Main Research Findings

1) Treatment with LL-37 was shown to exert antimicrobial activities by interacting with the phospholipid bilayer of bacterial cells.

2) LL-37 is a human antimicrobial peptide that has been shown to improve disease resistance in transgenic rice.

 

Selected Data

1) The study performed by the research team of Palusińska-Szysz et al investigated the interplay between L. micdadei metabolism, its lipid membrane composition, and its susceptibility to the human antimicrobial peptide LL-37. This comprehensive research employed a diverse array of advanced biochemical, biophysical, and microbiological techniques across in vitro settings to achieve its objectives [1].

The core of the study relied on the L. micdadei ATCC 33218 strain, which was cultured under specific conditions. Initial growth occurred in buffered yeast extract (YEB) medium supplemented with L-cysteine and iron pyrophosphate at 37 °C with continuous agitation to an OD600 of 3.0. A crucial aspect involved manipulating the choline availability in the growth medium, with choline chloride added at concentrations of 100, 400, and 800 µg/mL to investigate its impact on bacterial physiology. For deuterium-labeled lipid studies, L. micdadei was cultured on buffered charcoal-yeast extract (BCYE) agar, either with or without choline-trimethyl-d9 chloride, for three days at 37 °C under a 5% CO2 humid atmosphere. Post-culture, bacterial pellets were collected via centrifugation, extensively washed to remove external contaminants, and then lyophilized in preparation for lipid extraction. To ensure the robustness and reproducibility of the findings, six independent culturing experiments were performed for each condition involving lipid analysis [1].

Lipids were meticulously extracted from approximately 130 mg of dry bacterial mass using a modified Bligh and Dyer method, a standard protocol for lipid isolation. This involved homogenizing the bacterial mass, followed by a multi-step solvent extraction using a chloroform:methanol mixture, thorough shaking, and sonication to maximize lipid recovery. Phase separation was achieved through centrifugation, and the organic phase, rich in lipids, was collected. This extraction process was repeated multiple times to ensure maximal lipid yield, with the pooled organic phases subsequently dried under nitrogen and then resuspended in chloroform for further analyses.

Two primary mass spectrometry (MS) techniques were employed for detailed lipid characterization. Shotgun lipidomics was performed on an Apex Qe Fourier Transform Ion Cyclotron Resonance mass spectrometer, coupled with a TriVersa NanoMate for automated sample introduction. Measurements were acquired in negative ion mode, and peak lists were processed using Bruker Compass DataAnalysis 4.0. Lipid identification relied on LipidXplorer 1.2.6, utilizing a top-down lipidomic screen, with annotations following standardized shorthand nomenclature. This approach provided a broad overview of the lipidome. For precise fatty acid (FA) composition analysis, individual phospholipid (PL) classes were first separated using two-dimensional thin-layer chromatography (2D TLC). After visualization, individual spots were scraped, extracted, and saponified to release FAs, which were then derivatized into fatty acid methyl esters (FAME). These FAMEs were analyzed by gas chromatography with mass spectrometry (GC/MS) using an Agilent 7890A-5975C instrument equipped with an HP-5MS capillary column, allowing for qualitative and quantitative determination of FA profiles [1].

To complement MS data, 31P NMR spectroscopy was conducted on lipid extracts using 360 MHz or 700 MHz NMR spectrometers. Samples were prepared in a CDC13:CD3OD:Cs-EDTA solvent system with 18:0 lyso-PG as an internal standard and 85% phosphoric acid as an external reference. This technique provided quantitative data on the overall phospholipid class composition and was crucial for validating MS findings.

The expression pattern of the pcsA gene, encoding phosphatidylcholine synthase, was determined using real-time quantitative PCR (qPCR). Total RNA was extracted, treated to remove DNA contamination, and reverse-transcribed into cDNA. Specific primers for L. micdadei pcsA were used for qPCR, with the mip gene serving as an internal control. For detailed analysis of membrane lipid distribution, L. micdadei cells were subjected to sucrose density gradient ultracentrifugation to fractionate inner and outer membranes. The efficiency of separation was verified by measuring protein content and specific enzyme activities: NADH oxidase for the inner membrane and esterase activity for the outer membrane [1].

