Glycine fermentation by C. difficile promotes virulence and spore formation, and is induced by host cathelicidin.

Clostridioides difficile is a leading cause of antibiotic-associated diarrheal disease. C. difficile colonization, growth, and toxin production in the intestine is strongly associated with its ability to use amino acids to generate energy, but little is known about the impact of specific amino acids on C. difficile pathogenesis. The amino acid glycine is enriched in the dysbiotic gut and is suspected to contribute to C. difficile infection. We hypothesized that the use of glycine as an energy source contributes to colonization of the intestine and pathogenesis of C. difficile. To test this hypothesis, we deleted the glycine reductase (GR) genes grdAB, rendering C. difficile unable to ferment glycine, and investigated the impact on growth and pathogenesis. Our data show that the grd pathway promotes growth, toxin production, and sporulation. Glycine fermentation also had a significant impact on toxin production and pathogenesis of C. difficile in the hamster model of disease. Furthermore, we determined that the grd locus is regulated by host cathelicidin (LL-37) and the cathelicidin-responsive regulator, ClnR, indicating that the host peptide signals to control glycine catabolism. The induction of glycine fermentation by LL-37 demonstrates a direct link between the host immune response and the bacterial reactions of toxin production and spore formation.

CrrAB regulates PagP-mediated glycerophosphoglycerol palmitoylation in the outer membrane of Klebsiella pneumoniae.

The outer membrane (OM) of Gram-negative bacteria is an evolving antibiotic barrier composed of a glycerophospholipid (GP) inner leaflet and a lipopolysaccharide (LPS) outer leaflet. The two-component regulatory system CrrAB has only recently been reported to confer high-level polymyxin resistance and virulence in Klebsiella pneumoniae. Mutations in crrB have been shown to lead to the modification of the lipid A moiety of LPS through CrrAB activation. However, functions of CrrAB activation in the regulation of other lipids are unclear. Work here demonstrates that CrrAB activation not only stimulates LPS modification but also regulates synthesis of acyl-glycerophosphoglycerols (acyl-PGs), a lipid species with undefined functions and biosynthesis. Among all possible modulators of acyl-PG identified from proteomic data, we found expression of lipid A palmitoyltransferase (PagP) was significantly upregulated in the crrB mutant. Furthermore, comparative lipidomics showed that most of the increasing acyl-PG activated by CrrAB was decreased after pagP knockout with CRISPR-Cas9. These results suggest that PagP also transfers a palmitate chain from GPs to PGs, generating acyl-PGs. Further investigation revealed that PagP mainly regulates the GP contents within the OM, leading to an increased ratio of acyl-PG to PG species and improving OM hydrophobicity, which may contribute to resistance against certain cationic antimicrobial peptides resistance upon LPS modification. Taken together, this work suggests that CrrAB regulates the palmitoylation of PGs and lipid A within the OM through upregulated PagP, which functions together to form an outer membrane barrier critical for bacterial survival.

Effects of Medicinal Leech-Related Cationic Antimicrobial Peptides on Human Blood Cells and Plasma.

Cationic antimicrobial peptides (CAMPs) are considered as next-generation antibiotics with a lower probability of developing bacterial resistance. In view of potential clinical use, studies on CAMP biocompatibility are important. This work aimed to evaluate the behavior of synthetic short CAMPs (designed using bioinformatic analysis of the medicinal leech genome and microbiome) in direct contact with blood cells and plasma. Eight CAMPs were included in the study. Hemolysis and lactate dehydrogenase assays showed that the potency to disrupt erythrocyte, neutrophil and mononuclear cell membranes descended in the order pept_1 > pept_3 ~ pept_5 > pept_2 ~ pept_4. Pept_3 caused both cell lysis and aggregation. Blood plasma and albumin inhibited the CAMP-induced hemolysis. The chemiluminescence method allowed the detection of pept_3-mediated neutrophil activation. In plasma coagulation assays, pept_3 prolonged the activated partial thromboplastin time (APTT) and prothrombin time (at 50 µM by 75% and 320%, respectively). Pept_3 was also capable of causing fibrinogen aggregation. Pept_6 prolonged APTT (at 50 µM by 115%). Pept_2 was found to combine higher bactericidal activity with lower effects on cells and coagulation. Our data emphasize the necessity of investigating CAMP interaction with plasma.

