Cathelicidin Antimicrobial Peptide LL 37 - an overview

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Antibacterial Peptides

Antimicrobial peptides include defensins and cathelicidins and contribute to the first line of defense against microbes in the skin and at mucosal surfaces (Boman, 1991). Cationic antimicrobial peptides such as β-defensins and cathelicidin (LL-37 or CAMP) (Chromek et al., 2006) have variable antimicrobial activity. In the urinary tract, experimental evidence suggests that antimicrobial peptides may protect the bladder against the microbiome (Sorensen et al., 2008; Zasloff, 2007), especially Gram-positive bacteria (Morrison et al., 2002). Human β-defensin-1 and β-defensin-5 are constitutively expressed in the kidneys and female genital tract and β-defensin-2 levels are increased during infection (Valore et al., 1998). Cathelicidin-related antimicrobial peptide and LL-37 are expressed in human kidney uroepithelial cells. Camp–/– mice show elevated bacterial persistence (Chromek et al., 2006), increased mortality with septicemia, and elevated kidney size compared with wild-type mice. In an epidemiological study of sensitivity to the human cathelicidin LL-37, APN strains were found to be more resistant than lower urinary tract isolates (Chromek et al., 2006) and vitamin D supplementation in postmenopausal women was suggested to decrease the risk of UTI by increasing cathelicidin expression (Hertting et al., 2010). The antimicrobial peptide ribonuclease 7 was recently shown to be upregulated during pyelonephritis (Spencer et al., 2013).

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In contrast, the cationic cathelicidin LL37 has been shown to stimulate NADPH oxidase activity through FPRL1 signalling (Zheng et al., 2007). This illustrates that cationic peptides can exert very diverse and even opposite functions, despite their common physico-chemical properties such as amphipathicity and positive charge.

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IV Cathelicidin and Vitamin D

Among the various components of the innate immune system, antimicrobial peptides have a role as effectors and are involved in killing of a broad spectrum of microbes (Bals et al., 1998). One of the best characterized families of antimicrobial peptides is defensins which are small cationic cystine-rich peptides of broad antimicrobial activity (Ganz and Lehrer, 1994). A human homolog of these β-defensins called human β-defensin-1 (hBD-1) has been described (Bensch et al., 1995). Another family of peptide antibiotics that is receiving increased attention is the cathelicidins (Agerberth et al., 1995). The only cathelicidin present in human is hCAP-18, which contains a 30-residue signal region, a 103-residue polypeptide corresponding to the conserved cathelin-like proregion, and the 37-residue peptide LL-37 at the C terminus. LL-37 becomes antimicrobially active on release from the proregion by the action of proteinase 3 (Sorensen et al., 2001).

LL-37 is produced by various cell types including neutrophils, lung epithelial cells, keratinocytes, monocytes, mast cells, and γδ T cells (Agerberth et al., 2000; Di Nardo et al., 2003; Frohm et al., 1997). The synthetic peptide LL-37 is known to have antibiotic activity against a number of gram-negative and gram-positive organisms including Pseudomonas aeruginosa, Salmonella typhimurium, Escherichia coli, Listeria monocytogenes, Staphylococcus epidermidis, Staphylococcus aureus (Bals et al., 1998; Turner et al., 1998).

Apart from antimicrobial activities, host defense peptides have pleiotrophic immunomodulatory activities. LL-37 has been demonstrated to be a chemoattractant for neutrophils, monocytes, and subsets of T cells (De et al., 2000). LL-37 is also shown to induce IL-8, a potent chemokine for neutrophils and monocytes. Moreover, LL-37 also upregulated the chemokine receptors CXCR4, CCR2, and IL-8RB, suggesting that LL-37 is a multifunctional modulator of innate immune response (Scott et al., 2002).

