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In the complement system, the opsonin C3b binds to the bacterial cell surface and mediates the opsonophagocytosis. However, the cell-wall protein SdrE of Staphylococcus aureus inhibits the C3b activity by recruiting the complement regulatory protein factor H (fH). SdrE binds to fH via its N-terminal N2N3 domain, which are also found in six other staphylococcal cell-wall proteins. In this study, we report that not only the N2N3 domain of SdrE but also those of ClfA, FnbpA and FnbpB can bind to fH. When immobilized on a microplate, the N2N3 domains recruited fH and enhanced the factor I (fI)-mediated cleavage of C3b. When mixed with fH and S. aureus cells, the N2N3 domains inhibited the fH binding to S. aureus cells and reduced the fI-mediated C3b cleavage on the bacterial cell surface. The F(ab)′2 fragments of the rabbit N2N3 antibodies also inhibited the fH binding to the S. aureus cell surface. When added to human blood, the N2N3 antibodies or the N2N3 domain proteins significantly increased the bactericidal activity. Based on these results, we conclude that, in S. aureus , not only SdrE but also ClfA, FnbpA and FnbpB can contribute to the inhibition of C3b-mediated opsonophagocytosis. [ABSTRACT FROM AUTHOR]

Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent cause of difficult-to-treat, often fatal infections in humans1,2. Most humans have antibodies against S. aureus, but these are highly variable and often not protective in immunocompromised patients3. Previous vaccine development programs have not been successful4. A large percentage of human antibodies against S. aureus target wall teichoic acid (WTA), a ribitol-phosphate (RboP) surface polymer modified with N-acetylglucosamine (GlcNAc)5,6. It is currently unknown whether the immune evasion capacities of MRSA are due to variation of dominant surface epitopes such as those associated with WTA. Here we show that a considerable proportion of the prominent healthcare-associated and livestock-associated MRSA clones CC5 and CC398, respectively, contain prophages that encode an alternative WTA glycosyltransferase. This enzyme, TarP, transfers GlcNAc to a different hydroxyl group of the WTA RboP than the standard enzyme TarS7, with important consequences for immune recognition. TarP-glycosylated WTA elicits 7.5-40-fold lower levels of immunoglobulin G in mice than TarS-modified WTA. Consistent with this, human sera contained only low levels of antibodies against TarP-modified WTA. Notably, mice immunized with TarS-modified WTA were not protected against infection with tarP-expressing MRSA, indicating that TarP is crucial for the capacity of S. aureus to evade host defences. High-resolution structural analyses of TarP bound to WTA components and uridine diphosphate GlcNAc (UDP-GlcNAc) explain the mechanism of altered RboP glycosylation and form a template for targeted inhibition of TarP. Our study reveals an immune evasion strategy of S. aureus based on averting the immunogenicity of its dominant glycoantigen WTA. These results will help with the identification of invariant S. aureus vaccine antigens and may enable the development of TarP inhibitors as a new strategy for rendering MRSA susceptible to human host defences. Strains of methicillin-resistant Staphylococcus aureus use a prophage-encoded glycosyltransferase to alter the glycosylation of their wall teichoic acid and thereby evade antibody-mediated immune responses. [ABSTRACT FROM AUTHOR]

We generated a Burkholderia mutant, which is deficient of an N -acetylmuramyl- l -alanine amidase, AmiC, involved in peptidoglycan degradation. When non-motile Δ amiC mutant Burkholderia cells harboring chain form were orally administered to Riptortus insects, Δ amiC mutant cells were unable to establish symbiotic association. But, Δ amiC mutant complemented with amiC gene restored in vivo symbiotic association. Δ amiC mutant cultured in minimal medium restored their motility with single-celled morphology. When Δ amiC mutant cells harboring single-celled morphology were administered to the host insect, this mutant established normal symbiotic association, suggesting that bacterial motility is essential for the successful symbiosis between host insect and Burkholderia symbiont. [ABSTRACT FROM AUTHOR]