GraS Sensory Activity in Staphylococcus epidermidis Is Modulated by the "Guard Loop" of VraG and the ATPase Activity of VraF.

Antimicrobial peptides (AMPs) are one of the key immune responses that can eliminate pathogenic bacteria through membrane perturbation. As a successful skin commensal, Staphylococcus epidermidis can sense and respond to AMPs through the GraXRS two-component system and an efflux system comprising the VraG permease and VraF ATPase. GraS is a membrane sensor known to function in AMP resistance through a negatively charged, 9-residue extracellular loop, which is predicted to be linear without any secondary structure. An important question is how GraS can impart effective sensing of AMPs through such a small unstructured sequence. In this study, we verified the role of graS and vraG in AMP sensing in S. epidermidis, as demonstrated by the failure of the ΔgraS or ΔvraG mutants to sense. Deletion of the extracellular loop of VraG did not affect sensing but reduced survival with polymyxin B. Importantly, a specific region within the extracellular loop, termed the guard loop (GL), has inhibitory activity since sensing of polymyxin B was enhanced in the ΔGL mutant, indicating that the GL may act as a gatekeeper for sensing. Bacterial two-hybrid analysis demonstrated that the extracellular regions of GraS and VraG interact, but interaction appears dispensable to sensing activity. Mutation of the extracellular loop of VraG, the GL, and the active site of VraF suggested that an active detoxification function of VraG is necessary for AMP resistance. Altogether, we provide evidence for a unique sensory scheme that relies on the function of a permease to impart effective information processing. IMPORTANCE Staphylococcus epidermidis has become an important opportunistic pathogen that is responsible for nosocomial and device-related infections that account for considerable morbidity worldwide. A thorough understanding of the mechanisms that enable S. epidermidis to colonize human skin successfully is essential for the development of alternative treatment strategies and prophylaxis. Here, we demonstrate the importance of an AMP response system in a clinically relevant S. epidermidis strain. Furthermore, we provide evidence for a unique sensory scheme that would rely on the detoxification function of a permease to effect information processing.

Lactoferrin receptors in Gram-negative bacteria: an evolutionary perspective.

In this short review, we outline the major events that led to the development of iron acquisition systems in Gram-negative bacteria and mammals since the beginning of life on earth. Naturally, the interaction between these organisms led to the development of a wonderfully complex set of protein systems used for competition over a once prevalent (but no longer) biocatalytic cofactor. These events led to the appearance of the lactoferrin gene, which has since been exploited into adopting countless new functions, including the provision of highly bactericidal degradation products. In parallel to lactoferrin's evolution, evolving bacterial receptors have countered the bactericidal properties of this innate immunity protein.

Targeting antibiotic tolerance in anaerobic biofilms associated with oral diseases: Human antimicrobial peptides LL-37 and lactoferricin enhance the antibiotic efficacy of amoxicillin, clindamycin and metronidazole.

Antimicrobial peptides are receiving increasing attention as potential therapeutic agents for treating biofilm-related infections of the oral cavity. Many bacteria residing in biofilms exhibit an enhanced antibiotic tolerance, which grants intrinsically susceptible microorganisms to survive lethal concentrations of antibiotics. In this study, we examined the effects of two endogenous human antimicrobial peptides, LL-37 and human Lactoferricin, on the antibiotic drug efficacy of amoxicillin, clindamycin and metronidazole in two types of polymicrobial biofilms, which aimed to represent frequent oral diseases: (1) facultative anaerobic (Streptococcus mutans, Streptococcus sanguinis, Actinomyces naeslundii) and (2) obligate anaerobic biofilms (Veillonella parvula, Parvimonas micra, Fusobacterium nucleatum). LL-37 and Lactoferricin enhanced the anti-biofilm effect of amoxicillin and clindamycin in facultative anaerobic biofilms. Metronidazole alone was ineffective against facultative anaerobic biofilms, but the presence of LL-37 and Lactoferricin led to a greater biofilm reduction. Obligate anaerobic biofilms showed an increased drug tolerance to amoxicillin and clindamycin, presumably due to metabolic downshifts of the bacteria residing within the biofilm. However, when combined with LL-37 or Lactoferricin, the reduction of obligate anaerobic biofilms was markedly enhanced for all antibiotics, even for amoxicillin and clindamycin. Furthermore, our results suggest that antimicrobial peptides enhance the dispersion of matured biofilms, which may be one of their mechanisms for targeting biofilms. In summary, our study proves that antimicrobial peptides can serve as an auxiliary treatment strategy for combatting enhanced antibiotic tolerance in bacterial biofilms.

