Human Antimicrobial Peptide Research Articles

The matrix‐assisted laser desorption/ionisation in‐source decay of peptides using ion mobility enabled quadrupole‐time‐of‐flight mass spectrometry

In-source decay (ISD) matrix-assisted laser desorption/ionisation (MALDI) mass spectrometry with a 1,5-diaminonaphthalene (1,5-DAN) matrix is used for the structural characterisation of peptides. However, MALDI spectra are intrinsically complicated by the presence of matrix ions, which interfere with the peptide fragments. This may cause false-positive results or reduced sequence coverage. This paper reports investigations of ISD processes in an intermediate pressure MALDI ion source and a protocol for the removal of interfering ions using ion mobility separation (IMS). An intermediate pressure MALDI source of a Q-IMS-Q-TOF instrument (Synapt G2) has been employed for the ISD of selected peptides using a 1,5-DAN matrix. Successful coupling of the MALDI source tuned for ISD experiments using IMS is demonstrated. The IMS made it possible to remove interfering matrix ions effectively from the spectra and thus to increase the confidence of spectral interpretation. Extensive fragment series corresponding to N-Cα bond cleavages were observed under optimised conditions; on the other hand, weaker series of ions caused by peptide bond cleavages were prevalent for default conditions and/or the α-hydroxycinnamic acid matrix. Ion mobility has been used for the elimination of matrix ions. The technique has been applied to top-down sequencing of non-tryptic peptides, such as the human palmitoylated analogue of prolactin-releasing peptide used in recent obesity studies, and human and insect antimicrobial peptides.

Lung allograft donors with excessive alcohol use have increased levels of human antimicrobial peptide LL-37.

The relatively low long-term survival rate of lung transplant recipients as compared to other organ recipients serves as an impetus to identify potential lung dysfunction as early as possible. There is an association between donor heavy alcohol use and acute lung injury in the lung allograft after transplant, known as primary graft dysfunction. Excessive alcohol use (EAU) can induce pulmonary immune dysregulation in response to an infection. Antimicrobial peptides (AMPs) are an important component of the innate immune response to pulmonary infections, but the impact of EAU on AMPs in the allograft lung has not been evaluated. Our hypothesis is that specific lung AMPs, LL-37, α-defensin-1,2,3, and β-defensin-2, are dysregulated in the lungs from organ donors who had EAU. In this prospective observational investigation, we measured AMPs via ELISA and inflammatory cytokines via multiplex bead array, in bronchoalveolar lavage (BAL) fluid of lung allograft donors, comparing results based on their alcohol consumption. LL-37 levels in lung donors with EAU were found to be increased compared to nondrinker (ND) donors [median 7.7ng/mL (IQR 4.1-37.0) vs. 2.3ng/mL (IQR 1.1-7.9), p=0.004], whereas α-defensins-1,2,3 were decreased only in the presence of an infection in donors with EAU compared to ND donors [median 2.2ng/mL (IQR 1.6-2.4) vs. 3.2ng/mL (IQR 2.3-3.8), p=0.049]. There was no difference in β-defensin-2 levels. Gene expression levels of these AMPs were not different. Elevated levels of CXCL8 were noted in bronchial washings of donors with EAU compared to ND donors, [median 4372pg/mL (IQR 3352-13180) vs. 867.3pg/mL (IQR 163.6-3675), p=0.04], suggesting a potentially heightened inflammatory response. At 1 month post-transplant, LL-37 and CXCL8 levels are decreased compared to levels at time of transplant. In lung donors with EAU, LL-37 and α-defensins-1,2,3 dysregulated levels in the presence of an infection may be a harbinger of dysfunction of the lungs through the transplant process.

PU.1 and epigenetic signals modulate 1,25-dihydroxyvitamin D3 and C/EBPα regulation of the human cathelicidin antimicrobial peptide gene in lung epithelial cells.