The promoter region of the hCAP18 gene contains multiple VDR-response elements, and stimulation with 1,25-VitD3ex vivo enhances hCAP18 expression in keratinocytes, monocytes, and neutrophils (Wang et al., 2004). Further, the 1,25-VitD3-dependent induction of LL-37 contributes to the microbicidal activity of macrophages against M. tuberculosis (Segaert, 2008). The role of 1,25-VitD3 in the induction of hCAP18 and LL-37 in keratinocytes is brought out during wound healing process, with cytokine TGFβ1 upregulating the expression of the VitD3-activating enzyme CYP27B1, which subsequently leads to VitD3 activation and increased levels of hCAP18 and LL-37 in the wounded tissue (Schauber et al., 2007). Oral intake of vitamin D3 in rickets patients for 4 weeks significantly increased the hCAP18 expression in neutrophils compared to age-matched healthy controls without vitamin D3, indicating the potential role of vitamin D3 as a regulator of the innate immune response (Misawa et al., 2009). The hormonal form of vitamin D3 induced the expression of pro-LL-37 in isolated neutrophil progenitors and in Epstein–Barr virus-transformed B cells from patients with severe congenital neutropenia (Karlsson et al., 2008). 1,25(OH)2D3 induces expression of the human cathelicidin antimicrobial peptide (CAMP) gene in acute myeloid leukemia (AML), immortalized keratinocyte, and colon cancer cell lines as well as normal human bone marrow (BM)-derived macrophages and fresh BM cells from two normal individuals and one AML patient (Gombart et al., 2005). Moreover, 1,25(OH)2D3 enhances the induction of antimicrobial proteins and secretion of antimicrobial activity against pathogens including P. aeruginosa, revealing the potential role of its analogues in the treatment of opportunistic infections (Wang et al., 2004).

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Biological Functions

Cathelicidins can function as immunomodulators and/or microbicidal agents in the innate immune system. The biological effects exerted by cathelicidins at a given time and place are probably determined by the specific activities of the peptide and the physiological setting, including the concentration of the peptide, cellular environment, and soluble components of the extracellular milieu. Although many publications refer to LL-37 as an antimicrobial peptide, in the presence of physiological salt, heparin, and serum conditions, LL-37 is not antimicrobial at the concentrations (generally 2 µg/ml) normally found in adults at mucosal surfaces, although it does exhibit immunomodulatory functions under these conditions. It is possible that the release of LL-37 during degranulation from neutrophils can considerably elevate local concentrations.

The most studied cathelicidin peptides, human LL-37, porcine PR-39, and bovine BMAP-28, have been shown, when added exogenously, to control bacterial load and prevent sepsis and mortality in rats and mice after bacterial challenge (Table 2). Mice deficient in the murine cathelicidin CRAMP also exhibit significant but moderate defects in defense against bacterial infections of the skin and respiratory tract as well as decreased angiogenesis and delayed wound closure. In humans with atopic dermatitis, low expression of LL-37 (and possibly other peptides) in skin lesions from patients coincides with their enhanced susceptibility to skin infections, while individuals with Morbus Kostmann, suffer from frequent oral bacterial infections and severe periodontal disease that correlates most with a deficiency in LL-37. Conversely, LL-37 is highly expressed in psoriatic skin and rosacea. Together, these observations demonstrate, in vivo, a significant role for cathelicidins in mucosal host defenses and wound healing.3,15,22

Table 2. Functions of Selected Cathelicidins

Cathelicidin functions	Human LL-371,3,8,22,34	Murine CRAMP 15,29,34	Bovine BMAP-28, Bac2A or indolicidin 29	Porcine, PR-3926
Anti-infective effects observed in vivo
Protects against bacterial challenge and sepsis	✓	✓	✓
Controls bacterial load	✓	✓
Selectively chemotactic	✓	✓
Reduces amounts of proinflammatory mediators	✓		✓
Enhances adaptive immune response	✓	✓
Immunomodulatory effects observed in vitro
Induces and influences signal transduction	✓	✓		✓
Induces gene expression and enhances protein secretion	✓		✓	✓
Suppresses inflammatory stimuli-induced gene expression and protein secretion	✓		✓
Influences cell proliferation, differentiation, and migration	✓	✓	✓	✓
Promotes angiogenesis and/or wound healing	✓			✓
Cooperates with immune mediators	✓
Influences adaptive immune response	✓