Molecular basis of bile-salt- and iron-induced enterohaemorrhagic E. coli resistance to cationic antimicrobial peptides.

Colonization of the gastrointestinal tract by enterohaemorrhagic Escherichia coli (EHEC) is critically dependent on its ability to sense and respond to various microenvironments within the host. EHEC exposure to physiologically relevant levels of bile salts upregulates the two-component system, pmrAB, and the arnBCADTEF operon, resulting in lipopolysaccharide modification and increased resistance to the cationic antimicrobial peptide, polymyxin B (PMB). A similar pmrAB- and arn-dependent PMB resistance has been observed in Salmonella enterica in the presence of ferric iron. Limiting magnesium levels and mild acid can also induce Salmonella resistance to PMB through another two-component system, PhoPQ and the connector protein, PmrD. This study aims to evaluate the relative contributions of a bile-salt mix (BSM), iron, limiting magnesium as well as the roles of pmrAB, phoPQ and pmrD to EHEC's resistance to PMB. Killing assays show that EHEC treatment with the BSM or iron under excess magnesium and neutral pH conditions induces a pmrAB-dependent, phoP-independent PMB resistance. By contrast, exposure to limiting magnesium triggers a pmrB-, phoP- and pmrD-dependent PMB resistance. The iron-induced PMB resistance is independent of phoP and pmrD under limiting magnesium conditions while the bile-salt-induced PMB resistance is independent of pmrD only under non-PhoP-inducing conditions. GFP-pmrD transcriptional reporter studies reveal that the limiting magnesium enhances pmrD expression, which is repressed upon additional exposure to either BSM or iron. Our results also show that exposure to mild acid enhances PMB resistance in a pmrD-independent manner and GFP reporter results confirm minimal expression of pmrD at this pH regardless of the magnesium level. This study provides novel insights into how EHEC differentially employs PmrAB, PhoPQ and PmrD to monitor and respond to bile salts, iron, acidic pH and magnesium typically encountered within the gastrointestinal tract in order to modulate its survival against cationic antimicrobial peptides.

Synergistic Biophysical Techniques Reveal Structural Mechanisms of Engineered Cationic Antimicrobial Peptides in Lipid Model Membranes.

In the quest for new antibiotics, two novel engineered cationic antimicrobial peptides (eCAPs) have been rationally designed. WLBU2 and D8 (all 8 valines are the d-enantiomer) efficiently kill both Gram-negative and -positive bacteria, but WLBU2 is toxic and D8 nontoxic to eukaryotic cells. We explore protein secondary structure, location of peptides in six lipid model membranes, changes in membrane structure and pore evidence. We suggest that protein secondary structure is not a critical determinant of bactericidal activity, but that membrane thinning and dual location of WLBU2 and D8 in the membrane headgroup and hydrocarbon region may be important. While neither peptide thins the Gram-negative lipopolysaccharide outer membrane model, both locate deep into its hydrocarbon region where they are primed for self-promoted uptake into the periplasm. The partially α-helical secondary structure of WLBU2 in a red blood cell (RBC) membrane model containing 50 % cholesterol, could play a role in destabilizing this RBC membrane model causing pore formation that is not observed with the D8 random coil, which correlates with RBC hemolysis caused by WLBU2 but not by D8.

[Preliminary study of antimicrobial peptide cathelicidin-PY therapy in mice with acute liver failure].