Legionella pneumophila, a human intracellular pathogen, encodes about 290 effector proteins that are translocated into host cells through a secretion machinery. Some of these proteins have been shown to manipulate or subvert cellular processes during infection, but functional roles of a majority of them remain unknown. Lpg0393 is a newly identified Legionella effector classified as a hypothetical protein. Through X-ray crystallographic analysis, we show that Lpg0393 contains a Vps9-like domain, which is structurally most similar to the catalytic core of human Rabex-5 that activates the endosomal Rab proteins Rab5, Rab21 and Rab22. Consistently, Lpg0393 exhibited a guanine-nucleotide exchange factor activity toward the endosomal Rabs. This work identifies the first example of a bacterial guanine-nucleotide exchange factor that is active towards the Rab5 sub-cluster members, implying that the activation of these Rab proteins might be advantageous for the intracellular survival of Legionella. [ABSTRACT FROM AUTHOR]

In P seudomonas aeruginosa, quorum sensing ( QS) plays an essential role in pathogenesis and the QS response controls many virulence factors. Using a mealworm, T enebrio molitor as a host model, we found that Protease IV, a QS-regulated exoprotease of P . aeruginosa functions as a key virulence effector causing the melanization and death of T . molitor larvae. Protease IV was able to degrade zymogens of spätzle processing enzyme ( SPE) and SPE-activating enzyme ( SAE) without the activation of the antimicrobial peptide ( AMP) production. Since SPE and SAE function to activate spätzle, a ligand of Toll receptor in the innate immune system of T . molitor, we suggest that Protease IV may interfere with the activation of the Toll signaling. Independently of the Toll pathway, the melanization response, another innate immunity was still generated, since Protease IV directly converted T enebrio prophenoloxidase into active phenoloxidase. Protease IV also worked as an important factor in the virulence to brine shrimp and nematode. These results suggest that Protease IV provides P . aeruginosa with a sophisticated way to escape the immune attack of host by interfering with the production of AMPs. [ABSTRACT FROM AUTHOR]

The Riptortus-Burkholderia symbiotic system represents a promising experimental model to study the molecular mechanisms involved in insect-bacterium symbiosis due to the availability of genetically manipulated Burkholderia symbiont. Using transposon mutagenesis screening, we found a symbiosis-deficient mutant that was able to colonize the host insect but failed to induce normal development of host's symbiotic organ. The disrupted gene was identified as purL involved in purine biosynthesis. In vitro growth impairment of the purL mutant and its growth dependency on adenine and adenosine confirmed the functional disruption of the purine synthesis gene. The purL mutant also showed defects in biofilm formation, and this defect was not rescued by supplementation of purine derivatives. When inoculated to host insects, the purL mutant was initially able to colonize the symbiotic organ but failed to attain a normal infection density. The low level of infection density of the purL mutant attenuated the development of the host's symbiotic organ at early instar stages and reduced the host's fitness throughout the nymphal stages. Another symbiont mutant-deficient in a purine biosynthesis gene, purM, showed phenotypes similar to those of the purL mutant both in vitro and in vivo, confirming that the purL phenotypes are due to disrupted purine biosynthesis. These results demonstrate that the purine biosynthesis genes of the Burkholderia symbiont are critical for the successful accommodation of symbiont within the host, thereby facilitating the development of the host's symbiotic organ and enhancing the host's fitness values. [ABSTRACT FROM AUTHOR]

