Membrane Lysis Research Articles

Insights into the Mechanism of Action of the Two-Peptide Lantibiotic Lacticin 3147

Lacticin 3147 is a two peptide lantibiotc (LtnA1 and LtnA2) that displays nanomolar activity against many Gram-positive bacteria. Lacticin 3147 may exert its antimicrobial effect by several mechanisms. Isothermal titration calorimetry experiments show that only LtnA1 binds to the peptidoglycan precursor lipid II, which could inhibit peptidoglycan biosynthesis. An experimentally supported model of the resulting complex suggests that the key binding partners are the C-terminus of LtnA1 and pyrophosphate of lipid II. A combination of in vivo and in vitro assays indicates that LtnA1 and LtnA2 can induce rapid membrane lysis without the need for lipid II binding. However, the presence of lipid II substantially increases the activity of lacticin 3147. Furthermore, studies with synthetic LtnA2 analogues containing either desmethyl- or oxa-lanthionine rings confirm that the precise geometry of these rings is essential for this synergistic activity.

Physiometabolic effects of Agave salmiana fructans evaluated in Wistar rats

A prospective completely randomized experimental study was conducted using 48 animals to evaluate the physiometabolic effects of Agave salmiana fructans as a dietary supplement in healthy Wistar rats. Five fructans concentrations from 5 to 20% (w/w) and one control were used in the rats’ diet and were divided into six groups (n=8 rats/group). The treatments were carried out for 35days, during which glucose, cholesterol, triglycerides, body-weight gain, food intake, fecal excretion, organ weights, renal and hepatic functions and a histological analysis of the cecum were evaluated. Glucose, cholesterol, triglycerides, renal and hepatic functions were not significantly affected by any treatment. Body-weight gain and food intake were lower in the rat groups fed fructans than in the control group. Increased fecal excretion (p<0.05) was observed only in animals fed 12.5 and 20% fructans. Mice supplemented with fructans exhibited increased weight and length (p<0.05) in the cecum and colon. A histological analysis of the cecum showed cellular proliferation with a dose of 12.5% and membrane lysis at doses of 15 and 20%. In conclusion, the inclusion of 12.5% of Agave salmiana fructans in the animals’ diets exerts beneficial physiometabolic effects after the seventh treatment day.

Salt stress-induced FERROCHELATASE 1 improves resistance to salt stress by limiting sodium accumulation in Arabidopsis thaliana

Ferrochelatase-1 as a terminal enzyme of heme biosynthesis regulates many essential metabolic and physiological processes. Whether FC1 is involved in plant response to salt stress has not been described. This study shows that Arabidopsis overexpressing AtFC1 displays resistance to high salinity, whereas a T-DNA insertion knock-down mutant fc1 was more sensitive to salt stress than wild-type plants. AtFC1 conferred plant salt resistance by reducing Na+ concentration, enhancing K+ accumulation and preventing lysis of the cell membrane. Such observations were associated with the upregulation of SOS1, which encodes a plasma membrane Na+/H+ antiporter. AtFC1 overexpression led to a reduced expression of several well known salt stress-responsive genes such as NHX1 and AVP1, suggesting that AtFC1-regulated low concentration of Na+ in plants might not be through the mechanism for Na+ sequestration. To investigate the mechanism leading to the role of AtFC1 in mediating salt stress response in plants, a transcriptome of fc1 mutant plants under salt stress was profiled. Our data show that mutation of AtFC1 led to 490 specific genes up-regulated and 380 specific genes down-regulated in fc1 mutants under salt stress. Some of the genes are involved in salt-induced oxidative stress response, monovalent cation-proton (Na+/H+) exchange, and Na+ detoxification.

P2X-Receptor Antagonists Inhibit the Interaction of S. aureus Hemolysin A with Membranes.