Anti-infective

The protective effects of cathelicidin peptides against different microorganisms (bacteria, viruses, yeast, fungi) have often been attributed to their direct antimicrobial killing properties, which can be mediated by bacterial cytoplasmic membrane disruption.3,15 Indeed, some cathelicidin peptides have exceptionally good antimicrobial activity (e.g., pig protegrin 1). Most cathelicidins, however, are not nearly as potent as protegrin yet may exert microbicidal action by other mechanisms, for example, LL-37 inhibits microbial attachment capabilities and biofilm formation.5 LL-37 also exhibits antiviral effects by attacking viral membranes,7 or targeting the reverse transcriptase of HIV.32 Mouse cathelicidin CRAMP maintains colonic integrity and prevents inflammatory disease and infection in part by suppressing apoptosis and stimulating mucin gene expression and mucous production in the colon.34

Immunomodulatory

In addition to being antimicrobial, host defense peptides stimulate a broad range of biological effects in a innate immune cells (including neutrophils and epithelial cells), adaptive immune cells (T- and B-lymphocytes), and in cells that are involved in and bridge the innate and adaptive immune systems such as monocytes/macrophages and dendritic cells3,8,15,22 (Table 2). They promote effector mechanisms that initiate and support innate and adaptive immune responses, yet they also protect the host against the detrimental effects of excessive inflammatory responses and support the subsequent resolution of inflammation.

Immune Activation

Cathelicidins promote chemotaxis of effector cells, induce the transcription and secretion of chemokines and cytokines, and induce mast cell activation and degranulation. Together these effects enhance vascular permeabilization, promote leukocyte extravasation, and regulate cellular responses at the site of infection/injury. Human cathelicidin LL-37 activates effector monocytes through many signaling pathways (IκB-α/NF-κB, PI3 K and the MAP kinases p38, ERK1/2 and JNK) and transcription factors (including NF-κB, AP-1, AP-2, SP-1, E2F1, CREB, and EGR) that induce the expression of nearly 500 immune-related genes.18 LL-37 can also activate effector cells synergistically with proinflammatory stimuli (IL-1β, CpG, LTB4), cellular differentiation factors (GM-CSF) and some TLR agonists (e.g., flagellin, polyI:C, and viral and microbial self-DNA/RNA); the latter synergy is in line with the observed adjuventicity of cathelicidins LL-37 and CRAMP.1,3,22

Inflammatory Control

In a diverse range of cells exposed to proinflammatory bacterial products, cathelicidins suppress gene transcription and the release of proinflammatory mediators. The antiendotoxin (LPS) effect of cathelicidins has been well described in numerous immune cells and correlates with protection in several animal models of sepsis.20 Recently, it was shown that LL-37 inhibited both innate and adaptive cellular responses to IFNγ, the key cytokine of Th1-polarized immunity.23 Cathelicidins exert these effects through intracellaular and/or surface receptors that trigger intracellular signaling pathways that influence MAP kinases, Ca2+ moblilization and NF-κB.18 Cathelicidins can also directly bind to negatively charged microbial components (e.g., LPS) and can also control inflammation by preventing the release of toxic cellular components that cause excess tissue damage and inflammation. For example, PR-39 inhibits the production of reactive oxygen species and BMAP-28 induces cell death via apoptosis of activated (infected) lymphocytes, which, unlike necrosis, occurs without releasing toxic cytoplasmic molecules. LL-37 also modulates apoptosis, necrosis and secondary necrosis with differing effects that are cell type specific; affected cells include neutrophils, keratinocytes, epithelial cells, endothelial cells, CD4+ CD25+ FoxP3+ regulatory T cells, and cytotoxic, but not helper, T cells.3