ABSTRACT Bacterial infection can cause inflammatory bone diseases accompanied by the bone destruction resulting from excess generation of osteoclasts. Although lipoproteins are one of the major immunostimulating components of bacteria, little is known about their effects on bone metabolism. In this study, we investigated the role of lipoproteins in bacteria-induced bone destruction using Staphylococcus aureus wild type, its lipoprotein-deficient mutant, and synthetic lipopeptides Pam2CSK4 and Pam3CSK4 known to mimic bacterial lipoproteins. Formaldehyde-inactivated S. aureus or the synthetic lipopeptides induced severe bone loss in the femurs of mice after intraperitoneal administration and in a calvarial bone implantation model, whereas the lipoprotein-deficient S. aureus did not show such effects. Mechanism studies further identified three action mechanisms for the lipopeptide-induced osteoclast differentiation and bone resorption via (i) enhancement of osteoclast differentiation through Toll-like receptor 2 and MyD88-dependent signaling pathways; (ii) induction of pro-inflammatory cytokines, TNF-α and IL-6; and (iii) upregulation of RANKL expression with downregulation of osteoprotegerin expression in osteoblasts. Taken together, these results suggest that lipoprotein might be an important bacterial component responsible for bone destruction during bacterial infections through augmentation of osteoclast differentiation and activation. © 2013 American Society for Bone and Mineral Research. [ABSTRACT FROM AUTHOR]

Highlights: [•] We examine the action mechanism of antimicrobial peptides against mycoplasma. [•] The mechanism relies on interaction of antimicrobial peptides with membrane lipids. [•] Binding to phosphatidylcholine is critical for their anti-mycoplasma activity. [ABSTRACT FROM AUTHOR]

The objectives of this study were to investigate the immune response to intradermal immunization with wall teichoic acid (WTA) and the effect of MBL deficiency in a murine model of infection with methicillin-resistant Staphylococcus aureus (MRSA). WTA is a bacterial cell wall component that is implicated in invasive infection. We tested susceptibility to MRSA infection in wild type (WT) and MBL deficient mice using two strains of MRSA: MW2, a community-associated MRSA (CA-MRSA); and COL, a healthcare-associated MRSA (HA-MRSA). We also performed in vitro assays to investigate the effects of anti-WTA IgG containing murine serum on complement activation and bacterial growth in whole blood. We found that MBL knockout (KO) mice are relatively resistant to a specific MRSA strain, MW2 CA-MRSA, compared to WT mice, while both strains of mice had similar susceptibility to a different strain, COL HA-MRSA. Intradermal immunization with WTA elicited and augmented an anti-WTA IgG response in both WT and MBL KO mice. WTA immunization significantly reduced susceptibility to both MW2 CA-MRSA and COL HA-MRSA, independent of the presence of MBL. The protective mechanisms of anti-WTA IgG are mediated at least in part by complement activation and clearance of bacteria from blood. The significance of these findings is that 1) Intradermal immunization with WTA induces production of anti-WTA IgG; and 2) This anti-WTA IgG response protects from infection with both MW2 CA-MRSA and COL HA-MRSA even in the absence of MBL, the deficiency of which is common in humans. [ABSTRACT FROM AUTHOR]

The bean bug Riptortus pedestris possesses a specialized symbiotic organ in a posterior region of the midgut, where numerous crypts harbor extracellular betaproteobacterial symbionts of the genus Burkholderia. Second instar nymphs orally acquire the symbiont from the environment, and the symbiont infection benefits the host by facilitating growth and by occasionally conferring insecticide resistance. Here we performed comparative transcriptomic analyses of insect genes expressed in symbiotic and non-symbiotic regions of the midgut dissected from Burkholderia-infected and uninfected R. pedestris. Expression sequence tag analysis of cDNA libraries and quantitative reverse transcription PCR identified a number of insect genes expressed in symbiosis- or aposymbiosis-associated patterns. For example, genes up-regulated in symbiotic relative to aposymbiotic individuals, including many cysteine-rich secreted protein genes and many cathepsin protease genes, are likely to play a role in regulating the symbiosis. Conversely, genes up-regulated in aposymbiotic relative to symbiotic individuals, including a chicken-type lysozyme gene and a defensin-like protein gene, are possibly involved in regulation of non-symbiotic bacterial infections. Our study presents the first transcriptomic data on gut symbiotic organ of a stinkbug, which provides initial clues to understanding of molecular mechanisms underlying the insect-bacterium gut symbiosis and sheds light on several intriguing commonalities between endocellular and extracellular symbiotic associations. [ABSTRACT FROM AUTHOR]

