coli in serum, while three of the four natural cationic AMPs retained some serum activity. Notably, the SLAY-derived antimicrobials were identified in screens using a K12 laboratory strain of E.

coli grown in Luria Broth LB. Interestingly, both natural β-AMPs examined Protegrin-1 [PG-1] and Tachyplesin-1 retained the most activity in HS relative to MH, suggesting this is a promising class to examine under more in vivo relevant screening conditions.

To identify synthetic AMPs with greater activity under biologically relevant conditions, we designed a 99,peptide library mimicking natural β-AMP attributes BH including residue frequency, length, charge, and potential number of disulfide bonds.

To do this, we used codon variation to represent the residue frequencies found in three regions of the β-AMP structure tail, sheet, and loop Fig. We potentiated multiple lengths via early stop codons and included the possibility of one to three cysteine pairs Fig.

Inclusion of multiple arginine residues also allowed peptides in the library to range in charge from 4 to We had 24 randomly selected peptides from the library BHR synthesized to determine their antimicrobial activity in both MH and HS Table S2.

Of the 24 peptides, These data confirmed the majority of the peptides in the library have antimicrobial activity but lack strong activity in HS and are therefore ideal for testing our antimicrobial screening scheme in serum.

This library was cloned into a surface display plasmid and transformed into E. coli ATCC for testing. Synthetic discovery of serum-active AMPs.

A Diagram of the potential residues at each position of a peptide library based on natural β-AMP sequence frequencies. Potential disulfide bonds are indicated with a dotted line. Octagons potentiate early stop codons. B Diagram of how SLAY functions.

D Distribution of log 2 -fold change, charge, length, and number of potential S—S bonds for select BHS peptides examined in vitro.

SLAY functions through the expression of a plasmid-encoded fusion tethering a peptide to the outer membrane Fig. During expression of the library, sequences with antimicrobial activity cause self-killing and therefore are reduced within the bacterial population.

Differences between induced and uninduced plasmid copy numbers can be monitored, and a log 2 -fold change is generated by comparing induced and uninduced next-generation sequencing reads Fig. The resulting screen in E. We selected 41 peptides from this SLAY active group to examine in vitro BHS Fig.

This group had a diverse range of charge 3. All data relating to our SLAY screen can be found in the Supplementary material. The 41 selected SLAY active peptides were commercially synthesized and tested for their MBC in both MH and HS BHS.

coli in MH media, while This is a large improvement in serum activity compared to the randomly selected peptides BHR Table S2. BHS peptides with a length of 16 amino acids, three disulfide bonds, and a charge of 6—8 were most likely to retain serum activity, suggesting these features improve potency Table 1.

No BHS peptides with a length of less than 16 amino acids or charge greater than 9 retained any serum activity Table 1.

Aa, amino acids; HS: human serum; MBC, minimum bactericidal concentration; n, number of samples; S—S, number of disulfide bonds. The most potent serum-active peptide BHS was renamed SAP for s erum- a ctive p eptide and further optimized to increase serum potency with the above data in mind Table S4.

Optimization included both canonical and noncanonical residue modifications, truncations, C-terminal amidation, N-terminal lipidation, and end-to-end cyclization.

Truncation to 16 amino acids, C-terminal amidation, and use of 2,4-diaminobutyric acid Dab in the loop region all modestly improved serum potency, while the use of D-enantiomers, N-terminal lipidation, and end-to-end cyclization negatively impacted serum potency Table S4.

Ultimately, optimized variant SAP, with a four-fold improvement in serum potency over SAP, was selected for a more detailed study. Many cationic AMPs demonstrate either β-hairpin or α-helical secondary structure upon interaction with bacterial membranes or membrane mimics like lipopolysaccharide LPS To examine whether SAP demonstrates a similar change in secondary structure, we performed circular dichroism spectroscopy with and without the presence of 0.

S2A and B. SAP encodes six cystine residues. This is consistent with the presence of three disulfide bonds Fig.

S2C and D ; however, we did observe a change in expected isotope distribution suggesting the SAP molecular population contained a mixture of one to three disulfide bonds. Together, these data suggest that SAP is macrocyclic but lacks a strong secondary structure in solution with and without LPS present.

Most cationic AMPs kill bacteria via disruption of the bacterial membrane, so we compared SAP activity to two known membrane lytic cationic AMPs PG-1 and CP1 and two small-molecule antibiotics kanamycin and cefuroxime which kill through ribosome and cell wall synthesis inhibition, respectively Fig.

First, we performed two fluorescent membrane disruption assays using 1-N-phenylnaphthylamine NPN and propidium iodide PI. Normally the outer membrane excludes hydrophobic molecules such as NPN, but when damaged, NPN can bind hydrophobic fatty acids within the membrane and fluoresce.

PI fluoresces upon DNA binding but can only gain access to the cytoplasm if both the outer and inner membranes have been permeabilized.