Immune Resolution

Cathelicidins are strongly expressed during wound healing and have been shown to promote reepithelialization of human skin wounds and rat gastric ulcers. These effects may be mediated by cathelicidin-induced angiogenic effects, induction of epithelial cell migration and proliferation, and inflammatory modulation. LL-37-induced migration of keratinocytes and proliferation of epithelial cells is mediated by pathways involving EGFR/STAT3 and GPCR/EGFR/ERK.34

The neutralizing and resolving effects of cathelicidins provide a balance between the protective and destructive components of inflammation, protecting against lethal conditions, while promoting responses to eradicate the infectious agent. These neutralizing activities also implicate cathelicidins in maintaining homeostasis, particularly in regions of the gut rich in commensals that contain the same bacterial molecules that activate the innate immune response through TLRs.

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As well as the C-X-C chemokines, another potential link between activation of innate immunity and chronic allograft dysfunction is emerging from studies on antimicrobial peptides (AMPs), which are secreted by neutrophils and epithelial cells. The human cathelicidin antimicrobial peptide LL-37/hCAP-18 is expressed in leukocytes and epithelial cells and secreted into wound and airway surface fluid. There has been substantial interest in its angiogenic properties (Koczulla et al., 2003). In recent work in Newcastle, U.K. (Anderson et al., 2006), pulmonary expression of LL-37/hCAP-18 and also α-defensins (HND1-3) were measured in 30 stable lung transplant recipients and 14 with BOS. Presence of airway pathogens was associated with significantly increased levels of neutrophil-derived AMPs. Subjects with BOS had significantly elevated hCAP-18/LL-37 and HND 1-3, compared to stable recipients. Taken together, these findings demonstrate that LL-37/hCAP-18 could have a potential central role for linking host defence and inflammation with angiogenic airway remodelling and the chronic allograft dysfunction of BOS.

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2.3.2 Cathelicidins

A long α-helical structure is characteristic for the cathelicidins found in the skin and in neutrophils. Several peptides are derived by posttranslational peptide cleavage from a precursor protein encoded by a single gen. Specifically, a mature 37 amino acid cathelicidin cleavage product with a pair of leucines at the amino terminal (cathelicidin LL-37) is active against several Gram-positive and Gram-negative bacteria and cathelicidin LL-37 fragments with varying antibacterial and anti-Candida activity were isolated from human skin. Topical vitamin D application induces cathelicidin LL-37 production in human skin [17].

Whereas µM concentrations are needed for the membrane damage-related antimicrobial activity, hBDs and cathelidins enhance adaptive and innate immune responses in pM to nM concentrations by stimulation of host cell receptors. Both AMPs induce a chemotactic activity, e.g., for immature dendritic cells and memory T cells, and defensins activate also macrophages and neutrophils. hBD-2 enhances the production of Th2 cytokines (IL-4, IL-13, IL-31). Defensins and cathelicidin LL-37 induced mast cell degranulation results in the release of histamine and other pruritogenic substances, which aggravate allergic responses in atopic patients (for review see [18]). Moreover, these AMPs induce apoptosis and enhance wound healing.

Anticancer effects are less well characterized, they appear to depend on the tumor type. Besides an inhibition of tumor growth, a stimulation of tumor growth has also been described. Cathelicidin LL-37 can induce apoptosis in oral squamous cell carcinoma, but inhibits apoptosis in normal keratinocytes.

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The role of antimicrobial proteins (AMPs) as a defense has been reshaped. Human cathelicidin LL-37 displays wide antimicrobial spectrum and inhibits biofilm formation, but is strongly antagonized by physiological salt concentration, divalent cations, and mucins [12–15]. Probably its more important role is as an immunomodulator. LL-37 induces chemokines, suppresses apoptosis of neutrophils and lipopolysaccharide (LPS)-induced production of cytokines, permeabilizes apoptotic leukocytes, induces leukocyte secondary necrosis, stimulates colonic mucin synthesis, and promotes angiogenesis and wound healing [16–19].