Bacterial lipoproteins are characterized by the presence of a conserved N-terminal lipid-modified cysteine residue that allows the hydrophilic protein to anchor onto bacterial cell membranes. These proteins play important roles in a wide variety of bacterial physiological processes, including virulence, and induce innate immune reactions by functioning as ligands of the mammalian Toll-like receptor 2. We review recent advances in our understanding of bacterial lipoprotein structure, biosynthesis and structure-function relationships between bacterial lipoproteins and Toll-like receptor 2. Notably, 40 years after the first report of the triacyl structure of Braun's lipoprotein in Escherichia coli, recent intensive MS-based analyses have led to the discovery of three new lipidated structures of lipoproteins in monoderm bacteria: the lyso, N-acetyl and peptidyl forms. Moreover, the bacterial lipoprotein structure is considered to be constant in each bacterium; however, lipoprotein structures in Staphylococcus aureus vary between the diacyl and triacyl forms depending on the environmental conditions. Thus, the lipidation state of bacterial lipoproteins, particularly in monoderm bacteria, is more complex than previously assumed. [ABSTRACT FROM AUTHOR]

Bacterial lipoproteins are known to be diacylated or triacylated and activate mammalian immune cells via Toll-like receptor 2/6 or 2/1 heterodimer. Because the genomes of low G+C content Gram-positive bacteria, such as Staphylococcus aureus, do not contain Escherichia coli-type apolipoprotein N-acyltransferase, an enzyme converting diacylated lipoproteins into triacylated forms, it has been widely believed that native lipoproteins of S. aureus are diacylated. However, we recently demonstrated that one lipoprotein SitC purified from S. aureus RN4220 strain was triacylated. Almost simultaneously, another group reported that another lipoprotein SA2202 purified from S. aureus SA113 strain was diacylated. The determination of exact lipidated structures of S. aureus lipoproteins is thus crucial for elucidating the molecular basis of host-microorganism interactions. Toward this purpose, we intensively used MS-based analyses. Here, we demonstrate that SitC lipoprotein of S. aureus RN4220 strain has two lipoprotein lipase-labile O-esterified fatty acids and one lipoprotein lipase-resistant fatty acid. Further MS/MS analysis of the lipoprotein lipase digest revealed that the lipoprotein lipase-resistant fatty acid was acylated to α-amino group of the N-terminal cysteine residue of SitC. Triacylated forms of SitC with various length fatty acids were also confirmed in cell lysate of the RN4220 and Triton X-114 phase in three other S. aureus strains, including SA113 strain and one Staphylococcus epidermidis strain. Moreover, four other major lipoproteins including SA2202 in S. aureus strains were identified as N-acylated. These results strongly suggest that lipoproteins of S. aureus are mainly in the N-acylated triacyl form. The determination of exact lipidated structures of Staphylococcus aureus lipoproteins is crucial for elucidating the molecular basis of the interaction of host microorganisms. Based on analytical data of lipoprotein lipase treatment and tandem mass spectrometry (MS/MS), we demonstrate that major lipoproteins of S. aureus are mainly in the N-acylated triacyl form, suggesting that S. aureus has an unidentified enzyme catalyzing N-acylation of diacylated lipoproteins. [ABSTRACT FROM AUTHOR]

