Cationic antimicrobial peptides and biogenic silver nanoparticles kill mycobacteria without eliciting DNA damage and cytotoxicity in mouse macrophages.

With the emergence of multidrug-resistant mycobacterial strains, better therapeutic strategies are required for the successful treatment of the infection. Although antimicrobial peptides (AMPs) and silver nanoparticles (AgNPs) are becoming one of the popular antibacterial agents, their antimycobacterial potential is not fully evaluated. In this study, we synthesized biogenic-silver nanoparticles using bacterial, fungal, and plant biomasses and analyzed their antibacterial activities in combination with AMPs against mycobacteria. Mycobacterium smegmatis was found to be more susceptible to AgNPs compared to M. marinum. We found that NK-2 showed enhanced killing effect with NP-1 and NP-2 biogenic nanoparticles at a 0.5-ppm concentration, whereas LLKKK-18 showed antibacterial activity only with NP-2 at 0.5-ppm dose against M. smegmatis. In case of M. marinum NK-2 did not show any additive activity with NP-1 and NP-2 and LLKKK-18 alone completely inhibited the bacterial growth. Both NP-1 and NP-2 also showed increased killing of M. smegmatis in combination with the antituberculosis drug rifampin. The sizes and shapes of the AgNPs were determined by transmission electron microscopy and dynamic light scattering. AgNPs showed no cytotoxic or DNA damage effects on macrophages at the mycobactericidal dose, whereas treatment with higher doses of AgNPs caused toxicity and micronuclei formation in cytokinesis blocked cells. Macrophages actively endocytosed fluorescein isothiocyanate-labeled AgNPs resulting in nitric oxide independent intracellular killing of M. smegmatis. Apoptosis and cell cycle studies showed that treatment with higher dose of AgNPs arrested macrophages at the G1-phase. In summary, our data suggest the combined effect of biogenic-AgNPs and antimicrobial peptides as a promising antimycobacterial template.

Cathelicidin is involved in the intracellular killing of mycobacteria in macrophages.

Macrophages have been shown to kill Mycobacterium tuberculosis through the action of the antimicrobial peptide cathelicidin (CAMP), whose expression was shown to be induced by 1,25-dihydroxyvitamin D3 (1,25D3). Here, we investigated in detail the antimycobacterial effect of murine and human cathelicidin against Mycobacterium smegmatis and M. bovis BCG infections. We have synthesized novel LL-37 peptide variants that exhibited potent in vitro bactericidal activity against M. smegmatis, M. bovis BCG and M. tuberculosis H37Rv, as compared with parental peptide. We show that the exogenous addition of LL-37 or endogenous overexpression of cathelicidin in macrophages significantly reduced the intracellular survival of mycobacteria relative to control cells. An upregulation of cathelicidin mRNA expression was observed that correlated with known M. smegmatis killing phases in J774 macrophages. Moreover, RNAi-based Camp knock-down macrophages and Camp(-/-) bone marrow derived mouse macrophages were significantly impaired in their ability to kill mycobacteria. M. smegmatis killing in Camp(-/-) macrophages was less extensive than in Camp(+/+) cells following activation with FSL-1, an inducer of cathelicidin expression. Finally we show that LL-37 and 1,25D3 treatment results in increase in colocalization of BCG-containing phagosomes with lysosomes. Altogether, these data demonstrate that cathelicidin plays an important role in controlling intracellular survival of mycobacteria.

An investigation on the antibacterial, cytotoxic, and antibiofilm efficacy of starch-stabilized silver nanoparticles.

The increased emergence of drug resistant microbes creates a major challenge to the scientific community for successful development of effective therapeutics. The antimicrobial activities of silver ions are well known, but limited information is available on the effects of green silver-nanoparticles (AgNPs) on human pathogens. In this article, we evaluated the antibacterial activity of starch-stabilized AgNPs against a panel of human pathogens commonly associated with air, water and food borne infections. The shape and size distribution of AgNPs were characterized by transmission electron microscopy. We showed that AgNPs were more effective against Gram-positive and Gram-negative pathogens as compared with acid-fast bacteria. AgNPs were not cytotoxic to macrophages at the bactericidal concentration and can augment intracellular killing potential of macrophages. Furthermore, we showed that AgNPs disrupt biofilm formation and exhibit better antibacterial activity compared to human cationic antimicrobial peptide LL-37. In summary, our data suggest AgNPs as a promising template for the design of novel antibacterial agents.