Objective: To investigate the feasibility of cationic antimicrobial peptide cathelicidin-PY(PY) therapy through a mouse model of acute liver failure. Methods: The ability of different concentrations of antimicrobial peptide PY to neutralize endotoxin / lipopolysaccharide (LPS) in vitro was detected by Limulus Amebocyte Lysate (LAL) assay. Cell counting kit-8 (CCK-8) was used to detect the toxic effect of different concentrations of antimicrobial peptide PY on mouse monocyte macrophages (RAW264.7). An in vitro hemolysis experiment was used to evaluate the activity of antimicrobial peptide PY on healthy human erythrocytes. D-galactosamine combined with LPS- induced mouse model of acute liver failure was constructed. The antimicrobial peptide PY effect on survival rate of mouse model was observed. HE staining was used to observe the pathological changes of liver tissue. Immunohistochemistry and Western blotting were used to detect the expression of apoptosis-associated protein caspase-3. Intra-group comparisons were performed using t-test and analysis of variance. χ (2) test was used for the comparison of rates. Results: An in vitro experiment showed that the endotoxin neutralization rate was higher at very low dose (0.01 µmol/L), and exceeded 70% at medium-dose (10-40 µmol/L), and the difference between groups with different concentration was statistically significant (F = 569.22, P < 0.05). Medium-dose antimicrobial peptide PY had strong endotoxin neutralizing effect, low cytotoxicity and hemolytic activity. Moreover, in vivo experiments showed that the degree of liver injury and survival rate of mouse model was significantly improved with the medium-dose of antimicrobial peptide PY. Immunohistochemistry results showed that the expression of caspase-3 in the liver tissue was significantly depleted in the medium-dose group than that of the liver failure group, and the results were consistent with protein immunoblotting testing. Conclusion: Antimicrobial peptide PY possesses a strong ability to neutralize endotoxin and few toxic side effects. A specific dose of antimicrobial peptide PY can attenuate hepatocyte apoptosis and significantly improve the survival rate of animal model, and thus provides a new idea for the liver failure treatment.

Antimicrobial efficacy and toxicity of novel CAMPs against P. aeruginosa infection in a murine skin wound infection model.

BACKGROUND: Treatment of P. aeruginosa wound infection is challenging due to its inherent and acquired resistance to many conventional antibiotics. Cationic antimicrobial peptides (CAMPs) with distinct modes of antimicrobial action have been considered as the next-generation therapeutic agents. In the present study, a murine skin surgical wound infection model was used to evaluate the in vivo toxicity and efficacy of two newly designed antimicrobial peptides (CAMP-A and CAMP-B), as chemotherapeutic agents to combat P. aeruginosa infection. RESULTS: In the first trial, topical application of CAMPs on the wounds at a dose equivalent to 4 × MIC for 7 consecutive days did not cause any significant changes in the physical activities, hematologic and plasma biochemical parameters, or histology of systemic organs of the treated mice. Daily treatment of infected wounds with CAMP-A and CAMP-B for 5 days at a dose equivalent to 2× MIC resulted in a significant reduction in wound bacterial burden (CAMP-A: 4.3 log10CFU/g of tissue and CAMP-B: 5.8 log10CFU/g of tissue), compared to that of the mock-treated group (8.1 log10CFU/g of tissue). Treatment with CAMPs significantly promoted wound closure and induced epidermal cell proliferation. Topical application of CAMP-A on wounds completely prevented systemic dissemination of P. aeruginosa while CAMP-B blocked systemic infection in 67% of mice and delayed the onset of systemic infection by at least 2 days in the rest of the mice (33%). In a second trial, daily application of CAMP-A at higher doses (5× MIC and 50× MIC) didn't show any significant toxic effect on mice and the treatments with CAMP-A further reduced wound bacterial burden (5× MIC: 4.5 log10CFU/g of tissue and 50× MIC: 3.8 log10CFU/g of tissue). CONCLUSIONS: The data collectively indicated that CAMPs significantly reduced wound bacterial load, promoted wound healing, and prevented hepatic dissemination. CAMP-A is a promising alternative to commonly used antibiotics to treat P. aeruginosa skin infection.

Cationic antimicrobial peptides: alternatives and/or adjuvants to antibiotics active against methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa.