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4.1.6 Antimicrobial peptide LL37

Antimicrobial peptide LL37 is produced by neutrophils and is enriched on the chromatin fibers of NETs. LL37 not only induces autophagy in ECs, but exerts an adverse action on autophagy-dysfunctional ECs to induce cell death in the pathogenesis of AS. IL-1β, an AS-associated inflammatory cytokine, can result in cell death. LL37 further increases the IL-1β-induced expression of LC3-II NETs to promote cell death [143,144]. NETs-derived LL37 (named Cramp in mice) is anchored to ECs and connects to formyl peptide receptor 2 on monocytes, recruiting monocytes to ECs [47,104], to enable monocytes to attach to inflammatory blood vessels, and subsequently exacerbate inflammatory responses (Fig. 3). The immune modulator function of LL-37 induces the expression of inflammatory genes including adhesion molecules (e.g., ICAM-1) and chemokines(e.g., MCP-1) in human ECs, promoting the recruitment of leukocytes to the site of infection in the vessel wall, thus enhancing innate immunity and modulating the local inflammatory response of diseased vessels [145]. LL37 also plays an important role in the indirect pathway of EC inflammation mediated by NETs.

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3.1.1 Antimycobacterial Peptides Derived From Human Cells

The details of the mode of action of some important antimycobacterial peptides derived from human immune and nonimmune cells, microbes, and other natural sources are given in Table 2.

