On the Offensive: the Role of Outer Membrane Vesicles in the Successful Dissemination of New Delhi Metallo-ß-lactamase (NDM-1).

The emergence and worldwide dissemination of carbapenemase-producing Gram-negative bacteria are a major public health threat. Metallo-ß-lactamases (MBLs) represent the largest family of carbapenemases. Regrettably, these resistance determinants are spreading worldwide. Among them, the New Delhi metallo-ß-lactamase (NDM-1) is experiencing the fastest and largest geographical spread. NDM-1 ß-lactamase is anchored to the bacterial outer membrane, while most MBLs are soluble, periplasmic enzymes. This unique cellular localization favors the selective secretion of active NDM-1 into outer membrane vesicles (OMVs). Here, we advance the idea that NDM-containing vesicles serve as vehicles for the local dissemination of NDM-1. We show that OMVs with NDM-1 can protect a carbapenem-susceptible strain of Escherichia coli upon treatment with meropenem in a Galleria mellonella infection model. Survival curves of G. mellonella revealed that vesicle encapsulation enhances the action of NDM-1, prolonging and favoring bacterial protection against meropenem inside the larva hemolymph. We also demonstrate that E. coli cells expressing NDM-1 protect a susceptible Pseudomonas aeruginosa strain within the larvae in the presence of meropenem. By using E. coli variants engineered to secrete variable amounts of NDM-1, we demonstrate that the protective effect correlates with the amount of NDM-1 secreted into vesicles. We conclude that secretion of NDM-1 into OMVs contributes to the survival of otherwise susceptible nearby bacteria at infection sites. These results disclose that OMVs play a role in the establishment of bacterial communities, in addition to traditional horizontal gene transfer mechanisms. IMPORTANCE Resistance to carbapenems, last-resort antibiotics, is spreading worldwide, raising great concern. NDM-1 is one of the most potent and widely disseminated carbapenem-hydrolyzing enzymes spread among many bacteria and is secreted to the extracellular medium within outer membrane vesicles. We show that vesicles carrying NDM-1 can protect carbapenem-susceptible strains of E. coli and P. aeruginosa upon treatment with meropenem in a live infection model. These vesicles act as nanoparticles that encapsulate and transport NDM-1, prolonging and favoring its action against meropenem inside a living organism. Secretion of NDM-1 into vesicles contributes to the survival of otherwise susceptible nearby bacteria at infection sites. We propose that vesicles play a role in the establishment of bacterial communities and the dissemination of antibiotic resistance, in addition to traditional horizontal gene transfer mechanisms.

Review of antiviral peptides for use against zoonotic and selected non-zoonotic viruses.

Viruses remain one of the leading causes of animal and human disease. Some animal viral infections spread sporadically to human populations, posing a serious health risk. Particularly the emerging viral zoonotic diseases such as the novel, zoonotic coronavirus represent an actual challenge for the scientific and medical community. Besides human health risks, some animal viral infections, although still not zoonotic, represent important economic loses to the livestock industry. Viral infections pose a genuine concern for which there has been an increasing interest for new antiviral molecules. Among these novel compounds, antiviral peptides have been proposed as promising therapeutic options, not only for the growing body of evidence showing hopeful results but also due to the many adverse effects of chemical-based drugs. Here we review the current progress, key targets and considerations for the development of antiviral peptides (AVPs). The review summarizes the state of the art of the AVPs tested in zoonotic (coronaviruses, Rift Valley fever viruses, Eastern Equine Encephalitis Virus, Dengue and Junín virus) and also non-zoonotic farm animal viruses (avian and cattle viruses). Their molecular target, amino acid sequence and mechanism of action are summarized and reviewed. Antiviral peptides are currently on the cutting edge since they have been reported to display anti-coronavirus activity. Particularly, the review will discuss the specific mode of action of AVPs that specifically inhibit the fusion of viral and host-cell membranes for SARS-CoV-2, showing in detail some important features of the fusion inhibiting peptides that target the spike protein of these risky viruses.