The pore forming hemolysin A, Hla, is a major virulence factor of Staphylococcus aureus. Apparently, 1–2 pore(s) per cell suffice(s) to cause cell death. Accumulated experimental evidence points towards a major role of ATP-gated purinergic receptors (P2XR) for hemolysis caused by Hla, complement and other pore forming proteins, presumably by increasing membrane permeability. Indeed, in experiments employing rabbit erythrocytes, inhibitory concentrations of frequently employed P2XR-antagonists were in a similar range as previously reported for erythrocytes of other species and other toxins. However, Hla-dependent hemolysis was not enhanced by extracellular ATP, and oxidized adenosinetriphosphate (oxATP) had only a minor inhibitory effect. Unexpectedly, P2XR-inhibitors also prevented Hla-induced lysis of pure lipid membranes, demonstrating that the inhibition did not even depend on the presence of P2XR. Fluorescence microscopy and gel-electrophoresis clearly revealed that P2XR-inhibitors interfere with binding and subsequent oligomerisation of Hla with membranes. Similar results were obtained employing HaCaT-cells. Furthermore, calorimetric data and hemolysis experiments with Hla pre-treated with pyridoxal phosphate-6-azophenyl-2′,4′-disulfonic acid (PPADS) showed that this compound directly binds to Hla. Our results call for a critical re-assessment of the appealing concept, which suggests that P2XR are general amplifiers of damage by pore-forming proteins.

Mechanisms and functions of guanylate-binding proteins and related interferon-inducible GTPases: Roles in intracellular lysis of pathogens.

Guanylate-binding proteins (GBPs) are a group interferon-inducible GTPases within the constellation of the dynamin GTPase superfamily. These proteins restrict the replication of intracellular pathogens in both immune and non-immune cells. GBPs and their related family members immunity-related GTPases target and lyse the membrane of the pathogen-containing vacuole, destroying the residential niche of vacuolar protozoal and bacterial pathogens. They also prevent virion infectivity and target replication complexes of ribonucleic acid viruses. The exciting concept that GBPs and immunity-related GTPases can directly target the membrane of bacteria and protozoa has emerged. Rupture and lysis of the pathogen membrane mediates liberation of concealed microbial ligands for activation of innate immune sensing pathways and the inflammasome. Further studies have demonstrated a capacity of GBPs to recruit additional antimicrobial factors, highlighting the complexity of the molecular mechanisms involved in pathogen killing. In this mini-review, we discuss recent advances describing the localisation and functions of GBPs on the host and pathogen membrane. We also highlight unresolved questions related to the regulation of GBPs in cell-autonomous immunity to intracellular pathogens.

Total Synthesis and Biological Mode of Action of WAP-8294A2: A Menaquinone-Targeting Antibiotic.

WAP-8294A2 (lotilibcin, 1) is a potent antibiotic with superior in vivo efficacy to vancomycin against methicillin-resistant Staphylococcus aureus (MRSA). Despite the great medical importance, its molecular mode of action remains unknown. Here we report the total synthesis of complex macrocyclic peptide 1 comprised of 12 amino acids with a β-hydroxy fatty-acid chain, and its deoxy analogue 2. A full solid-phase synthesis of 1 and 2 enabled their rapid assembly and the first detailed investigation of their functions. Compounds 1 and 2 were equipotent against various strains of Gram-positive bacteria including MRSA. We present evidence that the antimicrobial activities of 1 and 2 are due to lysis of the bacterial membrane, and their membrane-disrupting effects depend on the presence of menaquinone, an essential factor for the bacterial respiratory chain. The established synthetic routes and the menaquinone-targeting mechanisms provide valuable information for designing and developing new antibiotics based on their structures.

A druggable secretory protein maturase of Toxoplasma essential for invasion and egress.

Micronemes and rhoptries are specialized secretory organelles that deploy their contents at the apical tip of apicomplexan parasites in a regulated manner. The secretory proteins participate in motility, invasion, and egress and are subjected to proteolytic maturation prior to organellar storage and discharge. Here we establish that Toxoplasma gondii aspartyl protease 3 (ASP3) resides in the endosomal-like compartment and is crucially associated to rhoptry discharge during invasion and to host cell plasma membrane lysis during egress. A comparison of the N-terminome, by terminal amine isotopic labelling of substrates between wild type and ASP3 depleted parasites identified microneme and rhoptry proteins as repertoire of ASP3 substrates. The role of ASP3 as a maturase for previously described and newly identified secretory proteins is confirmed in vivo and in vitro. An antimalarial compound based on a hydroxyethylamine scaffold interrupts the lytic cycle of T. gondii at submicromolar concentration by targeting ASP3.