Bimetallic gold-silver nanoparticles mediate bacterial killing by disrupting the actin cytoskeleton MreB.

The actin cytoskeleton is required for the maintenance of the cell shape and viability of bacteria. It remains unknown to which extent nanoparticles (NPs) can orchestrate the mechanical instability by disrupting the cytoskeletal network in bacterial cells. Our work demonstrates that Au-Ag NPs disrupt the bacterial actin cytoskeleton specifically, fluidize the inner membrane and lead to killing of bacterial cells. In this study, we have tried to emphasize on the key parameters important for NP-cell interactions and found that the shape, specific elemental surface localization and enhanced electrostatic interaction developed due to the acquired partial positive charge by silver atoms in the aggregated NPs are some of the major factors contributing towards better NP interactions and subsequent cell death. In vivo studies in bacterial cells showed that the NPs exerted a mild perturbation of the membrane potential. However, its most striking effect was on the actin cytoskeleton MreB resulting in morphological changes in the bacterial cell shape from rods to predominantly spheres. Exposure to NPs resulted in the delocalization of MreB patches from the membrane but not the tubulin homologue FtsZ. Concomitant with the redistribution of MreB localization, a dramatic increase of membrane fluid regions was observed. Our studies reveal for the first time that Au-Ag NPs can mediate bacterial killing and disrupt the actin cytoskeletal functions in bacteria.

Mycobacterium tuberculosis EsxO (Rv2346c) promotes bacillary survival by inducing oxidative stress mediated genomic instability in macrophages.

Mycobacterium tuberculosis (Mtb) survives inside the macrophages by modulating the host immune responses in its favor. The 6-kDa early secretory antigenic target (ESAT-6; esxA) of Mtb is known as a potent virulence and T-cell antigenic determinant. At least 23 such ESAT-6 family proteins are encoded in the genome of Mtb; however, the function of many of them is still unknown. We herein report that ectopic expression of Mtb Rv2346c (esxO), a member of ESAT-6 family proteins, in non-pathogenic Mycobacterium smegmatis strain (MsmRv2346c) aids host cell invasion and intracellular bacillary persistence. Further mechanistic studies revealed that MsmRv2346c infection abated macrophage immunity by inducing host cell death and genomic instability as evident from the appearance of several DNA damage markers. We further report that the induction of genomic instability in infected cells was due to increase in the hosts oxidative stress responses. MsmRv2346c infection was also found to induce autophagy and modulate the immune function of macrophages. In contrast, blockade of Rv2346c induced oxidative stress by treatment with ROS inhibitor N-acetyl-L-cysteine prevented the host cell death, autophagy induction and genomic instability in infected macrophages. Conversely, MtbΔRv2346c mutant did not show any difference in intracellular survival and oxidative stress responses. We envision that Mtb ESAT-6 family protein Rv2346c dampens antibacterial effector functions namely by inducing oxidative stress mediated genomic instability in infected macrophages, while loss of Rv2346c gene function may be compensated by other redundant ESAT-6 family proteins. Thus EsxO plays an important role in mycobacterial pathogenesis in the context of innate immunity.

Toxicity and antibacterial assessment of chitosan-coated silver nanoparticles on human pathogens and macrophage cells.