Interactions of "de novo" designed peptides with bacterial membranes: Implications in the antimicrobial activity.

Antimicrobial peptides are small molecules that display antimicrobial activity against a wide range of pathogens. In a previous work, by using model membranes we studied P6, a peptide that shows no antimicrobial activity, and P6.2, which exhibits antibacterial activity. In the present work we aimed to unravel the mode of action of these peptides by studying their interaction in vivo with Escherichia coli and Staphylococcus aureus. In this sense, to study the interactions with bacterial cells and their effect on the bacterial surface, zeta potential, spectroscopic, and microscopic methodologies were applied. P6.2 exhibits a higher affinity toward both bacterial envelopes. The ability of both peptides to disrupt afterwards the bacterial membrane was also studied. Both peptides were able to induce bacterial membrane damage, but higher concentrations of P6 were needed to obtain results comparable to those obtained for P6.2. Additionally, P6.2 exhibited faster damage kinetics. Altogether, these data allow postulating, in a physiologic model, that the lower affinity of P6 for bacterial envelope results in a minor final concentration of the peptide in the bacterial membrane unable to trigger the antimicrobial activity. Finally, the fact that the active P6.2 has the same MIC value for the Gram-positive and Gram-negative bacteria tested, but not the same profile in the permeabilization assays, reinforces the question of whether cell wall components act as electrostatic barriers preventing or minimizing membrane-active AMPs lethal action at the membrane level.

Unravelling the mechanism of action of "de novo" designed peptide P1 with model membranes and gram-positive and gram-negative bacteria.

In the last years, the decreasing effectiveness of conventional antimicrobial-drugs has caused serious problems due to the rapid emergence of multidrug-resistant pathogens. This situation has brought attention to other antimicrobial agents like antimicrobial peptides (AMPs), for being considered an alternative to conventional drugs. These compounds target bacterial membranes for their activity, which gives them a broad spectrum of action and less probable resistance development. That is why the peptide-membrane interaction is a crucial aspect to consider in the study of AMPs. The aim of this work was the characterization of the "de novo" designed peptide P1, studying its interactions with model membranes (i.e. liposomes of DMPC:DMPG 5:1) in order to evaluate the final position of the peptide upon interacting with the membrane. Also, we tested the effects of the peptide in gram-positive and gram-negative bacteria. Later, by spectroscopic methods, the ability of the peptide to permeabilize the inner and outer membrane of E. coli and plasmatic membrane of S. aureus was assessed. The results obtained confirmed that P1 can disrupt both membranes, showing some difference in its activity as a function of the nature of each bacterial cell wall, confirming higher effects on gram-positive S. aureus. Finally, we also showed the ability of P1 to inhibit biofilms of that gram-positive bacterium. All data obtained in this work allowed us to propose a model, where the first interactions of the peptide with the bacterial envelope, seem to depend on the gram-negative and gram-positive cell wall structure. After that first interaction, the peptide is stabilized by Trp residues depth inserted into the hydrocarbon region, promoting several changes in the organization of the lipid bilayer, following a carpet-like mechanism, which results in permeabilization of the membrane, triggering the antimicrobial activity.

New insights into novel Escherichia coli colistin-resistant strains isolated from Argentina.

Colistin is a polymyxin antibiotic (polymyxin E) that has in recent years re-emerged as an option for treatment of multidrug-resistant bacteria. Recently, the re-introduction of colistin resulted in the appearance of colistin-resistant bacteria, which is usually caused by LPS modifications. The fact that this modification is mediated by a plasmid carrying the mcr-1 gene, implies a horizontal transfer of colistin resistance. In Argentina, the National Reference Laboratory in Antimicrobial Resistance (NRLAR), has recently screened several bacteria for the MCR-1 plasmid, detecting nine Escherichia coli isolates carrying the plasmid with the mcr-1 gene, among others. In this context, we proposed to assess the effect of surface charge modifications induced by the plasmid MCR-1 and its impact on the resulting colistin resistance in two clinical isolates of colistin-resistant E. coli. Using zeta potential assays, we confirmed the reduction of negative charge exposure on clinical isolates compared to the reference strain of E. coli. In addition, through permeabilization assays, we were able to correlate this reduction in charge exposure with the extent of damage to the bacterial membrane. The fact that this surface charge modification through substitution of lipid A is plasmid encoded, represents an important concern for future antimicrobial peptide drug development.

