Microbial Killing Research Articles

Network Science and Group Fusion Similarity-Based Searching to Explore the Chemical Space of Antiparasitic Peptides.

Antimicrobial peptides (AMPs) have appeared as promising compounds to treat a wide range of diseases. Their clinical potentialities reside in the wide range of mechanisms they can use for both killing microbes and modulating immune responses. However, the hugeness of the AMPs' chemical space (AMPCS), represented by more than 1065 unique sequences, has represented a big challenge for the discovery of new promising therapeutic peptides and for the identification of common structural motifs. Here, we introduce network science and a similarity searching approach to discover new promising AMPs, specifically antiparasitic peptides (APPs). We exploited the network-based representation of APPs' chemical space (APPCS) to retrieve valuable information by using three network types: chemical space (CSN), half-space proximal (HSPN), and metadata (METN). Some centrality measures were applied to identify in each network the most important and nonredundant peptides. Then, these central peptides were considered as queries (Qs) in group fusion similarity-based searches against a comprehensive collection of known AMPs, stored in the graph database StarPepDB, to propose new potential APPs. The performance of the resulting multiquery similarity-based search models (mQSSMs) was evaluated in five benchmarking data sets of APP/non-APPs. The predictions performed by the best mQSSM showed a strong-to-very-strong performance since their external Matthews correlation coefficient (MCC) values ranged from 0.834 to 0.965. Outstanding MCC values (>0.85) were attained by the mQSSM with 219 Qs from both networks CSN and HSPN with 0.5 as similarity threshold in external data sets. Then, the performance of our best mQSSM was compared with the APPs prediction servers AMPDiscover and AMPFun. The proposed model showed its relevance by outperforming state-of-the-art machine learning models to predict APPs. After applying the best mQSSM and additional filters on the non-APP space from StarPepDB, 95 AMPs were repurposed as potential APP hits. Due to the high sequence diversity of these peptides, different computational approaches were applied to identify relevant motifs for searching and designing new APPs. Lastly, we identified 11 promising APP lead candidates by using our best mQSSMs together with diversity-based network analyses, and 24 web servers for activity/toxicity and drug-like properties. These results support that network-based similarity searches can be an effective and reliable strategy to identify APPs. The proposed models and pipeline are freely available through the StarPep toolbox software at http://mobiosd-hub.com/starpep.

Bacteria‐Responsive Self‐Assembly of Antimicrobial Peptide Nanonets for Trap‐and‐Kill of Antibiotic‐Resistant Strains

AbstractBacterial trapping using nanonets is a ubiquitous immune defense mechanism against infectious microbes. These nanonets can entrap microbial cells, effectively arresting their dissemination and rendering them more vulnerable to locally secreted microbicides. Inspired by this evolutionarily conserved anti‐infective strategy, a series of 15 to 16 residue‐long synthetic β‐hairpin peptides is herein constructed with the ability to self‐assemble into nanonets in response to the presence of bacteria, enabling spatiotemporal control over microbial killing. Using amyloid‐specific K114 assay and confocal microscopy, the membrane components lipoteichoic acid and lipopolysaccharide are shown to play a major role in determining the amyloid‐nucleating capacity as triggered by Gram‐positive and Gram‐negative bacteria respectively. These nanonets displayed both trapping and killing functionalities, hence offering a direct improvement from the trap‐only biomimetics in literature. By substituting a single turn residue of the non‐amyloidogenic BTT1 peptide, the nanonet‐forming BTT1‐3A analog is produced with comparable antimicrobial potency. With the same sequence manipulation approach, BTT2‐4A analog modified from BTT2 peptide showed improved antimicrobial potency against colistin‐resistant clinical isolates. The peptide nanonets also demonstrated robust stability against proteolytic degradation, and promising in vivo efficacy and biosafety profile. Overall, these bacteria‐responsive peptide nanonets are promising clinical anti‐infective alternatives for circumventing antibiotic resistance.