P066 Structural aspects of HLA class I epitopes reacting with human alloantiserum

Aim Interaction between complementarity determining regions (CDR) of the antibody (Ab) and HLA (contact surface area of 700–900 A2) can be detected by mean fluorescence intensity (MFI) or lysis of cell membrane by complement dependent cytotoxicity (CDC). These biological functions of Ab depend on amino acid residue (AAR) composition of functional and structural epitopes (Ep) of the antigen. The aim of this study was to analyze Ep structure of HLA in order to interpret reactivity of Abs in the serum of kidney recipient (KR). Methods Ab analysis (IgG) and complement binding activity (C1q test) of alloantibodies were analyzed using solid phase single antigen (One Lambda, Inc.) (positive cut off MFI = 280–500) and cell based CDCx3 washes assays. Ep analysis was conducted by using HLAMatchmaker computer algorithm (MFI = 1000 was used as a positive cut off). Results Various combinations of four (63EE, 82LR, 127K, and 113YH) eplet-defined Eps were detected in HLAs Abs in KR serum reacted with, when SAA was applied. Consensus of AAR within 15 A radius of aforementioned eplets comprised sixteen positions. The results of reactivity of specific eplet carrying alleles in three assays are presented in Fig. 1. Our findings indicate that Ab reactivity is associated with AAR composition of structural Eps involved in contact with CDR of IgG. Binding to HLA likely depends on AARs at positions 44, 152, 156, and 158, whereas C1q reactivity is affected by AARs at 9H/24T and 163E/167S positions, and CDC reactivity is abolished by tryptophan (W) at 95 position. Conclusions Biological function of anti-HLA Ab requires not only the specific recognition of eplets but also depends on the interactions of their CDRs with structural Eps within the contact surface area. C1q + test does not always predict a positive CDC assay. The results of this study represent a new insight into organ allocation, immunomodulation therapy, and post-transplant Ab monitoring in solid organ recipients. Download : Download high-res image (603KB) Download : Download full-size image

CRYOSURGERY IN THE TREATMENT OF MUCOCELE AND RANULAS IN PEDIATRICS PATIENTS

Oral mucoceles and ranulas are retention phenomena/mucus extravasation that occur in the lower lip on the buccal floor, probably traumatic etiology, manifesting itself as well-defined bubbles and circumscribed, with varying sizes and coloring ranging from translucent to purple. Their diagnoses are clinical and when there are doubts, puncture and aspiration assists in the clinical diagnosis, which is confirmed by histologic study after removal of the lesion. The treatment of both injuries is usually surgical, but in children cryosurgery is the recommended conservative treatment, which is based on the cell death caused by an osmotic imbalance because of successive freezing and thawing cycles that end up taking excess water to the interior of the cell, causing lysis of the cell membrane. The present work reports 2 cases of ranula and mucocele in 18-month-old clinically diagnosed and treated with cryosurgery.

Clathrin-Independent Killing of Intracellular Mycobacteria and Biofilm Disruptions Using Synthetic Antimicrobial Polymers.

Current membrane targeting antimicrobials fail to target mycobacteria due to their hydrophobic membrane structure, ability to form drug-resistant biofilms, and their natural intracellular habitat within the confines of macrophages. In this work, we describe engineering of synthetic antimicrobial polymers (SAMPs) derived from biocompatible polyamides that can target drug-sensitive and drug-resistant mycobacteria with high selectivity. Structure-activity relationship studies revealed that reduced hydrophobicity of cationic pendants induces enhanced and selective permeabilization of mycobacterial membranes. The least hydrophobic SAMP (TAC1) was found to be the most active with maximum specificity toward mycobacteria over E. coli, S. aureus, and mammalian cells. Membrane perturbation studies, scanning electron microscopy, and colony PCR confirmed the ability of TAC1 to induce membrane lysis and to bind to the genomic material of mycobacteria, thereby inducing mycobacterial cell death. TAC1 was most effective in perfusing and disrupting the mycobacterial biofilms and was also able to kill the intracellular mycobacteria effectively without inducing any toxicity to mammalian cells. Cellular uptake studies revealed clathrin independent uptake of TAC1, thereby allowing it to escape hydrolytic lysosomal degradation and effectively kill the intracellular bacteria. Therefore, this manuscript presents the design and selective antimycobacterial nature of polyamide polymers with charged hydrophobic pendants that have ability to disrupt the biofilms and kill intracellular mycobacteria.

Neutron scattering shows a droplet of oleic acid at the center of the BAMLET complex.

