Defensin Resistance Research Articles

The Pseudomonas aeruginosa 1244 pilin glycan increases susceptibility to human beta defensin 2 but enhances twitching motility.

SHANIA DAVIS & JOSEPH HORZEMPA, Department of Biomedical Sciences, West LibertyUniversity, West Liberty, WV USA. The Pseudomonas aeruginosa 1244 pilin glycan increasessusceptibility to human beta defensin 2 but enhances twitching motility. Pseudomonas aeruginosa is an ESKAPE (highly drug resistant) pathogen that is commonlyacquired during hospital stays. This bacterium produces type IV pili which are adhesins andmotor appendages that mediate a surface motility referred to as “twitching.” These pili arepolymers of protein subunits referred to as pilin. The pilin subunit of P. aeruginosa 1244 isglycosylated. Previous studies have shown that the pilin glycan affects the efficiency oftwitching motility under certain conditions and the surface polarity of these fibers. Becausemodulation of the bacterial surface charge has been shown to mediate defensin resistance inother organisms, we tested whether pilin glycosylation affected sensitivity to human betadefensin 2 (HBD-2). Interestingly, the isogenic mutant strain lacking the pilin glycan (1244G7)showed increased resistance to HBD-2 compared to wild type bacteria and those completelylacking a type IV pilus (1244.47). This increased resistance to HBD-2 could explain data from arecent study in which various P. aeruginosa clinical strains were isolated that had pilinglycosylation defects. We also confirmed and extended previous findings that indicated that the1244 pilin glycan enhances twitching motility and we showed that this is especially true onpositively charged surfaces (poly L-lysine coated plastic). However, the positively chargedsurface was also capable of enhancing twitching of strains producing non-glycosylated pili or piliwith an incomplete glycan (1244.2.1).

Phosphoethanolamine Transferase LptA in Haemophilus ducreyi Modifies Lipid A and Contributes to Human Defensin Resistance In Vitro.

Haemophilus ducreyi resists the cytotoxic effects of human antimicrobial peptides (APs), including α-defensins, β-defensins, and the cathelicidin LL-37. Resistance to LL-37, mediated by the sensitive to antimicrobial peptide (Sap) transporter, is required for H. ducreyi virulence in humans. Cationic APs are attracted to the negatively charged bacterial cell surface. In other gram-negative bacteria, modification of lipopolysaccharide or lipooligosaccharide (LOS) by the addition of positively charged moieties, such as phosphoethanolamine (PEA), confers AP resistance by means of electrostatic repulsion. H. ducreyi LOS has PEA modifications at two sites, and we identified three genes (lptA, ptdA, and ptdB) in H. ducreyi with homology to a family of bacterial PEA transferases. We generated non-polar, unmarked mutants with deletions in one, two, or all three putative PEA transferase genes. The triple mutant was significantly more susceptible to both α- and β-defensins; complementation of all three genes restored parental levels of AP resistance. Deletion of all three PEA transferase genes also resulted in a significant increase in the negativity of the mutant cell surface. Mass spectrometric analysis revealed that LptA was required for PEA modification of lipid A; PtdA and PtdB did not affect PEA modification of LOS. In human inoculation experiments, the triple mutant was as virulent as its parent strain. While this is the first identified mechanism of resistance to α-defensins in H. ducreyi, our in vivo data suggest that resistance to cathelicidin LL-37 may be more important than defensin resistance to H. ducreyi pathogenesis.

Molecular interactions and immune responses between Maize fine streak virus and the leafhopper vector Graminella nigrifrons through differential expression and RNA interference.

Graminella nigrifrons is the only known vector for Maize fine streak virus (MFSV). In this study, we used real-time quantitative PCR to compare the expression profiles of transcripts that putatively function in the insect immune response: four peptidoglycan recognition proteins (PGRP-SB1, -SD, -LC and LB), Toll, spaetzle, defensin, Dicer-2 (Dcr-2), Argonaut-2 (Ago-2) and Arsenic resistance protein 2 (Ars-2). Except for PGRP-LB and defensin, transcripts involved in humoral pathways were significantly suppressed in G.nigrifrons fed on MFSV-infected maize. The abundance of three RNA interference (RNAi) pathway transcripts (Dcr-2, Ago-2, Ars-2) was significantly lower in nontransmitting relative to transmitting G.nigrifrons. Injection with double-stranded RNA (dsRNA) encoding segments of the PGRP-LC and Dcr-2 transcripts effectively reduced transcript levels by 90 and 75% over 14 and 22 days, respectively. MFSV acquisition and transmission were not significantly affected by injection of either dsRNA. Knock-down of PGRP-LC resulted in significant mortality (greater than 90%) at 27 days postinjection, and resulted in more abnormal moults relative to those injected with Dcr-2 or control dsRNA. The use of RNAi to silence G.nigrifrons transcripts will facilitate the study of gene function and pathogen transmission, and may provide approaches for developing novel targets of RNAi-based pest control.

Host defense peptide resistance contributes to colonization and maximal intestinal pathology by Crohn's disease-associated adherent-invasive Escherichia coli.