Secretory Leucoprotease Inhibitor (SLPI) Promotes Survival during Acute Pseudomonas aeruginosa Infection by Suppression of Inflammation Rather Than Microbial Killing.

Secretory leucoprotease inhibitor (SLPI) has multifaceted functions, including inhibition of protease activity, antimicrobial functions, and anti-inflammatory properties. In this study, we show that SLPI plays a role in controlling pulmonary Pseudomonas aeruginosa infection. Mice lacking SLPI were highly susceptible to P. aeruginosa infection, however there was no difference in bacterial burden. Utilising a model of P. aeruginosa LPS-induced lung inflammation, human recombinant SLPI (hrSLPI) administered intraperitoneally suppressed the recruitment of inflammatory cells in the bronchoalveolar lavage fluid (BALF) and resulted in reduced BALF and serum levels of inflammatory cytokines and chemokines. This anti-inflammatory effect of hrSLPI was similarly demonstrated in a systemic inflammation model induced by intraperitoneal injection of LPS from various bacteria or lipoteichoic acid, highlighting the broad anti-inflammatory properties of hrSLPI. Moreover, in bone-marrow-derived macrophages, hrSLPI reduced LPS-induced phosphorylation of p-IkB-α, p-IKK-α/β, p-P38, demonstrating that the anti-inflammatory effect of hrSLPI was due to the inhibition of the NFκB and MAPK pathways. In conclusion, administration of hrSLPI attenuates excessive inflammatory responses and is therefore, a promising strategy to target inflammatory diseases such as acute respiratory distress syndrome or sepsis and could potentially be used to augment antibiotic treatment.

Modulation of Host Defense in Chronic Granulomatous Disease (CGD) Via Adoptive Transfer of Neutrophil Progenitor Cells

Ectopic expression of transcription factor HoxB8 in murine bone marrow stem cells programs cells to stably expand exponentially in culture. When HoxB8 expression is regulated through a conditional promoter, cells are capable of expansion through HoxB8 induction or differentiation into neutrophil-like cells through HoxB8 withdrawal. We have previously demonstrated that some derivations of HoxB8 progenitor cells are capable of engraftment in an unconditioned murine host leading to rapid formation of a transient population of donor neutrophils in the periphery. Donor derived neutrophils (ddN) function comparably to host wild-type (WT) neutrophils ex vivo. Based on these findings, we hypothesize that conditionally immortalized HoxB8 progenitor cells have the potential to serve as a novel neutrophil driven cellular therapy. To investigate the use of HoxB8 progenitor cells as a cellular therapy for disease, we utilized a well characterized murine model of the congenital disorder CGD. Individuals with CGD are particularly susceptible to infections with Staphylococcus aureus due to loss of reactive oxygen species (ROS) production via the NADPH oxidase. Impaired neutrophil function leads to increased inflammation and decreased microbial killing. We found that prophylactic treatment of CGD mice with an adoptive transfer (AT) of WT HoxB8 progenitor cells protected them from an otherwise lethal pulmonary infection (Figure 1). Mice which received an AT of progenitor cells derived from a CGD animal did not have improved outcomes. This suggests the effector functions of the WT ddN were responsible for the change in survival. To better understand the mechanism of protection derived from the AT, we developed a novel fluorescent killing assay. This assay demonstrated the positive influence of the WT ddN on the host CGD neutrophils intracellular killing capacity in vivo. In addition to prophylactic treatment, post infectious treatment with the progenitor cells improved survival to >50% (Figure 2). Interestingly, twelve hours post adoptive transfer, donor derived cells represent a small population of the BAL cellular environment (<1%). Evaluation of the BAL and serum cytokine environment at this time-point provide insight to the immuno-modulatory role of the donor derived cells. Results from this study demonstrate the promise of a neutrophil progenitor based cellular therapy. This approach is unique from those currently employed as progenitor cells engraft without conditioning, expand and differentiate in vivo unlike granulocyte transfusions, and may be genetically modified in culture to potentially address the challenges of microbial resistance genes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Minocycline intra-bacterial pharmacokinetic hysteresis as a basis for pharmacologic memory and a backbone for once-a-week pan-tuberculosis therapy.