Table 2. Natural Antimycobacterial Peptides and Their Mode of Action

S. No.	Name of the Antitubercular peptide	Source	Name of the M. tuberculosis Target/M. tuberculosis Strain	In Vitro Activity	In Vivo Activity	Reference(s)
1.	Cathelicidin antimicrobial peptide (CAMP); hCAP-18 and LL-37	Homo sapiens (human): neutrophils, macrophages, dendritic cells, epithelial cells, monocytes, mast cells, natural killer cells	Mycobacterium tuberculosis	Mycobacterial cell wall and immunomodulation activity	[44,68–72]
2.	Trichoderin A	Fungus Trichoderma sp.	M. smegmatis, M. bovis and M. tuberculosis H37Rv	MIC range 0.02–2.0 μg/mL under aerobic and dormancy inducing hypoxic conditions	N.D.	[73]
3.	Cyclomarin A	Marine Streptomyces spp. CNB-982	ClpC1 Subunit of the Caseinolytic Protease of M. tb	Tested on MDR and XDR M. tb as well as drug-susceptible M. tuberculosis. Please refer [74]
4.	Lassomycin (cyclic peptides from actinomycetes)	Lentzea kentuckyensis spp. IO0009804	ClpC1 Subunit of the Caseinolytic Protease of M. tb	MIC of 0.8–3 mg/mL and MBC of 1–4 mg/mL. Tested on MDR and XDR M. tb as well as drug-susceptible M. tuberculosis. Please refer [75]
5.	Ecumicin (macrocyclic tridecapeptide)	Nonomuraea spp. MJM5123	ClpC1 Subunit of the Caseinolytic Protease of M. tb	Tested on MDR and XDR M. tb as well as drug-susceptible M. tuberculosis. Please refer [76]
6.	Nocathiacins	Amycolatopsis fastidiosa	Clinical strains of Mycobacterium tuberculosis with MDR phenotypes.	Please refer [77]
7.	Thiazomycins	Amycolatopsis fastidiosa	Clinical strains of Mycobacterium tuberculosis with MDR phenotypes.
8.	Magainin-I	From skin secretions of species in the genera Xenopus	Mycobacterium tuberculosis	Please refer [78]
9.	Defensins	Homo sapiens: Macrophages, Eosinophils, Dendritic cells, Neutrophils and epithelial cells	Mycobacterium tuberculosis	Please refer [79–82]
10.	Granulysin	Homo sapiens: Lymphocytes	Mycobacterium tuberculosis	Mycobacterial cell lysis. Please refer [83,84]
11.	Calgranulin	Homo sapiens: Neutrophils, leucocytes, monocytes	Mycobacterium tuberculosis	Phagolysosomal fusion and cell lysis. Please refer [85,86]
12.	Ubiquitin derived peptides	Located in lysosomes of host cell of Homo sapiens	Mycobacterium tuberculosis	MIC: 5 μM Membrane targeting ability and mycobactericidal activity	Please refer [44,87]
13.	Hepcidin	Hepatocytes, macrophages, lymphocytes of host cell, Homo sapiens	Mycobacterium tuberculosis	Growth inhibition and cell lysis. Please Refer [44,88]
14.	Lactoferrin	Epithelial, neutrophils and leucocytes of host cell, Homo sapiens	Mycobacterium tuberculosis	Cell permeation and antiinflammatory. Please Refer [89–91]
15.	Lipocalin2	Neutrophils of host cell, Homo sapiens	Mycobacterium tuberculosis	Growth inhibition and immunomodulation. Please Refer [92]
16.	Human host defense ribonucleases (RNase 3 and RNase 7)	RNase 3 is secreted by eosinophil secondary granules (called ECP) and RNase 7 is abundantly secreted by keratinocytes.	Mycobacterium tuberculosis	MIC: 20 μM	Please refer [93–95]
17.	HCL2	Part of the human cytochrome c oxidase subunit3 (COX3) protein.	Mycobacterium tuberculosis	Please refer [96]
18.	Cathepsin	Neutrophils and monocytes of host cell, Homo sapiens	Mycobacterium tuberculosis	Immunomodulation and apoptosis of TB infected cells. Please refer [97]
19.	Elastases	Neutrophil azurophilic granules and macrophages	Mycobacterium tuberculosis	Cell lysis. Please refer [98,99]
20.	Glaucacyclopeptide A (cyclic heptapeptide)	Isolated from seeds of Annona glauca	Mycobacterium tuberculosis	Synthetic derivatives tested through in vitro and in silico assays	Please refer [100]
21.	Lariatins A and B	Rhodococcus jostii K01B0171	Mycobacterium tuberculosis and Mycobacterium smegmatis	Lariains A and B showed growth inhibition against Mycobacterium smegmatis with MIC values of 3.13 and 6.25 mg/mL, respectively (in agar dilution method). Lariains A showed growth inhibition against Mycobacterium tuberculosis with MIC: 0.39 mg/mL (in liquid microdilution method)	N.D.	Please refer [101,102]
22.	Callyaerins A-G	Indonesian sponge Callyspongia aerizusa	Mycobacterium tuberculosis	Callyaerins A and B showed MIC90 values of 2 and 5 Μm, respectively.	Please Refer [103]
23.	Bacteriocins	Ribosomally synthesized antimicrobial peptides produced by bacteria and class-I bacteriocin is called Lantibiotic (contains posttranslationally modified lanthionine and methyllanthionine amino acids)	Encapsulation of bacteriocins, particularly in liposome complexes, could offer potential for successful delivery of bacteriocins to specific sites of infection. Please refer [104]
24.	PR-39 (proline and Arginine rich) peptide	Isolated from porcine leucocytes	Effective on MDR strain of M. tuberculosis (resistant to both isoniazid and rifampicin). BACTEC radiometric method showed MDR strains E1380/94 and P34/95 inhibition between 39% and 49%. Please refer [105]
25.	Neamphamide B	Japanese marine sponge of Neamphius sp.	Mycobacterium tuberculosis	MIC values were 1.56 μg/mL for M. smegmatis and 6.25 to 12.5 μg/mL for M. bovis, BCG.	N.D.	Please refer [106]