Antibiotics tested retained antimicrobial activity under the assay conditions used Fig. This was in contrast to PG-1 and CP1 which caused strong fluorescence of both NPN and PI. As expected, kanamycin, which can freely bypass cell membranes, showed no NPN fluorescence or PI uptake.

These data support SAP that quickly permeates the outer membrane without strong inner membrane disruption. SAP functions differently from traditional cationic AMPs.

A Table describing the structure and activity of various antibiotics. NPN B and PI C fluorescence of E. D Kill curve of E. coli cells treated with antibiotic at 2× their listed MIC. E Growth curve of E. coli cells treated with antibiotic at 8× their listed MIC.

Listed MICs are the median of three replicates data points are an average of three replicates with error bars representing 1 SD. Membrane lytic cationic AMPs like PG-1 and CP1 kill cells rapidly, so we compared kill curves for E. coli treated with antibiotic at two-fold its MIC. SAP did not kill cells rapidly like PG-1 and CP1; instead killing was delayed past that of kanamycin but before cefuroxime Fig.

A growth curve performed with each antibiotic added at eight-fold its MIC showed a similar result, with growth ultimately being inhibited later than CP1, PG-1, and kanamycin but slightly before cefuroxime Fig. Together, these data further support that SAP is not killing through a rapid, inner membrane lytic mechanism like traditional cationic AMPs.

To determine how SAP kills bacterial cells, we first tested its spectrum of activity by determining its MIC against a panel of clinical and laboratory monoderm and diderm bacteria Table S5. SAP remained active against both groups, and there was no clear evolutionary relationship between bacterial susceptibility.

Interestingly a strain of Corynebacterium striatum was most susceptible while Enterobacter cloacae and Klebsiella pneumoniae strains examined were fully resistant. We also found SAP activity was unaffected by the expression of mobile colistin resistance mcr-1 , suggesting it likely has a different mechanism of outer membrane disruption from the polymyxins Fig.

Next, we attempted to identify SAP's target through the isolation of resistant mutants via both plating and subinhibitory liquid passage in MH supplemented with SAP To improve the likelihood of isolating a resistant mutant, we used the E. coli Keio parent and a mutS -deficient strain from the Keio collection with an increased mutation rate relative to wild type.

Plating and serial passaging of both strains in the presence of SAP resulted in no mutants with greater than two-fold resistance. In contrast, rifampicin-resistant mutants were easily isolated via both methods Fig.

These data suggest that resistance to SAP is not easily developed. To further evaluate whether SAP may target an essential periplasmic protein, a pull-down was performed. Active N-terminal biotinylated SAP was incubated with E.

Cells were then lysed, and SAP was pulled down with streptavidin beads. Biotin alone was used as a background binding control. A comparison of MS between the two samples revealed only very low levels of protein binding specific to the SAP sample and no obvious target.

A full list of results is in the Supplementary material. Since attempted isolation of resistant mutants and pull-downs failed to identify a clear SAP target, fluorescent microscopy with E.

coli expressing cytoplasmic GFP was used to visualize possible changes in cell morphology. A small percentage 5. Interruptions in cell wall synthesis caused by β-lactam antibiotics also cause cell elongation; however, knockout strains deficient in cell wall synthesis such as dacA::kan , cpoB::kan , and mrcB::kan showed no increased sensitivity to SAP, unlike β-lactams, suggesting SAP elongation is likely caused by a different process 27 , 28 Fig.

SAP causes cell elongation, is nontoxic, and functions in vivo. A Fluorescent microscopy images of E. mellonella larvae. Percentage of hemolysis error is 1 SD of triplicate samples. c Survival of G. mellonella larvae infected with E. Cationic AMPs commonly have mammalian cell toxicity making them difficult to develop as therapeutics.

SAP and nearly all its derivatives, including SAP, show almost no hemolytic activity, in contrast to cationic AMPs like PG-1, a peptide included in the BH library design Fig.

SAP was also less toxic to embryonic kidney cells HEKT than PG Peptide antibiotics also often lose functionality in vivo, so we were curious if SAP treatment could increase survival of G.

mellonella larvae infected with 1. coli cells Fig. Together, this demonstrates that SAP is both nontoxic and retains its antibacterial activity in vivo, in contrast to many cationic AMPs, suggesting it may be a promising lead for further mechanistic examination and clinical development.

SLAY is a promising synthetic method for peptide antibiotic discovery; however, it has thus far only identified membrane-active peptides lacking activity in blood serum Table S1 17 , We adapted SLAY to function in conditions more representative of infection, identifying multiple macrocyclic AMPs retaining activity in HS Fig.

Characteristics of many serum-active peptides mimicked characteristics of potent natural β-AMPs including length 16—18 , overall charge 6—8 , and number of disulfide bonds 2 , 3 Table 1. These same attributes are observed in Tachyplesin-1 and PG-1 which were also shown to be serum active Table S1.