BACKGROUND: Methicillin-resistant Staphylococcus aureus and multidrug-resistant Pseudomonas aeruginosa are becoming difficult to treat with antibiotics whereas Cationic Antimicrobial Peptides (CAMPs) represent promising alternatives. The effects of four CAMPs (LL-37: human cathelicidin, CAMA: cecropin(1-7)-melittin A(2-9) amide, magainin-II and nisin) were investigated against clinical and laboratory S. aureus (n = 10) and P. aeruginosa (n = 11) isolates either susceptible or resistant to antibiotics. Minimal Inhibitory Concentrations (MICs), Minimal Bactericidal Concentrations (MBCs), and bacterial survival rates (2 h post-treatment) were determined by microbroth dilution. The antipseudomonal effects of the antibiotics colistin or imipenem combined to LL-37 or CAMA were also studied. The toxicity of CAMPs used alone and in combination with antibiotics was evaluated on two human lung epithelial cell lines by determining the quantity of released cytoplasmic lactate dehydrogenase (LDH). Attempts to induce bacterial resistance to gentamicin, LL-37 or CAMA were also performed. RESULTS: The results revealed the rapid antibacterial effect of LL-37 and CAMA against both antibiotic susceptible and resistant strains with almost a total reduction in bacterial count 2 h post-treatment. Magainin-II and nisin were less active against tested strains. When antibiotics were combined with LL-37 or CAMA, MICs of colistin decreased up to eight-fold and MICs of imipenem decreased up to four-fold. Cytotoxicity assays revealed non-significant LDH-release suggesting no cell damage in all experiments. Induction of bacterial resistance to LL-37 was transient, tardive and much lower than that to gentamicin and induction of resistance to CAMA was not observed. CONCLUSION: This study showed the potent and rapid antibacterial activity of CAMPs on both laboratory and clinical isolates of S. aureus and P. aeruginosa either susceptible or resistant to antibiotics. Most importantly, CAMPs synergized the efficacy of antibiotics, had non toxic effects on human cells and were associated with transient and low levels of induced resistance.

Beta-defensin derived cationic antimicrobial peptides with potent killing activity against gram negative and gram positive bacteria.

BACKGROUND: Avian ß-defensins (AvBD) are cationic antimicrobial peptides (CAMP) with broad-spectrum antimicrobial activity, chemotactic property, and low host cytotoxicity. However, their bactericidal activity is greatly compromised under physiological salt concentrations which limits the use of these peptides as therapeutic agents. The length and the complex structure involving three conserved disulfide bridges are additional drawbacks associated with high production cost. In the present study, short linear CAMPs (11 to 25 a.a. residues) were developed based on the key functional components of AvBDs with additional modifications. Their biological functions were characterized. RESULTS: CAMP-t1 contained the CCR2 binding domain (N-terminal loop and adjacent α-helix) of AvBD-12 whereas CAMP-t2 comprised the key a.a. residues responsible for the concentrated positive surface charge and hydrophobicity of AvBD-6. Both CAMP-t1 and CAMP-t2 demonstrated strong antimicrobial activity against Pseudomonas aeruginosa, Staphylococcus aureus and Staphylococcus pseudintermedius. However, CAMP-t1 failed to show chemotactic activity and CAMP-t2, although superior in killing Staphylococcus spp., remained sensitive to salts. Using an integrated design approach, CAMP-t2 was further modified to yield CAMP-A and CAMP-B which possessed the following characteristics: α-helical structure with positively and negatively charged residues aligned on the opposite side of the helix, lack of protease cutting sites, C-terminal poly-Trp tail, N-terminal acetylation, and C-terminal amidation. Both CAMP-A and CAMP-B demonstrated strong antimicrobial activity against multidrug-resistant P. aeruginosa and methicillin-resistant S. pseudintermedius (MRSP) strains. These peptides were resistant to major proteases and fully active at physiological concentrations of NaCl and CaCl2. The peptides were minimally cytotoxic to avian and murine cells and their therapeutic index was moderate (≥ 4.5). CONCLUSIONS: An integrated design approach can be used to develop short and potent antimicrobial peptides, such as CAMP-A and CAMP-B. The advantageous characteristics, including structural simplicity, resistance to salts and proteases, potent antimicrobial activity, rapid membrane attacking mode, and moderate therapeutic index, suggest that CAMP-A and CAMP-B are excellent candidates for development as therapeutic agents against multidrug-resistant P. aeruginosa and methicillin-resistant staphylococci.

Cationic antimicrobial peptides do not change recombination frequency in Escherichia coli.