Fast Biofilm Penetration and Anti-PAO1 Activity of Nebulized Azithromycin in Nanoarchaeosomes.

Azithromycin (AZ) is a broad-spectrum antibiotic with anti-inflammatory and antiquorum sensing activity against biofilm forming bacteria such as Pseudomonas aeruginosa. AZ administered by oral or parenteral routes, however, neither efficiently accesses nor remains in therapeutic doses inside pulmonary biofilm depths. Instead, inhaled nanocarriers loaded with AZ may revert the problem of low accessibility and permanence of AZ into biofilms, enhancing its antimicrobial activity. The first inhalable nanovesicle formulation of AZ, nanoarchaeosome-AZ (nanoARC-AZ), is here presented. NanoARC prepared with total polar archaeolipids (TPAs), rich in 2,3-di-O-phytanyl-sn-glycero-1-phospho-(3'-sn-glycerol-1'-methylphosphate) (PGP-Me) from Halorubrum tebenquichense archaebacteria, consisted of ∼180 nm-diameter nanovesicles, loaded with 0.28 w/w AZ/TPA. NanoARC-AZ displayed lower minimal inhibitory concentration and minimal bactericidal concentration, higher preformed biofilm disruptive, and anti-PAO1 activity in biofilms than AZ. NanoARC penetrated and disrupted the structure of the PAO1 biofilm within only 1 h. Two milliliters of 15 µg/mL AZ nanoARC-AZ nebulized for 5 min rendered AZ doses compatible with in vitro antibacterial activity. The strong association between AZ and the nanoARC bilayer, combined with electrostatic attraction and trapping into perpendicular methyl groups of archaeolipids, as determined by Laurdan fluorescence anisotropy, generalized polarization, and small-angle X-ray scattering, was critical to stabilize during storage and endure shear forces of nebulization. NanoARC-AZ was noncytotoxic on A549 cells and human THP-1-derived macrophages, deserving further preclinical exploration as enhancers of AZ anti-PAO1 activity.

Synergistic and antibiofilm activity of the antimicrobial peptide P5 against carbapenem-resistant Pseudomonas aeruginosa.

In the search for new antimicrobial molecules, antimicrobial peptides (AMPs) offer a viable alternative to conventional antibiotics, as they physically disrupt the bacterial membranes, leading to membrane disruption and eventually cell death. In particular, the group of linear α-helical cationic peptides has attracted increasing research and clinical interest. The AMP P5 has been previously designed as a cationic linear α-helical sequence, being its antimicrobial and hemolytic properties also evaluated. In this work, we analyzed the feasibility of using P5 against a carbapenem-resistant clinical isolate of Pseudomonas aeruginosa, one of the most common and risky pathogens in clinical practice. After antimicrobial activity confirmation in in vitro studies, synergistic activity of P5 with meropenem was evaluated, showing that P5 displayed significant synergistic activity in a time kill curve assay. The ability of P5 to permeabilize the outer membrane of P. aeruginosa can explain the obtained results. Finally, the antibiofilm activity was investigated by viability analysis (MTT assay), crystal violet and confocal imaging, with P5 displaying mild biofilm inhibition in the range of concentrations tested. Regarding biofilm disruption activity, P5 showed a higher efficacy, interfering with biofilm structure and promoting bacterial cell death. Atomic force microscope images further demonstrated the peptide potential in P. aeruginosa biofilm eradication, confirming the promising application of P5 in multi-resistant infections therapeutics.

Antimicrobial Peptides: Interaction With Model and Biological Membranes and Synergism With Chemical Antibiotics.

