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Antimicrobial peptides: A new drug therapy for multidrug resistant bacteria

Author: Dr. Shamsher Singh, Dr. Darrel Singh, Dr. Nitin Sharma.

Objectives: This review presents some recent progresses in the study of antimicrobial peptides and its potential role in the oral environment.

Data: 52 research/review articles were selected initially. Out of which 26 articles were selected, found in relation to the oral cavity.
Sources: Data collection was done by searching the literature using keywords: antimicrobial peptides, its mechanism of action, therapeutic potential in PubMed, Medline, Google scholar and electronic databases and the search was restricted to English articles.

Study selection: Studies included in this present review were divided into four groups: In vivo- studies, In vitro- studies, observational studies and review articles. Eligibility criteria were fixed for each category. In vivo studies were included if they have clear outcome of the study as well as had a control group. For in vitro studies, biological studies were included, interaction with oral cells and microorganisms were reviewed. Exclusion criteria were fixed for the articles, who does not matched the inclusion criteria A total of 26 articles were selected after eliminating those articles that were found not in relation with present review article, which comprises of review articles: 14, In- vivo studies: 7, In- vitro studies: 4, and one observational study.

Conclusions:. The currently available antibiotics not only fail to remove the infection, but also ineffective in the presence of multidrug resistant bacteria. AMPs when administered alone or in combination with other molecules may solve this problem It has raised the interest of the researchers to use these evolutionary conserved components to combat resistant to current antibiotics. However still there are challenges to prove or examine the roles of AMPs in defence of the oral cavity and testing should be done to know more about the therapeutic role of these AMPs in the oral environment.

The development of resistance to the conventional antibiotics is a growing health problem worldwide and far exceeds the pace of new development of drugs1. Unlike other antibiotics it appears as though antimicrobial peptides may also have the ability to enhance immunity by functioning as immunomodulators2.The emergence of new antibiotics has stimulated the interest in the development of antimicrobial peptides. Many studies have found that antimicrobial peptides act effectively in synergy with currently used antibiotics against multidrug resistant bacteria3,4,5.

The antimicrobial peptides are small proteins, isolated from natural and synthetic sources like fungi, plants, invertebrates and vertebrates and it forms a defence against bacteria, protozoa, fungi, and viruses6. Most AMPs kill both gram-positive and gram-negative bacteria, while a significant numbers of these bactericidal peptides have been found to have anticancer and antiviral activities6,7. These AMPs are considered as powerful agents of healing by altering host immune response and important in diverse functions such as wound healing, chemotaxis and angiogenesis.

Antimicrobial peptides are made by many cells in skin and can be made by keratinocytes when activated. Resident and recruited bone marrow—derived cells such as mast cells and neutrophils also express high levels of antimicrobial peptides. Functions attributed to these peptides extend well beyond activity as simple antibiotic. Select molecules will influence cytokine release and synthesis of components of the extracellular matrix (ECM) and are important in diverse functions such as wound healing, chemotaxis and angiogenesis8. (Figure I)

Functionsand mechanismof

Figure I: Functions and Locations of the antimicrobial peptides

Figure II: Mechanism of membrane disruption by AMP (adapted from BMC Biochemistry 2005, 6:30)

Mechanism of action:
The mechanism of action of AMP is not fully understood, but it is believed that their major targets are cytoplasmic membrane and intercellular molecules9. AMPs are positively charged and have both a hydrophobic and hydrophilic side that enables the molecule to be soluble in aqueous environments yet have the capability to enter lipid-rich membranes of various microbes and also stimulate immune cells and leads to membrane disruption membrane disruption. (Figure II)

The outer layer of Gram-ve bacteria consists of lipopolysaccarides (LPS), which is essential for bacterial growth and viability. Bacterial LPS exhibits a number of important properties, such as immunogenicity, induction of pro-inflammatory cytokines, and protection against phagocytosis and complement killing10. Hence, LPS are an excellent target for AMP, because it has the potential to inhibit the growth of bacteria as well as to neutralize the action of released LPS that may lead to the release of pro-inflammatory cytokines into the blood, resulting in septic shock11. AMP bind to the divalent cation binding  sites and with the polyanionic outer moieties of LPS, disrupting and expanding the outer membrane and allows passage of AMPs through the outer membrane (termed as  the self-promoted uptake pathway12).When sufficient  concentration of AMPs are present, they aggregate within the membrane  causing depolarisation and permeabilisation(Table I).