The antibacterial activity of human LL-37 peptide against L. micdadei was quantified using a colony-counting assay. Bacteria, both with and without choline supplementation, were exposed to varying concentrations of LL-37 at 5, 10, and 20 µM doses, incubated, and plated to determine survival rates. To understand the molecular interactions, Langmuir trough measurements (π-A isotherms) were performed. This technique involved forming phospholipid monolayers from L. micdadei lipids, with or without choline at an air-liquid interface and compressing them while measuring surface pressure. LL-37 peptide was introduced into the subphase to observe its effects on monolayer properties, such as area per molecule, collapse pressure, and compression modulus, which reflect lipid packing and membrane stability. Finally, potassium iodide (KI) quenching on liposomes was used to assess LL-37’s ability to permeabilize lipid bilayers. Liposomes, mimicking bacterial membranes and containing a fluorescent reporter (NBD-PE), were exposed to KI and LL-37, with fluorescence spectroscopy measuring changes in membrane permeability [1].

2) The study completed by researchers Lee et al detailed the methods used to generate and characterize transgenic rice plants expressing the human antimicrobial peptide LL-37, and to evaluate their resistance against bacterial leaf blight and blast. The overarching approach involved genetic engineering, molecular verification, and in planta disease assays.

The rice cultivar Oryza sativa L. var. Japonica cv. Dongjinbyeo was selected as the host plant for transformation. To achieve stable expression and secretion of LL-37, a recombinant binary pPZP vector was constructed. This vector incorporated a synthetic SP-LL-37 gene (signal peptide-LL-37) designed with a vicilin fusion at its N-terminus to enable secretion into the intercellular space, thereby avoiding intracellular degradation. The expression of SP-LL-37 was driven by the pGD1 promoter, derived from Nakdongbyeo rice, ensuring inducible and stable expression. For selection purposes, the bar gene (confers glufosinate resistance) under the control of a P35s promoter and nos terminator was included as a selectable marker. The entire SP-LL-37 sequence, including the signal peptide, was subcloned into the KpnI and SmaI enzyme sites of the vector [2].

Transgenic rice plants were generated through Agrobacterium tumefaciens-mediated transformation, utilizing the EHA105 strain. Following transformation, embryogenic calli were cultured on 1/2 MS plates supplemented with 6 mg/L PPT (phosphinothricin) to select for PPT-resistant lines. A total of 472 T0 plants were regenerated and subjected to initial PCR analysis to confirm the integration of the SP-LL-37 and bar genes into the rice genome. To identify single-copy transgenic lines, TaqMan quantitative PCR (qPCR) was employed using specific probes for the actin gene and NOS gene, targeting both 5′ and 3′ regions of the ribosomal RNA gene. Copy numbers ranging from 0.42 to 0.65 were indicative of single-copy insertions [2].

To determine the precise insertion sites of the T-DNA and identify suitable intergenic lines, inverse PCR (IPCR) was performed on genomic DNA extracted from 121 T0 plants. This method involved digesting genomic DNA with BfaI, ligating the digested fragments to circularize them, and then using specific primers (AP1 and Fa1 for the left border (LB), AP1 and T-DNA for the right border (RB), along with nested primers) to amplify the regions flanking the T-DNA insert. The amplified products were separated by gel electrophoresis, and their sequences were analyzed by NCBI BLASTn to identify the insertion locations (genic, intergenic, or repeated regions). This comprehensive FST analysis was crucial for selecting stable, single-copy, intergenic lines for further characterization.

To confirm that the vicilin-fused SP-LL-37 was indeed secreted into the intercellular space, a pGWB5::SP-LL-37-GFP fusion vector was constructed, linking the C-terminus of SP-LL-37 to Green Fluorescent Protein (GFP). This construct, along with a GFP-only control, was transiently expressed in Nicotiana benthamiana (tobacco) leaves via Agrobacterium-mediated infiltration. Young apical tobacco leaves were infiltrated using a syringe, and the subcellular localization of the GFP signal was observed after 36–48 hours using a confocal microscope equipped with a combined laser (488 nm excitation, 520 nm emission). This allowed for visual confirmation of the target peptide’s secretion [2].