It appears that all AMPs examined lose activity in serum relative to broth; however, the extent of this loss in activity is variable, and highly potent peptides are more likely to retain serum activity. An increased number of disulfide bonds also correlated with increased serum activity which could be due to a reduction in proteolysis.

This has been observed with other cationic AMPs Reduced proteolysis could also explain the increased serum activity observed with SAP variants containing noncanonical residues and C-terminal amidation.

Our work strongly suggests that SAP functions uniquely from traditional cationic AMPs like PG-1 and CP1, which kill via rapid outer and inner membrane disruption. We demonstrate SAP permeates the outer membrane without strong inner membrane disruption and kills cells slowly Fig.

SAP also causes cells to elongate, some drastically so Fig. These attributes are more similar to nontraditional cationic AMPs like murepavadin, thanatin, and JB 29—31 , which have been suggested to target essential cell envelope processes other than the inner membrane.

Thanatin, which has been shown to permeate the outer membrane and target LPS transport, was included in the design of the β-hairpin library design screened here.

Additionally, SAP retains activity against both monoderm and diderm bacteria Table S5. Together, these data suggest that SAP targets a still unresolved, broadly conserved, cell envelope process; however, a delayed lysis of the inner membrane cannot be completely ruled out as possible.

Our inability to isolate SAPresistant mutants or pull down a strong protein interactor implicates a substrate rather than an enzyme as the SAP target.

We hope to elucidate a more detailed mechanism of action in future studies. Lastly, SAP does not appear to have significant erythrocyte, kidney tissue, or G. mellonella larvae toxicity and retains its function in vivo against G. coli Fig. SAP was especially active against a strain of C.

striatum , a growing nosocomial antimicrobial-resistant pathogen Unfortunately, the C. striatum strain examined here did not infect G.

mellonella larvae, so we were unable to evaluate SAP in vivo efficacy against this strain. Future therapeutic evaluation could be performed with clinically isolated antimicrobial-resistant C. striatum strains in murine or other appropriate models of infection. Peptides were then serial diluted two-fold down columns of the plate.

For crude peptides only BHR and BHS , acetic acid was added to 0. In cases where triplicate samples differed, the concentration supported by the median of the three replicates was reported.

Detailed methods for library creation have been previously reported 17 , Briefly, a library insert was generated by PCR using forward primer oJR with reverse primer oJR and 2× NR tether gBlock as the template Table S6. The ligated library was cleaned and transformed into E. coli competent cells via electroporation for further analysis via SLAY.

SLAY procedures have been detailed previously 17 , Briefly, E. The culture was then back diluted to an optical density OD 0. Plasmids from each triplicate culture were miniprepped, and Illumina sequencing primers were used to produce an amplicon via PCR Table S6. All possible codon combinations selected to generate the peptide sequences were encoded as nucleotide sequences for the reads to be mapped against.

Flexbar v3. The processed reads from flexbar were provided as input to Kallisto v0. The quantification file from Kallisto contained what would be akin to transcript-level quantification where there are numerous potential codon combinations for a given peptide in the library.

The R library tximport was used to take those values and transform them to peptide-level read counts. The peptide level read counts were then provided as input to DESEQ2 to determine which peptides had significantly more read counts between the two libraries All peptides used in this work were synthesized commercially by GenScript's custom peptide synthesis service and analyzed by reverse-phase HPLC and MS to confirm molecular weight.

Final concentrations of these peptides were also adjusted for purity. A full list of all of the peptides used and their reported commercial purity can be found in the Supplementary material. Briefly, peptides were diluted to 0.

Analysis was performed using Agilent MassHunter Qualitative software v10 and Agilent's Isotope Distribution Calculator. For SAP, this was performed in triplicate with a representative spectrum shown. For crude peptides, this was performed once, and the most abundant disulfide bond conformation was reported.

Ellipticity was then converted from mdeg to molar ellipticity. Reported spectra are an average of three separate spectra obtained from the same sample adjusted for molar concentration. A culture of E. coli was grown overnight in liquid media at 37°C. The following day, it was back diluted in LB media and grown at 37°C until an OD of 0.

PI uptake was measured for E. coli as previously described Briefly, single colonies from overnight growth on LB were inoculated into MH broth and grown to mid-log phase.

coli cells were back diluted from an overnight LB into MH to an OD of 0. Peptide stock solutions were diluted in MH to four times their reported MH MIC.

Ten microliters of each sample was then serially diluted fold at the indicated time points and plated on LB agar. Colony forming units were counted after overnight growth at 30°C.

Error bars represent 1 SD of triplicate samples. No peptide addition was used as a negative control. coli cells were back diluted fold from an overnight LB culture in MH and grown to an OD of 0. Peptide stock solutions were diluted in MH to 16 times their reported MH MIC.

Growth curves are reported as the mean representing triplicate samples. Cell length was calculated for individual cells using NIS-Elements AR software, and the mean was reported.