BACKGROUND: Pathogenic bacteria are able to develop various strategies to counteract the bactericidal action of antibiotics. Silver nanoparticles (AgNPs) have emerged as a potential alternative to conventional antibiotics because of their potent antimicrobial properties. The purpose of this study was to synthesize chitosan-stabilized AgNPs (CS-AgNPs) and test for their cytotoxic, genotoxic, macrophage cell uptake, antibacterial, and antibiofilm activities. METHODS: AgNPs were synthesized using chitosan as both a stabilizing and a reducing agent. Antibacterial activity was determined by colony-forming unit assay and scanning electron microscopy. Genotoxic and cytotoxic activity were determined by DNA fragmentation, comet, and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays. Cellular uptake and intracellular antibacterial activity were tested on macrophages. RESULTS: CS-AgNPs exhibited potent antibacterial activity against different human pathogens and also impeded bacterial biofilm formation. Scanning electron microscopy analysis indicated that CS-AgNPs kill bacteria by disrupting the cell membrane. CS-AgNPs showed no significant cytotoxic or DNA damage effect on macrophages at the bactericidal dose. Propidium iodide staining indicated active endocytosis of CS-AgNPs resulting in reduced intracellular bacterial survival in macrophages. CONCLUSION: The present study concludes that at a specific dose, chitosan-based AgNPs kill bacteria without harming the host cells, thus representing a potential template for the design of antibacterial agents to decrease bacterial colonization and to overcome the problem of drug resistance.

Azurophil granule proteins constitute the major mycobactericidal proteins in human neutrophils and enhance the killing of mycobacteria in macrophages.

Pathogenic mycobacteria reside in, and are in turn controlled by, macrophages. However, emerging data suggest that neutrophils also play a critical role in innate immunity to tuberculosis, presumably by their different antibacterial granule proteins. In this study, we purified neutrophil azurophil and specific granules and systematically analyzed the antimycobacterial activity of some purified azurophil and specific granule proteins against M. smegmatis, M. bovis-BCG and M. tuberculosis H37Rv. Using gel overlay and colony forming unit assays we showed that the defensin-depleted azurophil granule proteins (AZP) were more active against mycobacteria compared to other granule proteins and cytosolic proteins. The proteins showing antimycobacterial activity were identified by MALDI-TOF mass spectrometry. Electron microscopic studies demonstrate that the AZP disintegrate bacterial cell membrane resulting in killing of mycobacteria. Exogenous addition of AZP to murine macrophage RAW 264.7, THP-1 and peripheral blood monocyte-derived macrophages significantly reduced the intracellular survival of mycobacteria without exhibiting cytotoxic activity on macrophages. Immunofluorescence studies showed that macrophages actively endocytose neutrophil granular proteins. Treatment with AZP resulted in increase in co-localization of BCG containing phagosomes with lysosomes but not in increase of autophagy. These data demonstrate that neutrophil azurophil proteins may play an important role in controlling intracellular survival of mycobacteria in macrophages.

Membrane-active antimicrobial peptides and human placental lysosomal extracts are highly active against mycobacteria.

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, manifests discreet strategies to subvert host immune responses, which enable the pathogen to survive and multiply inside the macrophages. This problem is further worsened by the emergence of multidrug resistant mycobacterial strains, which make most of the anti-tuberculous drugs ineffective. It is thus imperative to search for and design better therapeutic strategies, including employment of new antibiotics. Recently, naturally produced antimicrobial molecules such as enzymes, peptides and their synthetic analogs have emerged as compounds with potentially significant therapeutical applications. Although, many antimicrobial peptides have been identified only very few of them have been tested against mycobacteria. A major limitation in using peptides as therapeutics is their sensitivity to enzymatic degradation or inactivity under certain physiological conditions such as relatively high salt concentration. Here, we show that NK-2, a peptide representing the cationic core region of the lymphocytic effector protein NK-lysin, and Ci-MAM-A24, a synthetic salt-tolerant peptide derived from immune cells of Ciona intestinalis, efficiently kill Mycobacterium smegmatis and Mycobacterium bovis-BCG. In addition, NK-2 and Ci-MAM-A24 showed a synergistic killing effect against M. smegmatis, no cytotoxic effect on mouse macrophages at bactericidal concentrations, and were even found to kill mycobacteria residing inside the macrophages. We also show that human placental lysosomal contents exert potent killing effect against mycobacteria under acidic and reducing growth conditions. Electron microscopic studies demonstrate that the lysosomal extract disintegrate bacterial cell membrane resulting in killing of mycobacteria.