclusions: The nanoaerosol-delivered dosage of liposome-encapsulated levofloxacin required to rescue mice is approximately 94× lower than the oral required dose and approximately 8× lower than the intraperitoneal dose required for rescue. In addition, treatment with nanoaerosols consumes less total volume of therapeutic solutions and is gentler on sprayed material than the aerosolization by a conventional three-jet Collison nebulizer as seen by the preservation of liposomes. This could represent a significant advance for the use of expensive therapeutics and lung directed therapies. [ABSTRACT FROM AUTHOR]

Cationic antimicrobial peptides and their therapeutic potential have garnered growing interest because of the proliferation of bacterial resistance. However, the discovery of new antimicrobial peptides from animals has proven challenging due to the limitations associated with conventional biochemical purification and difficulties in predicting active peptides from genomic sequences, if known. As an example, no antimicrobial peptides have been identified from the American alligator, Alligator mississippiensis, although their serum is antimicrobial. We have developed a novel approach for the discovery of new antimicrobial peptides from these animals, one that capitalizes on their fundamental and conserved physico-chemical properties. This sample-agnostic process employs custom-made functionalized hydrogel microparticles to harvest cationic peptides from biological samples, followed by de novo sequencing of captured peptides, eliminating the need to isolate individual peptides. After evaluation of the peptide sequences using a combination of rational and web-based bioinformatic analyses, forty-five potential antimicrobial peptides were identified, and eight of these peptides were selected to be chemically synthesized and evaluated. The successful identification of multiple novel peptides, exhibiting antibacterial properties, from Alligator mississippiensis plasma demonstrates the potential of this innovative discovery process in identifying potential new host defense peptides. [ABSTRACT FROM AUTHOR]

A device for the generation of nanoaerosols from biological substances was designed and the resulting nanoaerosols were characterized. The function of the generator is based on the electrospray-neutralization process in which nanoclusters, obtained by electrohydrodynamic atomization, are neutralized by counter-ions generated by the electrospraying of ethanol at the opposite potential. The device is capable of generating nanoaerosol from suspensions of nanoparticles and solutions of non-volatile organic substances (e.g., antibiotics), proteins, and other biomolecules. This device produces nanoaerosols (10–300nm in size) with a concentration up to 2×107 cm−3. A quartz crystal microbalance based device was designed to monitor the performance of the generator. Using magnetic Fe3O4 nanoparticles as reporters, it has been demonstrated that approximately 0.4µg of nanoaerosol was deposited into a mouse lung in 1h in a nose-only exposure system. Thus, we present an approach to both generate nanoaerosol from biological and therapeutic substances and to estimate the doses delivered to mice exposed to these nanoaersolized substances. [ABSTRACT FROM AUTHOR]

Biofilms, multicellular communities of bacteria, may be an environmental survival and transmission mechanism of Francisella tularensis. Chitinases of F. tularensis ssp. novicida (Fn) have been suggested to regulate biofilm formation on chitin surfaces. However, the underlying mechanisms of how chitinases may regulate biofilm formation are not fully determined. We hypothesized that Fn chitinase modulates bacterial surface properties resulting in the alteration of biofilm formation. We analyzed biofilm formation under diverse conditions using chitinase mutants and their counterpart parental strain. Substratum surface charges affected biofilm formation and initial attachments. Biophysical analysis of bacterial surfaces confirmed that the chi mutants had a net negative-charge. Lectin binding assays suggest that chitinase cleavage of its substrates could have exposed the concanavalin A-binding epitope. Fn biofilm was sensitive to chitinase, proteinase and DNase, suggesting that Fn biofilm contains exopolysaccharides, proteins and extracellular DNA. Exogenous chitinase increased the drug susceptibility of Fn biofilms to gentamicin while decreasing the amount of biofilm. In addition, chitinase modulated bacterial adhesion and invasion of A549 and J774A.1 cells as well as intracellular bacterial replication. Our results support a key role of the chitinase(s) in biofilm formation through modulation of the bacterial surface properties. Our findings position chitinase as a potential anti-biofilm enzyme in Francisella species. [ABSTRACT FROM AUTHOR]