Full genomes were downloaded from the NCBI database www. Phylogenetic reconstruction was generated using PhyloPhlAn version 3. Within the pipeline, Diamond was used for genome mapping, MAFFT was used to generate multisequence alignment, and IQ-Tree was specified for phylogeny building 40 , Output from PhyloPhlAn was visualized as a cladogram using the ggtree v 3.

Samples were then submitted to the UT Proteomics core for protein identification analysis. Results show total spectral counts for proteins found specifically in the SAP containing sample only with exception of streptavidin.

After the media was removed and serum-free, DMEM with 0. The media was removed, and the MTT crystals were dissolved with MTT solvent isopropanol, 0. Live G. mellonella larvae were purchased from DBDPet.

coli cells grown to an OD of 0. Injections were performed using a Hamilton µl pipet equipped with an autodispenser and gauge BD needle. Larvae were determined to be dead if they were unable to right themselves after being placed on their back. Also, thanks are due to Nancy Moran's lab for the use of their microscope and Despoina Mavridou's lab for sharing mcr-1 and GFP-expressing plasmids.

Supplementary material is available at PNAS Nexus online. This work was supported by the National Institutes of Health grants AI, AI, and AI; the Welch Foundation grant F; the Defense Threat Reduction Agency grant HDTRAC; and Tito's Handmade Vodka.

Conceptualization: J. and B. Methodology: J. Investigation: J. Visualization: J. and A. Funding acquisition: B. Project administration: J.

Supervision: J. Writing—original draft: J. Writing—review and editing: J. Raw sequencing data from the SLAY experiment are available in the SRA database accession number PRJNA All other data are available in the main text or the supplementary materials.

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Andolina G , et al. A peptidomimetic antibiotic interacts with the periplasmic domain of LptD from Pseudomonas aeruginosa. ACS Chem Biol. Kaur H , et al.

The antibiotic darobactin mimics a β-strand to inhibit outer membrane insertase. Luther A , Bisang C , Obrecht D. Advances in macrocyclic peptide-based antibiotics. Bioorg Med Chem. Brown JM , Dorman DC , Roy LP. Acute renal failure due to overdosage of colistin. Med J Aust.

Sabnis A , et al. Colistin kills bacteria by targeting lipopolysaccharide in the cytoplasmic membrane. Elife 10 : e Panteleev PV , Bolosov IA , Balandin SV , Ovchinnikova TV.

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Firstly, by binding iron, it limits the amount of free iron an essential growth factor for microorganisms available. For example, in one study Streptococcus mutans and Vibrio cholerae were killed by incubation with purified human apolactoferrin, the iron-depleted form of the protein.

Concentrations of lactoferrin below that necessary for total inhibition resulted in a marked reduction in viable colony-forming units. This bactericidal effect was contingent upon the metal-chelating properties of the lactoferrin molecule [ 13 ].

Lactoferrin contains two high-affinity ferric iron binding sites facilitating such iron sequestration from pathogenic microbes. Secondly, lactoferrin can destabilise the outer membrane of gram-negative bacteria by binding to it resulting in altered permeability that leads to microbial injury and death.

This activity has been attributed to the 17 amino acid N-terminal portion of lactoferrin. This portion is rich in arginine residues that give the molecule its highly cationic nature and has been termed lactoferricin. The related iron-binding protein transferrin lacks these arginine residues and is less cationic pI of 5—5.

Lactoferrin has been shown to kill clinical strains of E. coli, S. aureus and mucoid P. aeruginosa isolated from CF airways [ 15 ]. Lactoferrin acts synergistically with other innate immunity proteins such as lysozyme and SLPI in bacterial killing [ 16 , 17 ].

It has also been shown that lactoferrin enhances the antimicrobial effects of some antibiotics [ 18 ]. Lactoferrin reduces the minimum inhibitory concentration MIC and the minimum bactericidal concentration MBC of doxycycline for Burkholderia cepacia and P.

aeruginosa strains with MICs for B. cepacia falling from highly resistant to clinically achievable levels [ 19 ]. By binding iron, lactoferrin can also act as an anti-oxidant since iron bound to the protein is unable to participate as a catalyst for the generation of free hydroxyl radicals via the Haber-Weiss reaction [ 20 — 22 ].

Lactoferrin has recently been shown to inhibit Pseudomonas biofilm formation [ 12 ]. The inhibition of biofilm formation is a property unique to lactoferrin and thus it plays a pivotal role in host defense against this highly destructive mode of bacterial growth.

Biofilm bacteria are notoriously resistant to host killing and antibiotics. They may be up to one thousand times more resistant to antibiotics than their free-swimming, planktonic counterparts [ 23 , 24 ]. In the presence of lactoferrin, free iron levels decreased inducing a twitching motion in bacteria.

This twitching ensured that the bacteria wandered across a surface rather than stopping to form microcolonies, clusters and subsequent pillar and mushroom-shaped mature biofilms. Furthermore, Singh et al demonstrated increased susceptibility of biofilm bacteria to tobramycin in the presence of lactoferrin.