Selected single-copy, intergenic transgenic lines (T2 generation) were grown under paddy field conditions alongside wild-type (WT) Dongjinbyeo rice. At maturity, various agronomic traits were evaluated, including chlorophyll content, days to flowering, plant height, culm length, panicle length, panicles per plant, spikelets per panicle, percent ripened grain (PRG), and 1000-grain weight (TGW). These evaluations were performed using methods similar to those previously described for rice breeding.

The disease resistance of the transgenic rice lines was assessed against two major rice pathogens. First, bacterial leaf blight included twenty T2 generation plants per line were inoculated with Xanthomonas oryzae pv. oryzae (~1×10^7 cells/mL) using the “scissors clip-inoculation” method. Scissors dipped in the bacterial suspension were used to cut leaf tips. Inoculated plants were then covered with polythene bags and incubated under controlled conditions. Disease progression was monitored for 12 days, with water-soaked areas checked within 48–72 hours. Second, rice blast included artificial inoculation and field evaluations were carried out to test resistance against Magnaporthe oryzae. At the four-leaf stage, seedlings were inoculated by spraying with a spore suspension (100 spores in 100x visual field, 0.02% Tween-20). Inoculated plants were kept in the dark at 26–28°C for 24 hours, then transferred to an artificial climate tunnel. Disease reactions were scored approximately 7 days post-inoculation based on standard assessment protocols [2].

Protein extraction from untransformed control plants and transgenic rice was performed by homogenizing leaf tissue in an extraction buffer, and total soluble protein was quantified using the Bradford method. Recombinant LL-37 protein levels were specifically determined using a direct ELISA assay. ELISA plates were coated with total soluble protein, incubated with mouse IgG conjugated to horseradish peroxidase, and then with tetramethylbenzidine substrate. Absorbance was measured at 405 nm to quantify LL-37 levels [2].

 

Discussion

1) The study performed by Palusińska-Szysz et al elucidated the metabolic adaptability of Legionella micdadei regarding its membrane phospholipid composition and how these changes influence its susceptibility to the human antimicrobial peptide LL-37. The results underscored the crucial role of exogenous choline in shaping L. micdadei’s lipidome and its vulnerability.

Investigation into the pcsA gene, encoding phosphatidylcholine synthase, revealed a modest but significant upregulation of its transcript levels in L. micdadei when cultured with a low 100 µg/mL concentration of exogenous choline. However, higher choline concentrations did not induce further pcsA expression, suggesting a saturated response. This confirmed L. micdadei’s capacity to utilize exogenous choline for phosphatidylcholine (PC) synthesis [1].

Detailed FA analysis of individual phospholipid classes (PC, phosphatidylethanolamine (PE), phosphatidylglycerol (PG), and cardiolipin (CL)) showed a diverse FA profile with 26 distinct FAs ranging from 14 to 23 carbon atoms, including unbranched, branched (iso and anteiso), and cyclopropane types. PC was predominantly composed of straight-chain hexadecenoic and octadecanoic FAs, while PE was rich in anteiso branched FAs. PG displayed a mix of branched and unbranched FAs, including longer chains. CL was unique, showing the highest content of cyclopropyl 17:0 acid and being dominated by branched and monounsaturated FAs [1].

While initial 31P NMR spectroscopy indicated no statistically significant overall changes in the content of the four major phospholipid classes (PC, PE, CL, PG) when L. micdadei was grown with or without choline, a more sensitive top-down lipidomics screen revealed subtle yet significant alterations. Choline supplementation led to a significant increase in PG abundance, particularly specific species like PG 38:1, PG 37:0, and PG 37:1. Concurrently, increases were observed in specific PC species, while decreases occurred in certain CL, and PE species. These changes, cumulatively representing a 2.15% difference in the lipidome composition, suggested an enzymatic network adapting to choline availability, leading to a shift towards longer aliphatic chains in affected lipids. Deuterium-labeled choline studies confirmed that the Pcs pathway was overwhelmingly responsible for 98% of PC synthesis, with only 2% via the PmtA pathway. More d9-labeled PCs were directed to the outer membrane (OM) than the inner membrane (IM). PC profiles showed specific differences between IM and OM, with IM enriched in shorter-chain PCs and PE 30:0 being threefold higher in IM than OM [1].