Pneumonic tularemia is caused by inhalation of Francisella tularensis, one of the most infectious microbes known. We wanted to study the kinetics of the initial and early interactions between bacterium and host cells in the lung. To do this, we examined the infection of A549 airway epithelial cells with the live vaccine strain (LVS) of F. tularensis. A549 cells were infected and analyzed for global transcriptional response at multiple time points up to 16 h following infection. At 15 min and 2 h, a strong transcriptional response was observed including cytoskeletal rearrangement, intracellular transport, and interferon signaling. However, at later time points (6 and 16 h), very little differential gene expression was observed, indicating a general suppression of the host response consistent with other reported cell lines and murine tissues. Genes for macropinocytosis and actin/cytoskeleton rearrangement were highly up-regulated and common to the 15 min and 2 h time points, suggesting the use of this method for bacterial entry into cells. We demonstrate macropinocytosis through the uptake of FITC-dextran and amiloride inhibition of Francisella LVS uptake. Our results suggest that macropinocytosis is a potential mechanism of intracellular entry by LVS and that the host cell response is suppressed during the first 2-6 h of infection. These results suggest that the attenuated Francisella LVS induces significant host cell signaling at very early time points after the bacteria's interaction with the cell. [ABSTRACT FROM AUTHOR]

Background: The damaging effects of cigarette smoke on the lungs are well known in terms of cancer risks. Additional molecular changes within the lung tissue can also occur as a result of exposure to cigarette smoke. The human &#946-defensin (hBD) class of antimicrobial peptides is the focus of our research. In addition to antimicrobial activity, &#946-defensins also have immunomodulatory functions. Over 30 previously unrecognized &#946-defensin genes have recently been identified in the human genome, many with yet to be determined functions. We postulated that altered &#946-defensin production may play a role in the pathogenesis observed in the lungs of smokers. Our hypothesis is that cigarette smoke exposure will affect the expression of &#946-defensins in human lung alveolar epithelial cells (A549). Methods: We exposed A549 cells to cigarette smoke extract (CSE) and measured the changes in mRNA levels of several antimicrobial peptides by quantitative real-time PCR, and directly observed peptide expression in cells by immunofluorescence (IF) microscopy. Results: We found that hBD3, hBD5, and hBD9 gene expression was upregulated in A549 cells exposed to CSE. HBD1, hBD8, hBD18 and LL-37 gene expression did not significantly change upon exposure to CSE. Expression of hBD3 and hBD4 peptides was visualized by IF. Conclusions: This differential expression suggests that hBD3, hBD5, and hBD9 may play a role in the changes to the lung tissue observed in smokers. Establishing differential &#946-defensin expression following CSE treatment will add to our understanding of the molecular response of the lung alveolar epithelium to cigarette smoke exposure. [ABSTRACT FROM AUTHOR]

Bacteria, plants, and algae produce isoprenoids through the methylerythritol phosphate (MEP) pathway, an attractive pathway for antimicrobial drug development as it is present in prokaryotes and some lower eukaryotes but absent from human cells. The first committed step of the MEP pathway is catalyzed by 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR/MEP synthase). MEP pathway genes have been identified in many biothreat agents, including Francisella, Brucella, Bacillus, Burkholderia, and Yersinia. The importance of the MEP pathway to Francisella is demonstrated by the fact that MEP pathway mutations are lethal. We have previously established that fosmidomycin inhibits purified MEP synthase (DXR) from F. tularensis LVS. FR900098, the acetyl derivative of fosmidomycin, was found to inhibit the activity of purified DXR from F. tularensis LVS (IC50 = 230 nM). Fosmidomycin and FR900098 are effective against purified DXR from Mycobacterium tuberculosis as well, but have no effect on whole cells because the compounds are too polar to penetrate the thick cell wall. Fosmidomycin requires the GlpT transporter to enter cells, and this is absent in some pathogens, including M. tuberculosis. In this study, we have identified the GlpT homologs in F. novicida and tested transposon insertion mutants of glpT. We showed that FR900098 also requires GlpT for full activity against F. novicida. Thus, we synthesized several FR900098 prodrugs that have lipophilic groups to facilitate their passage through the bacterial cell wall and bypass the requirement for the GlpT transporter. One compound, that we termed "compound 1," was found to have GlpT-independent antimicrobial activity. We tested the ability of this best performing prodrug to inhibit F. novicida intracellular infection of eukaryotic cell lines and the caterpillar Galleria mellonella as an in vivo infection model. As a lipophilic GlpT-independent DXR inhibitor, compound 1 has the potential to be a broad-spectrum antibiotic, and should be effective against most MEP- dependent organisms. [ABSTRACT FROM AUTHOR]