table-1 table-2

Table I: Mechanism of action of AMP on Gram -ve bacteria

Table II: Mechanism of action of AMP on Gram +ve  Bacteria

Similarly outer layer of Gram +ve bacteria consists of peptidoglycans which are composed of polymers of sugars and amino acids13. Peptidoglycans are essential for the growth and replication of bacteria. AMP exerts antibacterial action through the interaction with cell wall and inhibits the biosynthesis of cell wall. AMP also disrupts the membrane by permeation through the cell wall and cytoplasmic membrane to reach their intracellular targets which are nucleic acids and causes the efflux of intracellular materials14 (Table II).A number of models for AMP membrane permeabilisation have been proposed like membrane disruptive (barrel stave, toroidal, carpet and micellar aggregate models) and non-membrane disruptive (intracellular targets) and still there is not universal consensus among investigators in this regard9.

Therapeutic potential of AMPs in health & disease:
An epithelial cell expresses antimicrobials peptides and delivers to the site of infection by circulating white cells. These amps act as epithelial preservatives on the keratinized epithelial sheets of the dry skin & tongue and can be isolated in humans and mammals. As in the mouth, the airways are protected by AMPs released from phagocytes and secreted by epithelial cells. Bals et.al in 1998, Agerbath et.al in 1999 found that the presence of cathelicidin LL-37 produced by epithelial & neutrophils and also α-defensins HNP (1-4) produced by neutrophils alone in airway surface fluid. Ashitani et.al 2001, Lee et.al 2002 confirmed that the levels of LL-37, β-defensins increased following infection and inflammation. The upper respiratory tract consists of organisms like staphylococcus, streptococcus, neisseria, hemophilus, mycoplasma and moraxcella species. AMPs protect sites that are colonised by these organisms such as nasopharynx and nasal cavity10.

In addition to defence against infection, Arabiou et.al. suggested that α-defensins enhances lung epithelial cells proliferation ,helps in epithelial cell repair15 and may act in synergy with wound healing factors such as TFF( Trefoil factor family) proteins expressed by respiratory epithelia16 and with other LPS binding molecules (i.e. bactericidal permeability inducing protein and LPS binding protein) to regulate responses to bacterial LPS. The homeostasis is disturbed by the genetic defect in cystic fibrosis cases, results in bacterial overgrowth and chronic tissue destruction17, 18, AMP fails to provide an effective barrier because LPS of AMP resistant cystic fibrosis pathogen such as pseudomonas aeruginosa, is a significant respiratory pathogen and is associated with high morbidity and mortality in patients suffering from cystic fibrosis10. P.aeruginosa and other pathogens produce extracellular and membrane bound proteases that degrade antimicrobial peptides.

Similarly in Gastrointestinal tract, the gastric mucosal cells of human & mice synthesises excess histone 2A, that accumulates within cytoplasmic secretory granules. It is converted to buforin II by pepsin on secretion, remains adhered to mucous membrane thus providing the stomach with a protective antimicrobial coat19. Also the GI tract of mammals consists of specialized granules cells called paneth cells & enterocytes. These cells secrete AMP either onto the GI surface or retain them in the superficial sheets of the epithelium. Paneth cells on stimulation also secrete α-defensins into the intestinal crypts, which eventually moved to the gut lumen and helps in maintenance of the bowel health20.

Role in oral cavity:
AMP performs many functions in the oral cavity. They may provide protection against microbial pathogens, assist in oral biofilm control, and function as an important part of the innate immune system in response to local and systemic infection. Synthetic versions of these peptides may be helpful to supplement natural antimicrobial peptides as therapeutics agents.