Cationic antimicrobial peptides are ubiquitous immune effectors of multicellular organisms. We previously reported, that in contrast to most of the classic antibiotics, cationic antimicrobial peptides (AMPs) do not increase mutation rates in E. coli Here, we provide new evidence showing that AMPs do not stimulate or enhance bacterial DNA recombination in the surviving fractions. Recombination accelerates evolution of antibiotic resistance. Our findings have implications for our understanding of host-microbe interactions, the evolution of innate immune defences, and shed new light on the dynamic of antimicrobial-resistance evolution.

Deciphering the tRNA-dependent lipid aminoacylation systems in bacteria: Novel components and structural advances.

tRNA-dependent addition of amino acids to lipids on the outer surface of the bacterial membrane results in decreased effectiveness of antimicrobials such as cationic antimicrobial peptides (CAMPs) that target the membrane, and increased virulence of several pathogenic species. After a brief introduction to CAMPs and the various bacterial resistance mechanisms used to counteract these compounds, this review focuses on recent advances in tRNA-dependent pathways for lipid modification in bacteria. Phenotypes associated with amino acid lipid modifications and regulation of their expression will also be discussed.

Design and synthesis of cell selective α/ß-diastereomeric peptidomimetic with potent in vivo antibacterial activity against methicillin resistant S. Aureus.

Design of therapeutically viable antimicrobial peptides with cell selectivity against microorganisms is an important step towards the development of new antimicrobial agents. Here, we report four de novo designed, short amphipathic sequences based on a α-helical template comprising of Lys, Trp and Leu or their corresponding D-and/or ß-amino acids. Sequence A-12 was protease susceptible whereas its α/ß-diastereomeric analogue UNA-12 was resistant to trypsin and proteinase K up to 24 h. A-12 and UNA-12 exhibited broad-spectrum antibacterial activity (MIC: 2-32 µg/mL) against pathogens including methicillin resistant S. aureus (MRSA) and methicillin-resistant S. epidermidis (MRSE). Interestingly, A-12 was found to be most toxic (>50% haemolytic at 250 µg/mL) whereas UNA-12 was found to be non cytotoxic among the all analogues against hRBCs and human keratinocytes. Interaction studies with artificial membranes by tryptophan fluorescence and acrylamide quenching assay demonstrated A-12 interacted equally in bacterial as well as mammalian mimic membrane whereas UNA-12 was found to be more selective towards bacterial mimic membrane. Further microscopic tool has revealed membrane damaging ability of A-12 and UNA-12 with bactericidal mode of action against MRSA. Encouragingly, peptidomimetics analogue UNA-12 showed remarkable safety and efficacy against MRSA in in-vivo neutropenic mice thigh infection model. In summary, simultaneous replacement of the natural amino acids with D-/ß-congeners is a promising strategy for designing of potent, cell selective and protease stable peptide based antibiotics.

In silico design of polycationic antimicrobial peptides active against Pseudomonas aeruginosa and Staphylococcus aureus.

Antimicrobial peptides (AMPs) have the potential to become valuable antimicrobial drugs in the coming years, since they offer wide spectrum of action, rapid bactericidal activity, and low probability for resistance development in comparison with traditional antibiotics. The search and improvement of methodologies for discovering new AMPs to treat resistant bacteria such as Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii and Pseudomonas aeruginosa are needed for further development of antimicrobial products. In this work, the software Peptide ID 1.0® was used to find new antimicrobial peptide candidates encrypted in proteins, considering the physicochemical parameters characteristics of AMPs such as positive net charge, hydrophobicity, and sequence length, among others. From the selected protein fragments, new AMPs were designed after conservative and semi-conservative modifications and amidation of the C-terminal region. In vitro studies of the antimicrobial activity of the newly designed peptides showed that two peptides, P3-B and P3-C, were active against P. aeruginosa Escherichia coli and A. baumannii with low minimum inhibitory concentrations. Peptide P3-C was also active against K. pneumoniae and S. aureus. Furthermore, bactericidal activity and information on the possible mechanisms of action are described according to the scanning electron microscopy studies.

A comparative, cross-species investigation of the properties and roles of transferrin- and lactoferrin-binding protein B from pathogenic bacteria.