A pivotal finding was the impact of choline-induced lipidome changes on L. micdadei’s susceptibility to the human antimicrobial peptide LL-37. The colony-counting assay demonstrated that LL-37 inhibited L. micdadei growth in a dose-dependent manner, with an LC50 of approximately 20 µM. Crucially, bacteria cultured with exogenous choline were significantly more sensitive to LL-37 compared to those grown without choline. At a 20 µM LL-37 concentration, there was a 47% mortality rate for choline-supplemented bacteria, versus only 29% for bacteria without choline. This indicated that choline supplementation directly increased the sensitivity of L. micdadei to LL-37.

Biophysical investigations using Langmuir trough measurements (π-A isotherms) provided mechanistic insights into LL-37’s interaction with . The monolayers formed from lipids of choline-supplemented bacteria exhibited smaller areas per molecule compared to those without choline, indicative of tighter packing. The presence of LL-37 in the subphase consistently increased the area per molecule and induced characteristic inflections in the isotherms (25-40 mN/m), a range typical for natural membrane lipid states. Higher LL-37 concentrations led to two distinct monolayer collapses, suggesting membrane destabilization. Compression modulus (Cs⁻¹) measurements indicated that both monolayers existed in a disordered liquid state. The interaction of LL-37 with choline-supplemented monolayers caused greater disturbances in molecular organization, evidenced by a higher relative area increase [1].

Figure 1: Changes in compression modules versus surface pressure determined for L. micdadei phospholipids

Further mechanistic exploration using potassium iodide (KI) quenching on NBD-PE labeled liposomes demonstrated that LL-37 induced a concentration-dependent increase in liposome permeabilization. This effect was significantly amplified in liposomes with a higher molar fraction of DPPC. The permeabilization by LL-37 was clearly amplified in the presence of higher PC content, which correlated with the increased PC levels observed in choline-supplemented membranes. Blank controls suggested LL-37 could induce lateral reorganization or solubilization of lipid domains, leading to reduced NBD self-quenching.

In conclusion, the study established that L. micdadei modifies its membrane lipid composition in response to exogenous choline, resulting in a more tightly packed membrane that is paradoxically more susceptible to the disruptive effects of the human antimicrobial peptide LL-37. The increased PC content in choline-supplemented membranes appears to be a key factor driving this enhanced susceptibility, suggesting that targeting choline metabolism could be a novel therapeutic strategy against L. micdadei infections [1].

2) The study conducted by Lee et al successfully generated and characterized transgenic rice plants expressing SP-LL-37, demonstrating enhanced disease resistance against bacterial leaf blight and blast while maintaining comparable agronomic traits to wild-type rice. These findings validate SP-LL-37 as a promising candidate for broad-spectrum disease resistance in rice [2].

Agrobacterium-mediated transformation yielded a regeneration rate of approximately 40%, resulting in 472 T0 transgenic plants. PCR analysis confirmed the successful integration of both the SP-LL-37 and bar genes into the rice genome. To ensure stable expression and avoid gene silencing issues, single-copy transgenic lines were identified using TaqMan PCR. A total of 181 lines exhibited single-copy insertions, making up 38.3% of the analyzed T0 plants, with an average of 2.1 T-DNA inserts per plant. Subsequent Flanking Sequence Tag (FST) analysis using inverse PCR was performed on 121 T0 plants to map the T-DNA insertion sites. The analysis revealed that 77.7% of insertions were genic, 11.6% were intergenic, and 10% were in repeated sequences. Based on this, 14 intergenic lines were selected, and five of these (T6, T12, T16, T17, T20) were used for detailed expression and performance analysis, with their exact insertion points on various chromosomes precisely mapped [2].