We have shown previously that lactoferrin levels are depleted in BAL from Cystic Fibrosis patients with active Pseudomonas infections compared to those with no active Pseudomonas infection and that this depletion results in impaired ability to prevent Pseudomonas biofilm formation [ 25 ].

Lactoferrin also has been shown to be active against a number of viruses including human immunodeficiency virus HIV and cytomegalovirus CMV [ 26 , 27 ]. Lactoferrin is known to prevent replication of hepatitis B and C viruses and is being investigated clinically together with interferon as a potential therapeutic agent in these conditions [ 28 , 29 ].

A recent study elucidated the potential of oral administration of lactoferrin to attenuate pneumonia in influenza-virus-infected mice through the suppression of infiltration of inflammatory cells in the lung [ 30 ]. In addition to antiviral properties, lactoferrin also possesses anti-fungal properties: it has been shown to be active against Candida species and is being investigated as a potential treatment for oral candidiasis [ 31 ].

Lactoferrin is also a key anti-inflammatory protein. It possesses two basic cradles at residues 1 to 5 and 28 to 34 of the N-terminal end that can bind anionic molecules such as lipopolysaccharide LPS , heparin and heparin sulfates.

Lactoferrin has been shown to inhibit the LPS-induced expression and proteoglycan binding ability of Interleukin-8 in human endothelial cells [ 32 ]. Lactoferrin protects against sublethal doses of LPS in mice and germfree piglets.

Animals that received pre-treatment with lactoferrin showed minimal effects when given an intra-peritoneal injection of LPS whereas the control group of animals that did not receive pre-treatment with lactoferrin succumbed rapidly to septic shock [ 33 ].

Lactoferrin has also been shown to lower the expression of adhesion molecules E-selectin and ICAM-1 on endothelial cells further modifying the immune response [ 34 ]. Another immunomodulatory function of lactoferrin stems from its ability to bind unmethylated CpG motifs, which are bacterial DNA products capable of stimulating various innate and acquired immune responses in human and murine models [ 20 ].

A schematic of lactoferrin's many antimicrobial and immunomodulatory roles is shown in figure 1. Multifunctional properties of lactoferrin. Lactoferrin is released from neutrophils and respiratory tract epithelium and has multiple activities including anti-inflammatory, anti-viral, anti-lipopolysaccharide, anti-biofilm, antibacterial and anti-fungal properties.

SLPI is a SLPI is the third most abundant innate immunity protein of respiratory secretions after lysozyme and lactoferrin. It is estimated that SLPI is present at concentrations of 0. SLPI through its C terminal domain is a serine protease inhibitor and provides significant protection against neutrophil elastase, a powerful elastolytic enzyme released from neutrophils during degranulation at areas of infection and inflammation.

SLPI also inhibits the serine protease cathepsin G as part of its anti-protease effects in the lung [ 41 ]. In addition to its potent anti-protease activity, SLPI has important anti-bacterial, anti-viral and anti-inflammatory properties.

The N-terminal domain of SLPI has modest antimicrobial activity in vitro against Gram-negative and Gram-positive bacteria [ 42 ]. SLPI has been shown to inhibit human immunodeficiency virus HIV infectivity of monocytes by blocking viral DNA synthesis.

Saliva is rich in SLPI and it is felt that this accounts for the low viral transmission rates via saliva [ 43 , 44 ]. Prior administration of SLPI to rats attenuated pulmonary recruitment of neutrophils in an immunoglobulin G IgG immune complex model of acute lung injury [ 45 ].

SLPI can inhibit LPS-induced NF-κB activation by inhibiting degradation of IRAK, IκBα and IκBβ [ 47 ] and can also impair lipoteichoic acid LTA and LPS induced pro-inflammatory gene expression in monocytes and macrophages in vitro [ 36 , 48 ]. In addition to its antiprotease activity, SLPI has been shown to exhibit anti-inflammatory properties, including down-regulation of tumor necrosis factor alpha expression by lipopolysaccharide LPS in macrophages and inhibition of nuclear factor NF -kappaB activation in a rat model of acute lung injury.

SLPI has recently been shown to enter cells, becoming rapidly localized to the cytoplasm and nucleus where it affects NF-kappaB activation by binding directly to NF-kappaB binding sites in a site-specific manner [ 47 ]. However once oxidised, the anti-inflammatory and anti-elastase effects of SLPI are diminished.

Greene et al. demonstrated cleavage of SLPI in infected lobes in community acquire pneumonia resulting in impaired anti-NE activity. SLPI was inactivated by cleavage, oxidation and complex formation paving the way for free neutrophil elastase to exacerbate pulmonary parenchymal inflammation and tissue damage [ 49 ].

SLPI is susceptible to protease cleavage. Cathepsins B, L, and S have been shown to cleave and inactivate SLPI. Analysis of epithelial lining fluid samples from individuals with emphysema indicated the presence of active cathepsin L and cleaved SLPI [ 50 ].