To elucidate the biochemical activation mechanism of the insect pro-phenoloxidase (pro-PO) system, we purified a 45-kDa protein to homogeneity from the hemolymph of Tenebrio molitor (mealworm) larvae, and cloned its cDNA. The overall structure of the 45-kDa protein is similar to Drosophila masquerade serine proteinase homologue, which is an essential component in Drosophila muscle development. This Tenebrio masquerade-like serine proteinase homologue (Tm-mas) contains a trypsin-like serine proteinase domain in the C-terminal region, except for the substitution of Ser to Gly at the active site triad, and a disulfide-knotted domain at the amino-terminal region. When the purified 45-kDa Tm-mas was incubated with CM-Toyopearl eluate solution containing pro-PO and other pro-PO activating factors, the resulting phenoloxidase (PO) activity was shown to be independent of Ca2+ . This suggests that the purified 45-kDa Tm-mas is an activated form of pro-PO activating factor. The55-kDa zymogen form of Tm-mas was detected in the hemolymph when PO activity was not evident. However, when Tenebrio hemolymph was incubated with Ca2+ , a 79-kDa Tenebrio pro-PO and the 55-kDa zymogen Tm-mas converted to 76-kDa PO and 45-kDa Tm-mas, respectively, with detectable PO activity. Furthermore, when Tenebrio hemolymph was incubated with Ca2+ and β-1,3-glucan, the conversion of pro-PO to PO and the 55-kDa zymogen Tm-mas to the 45-kDa protein, was faster than in the presence of Ca2+ only. These results suggest that the cleavage of the 55-kDa zymogen of Tm-mas by a limited proteolysis is necessary for PO activity, and the Tm-mas is a pro-PO activating cofactor. [ABSTRACT FROM AUTHOR]

Previously, we reported the molecular cloning of cDNA for the prophenoloxidase activating factor-I (PPAF-I) that encoded a member of the serine proteinase group with a disulfide-knotted motif at the N-terminus and a trypsin-like catalytic domain at the C-terminus [Lee, S.Y., Cho, M.Y., Hyun, J.H., Lee, K.M., Homma, K.I., Natori, S., Kawabata, S.I., Iwanaga, S. & Lee, B.L. (1998) Eur. J. Biochem. 257, 615–621]. PPAF-I is directly involved in the activation of pro-phenoloxidase (pro-PO) by limited proteolysis and the overall structure is highly similar to that of Drosophila easter serine protease, an essential serine protease zymogen for pattern formation in normal embryonic development. Here, we report purification and molecular cloning of cDNA for another 45-kDa novel PPAF from the hemocyte lysate of Holotrichia diomphalia larvae. The gene encodes a serine proteinase homologue consisting of 415 amino-acid residues with a molecular mass of 45 256 Da. The overall structure of the 45-kDa protein is similar to that of masquerade, a serine proteinase homologue expressed during embryogenesis, larval, and pupal development in Drosophila melanogaster. The 45-kDa protein contained a trypsin-like serine proteinase domain at the C-terminus, except for the substitution of Ser of the active site triad to Gly and had a disulfide-knotted domain at the N-terminus. A highly similar 45-kDa serine proteinase homologue was also cloned from the larval cDNA library of another coleopteran, Tenebrio molitor. By in vitro reconstitution experiments, we found that the purified 45-kDa serine proteinase homologue, the purified active PPAF-I and the purified pro-PO were necessary for expressing phenoloxidase activity in the Holotrichia pro-PO system. However, incubation of pro-PO with either PPAF-I or 45-kDa protein, no phenoloxidase activity was observed. Interestingly, when the 45-kDa protein was incubated with PPAF-I and pro-PO in the absence, but not in the presence of... [ABSTRACT FROM AUTHOR]