Epithelial tissues provide the first line of defence between an organism & the environment. If this delicate barrier disrupts, that may results in bacterial invasion followed by inflammation and can lead to bacterially induced periodontal disease  and to infections of the oral mucosa by bacteria, fungi and viruses. Dale et al. in 2001 and Marsh in 2003 concluded that tissues of the oral cavity are constantly exposed to innate defence components derived from saliva, GCF and epithelial cells10.

Normal commensal bacterial community acts in a manner to benefit the overall innate immune readiness of oral epithelium. Literature suggested that these oral commensals stimulates epithelial surfaces to provide protection and kill the pathogenic microbes by expressing antimicrobials pepetides.21 The epithelium expressed the β-defensins, neutrophils expressed α-defensins, similarly neutrophils & epithelium both expressed cathelicidin LL-37. These defensins & cathelicidin have broad antimicrobial activity against streptococcus mutans, porphyromonas gingivalis and actinobacillus actinomycetemcomitans22.

Murakami et al. studied that the role of histatins in the oral cavity is mainly anti-candidal, but they have been shown to inhibit the adherence of gram-positive bacteria and the periodontal pathogen Porphyromonas gingivalis to erythrocytes and streptococci23.

The epithelium surrounding the tooth is unique in its function by forming a firm seal around each tooth and is continuously exposed to the bacterial biofilm that forms on the tooth surface. This region is at significant risk of inflammation and bacterially induced infection. Whenever there is infection inside the oral cavity, particularly in the gingival sulcus AMPs like defensins stimulates mast cell degranulation (Befus et al, 1999), enhances macrophage phagocytosis (Ichinose et al, 1996) and result in activation of complement system (Prohaszka et al, 1997; van den Berg et al, 1998) placing both naive T- cells, memory cells and antigen presenting dendritic cells at infection sites. Hence these AMPs are important in clean up of infected region by promoting adaptive immunity22.

It has also been observed that there is increase in concentration of neutrophils following inflammation. Approximately 30,000 neutrophils per minute enter the oral cavity via the junctional epithelium surrounding the teeth required for periodontal health24 and any defects in neutrophils function and chemotaxis are associated with early onset periodontitis in children and Morbus Kostmann syndrome25, 26.

The expression of AMPs in saliva and in the oral cavity suggests that they may have a role in protecting the tooth from caries, either by prevention of biofilm formation on tooth surface or by direct killing of the bacteria. Hence oral AMPs can be considered as a natural antibiotic barrier by their broad spectrum antimicrobial activity, enhancing IgA as well as IgG production and synergistic action with other AMPs in saliva.

Future perspectives and conclusion:
The normal flora is extremely complex that requires multiple types of defences to prevent infections. AMP has provided insights into the innate defensive system that permits multicellular organisms like humans to maintain the balance between health and disease. There is increase in antibiotic resistance to conventional antibiotics, it has stimulated interest in the development of AMPs as human therapeutics. Now the time has come, when our future studied should put emphasizes on approaches to have a look on the evolutionary ancient weapons6 to combat resistance to current antibiotics. The fact remains that most animals, including insects and creatures like the octopus & starfish, heavily rely on AMPs for defence against microbes and to do so quite effectively without the help of the lymphocytes, a thymus or antibodies2. Such mechanisms can be effectively used to develop new therapeutic modalities. Both clinical and laboratory studies should be carried out to develop therapeutically used agents from natural and thousands of synthetic variants.

Devine and Hancock in 2002 concluded that, there is no clear cut relationship between pathogenic microbes or commensals and their interactions with AMPs and bacteria employ a range of strategies for surviving AMPs10. Despite being tested for so many years, still there are challenges to prove or examine the roles of AMPs in defence of the oral cavity, because of the complexity of the microbiota and the multiplicity of innate defence molecules produced.

Future investigations and testing should be done to know more about the therapeutic role of these AMPs in the oral environment. The emphasis will be on the development of new peptides of antimicrobials, their regulation and functional efficacy against oral pathogens. Therefore, it is expected that AMP will become the future drug of choice for emerging bacterial infections.

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