Pathogenic bacteria from the families Neisseriaeceae and Moraxellaceae acquire iron from their host using surface receptors that have the ability to hijack iron from the iron-sequestering host proteins transferrin (Tf) and lactoferrin (Lf). The process of acquiring iron from Tf has been well-characterized, including the role of the surface lipoprotein transferrin-binding protein B (TbpB). In contrast, the only well-defined role for the homologue, LbpB, is in its protection against cationic antimicrobial peptides, which is mediated by regions present in some LbpBs that are highly enriched in glutamic or aspartic acid. In this study we compare the Tf-TbpB and the Lf-LbpB interactions and examine the protective effect of LbpB against extracts from human and transgenic mouse neutrophils to gains insights into the physiological roles of LbpB. The results indicate that in contrast to the Tf-TbpB interaction, Lf-LbpB interaction is sensitive to pH and varies between species. In addition, the results with transgenic mouse neutrophils raise the question of whether there is species specificity in the cleavage of Lf to generate cationic antimicrobial peptides or differences in the potency of peptides derived from mouse and human Lf.

miR-663a regulates growth of colon cancer cells, after administration of antimicrobial peptides, by targeting CXCR4-p21 pathway.

BACKGROUND: Antimicrobial peptides (AMPs) play important roles in the innate immune system of all life forms and recently have been characterized as multifunctional peptides that have a variety of biological roles such as anticancer agents. However, detailed mechanism of antimicrobial peptides on cancer cells is still largely unknown. METHODS: miRNA array and real-time qPCR were performed to reveal the behavior of miRNA in colon cancer HCT116 cells during the growth suppression induced by the AMPs. Establishment of miR-663a over-expressing HCT116 cells was carried out for the evaluation of growth both in vitro and in vivo. To identify the molecular mechanisms, we used western blotting analysis. RESULTS: miR-663a is upregulated by administration of the human cathelicidin AMP, LL-37, and its analogue peptide, FF/CAP18, in the colon cancer cell line HCT116. Over-expression of miR-663a caused anti-proliferative effects both in vitro and in vivo. We also provide evidence supporting the view that these effects are attributed to suppression of the expression of the chemokine receptor CXCR4, resulting in the abrogation of phosphorylation of Akt and cell cycle arrest in G2/M via p21 activation. CONCLUSIONS: This study contributes to the understanding of the AMPs' mediated anti-cancer mechanisms in colon cancer cells and highlights the possibility of using AMPs and miRNAs towards developing future strategies for cancer therapy.

KR-12-a5 is a non-cytotoxic agent with potent antimicrobial effects against oral pathogens.

This study evaluated the cytotoxicity and antimicrobial activity of analogs of cationic peptides against microorganisms associated with endodontic infections. L-929 fibroblasts were exposed to LL-37, KR-12-a5 and hBD-3-1CV and chlorhexidine (CHX, control), and cell metabolism was evaluated with MTT. The minimal inhibitory concentration (MIC) and the minimal bactericidal/fungicidal concentration (MBC/MFC) of the peptides and CHX were determined against oral pathogens associated with endodontic infections. Enterococcus faecalis and Streptococcus mutans biofilms were cultivated in bovine dentin blocks, exposed to different concentrations of the most efficient antimicrobial peptide and analyzed by confocal laser scanning microscopy. CHX and peptides affected the metabolism of L-929 at concentrations > 31.25 and 500 µg ml-1, respectively. Among the peptides, KR-12-a5 inhibited growth of both the microorganisms tested with the lowest MIC/MBC/MFC values. In addition, KR-12-a5 significantly reduced E. faecalis and S. mutans biofilms inside dentin tubules. In conclusion, KR-12-a5 is a non-cytotoxic agent with potent antimicrobial and anti-biofilm activity against oral pathogens associated with endodontic infections.

Inhibition of phospho-MurNAc-pentapeptide translocase (MraY) by nucleoside natural product antibiotics, bacteriophage ϕX174 lysis protein E, and cationic antibacterial peptides.

This review covers recent developments in the inhibition of translocase MraY and related phospho-GlcNAc transferases WecA and TagO, and insight into the inhibition and catalytic mechanism of this class of integral membrane proteins from the structure of Aquifex aeolicus MraY. Recent studies have also identified a protein-protein interaction site in Escherichia coli MraY, that is targeted by bacteriophage ϕX174 lysis protein E, and also by cationic antimicrobial peptides containing Arg-Trp close to their N- or C-termini.