LL-37, the only known human cathelicidin which is encoded by the human antimicrobial peptide (CAMP) gene, plays a critical role in protection against bacterial infection. We previously demonstrated that cathelicidin is induced by 1,25-dihydroxyvitamin D3 (1,25(OH) 2 D 3 ) in human airway epithelial cells with a resultant increase in bactericidal activity. In this study we identify key factors that co-operate with 1,25(OH) 2 D 3 in the regulation of CAMP. Our results show for the first time that PU.1, the myeloid transcription factor (which has also been identified in lung epithelial cells), co-operates with the vitamin D receptor and CCAAT/enhancer binding protein α (CEBPα) to enhance the induction of CAMP in lung epithelial cells. Our findings also indicate that enhancement of 1,25(OH) 2 D 3 regulation of CAMP by histone deacetylase inhibitors involves co-operation between acetylation and chromatin remodeling through Brahma-related gene 1 (BRG1; a component of the SWItch/sucrose nonfermentable [SWI/SNF] complex). BRG1 can be an activator or repressor depending on BRG1-associated factors. Protein arginine methyltransferase 5 (PRMT5), a methlytransferase which interacts with BRG1, represses 1,25(OH) 2 D 3 induced CAMP in part through dimethylation of H4R3. Our findings identify key mediators involved in the regulation of the CAMP gene in lung epithelial cells and suggest new approaches for therapeutic manipulation of gene expression to increase the antibacterial capability of the airway.

Antimicrobial peptides activity in the skin.

This review presents the current state of knowledge regarding multifunctional role of human skin antimicrobial peptides (AMPs), including (a) protection from microbial infection, (b) improvement of skin barrier homoeostasis, (c) modulation of inflammation responses, and (d) promotion of wound healing. In addition, association of AMPs with skin diseases as well as challenges and future prospects for AMP therapeutics has also been discussed.

Statins influence epithelial expression of the anti-microbial peptide LL-37/hCAP-18 independently of the mevalonate pathway.

Anti-microbial resistance increases among bacterial pathogens and new therapeutic avenues needs to be explored. Boosting innate immune mechanisms could be one attractive alternative in the defence against infectious diseases. The cholesterol-lowering drugs, statins, have been demonstrated to also affect the immune system. Here we investigate the effect of statins on the expression of the human cathelicidin anti-microbial peptide (CAMP) LL-37/hCAP-18 [encoded by the CAMP gene] and explore the underlying mechanisms in four epithelial cell lines of different origin. Simvastatin induced CAMP expression in bladder epithelial cells telomerase-immortalized uroepithelial cells (TERT-NHUCs), intestinal cells HT-29 and keratinocytes HEKa, but not in airway epithelial cells A549. Gene induction in HEKa cells was reversible by mevalonate, while this effect was independent of the cholesterol biosynthesis pathway in TERT-NHUCs. Instead, inhibition of histone deacetylases by simvastatin seems to be involved. For HT-29 cells, both mechanisms may contribute. In addition, simvastatin increased transcription of the vitamin D-activating enzyme CYP27B1 which, in turn, may activate LL-37/hCAP-18 production. Taken together, simvastatin is able to promote the expression of LL-37/hCAP-18, but cell line-specific differences in efficacy and the involved signalling pathways exist.

LL-37 inhibits LPS-induced inflammation and stimulates the osteogenic differentiation of BMSCs via P2X7 receptor and MAPK signaling pathway