Background: There is need for shorter duration regimens for the treatment of tuberculosis, that can treat patients regardless of multidrug resistance status (pan-tuberculosis). Methods: We combined minocycline with tedizolid, moxifloxacin, and rifampin, in the hollow fiber system model of tuberculosis and mimicked each drugs' intrapulmonary pharmacokinetics for 28days. Minocycline-tedizolid was administered either as a once-a-week or a daily regimen. In order to explore a possible explanation for effectiveness of the once-a-week regimen, we measured systemic and intra-bacterial minocycline pharmacokinetics. Standard daily therapy (rifampin, isoniazid, pyrazinamide) was the comparator. We then calculated γ f or kill slopes for each regimen and ranked the regimens by time-to-extinction predicted in patients. Results: The steepest γ f and shortest time-to-extinction of entire bacterial population was with daily minocycline-rifampin combination. There was no difference in γ f between the minocycline-tedizolid once-a-week versus the daily therapy (p = 0.85). Standard therapy was predicted to cure 88% of patients, while minocycline-rifampin would cure 98% of patients. Minocycline concentrations fell below minimum inhibitory concentration after 2days of once-weekly dosing schedule. The shape of minocycline intra-bacterial concentration-time curve differed from the extracellular pharmacokinetic system and lagged by several days, consistent with system hysteresis. Hysteresis explained the persistent microbial killing after hollow fiber system model of tuberculosis concentrations dropped below the minimum inhibitory concentration. Conclusion: Minocycline could form a backbone of a shorter duration once-a-week pan-tuberculosis regimen. We propose a new concept of post-antibiotic microbial killing, distinct from post-antibiotic effect. We propose system hysteresis as the basis for the novel concept of pharmacologic memory, which allows intermittent dosing.

Non-Canonical Functions of Myeloperoxidase in Immune Regulation, Tissue Inflammation and Cancer.

Myeloperoxidase (MPO) is one of the most abundantly expressed proteins in neutrophils. It serves as a critical component of the antimicrobial defense system, facilitating microbial killing via generation of reactive oxygen species (ROS). Interestingly, emerging evidence indicates that in addition to the well-recognized canonical antimicrobial function of MPO, it can directly or indirectly impact immune cells and tissue responses in homeostatic and disease states. Here, we highlight the emerging non-canonical functions of MPO, including its impact on neutrophil longevity, activation and trafficking in inflammation, its interactions with other immune cells, and how these interactions shape disease outcomes. We further discuss MPO interactions with barrier forming endothelial and epithelial cells, specialized cells of the central nervous system (CNS) and its involvement in cancer progression. Such diverse function and the MPO association with numerous inflammatory disorders make it an attractive target for therapies aimed at resolving inflammation and limiting inflammation-associated tissue damage. However, while considering MPO inhibition as a potential therapy, one must account for the diverse impact of MPO activity on various cellular compartments both in health and disease.

PENICILLIN: A JOURNEY FROM A MAGIC BULLET TO A RESISTANT DRUG

PENICILLIN: A JOURNEY FROM A MAGIC BULLET TO A RESISTANT DRUG

Effect of negative air ionization technology on microbial reduction of food-related microorganisms