The anti-cancer complex, Bovine Alpha-lactalbumin Made LEthal to Tumors (BAMLET), has intriguing broad-spectrum anti-cancer activity. Although aspects of BAMLET's anti-cancer mechanism are still not known, it is understood that it involves the oleic acid or oleate component of BAMLET being preferentially released into cancer cell membranes leading to increased membrane permeability and lysis. The structure of the protein component of BAMLET has previously been elucidated by small angle X-ray scattering (SAXS) to be partially unfolded and dramatically enlarged. However, the structure of the oleic acid component of BAMLET and its disposition with respect to the protein component was not revealed as oleic acid has the same X-ray scattering length density (SLD) as water. Employing the difference in the neutron SLDs of hydrogen and deuterium, we carried out solvent contrast variation small angle neutron scattering (SANS) experiments of hydrogenated BAMLET in deuterated water buffers, to reveal the size, shape, and disposition of the oleic acid component of BAMLET. Our resulting analysis and models generated from SANS and SAXS data indicate that oleic acid forms a spherical droplet of oil incompletely encapsulated by the partially unfolded protein component. This model provides insight into the anti-cancer mechanism of this cache of lipid. The model also reveals a protein component "tail" not associated with the oleic acid component that is able to interact with the tail of other BAMLET molecules, providing a plausible explanation of how BAMLET readily forms aggregates. Proteins 2017; 85:1371-1378. © 2017 Wiley Periodicals, Inc.

One-dimensional poly(L-lysine)-block-poly(L-threonine) assemblies exhibit potent anticancer activity by enhancing membranolysis.

We report that cationic, one-dimensional (1D) fibril assemblies formed by coil-sheet poly(L-lysine)-block-poly(L-threonine) (PLL-b-PLT) block copolypeptides exhibited potent anticancer activity by enhancing membranolysis. The 1D fibril assemblies can efficiently interact with negatively charged cellular and mitochondrial membranes via electrostatic interactions, leading to necrosis via membrane lysis and apoptosis via mitochondria-lytic effect. Moreover, the fibril assemblies exhibited low hemolytic activity and selective cytotoxicity toward cancer cell, which is advantageous as compared to PLL and most antimicrobial/anticancerous peptides. This study provides a new concept of using cationic, 1D fibril assemblies for cancer therapy.

Antimicrobial Resistance and the Alternative Resources with Special Emphasis on Plant-Based Antimicrobials-A Review.

Indiscriminate and irrational use of antibiotics has created an unprecedented challenge for human civilization due to microbe’s development of antimicrobial resistance. It is difficult to treat bacterial infection due to bacteria’s ability to develop resistance against antimicrobial agents. Antimicrobial agents are categorized according to their mechanism of action, i.e., interference with cell wall synthesis, DNA and RNA synthesis, lysis of the bacterial membrane, inhibition of protein synthesis, inhibition of metabolic pathways, etc. Bacteria may become resistant by antibiotic inactivation, target modification, efflux pump and plasmidic efflux. Currently, the clinically available treatment is not effective against the antibiotic resistance developed by some bacterial species. However, plant-based antimicrobials have immense potential to combat bacterial, fungal, protozoal and viral diseases without any known side effects. Such plant metabolites include quinines, alkaloids, lectins, polypeptides, flavones, flavonoids, flavonols, coumarin, terpenoids, essential oils and tannins. The present review focuses on antibiotic resistance, the resistance mechanism in bacteria against antibiotics and the role of plant-active secondary metabolites against microorganisms, which might be useful as an alternative and effective strategy to break the resistance among microbes.

The Mechanism of Killing by the Proline-Rich Peptide Bac7(1–35) against Clinical Strains of Pseudomonas aeruginosa Differs from That against Other Gram-Negative Bacteria

Pseudomonas aeruginosa infections represent a serious threat to worldwide health. Proline-rich antimicrobial peptides (PR-AMPs), a particular group of peptide antibiotics, have demonstrated in vitro activity against P. aeruginosa strains. Here we show that the mammalian PR-AMP Bac7(1-35) is active against some multidrug-resistant cystic fibrosis isolates of P. aeruginosa By confocal microscopy and cytometric analyses, we investigated the mechanism of killing against P. aeruginosa strain PAO1 and three selected isolates, and we observed that the peptide inactivated the target cells by disrupting their cellular membranes. This effect is deeply different from that previously described for PR-AMPs in Escherichia coli and Salmonella enterica serovar Typhimurium, where these peptides act intracellularly after having been internalized by means of the transporter SbmA without membranolytic effects. The heterologous expression of SbmA in PAO1 cells enhanced the internalization of Bac7(1-35) into the cytoplasm, making the bacteria more susceptible to the peptide but at the same time more resistant to the membrane lysis, similarly to what occurs in E. coli The results evidenced a new mechanism of action for PR-AMPs and indicate that Bac7 has multiple and variable modes of action that depend on the characteristics of the different target species and the possibility to be internalized by bacterial transporters. This feature broadens the spectrum of activity of the peptide and makes the development of peptide-resistant bacteria a more difficult process.