Oral diseases, such as periapical periodontitis and periodontitis, are characterized by inflammation-induced bone loss. LL-37, a human antimicrobial peptide (AMP), has multiple biological functions and the potential to promote osteogenesis. Therefore, this study aimed to investigate the regulatory effects of LL-37 within normal and inflammatory microenvironments. The roles of P2X7 receptor (P2X7R) and mitogen-activated protein kinase (MAPK) signaling pathway were also demonstrated. The results showed that LL-37 promoted bone marrow stromal cell (BMSC) proliferation, migration and osteogenic differentiation. LL-37 inhibited the expression of the inflammatory cytokines interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and receptor activator of nuclear factor kappa-B ligand (RANKL) at both protein and gene levels, and attenuated the lipopolysaccharide (LPS)-induced inhibition of osteogenesis. Immunofluorescence (IF) confirmed P2X7R expression in BMSCs. BBG, a P2X7R antagonist, significantly attenuated LL-37-promoted osteogenesis. The phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun NH2-terminal kinase (JNK) increased after LL-37 stimulation, which did not affect p38 phosphorylation. The effects of LL-37 on osteogenesis-related gene expression were markedly attenuated by selective inhibitors of ERK1/2 and JNK. Furthermore, a mouse model of LPS-stimulated calvarial osteolysis was established, and results showed that LL-37 markedly inhibited osteoclastic bone resorption. In conclusion, we speculate that LL-37 inhibits inflammation and promotes BMSC osteogenesis via P2X7R and MAPK signaling pathway.

Natural molecules induce and synergize to boost expression of the human antimicrobial peptide β-defensin-3

Antimicrobial peptides (AMPs) are mucosal defense effectors of the human innate immune response. In the intestine, AMPs are produced and secreted by epithelial cells to protect the host against pathogens and to support homeostasis with commensals. The inducible nature of AMPs suggests that potent inducers could be used to increase their endogenous expression for the prevention or treatment of diseases. Here we aimed at identifying molecules from the natural pharmacopoeia that induce expression of human β-defensin-3 (HBD3), one of the most efficient AMPs, without modifying the production of proinflammatory cytokines. By screening, we identified three molecules isolated from medicinal plants, andrographolide, oridonin, and isoliquiritigenin, which induced HBD3 production in human colonic epithelial cells. This effect was observed without activation of the NF-κB pathway or the expression of associated proinflammatory cytokines. We identified the EGF receptor as the target of these compounds and characterized the downstream-activated MAPK pathways. At the chromatin level, molecules increased phosphorylation of histone H3 on serine S10 and recruitment of the c-Fos, c-Jun, and Elk1 or c-Myc transcription factors at the HBD3 promoter. Interestingly, stimulating cells with a combination of andrographolide and isoliquiritigenin synergistically enhanced HBD3 induction 10-fold more than observed with each molecule alone. Finally, we investigated the molecular basis governing the synergistic effect, confirmed our findings in human colonic primary cells, and demonstrated that synergism increased cellular antimicrobial activity. This work shows the capability of small molecules to achieve induction of epithelial antimicrobial defenses while simultaneously avoiding the deleterious risks of an inflammatory response.

Localized expression of antimicrobial proteins mitigates huanglongbing symptoms in Mexican lime

Citrus huanglongbing (HLB) is a devastating disease associated with Candidatus Liberibacter asiaticus spp. (CLas), a bacterium restricted to the sieve tube system of the phloem that is transmitted by the psyllid vector, Diaphorina citri. In this study, the human antimicrobial peptides, lysozyme and β-defensin 2, were targeted to the vascular tissue of Mexican lime (Citrus x aurantifolia [Christm.] Swingle) by fusion to a phloem-restricted protein. Localized expression was achieved, via Agrobacterium tumefaciens-mediated transformation of the stem, which led to protein expression and mobilization within the vascular tissue of heterotrophic tissues. HLB-infected plants were monitored for 360 days. Lower bacteria titers were observed in plants expressing either β-defensin 2, lysozyme, or the combination thereof, and these plants had increased photosynthesis, compared to untreated control trees. Thus, targeting of antimicrobial proteins to the vascular tissue was effective in decreasing CLas titer, and alleviating citrus greening symptoms. Based on these findings, this strategy could be used to effectively treat plants that are already infected with bacterial pathogens that reside in the phloem translocation stream.

A Commensal Dipeptidyl Aminopeptidase with Specificity for N-Terminal Glycine Degrades Human-Produced Antimicrobial Peptides in Vitro.