Negative air ions (NAI) have been shown to have bactericidal effects on various microbial species.Different concentrations of NAI and ozone (106-7x106 NAI cm-3 s−1, 2.20 mg h-1 of ozone or 8 × 106-2x107 NAI cm−3 s−1, 0.02 mg h−1 of ozone, respectively) were studied to determine whether the effect of these treatments on six food-related microorganisms was due to NAI or also to the ozone that forms.Bacillus subtilis, Escherichia coli, and Listeria monocytogenes showed a significantly microbial reduction after the treatments of NAI with a low concentration of ozone, while Pseudomonas fluorescens, Penicillium roqueforti, and Saccharomyces cerevisiae were affected mainly by NAI and higher ozone concentration. It should be noticed that the reduction of the pathogen L. monocytogenes has been 2.17 Log after 72 h treatment to NAI and low ozone concentration.The results proved that ions (NAI) contribute to microbial killing. However, the sensitivity of microbial species is different and depends on individual parameters of the different microorganisms.NAI technology with low ozone production can be rapid, green, and low-cost and it can represent an alternative to the use of chemicals for the sanitation of air and surfaces in the food sector in order to reduce microbial contamination.

An overview of complement systems in teleosts

Complement plays an important role in the innate immune system, and it comprises about 35 individual proteins. In mammals, complement is activated via three different pathways, the classical pathway, the alternative pathway, and the lectin pathway. All three activation pathways produce C3-convertase in different forms. C3-convertase cleaves C3 to C3a and C3b and initiates a cascade of cleavage and activation, eventually resulting in the formation of the membrane attack complex. Complement activation results in the generation of activated fragments that are involved in microbial killing, phagocytosis, inflammatory reactions, immune complex clearance, and antibody production. Although the complement system has been studied extensively in mammals, complement is less well understood in teleosts. This review summarizes the current knowledge of the teleost complement components involved in phagocytosis, chemotaxis, and cell lysis. We report the characterized complement components in various teleost species. In addition, we provide a comprehensive compilation of complement regulators, and this information is used to analyze the role of complement regulators in pathogen infection. The influence of complement receptors on the immune responses of teleosts is reviewed. Finally, we propose directions for future study of the molecular evolution, structure, and function of complement components in teleosts. This review provides new insights into the complement system of recognition and defense, and such knowledge is essential for the development of new immune strategies in aquaculture.

Niclosamide-loaded nanoparticles disrupt Candida biofilms and protect mice from mucosal candidiasis.

Candida albicans biofilms are a complex multilayer community of cells that are resistant to almost all classes of antifungal drugs. The bottommost layers of biofilms experience nutrient limitation where C. albicans cells are required to respire. We previously reported that a protein Ndu1 is essential for Candida mitochondrial respiration; loss of NDU1 causes inability of C. albicans to grow on alternative carbon sources and triggers early biofilm detachment. Here, we screened a repurposed library of FDA-approved small molecule inhibitors to identify those that prevent NDU1-associated functions. We identified an antihelminthic drug, Niclosamide (NCL), which not only prevented growth on acetate, C. albicans hyphenation and early biofilm growth, but also completely disengaged fully grown biofilms of drug-resistant C. albicans and Candida auris from their growth surface. To overcome the suboptimal solubility and permeability of NCL that is well known to affect its in vivo efficacy, we developed NCL-encapsulated Eudragit EPO (an FDA-approved polymer) nanoparticles (NCL-EPO-NPs) with high niclosamide loading, which also provided long-term stability. The developed NCL-EPO-NPs completely penetrated mature biofilms and attained anti-biofilm activity at low microgram concentrations. NCL-EPO-NPs induced ROS activity in C. albicans and drastically reduced oxygen consumption rate in the fungus, similar to that seen in an NDU1 mutant. NCL-EPO-NPs also significantly abrogated mucocutaneous candidiasis by fluconazole-resistant strains of C. albicans, in mice models of oropharyngeal and vulvovaginal candidiasis. To our knowledge, this is the first study that targets biofilm detachment as a target to get rid of drug-resistant Candida biofilms and uses NPs of an FDA-approved nontoxic drug to improve biofilm penetrability and microbial killing.

Multidrug-Resistant Microbial Therapy Using Antimicrobial Peptides and the CRISPR/Cas9 System.