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Cathelicidins: The precursor human cationic antimicrobial protein (hCAP-18) is composed of two regions: (1) N-terminal sequence, called cathelin, and (2) the bactericidal C-terminal region, called LL-37. LL-37 is released by proteinase-3 proteolysis. Pore formation and membrane disruption by LL-37 was observed in Mycobacterium tuberculosis through in vitro experiments. In addition to this, toll like receptor (TLR) activation of monocytes and vitamin D–mediated antimicrobial responses through in vivo experiments was observed by human host to kill the Mycobacteria. LL-37 is the only member of the cathelicidin family that is expressed in humans. The azurophilic granules of neutrophil is the primary source of cathelicidin in the human body. However, during the inflammation process LL-37 can be located in the mucosal surfaces and in keratinocytes [71]. Two derivatives of LL-37 peptide were found to have improved antimycobacterial activity. The truncated variant of LL-37 is the first derivative and is called LLKKK-18. This peptide contains polar uncharged residues, glutamine (Q22), asparagine (N30), and negatively charged aspartic acid (D26), which is replaced with positively charged lysine. This derivative was found to be more active against M. tuberculosis without cytotoxic effects on macrophages. In addition, this derivative was more successful in killing M. tuberculosis in combination with biogenic silver nanoparticles. Furthermore, there is a second derivative of LL-37 called D5 (a D-enantiomer). This second LL-37 variant peptide contains hydrophobic leucine residue as a substitution for three alanine residues and at position 16 valine is substituted with lysine to avoid self-association. This derivative showed better hemolytic activity and therapeutic index value than the original LL-37. Recent studies have observed that both the exogenous addition of LL-37 and endogenous overexpression of cathelicidin within macrophages could potentially affect the survival of mycobacteria. The intracellular survival of mycobacteria was found to reduce when compared with control cells [72,79–82].

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Defensins: Certain epithelial defensins are always expressed, whereas some defensins are induced by external stimuli such as tissue injury [83]. Defensins contains six conserved cysteine residues and this builds three disulfide linkages [84]. Based on structural differences, mammalian defensins are further classified as α-defensins, β-defensins, and θ-defensins. Alpha-defensins are found in the azurophilic granules of polymorphonuclear neutrophils and are named human neutrophil peptide 1 (HNP-1). This alpha-defensin (HNP-1) in combination with first-line drugs was found to possess significantly better clearance of M. tuberculosis H37Rv when compared with the same dose of first-line drugs without alpha-defensin. Beta-defensins are of four different types (HBD1–4) and are expressed in both leukocytes and epithelial cells. All beta-defensins are expressed upon induction except HBD-1 (expressed constitutively). HBD-2 defensin was found to suppress the growth of M. tuberculosis intracellularly [85]. Recent studies found improved immune response against M. tuberculosis strain using DNA vaccines containing beta-defensins-2 sequences, when used as adjuvant in BCG vaccination [86].

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Granulysin: This is a small cationic glycoprotein (9 kDa) and is synthesized as a secretory precursor (15 kDa). This precursor form of granulysin is enzymatically cleaved into granular glycoprotein (9 kDa). The cytolytic activity was observed with this form of glycoprotein against extracellular as well as intracellular M. tuberculosis. It was observed in research studies that granulysin levels are high in active or latent TB-infected individuals as compared to controls. Hence, granulysin can be used as an immune marker for the presence of M. tuberculosis infection. Studies have found that granulysin-derived peptides named, granF2 and G13 were effective against clinical isolates of multidrug-resistant M. tuberculosis including drug-susceptible mycobacteria [87]. Granulysins were found to: act on mycobacterial membranes, disrupt synthetic liposomes, and induce apoptosis of mammalian cells. In vitro efficacy was found to improve by attaching the TB5 antitubercular drug candidate to peptide carriers. It was found that the peptide conjugates, OT20 tuftsin and GranF2 have increased uptake into M. tuberculosis H37Rv infected human MonoMac6 cells followed by inhibition of intracellular bacteria [88].

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Calgranulin (calprotectin): This is a calcium-binding protein but it is also reported to bind with Zn2 + cations. Calgranulin binding to Zn2 + is found to activate the peptide antimicrobial activity. Calgranulin was found to be associated with antimycobacterial activity through phagolysosomal fusion [89,90].