Proteases within the C1B hydrolase family are encoded by many organisms. We subjected a putative C1B-like cysteine protease secreted by the human gut commensal Parabacteroides distasonis to mass spectrometry-based substrate profiling to find preferred peptide substrates. The P. distasonis protease, which we termed Pd_dinase, has a sequential diaminopeptidase activity with strong specificity for N-terminal glycine residues. Using the substrate sequence information, we verified the importance of the P2 glycine residue with a panel of fluorogenic substrates and calculated kcat and KM for the dipeptide glycine-arginine-AMC. A potent and irreversible dipeptide inhibitor with a C-terminal acyloxymethyl ketone warhead, glycine-arginine- AOMK, was then synthesized and demonstrated that the Pd_dinase active site requires a free N-terminal amine for potent and rapid inhibition. We next determined the homohexameric Pd_dinase structure in complex with glycine-arginine- AOMK and uncovered unexpected active site features that govern the strict substrate preferences and differentiate this protease from members of the C1B and broader papain-like C1 protease families. We finally showed that Pd_dinase hydrolyzes several human antimicrobial peptides and therefore posit that this P. distasonis enzyme may be secreted into the extracellular milieu to assist in gut colonization by inactivation of host antimicrobial peptides.

Designing effective anticancer-radiopeptides. A Molecular Dynamics study of their interaction with model tumor and healthy cell membranes

One of the greatest merit of the use of radiopeptides in oncology is their selectivity which, however, brings about the drawback that each radiopeptide is specific for a given tumor type. To overcome this problem the direction currently taken in drug design is that of radiolabelling peptide hormones (or their analogues), relying on their intrinsic ability to bind to specific receptors in precise areas of the human body, at the cost, however, of a poor selectivity against healthy cells. We present here an extensive Molecular Dynamics study of a promising alternative inspired by the mechanism through which antimicrobial peptides interact with the negatively charged bacterial membranes. Appropriately modifying the human antimicrobial peptide, LL-37, we designed a functionalized radionuclide carrier capable of binding more strongly to the negatively charged (model) tumor membranes than to the neutral healthy ones. The mechanism behind this behaviour relies on the fact that at the slight acidic pH surrounding tumor tissues the histidines belonging to the peptide get protonated thus making it positively charged. We have investigated by an extended numerical study the way in which this artificial peptide interacts with models of tumor and healthy cell membranes, proving by Potential Mean Force calculations that the affinity of the peptide to model tumor membranes is significantly larger than to healthy ones. These features (high affinity and generic tumor selectivity) recommend antimicrobial derived customized carriers as promising theranostic constructs in cancer diagnostic and therapy.

C10orf99 contributes to the development of psoriasis by promoting the proliferation of keratinocytes

Psoriasis is a chronic, relapsing inflammatory skin disease. The pathogenesis of psoriasis is complex and has not been fully understood. C10orf99 was a recently identified human antimicrobial peptide whose mRNA expression is elevated in psoriatic human skin samples. In this study, we investigated the functional roles of C10orf99 in epidermal proliferation under inflammatory condition. We showed that C10orf99 protein was significantly up-regulated in psoriatic skin samples from patients and the ortholog gene expression levels were up-regulated in imiquimod (IMQ)-induced psoriasis-like skin lesions in mice. Using M5-stimulated HaCaT cell line model of inflammation and a combinational approach of knockdown and overexpression of C10orf99, we demonstrated that C10orf99 could promote keratinocyte proliferation by facilitating the G1/S transition, and the pro-proliferation effect of C10orf99 was associated with the activation of the ERK1/2 and NF-κB but not the AKT pathways. Local depletion of C10orf99 by lentiviral vectors expressing C10orf99 shRNA effectively ameliorated IMQ-induced dermatitis. Taken together, these results indicate that C10orf99 plays a contributive role in psoriasis pathogenesis and may serve as a new target for psoriasis treatment.

Human Oral Defensins Antimicrobial Peptides: A Future Promising Antimicrobial Drug.