Biodegradable cationic poly(carbonates): Effect of varying side chain hydrophobicity on key aspects of gene transfection.

Owing to their ease of synthesis and well-controlled polymerization, biodegradable cationic poly(carbonates) have emerged as a highly promising class of biomaterials for gene delivery. The hydrophobicity of side chains in cationic polymers plays an important but often underappreciated role in influencing key aspects of gene transfection. In our efforts to improve gene transfection and understand structure-activity relationships, we synthesized a series of cationic polymers bearing a common poly(carbonate) backbone, and with side chains containing various hydrophobic spacers (propyl, hexyl, 4-methyl benzyl or nonyl) before the cationic moiety. A moderate degree of hydrophobicity was optimal as the cationic poly(carbonate) with hexyl side chains mediated high gene transfection efficiencies while causing low cytotoxicities.

TC38, a teleost TFPI-2 peptide that kills bacteria via penetration of the cell membrane and interaction with nucleic acids

Tissue factor pathway inhibitor 2 (TFPI-2) is an analog of TFPI-1 and a potent endogenous inhibitor of tissue factor (TF)-mediated blood coagulation. Recent reports have proven that the C-terminal of TFPI-2 peptides in humans and several other vertebrates possesses antibacterial activity against Gram-positive and Gram-negative bacteria. In our previous study, we reported that the TFPI-2 peptide, TC38 in tongue sole (Cynoglossus semilaevis) was active against Micrococcus luteus. In this study, we further examine the antimicrobial spectrum, mechanism of action, and function of TC38 in tongue sole. Our results indicate that TC38 is active against the Gram-negative bacteria Vibrio ichthyoenteri, Vibrio litoralis, Vibrio parahaemolyticus, and Vibrio vulnificus, as well as the fish Megalocytivirus, infectious spleen and kidney necrosis virus (ISKNV). The mechanism of action of TC38 against V. vulnificus was explored. The results showed that TC38 killed V. vulnificus cells without lysis of the cell membrane. FITC-labeled TC38 was able to penetrate the cell membrane and bind to DNA and RNA, then disrupt cellular function, eventually leading to cell death. Administration of TC38 to tongue sole significantly improved its defense against V. vulnificus infection. Overall, these results indicate that TC38 is a novel peptide with a broad antimicrobial spectrum. Furthermore, the unique action of TC38 against V. vulnificus adds new insights to the mechanism of action of vertebrate TFPI peptides. Moreover, TC38 is an interesting antimicrobial agent that could be useful in the fight against pathogenic invasion in aquaculture.

Antimicrobial Effect of Biocompatible Silicon Nanoparticles Activated Using Therapeutic Ultrasound.

In this study, we report a method for the suppression of Escherichia coli (E. coli) vitality by means of therapeutic ultrasound irradiation (USI) using biocompatible silicon nanoparticles as cavitation sensitizers. Silicon nanoparticles without (SiNPs) and with polysaccharide (dextran) coating (DSiNPs) were used. Both types of nanoparticles were nontoxic to Hep 2 cells up to a concentration of 2 mg/mL. The treatment of bacteria with nanoparticles and application of 1 W/cm2 USI resulted in the reduction of their viabilities up to 35 and 72% for SiNPs and DSiNPs, respectively. The higher bacterial viability reduction for DSiNPs as compared with SiNPs can be explained by the fact that the biopolymer shell of the polysaccharide provides a stronger adhesion of nanoparticles to the bacterial surface. Transmission electron microscopy (TEM) studies showed that the bacterial lipid shell was partially perforated after the combined treatment of DSiNPs and USI, which can be explained by the lysis of bacterial membrane due to the cavitation sensitized by the SiNPs. Furthermore, we have shown that 100% inhibition of E. coli bacterial colony growth is possible by coupling the treatments of DSiNPs and USI with an increased intensity of up to 3 W/cm2. The observed results reveal the application of SiNPs as promising antimicrobial agents.