Stable expression of the SP-LL-37 gene was confirmed in the T1 generation of transgenic lines. Basta selection, which utilizes the glufosinate resistance conferred by the bar gene, effectively differentiated transgenic plants from wild-type controls, with transgenic lines maintaining green color while wild-type plants yellowed and died. RT-PCR confirmed the presence of SP-LL-37 transcripts in the selected homozygous T1 lines. ELISA analysis further demonstrated the stable expression of the SP-LL-37 protein in all tested transgenic lines, with line 17-4 exhibiting the highest expression levels. Crucially, the subcellular localization of the SP-LL-37 protein was confirmed using a GFP fusion construct in a tobacco transient expression assay. Confocal microscopy revealed that the SP-LL-37-GFP fusion protein was successfully secreted and located in the intercellular space, as intended. This strategic localization helps avoid degradation by intracellular plant proteases, enhancing the peptide’s stability and efficacy.

The most significant finding was the markedly enhanced resistance of SP-LL-37 transgenic rice to bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae. Inoculation experiments on T2 homozygous lines showed substantially reduced disease symptoms compared to wild-type plants. Three weeks post-inoculation, wild-type leaves exhibited extensive lesions, covering approximately 10.8 cm. In stark contrast, the transgenic lines displayed significantly smaller lesions, ranging from a mere 0.1 cm to 2.2 cm. Furthermore, the transgenic plants did not show any signs of disease progression, indicating a robust and sustained resistance [2].

Beyond bacterial pathogens, the transgenic SP-LL-37 rice also demonstrated a broad spectrum of resistance by showing high resistance to rice blast, caused by the fungus Magnaporthe oryzae. While wild-type plants developed significant fusiform lesions and extensive diseased leaf area (approximately 73.2% of total leaf area), transgenic lines exhibited only minimal lesions, with diseased leaf area ranging from 3.1% to 9.0%. This further underscores the broad protective capabilities of the SP-LL-37 peptide.

Importantly, the introduction and expression of SP-LL-37 did not negatively impact the key agronomic traits of the rice plants. Comparative analysis of various traits (chlorophyll content, days to flowering, plant height, culm length, panicle length, panicles per plant, spikelets per panicle, percent ripened grain, and 1000-grain weight) between transgenic lines and wild-type Dongjinbyeo showed no statistically significant differences overall. Although slight variations were noted in parameters like plant height and grain weight for some individual lines, these differences were minimal and within a very narrow range, confirming the stability and suitability of these transgenic lines for agricultural application without yield penalties [2].

In conclusion, the study successfully developed transgenic rice expressing SP-LL-37, which showed stable expression, correct intercellular localization, and significantly enhanced resistance against two major rice diseases, bacterial leaf blight and blast, all while maintaining desirable agronomic traits. These results highlight the potential of SP-LL-37 as a valuable genetic resource for developing disease-resistant rice varieties [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Palusińska-Szysz M, Jurak M, Gisch N, et al. The human LL-37 peptide exerts antimicrobial activity against Legionella micdadei interacting with membrane phospholipids. Biochim Biophys Acta Mol Cell Biol Lipids. 2022;1867(6):159138. doi:10.1016/j.bbalip.2022.159138

[2] Lee IH, Jung YJ, Cho YG, et al. SP-LL-37, human antimicrobial peptide, enhances disease resistance in transgenic rice. PLoS One. 2017;12(3):e0172936. Published 2017 Mar 10. doi:10.1371/journal.pone.0172936

Characteristics of LL-37

LL-37, also referred to as cathelicidin, is an antimicrobial peptide (AMP) shown to be able to ward off various pathogens and help reduce inflammation. One of the leading concerns in medicine is the increase in antimicrobial resistant bacteria. Since their discovery in the 1980s,
AMPs have shown promise in treating the antimicrobial resistant bacteria and are generally less prone to resistance than traditional medications. LL-37 works by interacting with the cell wall in order to perforate the cytoplasmic membrane, causing cell death. The peptide is created by cleavage of the C-terminal of the 18 kDa hCAP18 protein. In this case, the extracellular cleavage is driven by serine proteases in keratinocytes and proteinase 3, found in neutrophils. Unlike other antimicrobial peptides LL-37 is protected from proteolytic degradation due to its ability to aggregate in the lipid bilayers and numerous other solutions.