The emergence and spread of multidrug-resistant microbes become a serious threat to animal and human health globally because of their less responsiveness to conventional antimicrobial therapy. Multidrug-resistant microbial infection poses higher morbidity and mortality rate with significant economic losses. Currently, antimicrobial peptides and the CRISPR/Cas9 system are explored as alternative therapy to circumvent the challenges of multidrug-resistant organisms. Antimicrobial peptides are small molecular weight, cationic peptides extracted from all living organisms. It is a promising drug candidate for the treatment of multidrug-resistant microbes by direct microbial killing or indirectly modulating the innate immune system. The CRISPR/Cas9 system is another novel antimicrobial alternative used to manage multidrug-resistant microbial infection. It is a versatile gene-editing tool that uses engineered single guide RNA for targeted gene recognition and the Cas9 enzyme for the destruction of target nucleic acids. Both the CRISPR/Cas9 system and antimicrobial peptides were used to successfully treat nosocomial infections caused by ESKAPE pathogens, which developed resistance to various antimicrobials. Despite, their valuable roles in multidrug-resistant microbial treatments, both the antimicrobial peptides and the CRISPR/Cas systems have various limitations like toxicity, instability, and incurring high manufacturing costs. Thus, this review paper gives detailed explanations of the roles of the CRISPR/Cas9 system and antimicrobial peptides in circumventing the challenges of multidrug-resistant microbial infections, its limitation and prospects in clinical applications.

Antimicrobial Properties of Highly Elastic Conductive Poly(ethylene terephthalate)/Multiwalled Carbon Nanotube Fabrics

This research focused on understanding the microbe killing mechanism of the antimicrobial properties of highly elastic conductive poly(ethylene terephthalate) (PET)/multi-walled carbon nanotube (MWCNT) powernet fabrics with the help of electric fields and highly elastic structure. In this study, PET/MWCNT fabrics containing three different percentages of MWCNT were knitted and characterized with antimicrobial activity, cytotoxicity, electromagnetic shielding properties, DSC analyses, stiffness tests, and pressure measurements using wireless pressure sensors. Results show that MWCNT has a statistically significant effect on antibacterial activity, cytotoxicity, electromagnetic shielding, stiffness, and exerted pressures. PET/MWCNT fabrics showed excellent antibacterial activity against tested germs; S. aureus and E. coli. A statistically significant increase in percentage reduction of bacteria was observed with the increase in carbon nanotube nanoparticle concentration, accompanied by a good laudering durability even after 20 washing cycle. In terms of cytotoxicity tests, all PET/MWCNT samples were found to have over 70% average relative cell viability, indicating that they are noncytotoxic without interrupting the cell line and primary cell growth in vitro. The materials have provided longer conductive networks and formed electromagnetic shielding at the range of 22.79 dB-25.77 dB at the frequency of 0–1.30 GHz. Moreover, MWCNT concentration did not lead to considerable changes in physical properties like stiffness and exerted pressure properties. Characterization analyses confirmed the changes in the chemical structure of the PET/MWCNT.

Procoagulant Activity of Blood and Microvesicles Is Disturbed by Pneumococcal Pneumolysin, Which Interacts with Coagulation Factors

The coagulation and contact systems are parts of the innate immune system as they prevent bleeding and dissemination of pathogens and also contribute to microbial killing by inflammatory reactions and the release of antimicrobial peptides. Here, we investigated the influence of Streptococcus pneumoniae on the coagulation and contact system. S. pneumoniae (pneumococci), but no other investigated streptococcal species, impairs coagulation of blood by autolysis and release of pneumolysin. Defective blood coagulation results from the lysis of tissue factor-producing mononuclear cells and their procoagulant microvesicles, which are the main trigger for blood coagulation during sepsis. In addition, pneumolysin binds coagulation and contact system factors, but this does not result in activation. Thus, pneumococci modulate activation of the coagulation system by releasing pneumolysin, which could potentiate lung injury during pneumonia.