One of the biological functions of activated phenoloxidase in arthropods is the synthesis of melanin around invaded foreign materials. However, little is known about how activated phenoloxidase synthesizes melanin at the molecular level. Even though it has been suggested that the quinone derivatives generated by activated phenoloxidase might use endogenous protein components for melanin synthesis in arthropods, there is no report of protein components engaged in melanin synthesis induced by activated phenoloxidase. In this study, to isolate and characterize proteins involved in melanin synthesis, we prepared in vitro prophenoloxidase activating solution (designated G-100 solution), specifically showing phenoloxidase activity in the presence of Ca2+ and β-1,3-glucan, from the hemolymph of larvae of the coleopteran Tenebrio molitor by using a Sephadex G-100 column. When G-100 solution was incubated with dopamine to induce melanin synthesis in the presence of Ca2+ and β-1,3-glucan, four types of protein (160 kDa, prophenoloxidase, phenoloxidase and 45 kDa) disappeared from SDS/PAGE under reducing conditions. Under identical conditions, but including phenylthiourea as a phenoloxidase inhibitor added to the G-100 solution, three of these proteins (160 kDa, phenoloxidase and 45 kDa) did not disappear. To characterize these melanization-engaging proteins, we first purified the 160-kDa melanization-engaging protein to homogeneity and raised a polyclonal antibody against it. Analysis of the cDNA revealed that it consisted of 1439 amino-acid residues and showed partial homology with Caenorhabditis elegans vitellogenin precursor-6 (19.7%). Western blot analysis showed that it disappeared when active phenoloxidase induced melanin synthesis. Furthermore, when the purified 160-kDa melanization-engaging protein was added to a G-100 solution deficient in it, melanin synthesis was enhanced compared with the same solution without the protein. These... [ABSTRACT FROM AUTHOR]

Previously, we identified two pro-phenol oxidase-activating factors, named PPAF-I and PPAF-II, directly involved in the activation of the purified pro-phenol oxidase (pro-PO) from the hemolymph of the coleopteran, Holotrichia diomphalia larvae [Lee, S. Y., Kwon, T. H., Hyun, J. H., Choi, J. S., Kawabata, S. I., Iwanga, S, & Lee, B. L. (1998) Eur. J. Biochem. 254, 90-97]. Here, we report molecular cloning of cDNA for PPAF-I. Based on the sequence of the cloned cDNA, the PPAF-I gene appears to encode a member of serine protease zymogen consisting of 365 amino acid residues with a molecular mass of 40 193 Da. The 109 amino acid residues preceding the amino-terminus Ile residue of the mature protein seem to constitute a prepro-sequence. The mature protein is a serine protease composed of 256 amino acids with a calculated molecular mass of 28 009 Da. The overall structure is highly similar to that of Drosophila easter serine protease (42.9 % identity), an essential serine protease zymogen for pattern formation in normal embryonic development. The locations of disulfide linkages in the pro-segment of PPAF-I were similar to those of Tachypleus proclotting enzyme and the mammalian neutrophil-derived defensin. Furthermore, [3H]diisopropylphosphate (iPr2P)-labeled PPAF-I was specifically produced from the crude preparation of PPAF-I zymogen by incubation with lipopolysaccharide or 1,3-β-glucan, whereas [3H]iPr2P-labeled PPAF-I was not produced under the same conditions in the absence of these microbial polysaccharides. These results indicate that the pro-PO-activation system in H. diomphalia larvae may proceed with the activation of PPAF-I zymogen by microbial polysaccharides. [ABSTRACT FROM AUTHOR]

Previously, we purified and characterized a pro-phenol-oxidase (pro-PO) of 79 kDa from coleopteran insect, Holotrichia diomphalia larvae [Kwon et al. (1997) Mol. Cells 7, 90-97]. Here, we describe the identification of two pro-PO-activating factors (PPAF), named PPAF-I and PPAF-II, directly involved in the activation of the isolated pro-PO. When pro-PO was incubated with either PPAF-I or PPAF-II, no phenol oxidase activity was observed. However, incubation of pro-PO with both PPAF-I and PPAF-II specifically exhibited phenol oxidase activity. The purified PPAF-I with a molecular mass of 33 kDa on SDS/PAGE had characteristics of a serine protease. It exhibited amidase activity against fluorogenic peptide substrates, tert-butoxycarbonyl-phenylalanyl-seryl-arginyl-4-methylcoumaryl-7-amide being the best among the substrates examined. The activity was completely inhibited by 0.02 mM p-nitrophenyl-p′-guanidinobenzoate ⨰ HCl and diisopropylflurophosphate. The NH2-terminal sequence of PPAF-I had significant sequence similarity to those of serine proteases. On the other hand, the purified PPAF-II had a molecular mass of 40 kDa on SDS/PAGE and 400 kDa determined by gel filtration, indicating an oligomeric protein. The NH2-terminal sequence of PPAF-II showed no similarity to known proteins. PPAF-II exhibited no amidase activity against the fluorogenic substrates. Reconstitution experiments and immunoblotting analysis using affinity-purified antibody against pro-PO demonstrated that PPAF-I first cleaves the intact pro-PO to an intermediate of 76 kDa with no phenol oxidase activity, and then, PPAF-I converts the intermediate to the active phenol oxidase of 60 kDa in the presence of PPAF-II. These results indicate that the activation of pro-PO system in hemolymph of H. diomphalia larvae is accomplished by at least two activating factors, a serine protease and a protein cofactor. [ABSTRACT FROM AUTHOR]