Highly potent antimicrobial polyionenes with rapid killing kinetics, skin biocompatibility and invivo bactericidal activity.

Effective antimicrobial agents are important arsenals in our perennial fight against communicable diseases, hospital-acquired and surgical site multidrug-resistant infections. In this study, we devise a strategy for the development of highly efficacious and skin compatible yet inexpensive water-soluble macromolecular antimicrobial polyionenes by employing a catalyst-free, polyaddition polymerization using commercially available monomers. A series of antimicrobial polyionenes are prepared through a simple polyaddition reaction with both polymer-forming reaction and charge installation occurring simultaneously. The compositions and structures of polymers are modulated to study their effects on antimicrobial activity against a broad spectrum of pathogenic microbes. Polymers with optimized compositions have potent antimicrobial activity with low minimum inhibitory concentrations of 1.95-7.8μg/mL and high selectivity over mammalian cells. In particular, a killing efficiency of more than 99.9% within 2min is obtained. Moreover, the polymers demonstrate high antimicrobial efficacy against various clinically-isolated multidrug-resistant microbes, yet exhibit vastly superior skin biocompatibility in mice as compared to other clinically used surgical scrubs (chlorhexidine and betadine). Microbicidal activity of the polymer is mediated via membrane lysis as demonstrated by confocal microscopy. Unlike small molecular antibiotics, repeated use of the polymer does not induce drug resistance. More importantly, the polymer shows excellent bactericidal activity in a P.aeruginosa-contaminated mouse skin model. Given their rapid and efficacious microbicidal activity and skin compatibility, these polymers have tremendous potential to be developed as surgical scrubs/hand sanitizers to prevent multidrug-resistant infections.

Mycobacterial ESX-1 secretion system mediates host cell lysis through bacterium contact-dependent gross membrane disruptions

Mycobacterium tuberculosis and Mycobacterium marinum are thought to exert virulence, in part, through their ability to lyse host cell membranes. The type VII secretion system ESX-1 [6-kDa early secretory antigenic target (ESAT-6) secretion system 1] is required for both virulence and host cell membrane lysis. Both activities are attributed to the pore-forming activity of the ESX-1-secreted substrate ESAT-6 because multiple studies have reported that recombinant ESAT-6 lyses eukaryotic membranes. We too find ESX-1 of M. tuberculosis and M. marinum lyses host cell membranes. However, we find that recombinant ESAT-6 does not lyse cell membranes. The lytic activity previously attributed to ESAT-6 is due to residual detergent in the preparations. We report here that ESX-1-dependent cell membrane lysis is contact dependent and accompanied by gross membrane disruptions rather than discrete pores. ESX-1-mediated lysis is also morphologically distinct from the contact-dependent lysis of other bacterial secretion systems. Our findings suggest redirection of research to understand the mechanism of ESX-1-mediated lysis.

Replacement of fishmeal with processed meal from knife fish Chitala ornata in diets of juvenile Nile tilapia Oreochromis niloticus

A 60-day feeding trial was conducted to assess the effects of processed meal from knife fish Chitala ornata (KFM) as fishmeal replacement in diets of Nile tilapia Oreochromis niloticus juveniles. Five iso-nitrogenous (36.4% in dry matter) and isolipidic diets (8.6% in dry matter) with 0 (D1), 25 (D2), 50 (D3), 75 (D4) and 100% (D5) KFM inclusions were prepared. With a stocking density of 15 fish (0.59±0.01g) per tank, tilapia juveniles were distributed randomly in fifteen 30-L tanks. Results indicate a significant increase (P<0.05) in the percent average weight gain, specific growth rate, and feed intake with increasing KFM inclusion up to the level of 75%. There were no adverse effects observed in both blood profile and carcass composition of the tilapia. Hepatosomatic index of fish significantly increased (P<0.05) when KFM was included into the diet of tilapia, compared to those fed D1. Viscerosomatic indices were not significantly different (P>0.05) among treatments. Results of hepatic histopathology showed absence of tumors, lesions and parenchymal inflammation in all treatments. However, mild cell membrane lysis and mild and mild to moderate apoptosis were evident in liver samples. Based on the results, KFM can partially and completely replace dietary protein from fishmeal. Moreover, D4 (75% KFM) is considered the optimal KFM replacement level for Nile tilapia juveniles.