The nature and structural composition of antimicrobial peptides are derived from their innate immune response and they are active against various bacteria, fungi and other microorganisms. The aim of this paper was to pool up the literature on the features of human oral defensins antimicrobial peptides. The defensins showed antimicrobial activity against Gram-positive and Gram-negative bacteria and various fungi and viruses. As with their other properties like antiviral, antifungal and antibacterial, human defensins peptides are thought to have a unique amino acid-based structure with Disulphide Bridge which makes them synthesize chemically or naturally with the help of these bacteria. The data contributing in this study was gathered from the research papers published in English language in the last twenty-five years. This literature mainly elaborates the general and analytical characteristics of antimicrobial peptides in the human oral cavity; focusing on the types, biochemistry, and mechanism of action of defensins with its clinical importance.

Signaling by a Conserved Quorum Sensing Pathway Contributes to Growth Ex Vivo and Oropharyngeal Colonization of Human Pathogen Group A Streptococcus

Bacterial virulence factor production is a highly coordinated process. The temporal pattern of bacterial gene expression varies in different host anatomic sites to overcome niche-specific challenges. The human pathogen group A streptococcus (GAS) produces a potent secreted protease, SpeB, that is crucial for pathogenesis. Recently, we discovered that a quorum sensing pathway comprised of a leaderless short peptide, SpeB-inducing peptide (SIP), and a cytosolic global regulator, RopB, controls speB expression in concert with bacterial population density. The SIP signaling pathway is active in vivo and contributes significantly to GAS invasive infections. In the current study, we investigated the role of the SIP signaling pathway in GAS-host interactions during oropharyngeal colonization. The SIP signaling pathway is functional during growth ex vivo in human saliva. SIP-mediated speB expression plays a crucial role in GAS colonization of the mouse oropharynx. GAS employs a distinct pattern of SpeB production during growth ex vivo in saliva that includes a transient burst of speB expression during early stages of growth coupled with sustained levels of secreted SpeB protein. SpeB production aids GAS survival by degrading LL37, an abundant human antimicrobial peptide. We found that SIP signaling occurs during growth in human blood ex vivo. Moreover, the SIP signaling pathway is critical for GAS survival in blood. SIP-dependent speB regulation is functional in strains of diverse emm types, indicating that SIP signaling is a conserved virulence regulatory mechanism. Our discoveries have implications for future translational studies.

Astragalus polysaccharides exerts anti-infective activity by inducing human cathelicidin antimicrobial peptide LL-37 in respiratory epithelial cells.

Astragalus polysaccharides (APS), one of the major active components in Astragalus membranaceus, is an effective immunomodulator used in the treatment of immunological diseases in China. However, the anti-infective action and mechanism of APS is not fully known. In the present study, we found that APS induced the expression of human cathelicidin antimicrobial peptide LL-37, a key host anti-infective molecule, in both mRNA and protein levels in respiratory epithelial cells HBE16 and A549. Furthermore, the lysate and supernatant from APS-treated HBE16 cells both exhibited an obvious antibacterial action, which was partially neutralizated by LL-37 monoclonal antibody. In addition, APS also significantly elevated the phosphorylation of p38 MAPK and JNK and caused the degradation of IκBα. Specific inhibitors of p38 MAPK, JNK, or NF-κB obviously abolished APS-induced LL-37 synthesis and antibacterial activity, respectively. Taken together, our results confirmed the enhancement of APS on LL-37 induction and antibacterial action in respiratory epithelial cells, which may be attributed to activation of p38 MAPK/JNK and NF-κB pathways. Furthermore, these results also supported the clinical application of APS in the treatment of infectious diseases.

Molecular Dynamics Simulations of Human Antimicrobial Peptide LL-37 in Model POPC and POPG Lipid Bilayers.