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Ubiquitin-derived peptides: These peptides were found in lysosomes and were reported to have antimycobacterial activity. Mycobacterial infection activates the macrophages and the mycobacteria are then delivered to the lysosome. Upon lysosomal entry, the mycobacteria can be killed through both oxidative and nonoxidative mechanisms. Oxidative mechanisms are performed through the action of reactive oxygen and nitrogen intermediates on mycobacteria. Whereas, nonoxidative mechanisms are associated with the ubiquitin-derived peptides. Autophagy is an important step for the mycobactericidal activity of ubiquitin peptides [72,91].

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Hepcidin: This peptide was first detected in serum and urine. This is known as a key component for the recycling of iron and inhibition of iron absorption in response to iron overload. The precursor (84 residues) is synthesized in macrophages and liver hepatocytes. This precursor is then cleaved to yield the mature hepcidin (25 residues) and proregion (35 residues). The two byproducts of precursor cleavage showed antimycobacterial activity against mycobacteria. Both the host macrophage and the pathogen compete for iron during pathogenesis. Hepcidin is reported to be secreted by the macrophage to restrict the growth of mycobacteria, although it also inhibits the release of the recycled iron by macrophages. In vitro experiments showed that hepcidin could inhibit M. tuberculosis growth and it has been shown to cause structural damage to the mycobacteria [72,92].

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Lactoferrin: This is an iron-binding glycoprotein (80 kDa) of transferrin family. This is present in most tissues and body fluids and has a multifunctional role related to iron homeostasis. Research studies have proved that this protein is an immunomodulator and has improved the efficacy of BCG vaccine when used as an adjuvant [93–95].

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Lipocalin2: This is also involved in iron homeostasis. This protein has shown expression in neutrophils with antitubercular activity. Hence, Lipocalin2 is also called a neutrophil gelatinase-associated lipocalin [96].

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Human host defense ribonucleases (RNase; RNase 3 and RNase 7): These are small cationic proteins with cytotoxic response towards mycobacterial infections. RNase 3 is called the eosinophil cationic protein (ECP) due to its secretion from eosinophil secretory granules. RNase 7 is functionally important in a skin-defense mechanism and it is secreted by keratinocytes for action against human pathogens [97–99].

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HCL2: This is a portion of the human cytochrome c oxidase subunit3 (COX3) proteins and possesses a helical structure. This protein is similar to an early secreted antigenic target protein (ESAT-6). Bacterial three-hybrid experiments showed that this HCL2 peptide disrupts the heterodimeric association of ESAT6-CFP10 complex. This was confirmed by the change of colony color from blue to white. Arabinose gradient liquid β-galactosidase assay also decreased β-galactosidase activity through increased expression of HCL2 peptide. M. tuberculosis showed remarkable changes in cell morphology and cell-wall degeneration upon endogenous and exogenous HCL2 treatment. This peptide also extensively reduced mycobacterial infection in the preclinical mouse infection model [73].

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Cathepsin and elastases (serine proteases): These proteins are expressed in neutrophils and macrophages. The precursor protein procathepsin is converted to the mature cathepsin under acidic conditions. However, the mycobacteria can downregulate the expression of cathepsin within macrophages for its intracellular survival. The macrophage lysosomes cathepsins has been proved to control the M. tuberculosis infection, i.e., granuloma formation using the zebrafish/M. marinum model. Elastase protein also showed colocalization with the neutrophil to facilitate the degradation of pathogen associated virulence factors [100–102].

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Resistance to Antimicrobial Peptides

In response to infectious stimuli, skin keratinocytes, mucosal epithelial cells and neutrophils produce high levels of antimicrobial peptides (AMPs) known as cathelicidins (LL-37) and defensins. The Staph. aureus metalloproteinase aureolysin cleaves LL-37, while staphylokinase (SAK) inhibits the bactericidal effect of α-defensins.22 Furthermore, modification of cell wall teichoic acids promotes Staph. aureus resistance to AMPs.23

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