Background: Macrolide antibiotics are commonly administered for bacterial respiratory illnesses. Azithromycin (Az) is especially noted for extremely high intracellular concentrations achieved within macrophages which is far greater than the serum concentration. Clinical strains of Type B Francisella (F.) tularensis have been reported to be resistant to Az, however our laboratory Francisella strains were found to be sensitive. We hypothesized that different strains/species of Francisella (including Type A) may have different susceptibilities to Az, a widely used and well-tolerated antibiotic. Results: In vitro susceptibility testing of Az confirmed that F. tularensis subsp. holarctica Live Vaccine Strain (LVS) (Type B) was not sensitive while F. philomiragia, F. novicida, and Type A F. tularensis (NIH B38 and Schu S4 strain) were susceptible. In J774A.1 mouse macrophage cells infected with F. philomiragia, F. novicida, and F. tularensis LVS, 5 μg/ml Az applied extracellularly eliminated intracellular Francisella infections. A concentration of 25 μg/ml Az was required for Francisellainfected A549 human lung epithelial cells, suggesting that macrophages are more effective at concentrating Az than epithelial cells. Mutants of RND efflux components (tolC and ftlC) in F. novicida demonstrated less sensitivity to Az by MIC than the parental strain, but the tolC disc-inhibition assay demonstrated increased sensitivity, indicating a complex role for the outer-membrane transporter. Mutants of acrA and acrB mutants were less sensitive to Az than the parental strain, suggesting that AcrAB is not critical for the efflux of Az in F. novicida. In contrast, F. tularensis Schu S4 mutants ?acrB and Δ?acrA were more sensitive than the parental strain, indicating that the AcrAB may be important for Az efflux in F. tularensis Schu S4. F. novicida LPS O-antigen mutants (wbtN, wbtE, wbtQ and wbtA) were found to be less sensitive in vitro to Az compared to the wild-type. Az treatment prolonged the survival of Galleria (G.) mellonella infected with Francisella. Conclusion: These studies demonstrate that Type A Francisella strains, as well as F. novicida and F. philomiragia, are sensitive to Az in vitro. Francisella LPS and the RND efflux pump may play a role in Az sensitivity. Az also has antimicrobial activity against intracellular Francisella, suggesting that the intracellular concentration of Az is high enough to be effective against multiple strains/species of Francisella, especially in macrophages. Az treatment prolonged survival an in vivo model of Francisella-infection. [ABSTRACT FROM AUTHOR]

Deliberate and natural outbreaks of infectious disease underscore the necessity of effective vaccines and antimicrobial/ antiviral therapeutics. The prevalence of antibiotic resistant strains and the ease by which antibiotic resistant bacteria can be intentionally engineered further highlights the need for continued development of novel antibiotics against new bacterial targets. Isoprenes are a class of molecules fundamentally involved in a variety of crucial biological functions. Mammalian cells utilize the mevalonic acid pathway for isoprene biosynthesis, whereas many bacteria utilize the methylerythritol phosphate (MEP) pathway, making the latter an attractive target for antibiotic development. In this report we describe the cloning and characterization of Francisella tularensis MEP synthase, a MEP pathway enzyme and potential target for antibiotic development. In vitro growth-inhibition assays using fosmidomycin, an inhibitor of MEP synthase, illustrates the effectiveness of MEP pathway inhibition with F. tularensis. To facilitate drug development, F. tularensis MEP synthase was cloned, expressed, purified, and characterized. Enzyme assays produced apparent kinetic constants (KMDXP = 104 μM, KMNADPH=13 μM, kcatDXP=2 s-1, kcatNADPH = 1.3 s-1, an IC50 for fosmidomycin of 247 nM, and a Ki for fosmidomycin of 99 nM. The enzyme exhibits a preference for Mg+2 as a divalent cation. Titanium dioxide chromatography-tandem mass spectrometry identified Ser177 as a site of phosphorylation. S177D and S177E site-directed mutants are inactive, suggesting a mechanism for post-translational control of metabolic flux through the F. tularensis MEP pathway. Overall, our study suggests that MEP synthase is an excellent target for the development of novel antibiotics against F. tularensis. [ABSTRACT FROM AUTHOR]