Cathelicidins are a large family of cationic antimicrobial peptides (AMPs) found in mammals with broad spectrum antimicrobial activity. LL-37 is the sole amphipathic α-helical AMP from human Cathelicidins family. In addition to its bactericidal capability, LL-37 has antiviral, anti-tumor, and immunoregulatory activity. Despite many experimental studies, its molecular mechanism of action is not yet fully understood. Here, we performed three independent molecular dynamics simulations (600 ns or more) of a LL-37 peptide in the presence of 256 lipid bilayers with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) mimicking bacterial and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) mimicking mammalian membranes. We found that LL-37 can be quickly absorbed onto the POPG bilayer without loss of its helical conformation in the core region and with the helix lying in parallel to the bilayer. The POPG bilayer was deformed. In contrast, LL-37 is slower in reaching the POPC surface and loss much of its helical conformation during the interaction with the bilayer. LL-37 only partially entered the POPC bilayer without significant deformation of the membrane. The observed difference for different bilayers is largely due to the fact that LL-37 is positively charged, POPG is negatively charged, and POPC is neutral. Our simulation results demonstrated the initial stage of disruption of the bacterial membrane by LL-37 in atomic details. Comparison to experimental results on LL-37 and simulation studies in other systems was made.

A QCM-D study of the concentration- and time-dependent interactions of human LL37 with model mammalian lipid bilayers.

The human antimicrobial peptide LL37 is promising as an alternative to antibiotics due to its biophysical interactions with charged bacterial lipids. However, its clinical potential is limited due to its interactions with zwitterionic mammalian lipids leading to cytotoxicity. Mechanistic insight into the LL37 interactions with mammalian lipids may enable rational design of less toxic LL37-based therapeutics. To this end, we studied concentration- and time-dependent interactions of LL37 with zwitterionic model phosphatidylcholine (PC) bilayers with quartz crystal microbalance with dissipation (QCM-D). LL37 mass adsorption and PC bilayer viscoelasticity changes were monitored by measuring changes in frequency (Δf) and dissipation (ΔD), respectively. The Voigt-Kelvin viscoelastic model was applied to Δf and ΔD to study changes in bilayer thickness and density with LL37 concentration. At low concentrations (0.10-1.00 μM), LL37 adsorbed onto bilayers in a concentration-dependent manner. Further analyses of Δf, ΔD and thickness revealed that peptide saturation on the bilayers was a threshold for interactions observed above 2.00 μM, interactions that were rapid, multi-step, and reached equilibrium in a concentration- and time-dependent manner. Based on these data, we proposed a model of stable transmembrane pore formation at 2.00-10.0 μM, or transition from a primarily lipid to a primarily protein film with a transmembrane pore formation intermediate state at concentrations of LL37 > 10 μM. The concentration-dependent interactions between LL37 and PC bilayers correlated with the observed concentration-dependent biological activities of LL37 (antimicrobial, immunomodulatory and non-cytotoxic at 0.1-1.0 μM, hemolytic and some cytotoxicity at 2.0-13 μM and cytotoxic at >13 μM).

Polyplex System Versus Nucleofection for Human Skin Cell Transfection and Effect of Internal Ribosome Entry Site Sequence.

Nonviral transfection has important implications on gene therapy because of its safety. In particular, polyfection and nucleofection are two widely used systems for nonviral gene delivery. Their potential depends on the transfection efficiency achieved, which is influenced in turn by the type of cells transfected and by the plasmid that carries the gene of interest. The efficiency of transfection by polyfection or nucleofection in human fibroblasts and keratinocytes was evaluated in this study. Transfections were performed with plasmids containing a gene of interest (human cathelicidin antimicrobial peptide) and two reporter genes (red or green fluorescent protein) that included or not an internal ribosome entry site (IRES). The efficiency was measured by flow cytometry in terms of percentage of cells expressing the reporter gene; viability of transfected cells was also evaluated. It was found that nucleofection was more efficient than polyplexes for transfecting fibroblasts, while no significant differences were found between both systems of transfection when applied to keratinocytes. Regarding the viability of fibroblasts after transfection, values were high in both systems. In contrast, keratinocytes were more sensitive to nucleofection. It was also noted that both types of cells decreased reporter gene expression when IRES sequence was located upstream of the reporter gene, suggesting a negative effect on the expression of this gene. These results confirm that the transfection efficiency depends on the type of cells and the system used.