Antimicrobial peptides (AMPs) are promising novel antibiotics since they have shown antimicrobial activity against a wide range of bacterial species, including multiresistant bacteria; however, toxicity is the major barrier to convert antimicrobial peptides into active drugs. A profound and proper understanding of the complex interactions between these peptides and biological membranes using biophysical tools and model membranes seems to be a key factor in the race to develop a suitable antimicrobial peptide therapy for clinical use. In the search for such therapy, different combined approaches with conventional antibiotics have been evaluated in recent years and demonstrated to improve the therapeutic potential of AMPs. Some of these approaches have revealed promising additive or synergistic activity between AMPs and chemical antibiotics. This review will give an insight into the possibilities that physicochemical tools can give in the AMPs research and also address the state of the art on the current promising combined therapies between AMPs and conventional antibiotics, which appear to be a plausible future opportunity for AMPs treatment.

Cementoin-SLPI fusion protein binds to human monocytes and epithelial cells and shows higher biological activity than SLPI.

Secretory Leukocyte Proteinase Inhibitor (SLPI) is an antiinflammatory peptide that blocks the activity of serine proteases, primarily the neutrophil elastase. In an attempt to direct the activity of SLPI on inflamed sites, a chimera consisting of the transglutaminase II substrate domain of trappin 2 (cementoin), and the mature SLPI protein was constructed. Cell attachment and biological activity were compared between SLPI and this chimera. By using whole cell ELISA, fluorescence microscopy and flow cytometry assays we observed that the cementoin-SLPI fusion protein (FP) but not SLPI attached to a human lung epithelial cell line and monocytes. A maximum attachment was achieved 15 min after FP was added to the cell cultures. In an elastase activity assay, we observed that FP retained its antiprotease activity and that at equimolar amount of proteins, FP was more efficient than SLPI in the inhibition. Both, FP and SLPI inhibits IL-2-induced lymphocyte proliferation, however, lower amounts of FP were required to achieve this inhibition. Furthermore, FP binds to mycobacteria and maintained the bactericidal activity observed for SLPI. Overall, these results show that this new chimera is able to attach to the cell surfaces retaining and improving some biological activities described for SLPI.

Cationic Antimicrobial Peptides Inactivate Shiga Toxin-Encoding Bacteriophages.

Shiga toxin (Stx) is the principal virulence factor during Shiga toxin-producing Escherichia coli (STEC) infections. We have previously reported the inactivation of bacteriophage encoding Stx after treatment with chitosan, a linear polysaccharide polymer with cationic properties. Cationic antimicrobial peptides (cAMPs) are short linear aminoacidic sequences, with a positive net charge, which display bactericidal or bacteriostatic activity against a wide range of bacterial species. They are promising novel antibiotics since they have shown bactericidal effects against multiresistant bacteria. To evaluate whether cationic properties are responsible for bacteriophage inactivation, we tested seven cationic peptides with proven antimicrobial activity as anti-bacteriophage agents, and one random sequence cationic peptide with no antimicrobial activity as a control. We observed bacteriophage inactivation after incubation with five cAMPs, but no inactivating activity was observed with the random sequence cationic peptide or with the non-alpha helical cAMP Omiganan. Finally, to confirm peptide-bacteriophage interaction, zeta potential was analyzed by following changes on bacteriophage surface charges after peptide incubation. According to our results we could propose that: (1) direct interaction of peptides with phage is a necessary step for bacteriophage inactivation, (2) cationic properties are necessary but not sufficient for bacteriophage inactivation, and (3) inactivation by cationic peptides could be sequence (or structure) specific. Overall our data suggest that these peptides could be considered a new family of molecules potentially useful to decrease bacteriophage replication and Stx expression.

-759C>T Polymorphism of the HTR2C Gene is Associated with Second Generation Antipsychotic-Induced Weight Gain in Female Patients with Schizophrenia.