Rational Use of Antibiotics and Requisition of Pharmacist

Background: Direct consequence of microbes developing resistance against antibiotics is prolonged hospitalization, increased treatment cost and duration. Increased duration of hospitalization causes prolonged use antibiotics which results towards side effects. Primary Study Objective: The purpose of antibiotics use is either to kill the microbes (bactericidal) or slows down their growth (bacteriostatic). Irrational use of antibiotics makes them do their exact opposite. Instead of killing microbes or slowing down their growth, it allows microbes to develop resistance against antibiotics. Methods/Design: An observational concurrent study was performed in which data was collected. Individuals were interviewed using structured questionnaire. Percentage of rationality and irrationality of antibiotics use was observed. Additionally percentage of prescribed class of antibiotics was also observed in patients. Setting: Health care system includes: Quaid e Azam International Hospital Islamabad, Benazir Hospital Rawalpindi, CMH Rawalakot AJK, Amna Hospital Rawalakot AJK, DHQ Kotli AJK Pakistan. Participants: This study was conducted on 100 patients of different health care systems, to whom antibiotics were prescribed. Most patients interviewed were female with UTI aged above 30 and adults male with RTI or chest infection. Intervention: Different patients were interviewed in different hospital settings to evaluate the rational use of antibiotics Primary Outcome Measures: Rational use of antibiotics is using antibiotics according to the guidelines provided by WHO. Major guidelines include:&nbsp; patient receives medication according to their clinical needs, dosage appropriate to the individual requirements and for specific duration of time with low cost. Results: 47 percent of these patients were using antibiotics rationally and 53 percent were taking antibiotics medication irrationally. The most prevalent form of irrational use was patients without counseling about the antibiotics use. Patients with poor knowledge about the use of antibiotics were 84 among the 100. Irrational use of antibiotics in the form of pre-mature discontinuation was reported 56 patients among the 100. Patient who irrationally use antibiotics as OTC or Self medications were 38 among the 100. Conclusion: Major form of irrationality found in the study was no proper counseling provided to the patients by specialist physician or Pharmacists. The reason we critically observed for this is the burden of patients on physicians due to which they are not able to properly guide patients. The community on which this study was conducted has a major absence of pharmacist. Dispensers and pharmacy technicians are performing pharmacist’s tasks. Absence of pharmacist leads to dosage dispensing errors, lack of professional medical counseling, and no drug utilization review

Lactoferricins impair the cytosolic membrane of Escherichia coli within a few seconds and accumulate inside the cell.

We report the real-time response of Escherichia coli to lactoferricin-derived antimicrobial peptides (AMPs) on length scales bridging microscopic cell sizes to nanoscopic lipid packing using millisecond time-resolved synchrotron small-angle X-ray scattering. Coupling a multiscale scattering data analysis to biophysical assays for peptide partitioning revealed that the AMPs rapidly permeabilize the cytosolic membrane within less than 3 s-much faster than previously considered. Final intracellular AMP concentrations of ∼80-100 mM suggest an efficient obstruction of physiologically important processes as the primary cause of bacterial killing. On the other hand, damage of the cell envelope and leakage occurred also at sublethal peptide concentrations, thus emerging as a collateral effect of AMP activity that does not kill the bacteria. This implies that the impairment of the membrane barrier is a necessary but not sufficient condition for microbial killing by lactoferricins. The most efficient AMP studied exceeds others in both speed of permeabilizing membranes and lowest intracellular peptide concentration needed to inhibit bacterial growth.