The Toll signalling pathway, which is crucial for innate immunity, is transduced in insect haemolymph via a proteolytic cascade consisting of three serine proteases. The proteolytic cascade is downregulated by a specific serine protease inhibitor (serpin). Recently, the serpin SPN48 was found to show an unusual specific reactivity towards the terminal serine protease, Spätzle-processing enzyme, in the beetle Tenebrio molitor. In this study, the mature form of SPN48 was overexpressed in Escherichia coli and purified. The purified SPN48 protein was crystallized using 14% polyethylene glycol 8000 and 0.1 M 2-( N-morpholino)ethanesulfonic acid pH 6.0 as the precipitant. The crystals diffracted X-rays to 2.1 Å resolution and were suitable for structure determination. The crystals belonged to space group P21. The crystal structure will provide information regarding how SPN48 achieves its unusual specificity for its target protease. [ABSTRACT FROM AUTHOR]

Adhesion of nanoparticles (NPs) to the bacterial cell wall by modifying their physicochemical properties can improve the antibacterial activity of antibiotic. In this study, we prepared positively charged clindamycin-loaded poly (lactic-co-glycolic acid)-polyethylenimine (PLGA-PEI) nanoparticles (Cly/PPNPs) and negatively charged clindamycin-loaded PLGA NPs (Cly/PNPs) and investigated the effect of NP adhesion to bacteria on the treatment of methicillin-resistant Staphylococcus aureus (MRSA)-infected wounds. The Cly/PPNPs and Cly/PNPs were characterized according to particle size, polydispersity index, surface charge, and drug loading. Both Cly/PPNPs and Cly/PNPs exhibited sustained drug release over 2 days. The Cly/PPNPs bind to the MRSA surface, thereby enhancing bactericidal efficacy against MRSA compared with the Cly/PNPs. Furthermore, compared with other groups, Cly/PPNPs significantly accelerated the healing and re-epithelialization of wounds in a mouse model of a MRSA-infected wounds. We also found that both NPs are harmless to healthy fibroblast cells. Therefore, our results suggest that the Cly/PPNPs developed in this study improve the efficacy of clindamycin for the treatment of MRSA-infected wounds. [ABSTRACT FROM AUTHOR]

CpG-DNA activates various immune cells, contributing to the host defense against bacteria. Here, we examined the biological function of CpG-DNA in the production of bacteria-reactive antibodies. The administration of CpG-DNA increased survival in mice following infection with methicillin-resistant S. aureus and protected immune cell populations in the peritoneal cavity, bone marrow, and spleen. CpG-DNA injection likewise increased bacteria-reactive antibodies in the mouse peritoneal fluid and serum, which was dependent on TLR9. B cells isolated from the peritoneal cavity produced bacteria-reactive antibodies in vitro following CpG-DNA administration that enhanced the phagocytic activity of the peritoneal cells. The bacteria-reactive monoclonal antibody enhanced phagocytosis in vitro and protected mice after S. aureus infection. Therefore, we suggest that CpG-DNA enhances the antibacterial activity of the immune system by protecting immune cells and triggering the production of bacteria-reactive antibodies. Consequently, we believe that monoclonal antibodies could aid in the treatment of antibiotic-resistant bacterial infections. [ABSTRACT FROM AUTHOR]