Serine and cysteine proteases produced by the house dust mite in asthma have been shown to cleave SLPI and may increase the susceptibility of patients with allergic inflammation to infection [ 36 ].

Lysozyme is a basic protein with a pI of It is stored in both primary and secondary neutrophil granules. In addition to enzymatic lysis of bacterial cell walls, lysozyme can also kill bacteria by a non-enzymatic mechanism [ 52 ]. Lysozyme is highly active against many Gram-positive species, including Bacillus megaterium , Micrococcus luteus , and many streptococci.

Lysozyme also has an important role against Gram-negative organisms. Its ability to kill Gram-negative organisms may be influenced by ionic concentration, osmolarity, and the presence of synergistic cofactors [ 2 , 15 , 17 ]. As lactoferrin and lysozyme are present together in high levels in mucosal secretions and neutrophil granules, it is probable that their interaction contributes to host defense [ 17 ].

Lactoferrin in concert with other cofactors presumably disrupt the outer membrane of Gram-negative bacteria and allow lysozyme access to the sensitive peptidoglycan layer. Lysozyme is a component of both phagocytic and secretory granules of neutrophils and is also produced by monocytes, macrophages, and epithelial cells.

Both lysozyme and lactoferrin arise in the lower respiratory tract within the airways and their levels are elevated in association with chronic bronchitis suggesting that lactoferrin and lysozyme may contribute to the modulation of airway inflammation in chronic bronchitis.

Lysozyme is about tenfold more abundant in the initial "airway" aliquot than in subsequent aliquots of bronchoalveolar lavage [ 53 ], and its concentration correlates poorly with neutrophil concentrations, suggesting that, in general, airway epithelium and its glands are the major sources of lysozyme in airway secretions.

Lysozyme is present at concentrations of between 0. In one study to assess the role of lysozyme in pulmonary host defense in vivo , transgenic mice expressing rat lysozyme cDNA in distal respiratory epithelial cells were generated.

Two transgenic mouse lines were established in which the level of lysozyme protein in bronchoalveolar lavage BAL fluid was increased 2- or 4-fold relative to that in wild type WT mice. Lysozyme activity in BAL was significantly increased 6. Killing of group B streptococc i was significantly enhanced 2- and 3-fold in the mouse transgenic lines at 6 h following infection and was accompanied by a decrease in systemic dissemination of pathogen.

Killing of Pseudomonas aeruginosa was also enhanced in the transgenic lines 5- and fold. The authors concluded that increased production of lysozyme in respiratory epithelial cells of transgenic mice enhanced bacterial killing in the lung in vivo , and was associated with decreased systemic dissemination of pathogen and increased survival following infection [ 54 ].

A recent in vivo study of lysozyme derived from submucosal glands in ferret trachea demonstrated that lysozyme-depleted secretions were much less effective at inhibiting bacterial growth than mock-depleted samples, suggesting that lysozyme is partially responsible for the antibacterial activity of the glandular airway secretions.

Defensins are 3- to 5-kDa peptide members of a widely distributed family with characteristic three-dimensional folding with six cysteine-three disulfide patterns. Defensins have broad cytotoxic activity against bacteria, fungi, parasites, viruses and even host cells [ 56 ].

Defensins are subdivided into different classes that include alpha and beta defensins. Alpha defensins are also known as human neutrophil peptides HNPs. HNP-5 and -6 have been identified in Paneth cells in the crypts of the small intestinal mucosa and also in the female reproductive tract.

The alpha defensins have a wide variety of actions including mitogenic and chemotactic activities [ 58 , 59 ]. Alpha defensins contribute to epithelial repair in the lung by enhancing epithelial cell proliferation [ 60 ].

The more recently identified human beta defensins HBDs 1—4 differ slightly from classical alpha defensins in the spacing and connectivity of their cysteines [ 2 ].

Beta defensins have five to eight positively charged residues resulting in quite similar calculated isoelectric points of 8.

HBD-2 and -3 are secreted in response to LPS and cytokines TNFα, interleukin-1 beta and are active against Gram-positive HBD-3 and Gram-negative HBD-1, -2 and -3 bacteria whilst HBD-1 is upregulated by interferon-gamma IFN-γ [ 62 — 64 ].

We have previously demonstrated that human lung epithelial cells express Toll-like receptor four TLR4 on their surface and that stimulation of these cells with Pseudomonas LPS results in increased HBD2 gene and protein expression [ 65 ]. All three beta defensins have been identified in lung and they have been shown to act synergistically with other innate immunity proteins in bacterial killing [ 15 ].

Individual beta defensins have differential antimicrobial activity. Staphylococcus aureus is resistant to killing by HBD-1 and HBD-2 but even strains of this organism that are multi-drug resistant are susceptible and sensitive to killing by HBD-3 [ 66 ].