Study of the synergistic effect of singlet oxygen with other plasma-generated ROS in fungi inactivation during water disinfection

Cold atmospheric plasma (CAP) possesses the ability of high-efficiency disinfection. It is reported that mixtures of reactive oxygen species (ROS) including ·OH, 1O2, O2− and H2O2 generated from CAP have better antimicrobial ability than mimicked solution of mixture of single ROS type, but the reason is not clear. In this study, CAP was applied to treat yeasts in water in order to investigate the fungal inactivation efficiency and mechanism. The results showed that plasma treatment for 5 min could result in >2-log reduction of yeast cells, and application of varied ROS scavengers could significantly increase the yeast survival rate, indicating that ·OH and 1O2 played the pivotal role in yeast inactivation. Moreover, the synergistic effect of 1O2 with other plasma-generated ROS was revealed. 1O2 could diffuse into cells and induce the depolarization of mitochondrial membrane potential (MMP), and different levels of MMP depolarization determined different cell death modes. Mild damage of mitochondria during short-term plasma treatment could lead to apoptosis. For long-term plasma treatment, the cell membrane could be severely damaged by the plasma-generated ·OH, so a large amount of 1O2 could induce more depolarization of MMP, leading to increase of intracellular O2− and Fe2+ which subsequently caused cell inactivation. 1O2 could also induce protein aggregation and increase of RIP1/RIP3 necrosome, leading to necroptosis. With participation of 1O2, endogenous ·OH could also be generated via Fenton and Haber-Weiss reactions during plasma treatment, which potentiated necroptosis. Adding l-His could mitigate membrane damage, inhibit the drop of MMP and the formation of necrosome, and thus prevent the happening of necroptosis. These findings may deepen the understanding of plasma sterilization mechanisms and provide guidance for microbial killing in the environment.

Identification and Functional Characterization of Peptides With Antimicrobial Activity From the Syphilis Spirochete, Treponema pallidum.

The etiological agent of syphilis, Treponema pallidum ssp. pallidum, is a highly invasive “stealth” pathogen that can evade the host immune response and persist within the host for decades. This obligate human pathogen is adept at establishing infection and surviving at sites within the host that have a multitude of competing microbes, sometimes including pathogens. One survival strategy employed by bacteria found at polymicrobial sites is elimination of competing microorganisms by production of antimicrobial peptides (AMPs). Antimicrobial peptides are low molecular weight proteins (miniproteins) that function directly via inhibition and killing of microbes and/or indirectly via modulation of the host immune response, which can facilitate immune evasion. In the current study, we used bioinformatics to show that approximately 7% of the T. pallidum proteome is comprised of miniproteins of 150 amino acids or less with unknown functions. To investigate the possibility that AMP production is an unrecognized defense strategy used by T. pallidum during infection, we developed a bioinformatics pipeline to analyze the complement of T. pallidum miniproteins of unknown function for the identification of potential AMPs. This analysis identified 45 T. pallidum AMP candidates; of these, Tp0451a and Tp0749 were subjected to further bioinformatic analyses to identify AMP critical core regions (AMPCCRs). Four potential AMPCCRs from the two predicted AMPs were identified and peptides corresponding to these AMPCCRs were experimentally confirmed to exhibit bacteriostatic and bactericidal activity against a panel of biologically relevant Gram-positive and Gram-negative bacteria. Immunomodulation assays performed under inflammatory conditions demonstrated that one of the AMPCCRs was also capable of differentially regulating expression of two pro-inflammatory chemokines [monocyte chemoattractant protein-1 (MCP-1) and interleukin-8 (IL-8)]. These findings demonstrate proof-of-concept for our developed AMP identification pipeline and are consistent with the novel concept that T. pallidum expresses AMPs to defend against competing microbes and modulate the host immune response.