Structure-Activity Relationship Study of Synthetic Variants Derived from the Highly Potent Human Antimicrobial Peptide hLF(1-11)

Structure-Activity Relationship Study of Synthetic Variants Derived from the Highly Potent Human Antimicrobial Peptide hLF(1-11)

Single Molecule Study of the Mechanism of Attack of the Human Antimicrobial Peptide LL-37 on E. coli

Evolutionarily conserved anti-microbial peptides (AMPs) are useful prototypes and promising drugs due to increasing antibiotic resistance. We have used single-cell, time-resolved fluorescence microscopy and single-molecule fluorescence microscopy to study the effects of human cathelicidin LL-37 on the E. coli chromosome (visualized through ParB-GFP), RNA polymerase (RNAP), ribosome and cytoplasmic protein Kaede. This enables us to correlate in real time the motion reduction of chromosome and RNAP, the halting of cell growth, and the permeabilization of the cytoplasmic membrane (CM) by LL-37. We have found that LL-37 could change the spatial distribution of ribosome and reduce the motion of chromosomal loci, RNAP, ribosome and Kaede. For chromosome, the apparent diffusion coefficient D decreased by 10 times on both long (∼ min) and short (∼ s) timescales 10 min after injecting 1x MIC (minimum inhibitory concentration) LL-37, larger than those treated with the ATP-depleting drugs NaN3 and CCCP, suggesting LL-37 might have effects on chromosome beyond energy depletion. Besides, some of the sharp clear foci of ParB complex dissolved into broader but dimmer foci. We speculate that LL-37 dissociates the ParB complex by disrupting the protein-protein or protein-DNA interactions. The general ability of a drug to disrupt protein-DNA interactions would be a catastrophe for infectious bacteria. It indicates that LL-37 has multi-target effects and its damage to bacterial cells goes far beyond the membrane permeabilization. This may make it harder for bacteria to gain resistance. A comprehensive understanding of all these effects and how they are interlinked with each other could be of medical significance.

Virulence Role of the GlcNAc Side Chain of the Lancefield Cell Wall Carbohydrate Antigen in Non-M1-Serotype Group A Streptococcus.

ABSTRACTClassification of streptococci is based upon expression of unique cell wall carbohydrate antigens. All serotypes of group A Streptococcus (GAS; Streptococcus pyogenes), a leading cause of infection-related mortality worldwide, express the group A carbohydrate (GAC). GAC, the classical Lancefield antigen, is comprised of a polyrhamnose backbone with N-acetylglucosamine (GlcNAc) side chains. The immunodominant GlcNAc epitope of GAC is the basis of all rapid diagnostic testing for GAS infection. We previously identified the 12-gene GAC biosynthesis gene cluster and determined that the glycosyltransferase GacI was required for addition of the GlcNAc side chain to the polyrhamnose core. Loss of the GAC GlcNAc epitope in serotype M1 GAS resulted in attenuated virulence in two animal infection models and increased GAS sensitivity to killing by whole human blood, serum, neutrophils, and antimicrobial peptides. Here, we report that the GAC biosynthesis gene cluster is ubiquitous among 520 GAS isolates from global sources, representing 105 GAS emm serotypes. Isogenic ΔgacI mutants were constructed in M2, M3, M4, M28, and M89 backgrounds and displayed an array of phenotypes in susceptibility to killing by whole human blood, baby rabbit serum, human platelet releasate, human neutrophils, and antimicrobial peptide LL-37. The contribution of the GlcNAc side chain to GAS survival in vivo also varied by strain, demonstrating that it is not a prerequisite for virulence in the murine infection model. Thus, the relative contribution of GAC to virulence in non-M1 serotypes appears to depend on the quorum of other virulence factors that each strain possesses.