We have shown previously that HBD 2 and 3 are susceptible to proteolytic cleavage by cysteinyl cathepsins that are present at elevated concentrations in the Cystic Fibrosis and COPD airways [ 49 ]. New families of defensins and individual defensins are being discovered.

A novel family of antimicrobial peptides termed "theta defensins" has been described in monocytes and macrophages of macaque monkeys. Theta defensins are naturally produced by a unique ligation of two truncated alpha defensins [ 67 ]. Whilst they are important in their own right, β-defensins are present at much lower concentrations than lysozyme, SLPI or lactoferrin and thus their overall contribution to host defense must be taken in context [ 15 ].

Cathelicidins are a family of antimicrobial proteins found in neutrophil specific granules of which LL is the only human member identified to date [ 9 , 68 , 69 ]. LL is also present in certain lymphocytes, testicular tissue and airway epithelium [ 9 , 68 ].

Cathelicidins are stored as inactive pro-peptides precursors and require processing to become active peptides [ 70 ].

LL is activated when proteinase 3 cleaves its precursor, hCAP Some cathelicidin genes possess upstream DNA motifs eg. NF-κB predicted to convey inducibility during acute phase responses [ 71 ]. LL has been shown to reduce bacterial load by Pseudomonas when over-expressed in murine models [ 72 ].

Furthermore it conveys improved survival following administration of lethal doses of LPS [ 72 ]. LL also reduced production of the pro-inflammatory cytokine TNF-α from macrophages stimulated with LPS and may be responsible for the migration of immune cells to areas of inflammation and infection [ 73 ].

LL activity may be impaired in Cystic Fibrosis where anionic filaments of F-actin and DNA bind to it and inhibit its bactericidal action. Addition of the actin filament-fragmenting protein gelsolin frees LL from this binding and restores antimicrobial activity [ 72 ].

BPI is a kDa protein that is predominantly active against Gram-negative bacteria [ 2 , 74 ]. It is stored in neutrophil primary granules and exerts its effects through three distinct mechanisms: firstly, it is directly cytotoxic via its effects on bacterial membranes; secondly, it acts as an opsonin to enhance neutrophil phagocytosis and thirdly, it can neutralise bacterial LPS [ 75 ].

In common with many innate immunity proteins, BPI possesses a highly cationic N-terminal end which contains its bactericidal and endotoxin neutralising zones [ 75 , 76 ].

As with many of these defensive proteins, BPI acts synergistically with other members of the innate immune system such as cathelicidins and defensins in bacterial killing. It also acts in concert with the complement system [ 76 ]. BPI is thought to have a role in down-regulating the pro-inflammatory effects of gram-negative bacteria and endotoxins in vivo [ 77 ].

Pulmonary surfactant is a lipoprotein complex that is synthesised by type II pneumocytes and by airway Clara cells. It is secreted into the epithelial lining fluid where it modulates surface tension.

More recently surfactant has been shown to play a role in host defense against infection and inflammation. Surfactant proteins belong to the collagen-like-lectin or collectin family that also includes mannose binding protein bovine coglutinin and CL [ 78 , 79 ].

Collectins share an N-terminal collagen-like domain and a C-terminal lectin or Carbohydrate recognition domain CRD domain capable of binding carbohydrates in a calcium-dependent manner. These C-type lectin domains can bind oligosaccharides found on bacterial, non-encapsulated fungal as well as viral envelope surfaces [ 79 ].

Surfactant consists of the four surfactant proteins 1—4 bound to phospholipids and is responsible for reducing surface tension at the air liquid interface within the alveoli in lung. Surfactant proteins are key opsonins facilitating phagocytosis of bacteria and viruses by macrophages and monocytes [ 80 ] Both SP-A and SP-D have been shown to be directly bactericidal against E.

coli , while SP-A and SP-D are fungicidal against Histoplasma Capsulatum. SP-A and D deficient mice were unable to kill this fungus [ 81 ].

Both SP-A and SP-D have the capacity to modulate multiple leucocyte functions [ 78 , 82 ]. The addition of SP-A to cultures of Mycoplasma. pneumoniae markedly attenuated the growth of the organism assessed by colony formation, metabolic activity, and DNA replication.

The bacteriostatic effects of SP-A were reversed by dipalmitoylphosphatidylglycerol. These findings demonstrate that human SP-A can play a direct role in antibody-independent immunity to M. pneumoniae by interacting with lipid ligands expressed on the surface of the organism and implicate SP-A in the immediate host response to the bacteria [ 83 ].

Recent research has focused on the proteolytic cleavage of SP-A and D by various proteases in the lung. Proteolytic damage to surfactant protein by neutrophil elastase and cathepsin G was demonstrated in bronchoalveolar lavage fluid of cystic fibrosis patients [ 84 ].

The bacterial protease, Pseudomonas aeruginosa elastase was shown to degrade SP-A and SP-D [ 85 ]. Furthermore, cleavage of SP-D by this enzyme results in failure of the surfactant to bind or aggregate bacteria that are aggregated by intact SP-D.