Chemokines and anionic phospholipids: new binding partners for microbial killing and apoptotic cell clearance

Abstract Chemokines constitute a large family of small cytokines that regulate leukocyte migration in immunity and inflammation, some of which also have direct bacteria-killing properties. While the molecular mechanism by which chemokines kill bacteria remains poorly understood, chemokines mediate cell migration by interacting with cell surface glycosaminoglycans (GAGs) and G protein-coupled receptors (GPCRs). We have discovered that many chemokines interact with high affinity with anionic membrane phospholipids, including phosphatidylserine (PS) and cardiolipin (CL) which are selectively exposed on the membrane of apoptotic cells and apoptotic extracellular vesicles (ApoEV), and bacteria, respectively. This adds to GPCRs and GAGs a third class of cell surface binding site for chemokines and expands the scope of chemokine action to the biological niches occupied by PS and CL. For instance, we found that ApoEV induce phagocyte migration for apoptotic cell clearance by presenting PS-binding chemokines on the vesicle surface to GPCRs expressed by phagocytes. This is a novel PS-dependent mechanism for chemokine delivery by extracellular vesicles potentially relevant to other areas of immunology and indicates that chemokines may play a more prominent role in the removal of dying cells than previously reported. On the other hand, we discovered that only CL-binding chemokines are antimicrobial and that CL-deficient bacteria are resistant to chemokine treatment, indicating that chemokines kill bacteria by targeting CL on the bacterial membrane. In short, we have identified chemokines as the first family of soluble cytokines whose bioactivities can be regulated by direct interaction with membrane phospholipids. Supported by NIAID/NIH intramural research program

Development of a Microenvironment-Responsive Hydrogel Promoting Chronically Infected Diabetic Wound Healing through Sequential Hemostatic, Antibacterial, and Angiogenic Activities.

Microenvironment-responsive hydrogels present high potential in treating refractory wounds due to their capability of on-demand drug release. In this study, a specially designed hydrogel with smart targeting of refractory wound characteristics was designed to treat chronically infected diabetic wounds. Aminated gelatin reacted with oxidized dextran, forming a hydrogel cross-linked with a dynamic Schiff base, which is sensitive to the low-pH environment in refractory wounds. Nano-ZnO was loaded into the hydrogel for killing microbes. A Paeoniflorin-encapsulated micelle with a ROS-responsive property was fixed to the skeleton of the hydrogel via a Schiff base bond for low-pH- and ROS-stimulated angiogenic activity. The sequential responsiveness of the novel hydrogel enabled smart rescue of the deleterious microenvironment in refractory wounds. This highly biocompatible hydrogel demonstrated antibacterial and angiogenic abilities in vitro and significantly promoted healing of chronically infected diabetic wounds via sequential hemostatic, microbe killing, and angiogenic activities. This microenvironment-responsive hydrogel loaded with nZnO and Pf-encapsulated micelles holds great potential as a location-specific dual-response delivery platform for curing refractory, chronically infected diabetic wounds.

Promising Photocytotoxicity of Water-Soluble Phtalocyanine against Planktonic and Biofilm Pseudomonas aeruginosa Isolates from Lower Respiratory Tract and Chronic Wounds

Alternative methods of killing microbes have been extensively researched in connection with the widespread appearance of antibiotic resistance among pathogenic bacteria. In this study, we report on in vitro antimicrobial phototoxicity research of cationic phthalocyanine with 2-(4-N-methylmorpholin-4-ium-4-yl)ethoxy substituents against selected clinical strains of Pseudomonas aeruginosa isolated from the lower respiratory tract and chronic wounds. The microorganisms tested in the research were analyzed in terms of drug resistance and biofilm formation. The photocytotoxic effect of phthalocyanine was determined by the reduction factor of bacteria. The studied cationic phthalocyanine at a concentration of 1.0 × 10−4 M, when activated by light, revealed a significant reduction factor, ranging from nearly 4 to 6 log, of P. aeruginosa cells when compared to the untreated control group. After single irradiation, a decrease in the number of bacteria in biofilm ranging from 1.3 to 4.2 log was observed, whereas the second treatment significantly improved the bacterial reduction factor from 3.4 to 5.5 log. It is worth mentioning that a boosted cell-death response was observed after the third irradiation, with a bacterial reduction factor ranging from 4.6 to 6.4 log. According to the obtained results, the tested photosensitizer can be considered as a potential antimicrobial photodynamic therapy against multidrug-resistant P. aeruginosa.