Introduction: The HTR2C gene is an important candidate in pharmacogenetic studies of antipsychotic-induced weight gain (AIWG). However, inconsistent results have been obtained. The present study investigated the association between -759C>T, functional polymorphism of the HTR2C receptor, and AIWG. Methods: A prospective cohort of 48 female inpatients with schizophrenia and related illness treated according to normal clinical practice with second generation antipsychotics (SGAs) risperidone, clozapine, quetiapine, and olanzapine were evaluated. Patients were weighted at admission and again at 6 weeks of hospitalization. Weight gain was defined as an increase≥7% of baseline weight. The association between polymorphisms HTR2C and weight gain was evaluated. Multiple logistic regression was run to determine potential confounders. Results: Patients with the T allele at position -759 (TT or CT) gained less weight as compared to patients who did not have the allele. This association was not affected by possible confounding factors such as age, baseline BMI, and prior psychopharmacological treatment. Discussion: The T allele at position -759 protects against AIWG in female patients with schizophrenia.

Role of amphipathicity and hydrophobicity in the balance between hemolysis and peptide-membrane interactions of three related antimicrobial peptides.

Cationic antimicrobial peptides (CAMPs) represent important self defense molecules in many organisms, including humans. These peptides have a broad spectrum of activities, killing or neutralizing many Gram-negative and Gram-positive bacteria. The emergence of multidrug resistant microbes has stimulated research on the development of alternative antibiotics. In the search for new antibiotics, cationic antimicrobial peptides (CAMPs) offer a viable alternative to conventional antibiotics, as they physically disrupt the bacterial membranes, leading to lysis of microbial membranes and eventually cell death. In particular, the group of linear α-helical cationic peptides has attracted increasing interest from clinical as well as basic research during the last decade. In this work, we studied the biophysical and microbiological characteristics of three new designed CAMPs. We modified a previously studied CAMP sequence, in order to increase or diminish the hydrophobic face, changing the position of two lysines or replacing three leucines, respectively. These mutations modified the hydrophobic moment of the resulting peptides and allowed us to study the importance of this parameter in the membrane interactions of the peptides. The structural properties of the peptides were also correlated with their membrane-disruptive abilities, antimicrobial activities and hemolysis of human red blood cells.

Anti-tumor effect of SLPI on mammary but not colon tumor growth.

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor that was related to cancer development and metastasis dissemination on several types of tumors. However, it is not known the effect of SLPI on mammary and colon tumors. The aim of this study was to examine the effect of SLPI on mammary and colon tumor growth. The effect of SLPI was tested on in vitro cell apoptosis and in vivo tumor growth experiments. SLPI over-expressing human and murine mammary and colon tumor cells were generated by gene transfection. The administration of murine mammary tumor cells over-expressing high levels of SLPI did not develop tumors in mice. On the contrary, the administration of murine colon tumor cells over-expressing SLPI, developed faster tumors than control cells. Intratumoral, but not intraperitoneal administration of SLPI, delayed the growth of tumors and increased the survival of mammary but not colon tumor bearing mice. In vitro culture of mammary tumor cell lines treated with SLPI, and SLPI producer clones were more prone to apoptosis than control cells, mainly under serum deprivation culture conditions. Herein we demonstrated that SLPI induces the apoptosis of mammary tumor cells in vitro and decreases the mammary but not colon tumor growth in vivo. Therefore, SLPI may be a new potential therapeutic tool for certain tumors, such as mammary tumors.

Serine leucocyte proteinase inhibitor-treated monocyte inhibits human CD4(+) lymphocyte proliferation.