Thus, cleavage eliminates many of SP-D's normal immune functions [ 86 ]. Early animal studies showed airway mucosa secretes an enzyme known as peroxidase that was active in preventing infection of the airway[ 87 ].

Gerson et al subsequently demonstrated production of the biocidal compound hypothiocyanite in vitro by airway lactoperoxidase LPO. They also showed in vivo inhibition of airway LPO in sheep leads to a significant decrease in bacterial clearance from the airways.

Their data suggest that the LPO system is a major contributor to airway defenses [ 88 ]. The airway LPO system may provide additional protection against viral [ 89 — 91 ] and fungal infections [ 92 , 93 ].

LPO was subsequently demonstrated in human airways airway secretions with activity against Pseudomonas aeruginosa, Burkholderia cepacia and Haemophilus influenzae [ 94 ]. Chemokine ligand 20 CCL20 shares similar structural and functional properties with human beta-defensins HBDs Airway epithelial cells have been shown to express CCL20 [ 95 ].

The inflammatory cytokines interleukin IL -1β and tumor necrosis factor-α TNF-α upregulate expression of this protein via the nuclear factor NF -κB pathway [ 96 , 97 ]. CCL20 is also produced by neutrophils [ 98 ]. It has been shown to exert antimicrobial activity against a wide spectrum of mainly Gram-negative bacteria [ 99 ].

Starner et al demonstrated that CF BAL contains elevated concentrations of CCL20 compared to normal BAL and that CCL20 exhibited salt-sensitive bactericidal activity [ 95 ]. The use of antimicrobial peptides and proteins as potential therapeutic targets is an attractive concept. Theoretically, such compounds would have low immunogenicity and high bioavailability with minimal toxicity [ 2 ].

The costs involved in developing, producing and administering such compounds should be outweighed by the enormous potential benefits in an era where antibiotic resistant has reached crisis proportions.

Gene therapy as a means of augmenting levels of active antimicrobial proteins cathelicidins was previously investigated [ 72 ] although there has been little progression in developing this early work. Alternatively, innate immunity proteins such as lactoferrin could potentially be aerosolized directly into the lungs in CF as is currently being evaluated with protegrin, a porcine-derived cathelicidin [ 3 ].

Recombinant human lactoferrin rHLF is relatively cheap to manufacture and of low antigenic potential. Inhaled rHLF has been shown to reduce the late phase response to antigenic stimuli in sheep [ ].

It has been shown to be safe in a phase II clinical trial based on its immunomodulatory effects in asthma in humans [ ]. Another strategy could involve protecting native antimicrobials from proteolytic degradation by proteases.

Surfactant proteins are susceptible to protease cleavage by bacteria-derived proteases [ 86 ]. We have shown previously that lactoferrin [ 25 ], SLPI [ 50 ] and human β-Defensins [ 49 ] are all rapidly degraded by cysteinyl cathepsins that are over-expressed in chronic lung diseaes such as Cystic Fibrosis and COPD.

Cleavage of innate immunity proteins results in loss of their antimicrobial effects. Cystatins are naturally occurring cathepsin inhibitors that appear to be overwhelmed by the sheer cathepsin burden in these conditions.

Design of a selective pharmaceutical cathepsin inhibitor based on cystatins or small synthetic cathepsin inhibitors could minimize cleavage of these innate immunity proteins, thereby optimizing the pulmonary antimicrobial screen. Perhaps the ideal combination would be a nebulised innate immunity protein eg lactoferrin to augment depleted natural levels coupled with a synthetic cathepsin inhibitor to prevent proteolytic degradation.

Implementation of such a regime early in CF before biofilms have taken hold could potentially reduce morbidity and mortality from P.

aeruginosa infections in CF significantly. Strategies to inhibit innate immunity protein cleavage by other proteases such as NE and bacterial proteases may also have therapeutic potential. The emergence of widespread resistance to many conventional antibiotics and the selecting out of multi-drug resistant "super-bugs" should prompt further investigation into potential roles for innate immunity proteins in the clinical arena.

Synergistic combinations of innate immunity proteins with existing antibacterial and antifungal agents should continue to be evaluated [ ]. Microbes are capable of rapid adaptation to changing environmental conditions to maximise survival and increase pathogenicity.

Species diversity and genetic heterogeneity lead to multiple virulence factors among microorganisms in the respiratory tract. In order to combat this threat, the lung is endowed with incredibly powerful and potent antimicrobial and anti-inflammatory proteins that also have rolls in epithelial repair.

Despite recent advances in our knowledge of the complex roles and functions of the various innate immunity proteins in pulmonary infection and inflammation, further research is needed to characterize and elucidate specific biological functions and pathways of individual proteins.

Strategies to augment innate immunity proteins or to prevent their degradation may provide future therapeutic options. As our understanding of this key area grows, we must learn to harness these "natural born killers" and derive maximum clinical benefit from them.

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