LL-37 is successful in crossing membranes primarily due to the positive charge it carries and its 𝞪-helical structure. The positive charge allows for the peptide to favorably interact with the negative charges found in the phospholipid membrane. The 𝞪-helical structure is beneficial
as it results in unilateral segregation of hydrophobic residues, which allows for penetration of the membrane, bacterial lysis, and the formation of transmembrane pores. LL-37 works primarily throughout the immune system by interacting with the membranes of various viruses, bacteria, and other abnormal compounds.

 

Effects of LL-37 on Autoimmune Disorders

AMPs are commonly produced by neutrophils, macrophages, and monocytes making them a primary variable in warding off infections. LL-37 in particular has vast immunomodulatory effects due to its ability to induce cytokine production, as well as regulate immune cell activity. Researcher Pahar et. Al conducted a study regarding how the immunomodulatory effects of LL-37 are able to assist in treating autoimmune diseases, in this case, psoriasis.

Psoriasis is caused due to disturbances of the immune system leading to proliferation of keratinocytes and differentiation dysfunction. In the early development stages of psoriasis keratinocytes produce high levels of AMPs LL-37, HBD-2, and psoriasin, which all help to recruit immune cells. Studies show that in psoriatic skin, LL-37 is the primary trigger for the pathogenic immune responses, leading researchers to believe there is a link between antimicrobial systems and psoriasis.

That being said, LL-37 was shown to have the ability to convert non-stimulatory self-DNA into a trigger of plasmacytoid dendritic cells (pDCs). This process begins by the binding of LL-37 to DNA and proceeds with the translocation of the LL-37 DNA complexes into the pDC pathway.
The complexes are then able to bypass the safety mechanism of the differentiation process between viruses and nucleic acids. LL-37 retains the DNA complex leading to the production of pDCs. An activated pDC releases tumor necrosis factor-𝞪 (TNF-𝞪) prompting the maturation of myeloid dendritic cells and allowing for the initiation of autoimmune responses. Following this pathway indicates that LL-37 released during psoriasis-related skin injury will elicit adaptive and innate immune responses, much like a virus does (https://www.mdpi.com/2076-393X/8/3/517).

 

Effects of LL-37 on Inflammation Levels

In addition to the role it plays in the immune system, LL-37 has many antiinflammatory effects stemming from its antagonist actions on compounds such as IFN-𝞬, TNF-𝞪 IL-4, and IL-12, as well as its ability to regulate toll-like receptor (TLR) signaling. TLRs are receptors that respond to various pathogen-associated molecular patterns (PAMPs). TLR signaling is disturbed through the binding of TLR4 to the ligand LPS, as well as the interruption of the role of TLR4 receptor complexes on generating macrophages and dendritic cells. This indicates that when LL-37 and LPS are present, levels of inflammatory cytokines are decreased.

LL-37 plays an important role in the regulation of several inflammatory pathways. An interesting function of LL-37 is its ability to activate P2X7R, which assists in inducing inflammasome activation in monocytes and macrophages. Further studies show that by activating P2X7R, LL-37 goes on to enhance expression of COX2, prostaglandin E2, and c-Jun-N-terminal kinase.. Due to its ability to activate P2X7R, the compound that drives the expression of these pro-inflammatory cytokines, LL-37 is easily able to help modulate the various inflammatory pathways and downregulate the expression of these compounds.

Overall, the researchers Kahlenberg et. Al were able to provide support for the theory that dysfunction of the TLR pathways leads to the development of autoimmunity. Furthermore, when TLR pathways are directly modulated by the presence of LL-37 it is considered an anti-inflammatory effect of the peptide (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3836506/).

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