Serine leucocyte proteinase inhibitor (SLPI) is the main serine proteinase inhibitor produced by epithelial cells and has been shown to be a pleiotropic molecule with anti-inflammatory and microbicidal activities. However, the role of SLPI on the adaptive immune response is not well established. Therefore, we evaluated the effect of SLPI on lymphocyte proliferation and cytokine production. Human peripheral blood mononuclear cells (PBMC) were treated with mitogens plus SLPI and proliferation was assessed by [(3) H]thymidine uptake. The SLPI decreased the lymphocyte proliferation induced by interleukin-2 (IL-2) or OKT3 monoclonal antibodies in a dose-dependent manner. Inhibition was not observed when depleting monocytes from the PBMC and it was restored by adding monocytes and SLPI. SLPI-treated monocyte slightly decreased MHC II and increased CD18 expression, and secreted greater amounts of IL-4, IL-6 and IL-10 in the cell culture supernatants. SLPI-treated monocyte culture supernatant inhibited the CD4(+) lymphocyte proliferation but did not affect the proliferation of CD8(+) cells. Moreover, IL-2 increased T-bet expression and the presence of SLPI significantly decreased it. Finally, SLPI-treated monocyte culture supernatant dramatically decreased interferon-γ but increased IL-4, IL-6 and IL-10 in the presence of IL-2-treated T cells. Our results demonstrate that SLPI target monocytes, which in turn inhibit CD4 lymphocyte proliferation and T helper type 1 cytokine secretion. Overall, these results suggest that SLPI is an alarm protein that modulates not only the innate immune response but also the adaptive immune response.

Secretory leukocyte protease inhibitor: a secreted pattern recognition receptor for mycobacteria.

RATIONALE: Human secretory leukocyte protease inhibitor (SLPI) displays bactericidal activity against pathogens such as Escherichia coli and Streptococcus. Furthermore, it has been reported that murine SLPI shows potent antimycobacterial activity. OBJECTIVES: The aim of the present study was to investigate whether human recombinant SLPI not only kills mycobacteria but also acts as a pattern recognition receptor for the host immune system. METHODS: For the in vivo experiment, BALB/c mice were infected by intranasal instillation with Mycobacterium bovis BCG and viable BCG load in lung homogenates was later determined. For the in vitro experiments, SLPI was incubated overnight with a suspension of M. bovis BCG or the virulent strain Mycobacterium tuberculosis H37Rv, and the percentage survival as well as the binding of SLPI to mycobacteria was determined. Furthermore, bacteria phagocytosis was also determined by flow cytometry. MEASUREMENTS AND MAIN RESULTS: Intranasal SLPI treatment decreased the number of colony-forming units recovered from lung homogenates, indicating that SLPI interfered with M. bovis BCG infection. Moreover, SLPI decreased the viability of both M. bovis BCG and H37Rv. We demonstrated that SLPI attached to the surface of the mycobacteria by binding to pathogen-associated molecular pattern mannan-capped lipoarabinomannans and phosphatidylinositol mannoside. Furthermore, we found that in the sputum of patients with tuberculosis, mycobacteria were coated with endogenous SLPI. Finally, we showed that phagocytosis of SLPI-coated mycobacteria was faster than that of uncoated bacteria. CONCLUSIONS: The present results demonstrate for the first time that human SLPI kills mycobacteria and is a new pattern recognition receptor for them.

Platelet-endothelial cell adhesion molecule-1 (CD31) recycles and induces cell growth inhibition on human tumor cell lines.

CD31 (PECAM-1) is a 130-kDa member of the immunoglobulin gene superfamily expressed on endothelial cells, platelets, and most leukocytes. This report demonstrates by Western Blot and immunofluorescence that some human melanoma and adenocarcinoma cell lines express CD31 on the cell surface. The surface expression of CD31 was regulated by cell-cell contact, being higher on sparse and spontaneously detached cells. Indeed, fixing and permeabilizing tumor cells revealed a cytoplasmic pool, which was confirmed by confocal microscopy. Some of the plasma surface molecule is endocytosed following mAb binding. Engagement of CD31 on tumor cells via domain-3 inhibited proliferation by inducing cell apoptosis. On the other hand, apoptosis does not increase CD31 expression. Overall, these results indicate that there is an intracellular pool of CD31 on some tumor cells, which modulates CD31 surface expression and its role in cancer cell growth and metastasis. Thus, the expression of CD31 and its role in cell survival in some tumor cells appears to differ from endothelial cells.