Host defense peptides secreted by colonocytes and Paneth cells play a key role in innate host defenses in the gut. In Crohn's disease, the burden of tissue-associated Escherichia coli commonly increases at epithelial surfaces where host defense peptides concentrate, suggesting that this bacterial population might actively resist this mechanism of bacterial killing. Adherent-invasive E. coli (AIEC) is associated with Crohn's disease; however, the colonization determinants of AIEC in the inflamed gut are undefined. Here, we establish that host defense peptide resistance contributes to host colonization by Crohn's-associated AIEC. We identified a plasmid-encoded genomic island (called PI-6) in AIEC strain NRG857c that confers high-level resistance to α-helical cationic peptides and α- and β-defensins. Deletion of PI-6 sensitized strain NRG857c to these host defense molecules, reduced its competitive fitness in a mouse model of infection, and attenuated its ability to induce cecal pathology. This phenotype is due to two genes in PI-6, arlA, which encodes a Mig-14 family protein implicated in defensin resistance, and arlC, an OmpT family outer membrane protease. Implicit in these findings are new bacterial targets whose inhibition might limit AIEC burden and disease in the gut.

Deletion of mtrC in Haemophilus ducreyi Increases Sensitivity to Human Antimicrobial Peptides and Activates the CpxRA Regulon

Haemophilus ducreyi resists killing by antimicrobial peptides encountered during human infection, including cathelicidin LL-37, α-defensins, and β-defensins. In this study, we examined the role of the proton motive force-dependent multiple transferable resistance (MTR) transporter in antimicrobial peptide resistance in H. ducreyi. We found a proton motive force-dependent effect on H. ducreyi's resistance to LL-37 and β-defensin HBD-3, but not α-defensin HNP-2. Deletion of the membrane fusion protein MtrC rendered H. ducreyi more sensitive to LL-37 and human β-defensins but had relatively little effect on α-defensin resistance. The mtrC mutant 35000HPmtrC exhibited phenotypic changes in outer membrane protein profiles, colony morphology, and serum sensitivity, which were restored to wild type by trans-complementation with mtrC. Similar phenotypes were reported in a cpxA mutant; activation of the two-component CpxRA regulator was confirmed by showing transcriptional effects on CpxRA-regulated genes in 35000HPmtrC. A cpxR mutant had wild-type levels of antimicrobial peptide resistance; a cpxA mutation had little effect on defensin resistance but led to increased sensitivity to LL-37. 35000HPmtrC was more sensitive than the cpxA mutant to LL-37, indicating that MTR contributed to LL-37 resistance independent of the CpxRA regulon. The CpxRA regulon did not affect proton motive force-dependent antimicrobial peptide resistance; however, 35000HPmtrC had lost proton motive force-dependent peptide resistance, suggesting that the MTR transporter promotes proton motive force-dependent resistance to LL-37 and human β-defensins. This is the first report of a β-defensin resistance mechanism in H. ducreyi and shows that LL-37 resistance in H. ducreyi is multifactorial.

The Bacterial Defensin Resistance Protein MprF Consists of Separable Domains for Lipid Lysinylation and Antimicrobial Peptide Repulsion

Many bacterial pathogens achieve resistance to defensin-like cationic antimicrobial peptides (CAMPs) by the multiple peptide resistance factor (MprF) protein. MprF plays a crucial role in Staphylococcus aureus virulence and it is involved in resistance to the CAMP-like antibiotic daptomycin. MprF is a large membrane protein that modifies the anionic phospholipid phosphatidylglycerol with l-lysine, thereby diminishing the bacterial affinity for CAMPs. Its widespread occurrence recommends MprF as a target for novel antimicrobials, although the mode of action of MprF has remained incompletely understood. We demonstrate that the hydrophilic C-terminal domain and six of the fourteen proposed trans-membrane segments of MprF are sufficient for full-level lysyl-phosphatidylglycerol (Lys-PG) production and that several conserved amino acid positions in MprF are indispensable for Lys-PG production. Notably, Lys-PG production did not lead to efficient CAMP resistance and most of the Lys-PG remained in the inner leaflet of the cytoplasmic membrane when the large N-terminal hydrophobic domain of MprF was absent, indicating a crucial role of this protein part. The N-terminal domain alone did not confer CAMP resistance or repulsion of the cationic test protein cytochrome c. However, when the N-terminal domain was coexpressed with the Lys-PG synthase domain either in one protein or as two separate proteins, full-level CAMP resistance was achieved. Moreover, only coexpression of the two domains led to efficient Lys-PG translocation to the outer leaflet of the membrane and to full-level cytochrome c repulsion, indicating that the N-terminal domain facilitates the flipping of Lys-PG. Thus, MprF represents a new class of lipid-biosynthetic enzymes with two separable functional domains that synthesize Lys-PG and facilitate Lys-PG translocation. Our study unravels crucial details on the molecular basis of an important bacterial immune evasion mechanism and it may help to employ MprF as a target for new anti-virulence drugs.

Antibacterial activity of human defensins on anaerobic intestinal bacterial species: a major role of HBD-3

Defensins are natural mucosal antimicrobial peptides and their broad spectrum activity against aerobic or facultative anaerobic bacteria has been well investigated. The aim of this study was to systematically examine the antibacterial activity of the small intestinal Paneth cell derived α-defensin HD5 and the major colonic β-defensins HBD-1–3 against strict anaerobic intestinal bacteria. The antibacterial activity was assessed with a flow cytometric assay employing a membrane potential sensitive dye as marker for loss of cell viability. The majority of the tested strains belonging to the dominant anaerobe genera of the gut, Bacteroides and Parabacteroides, were only minimally affected by the constitutively expressed defensins HD5 and HBD-1. The inducible defensin HBD-2 had a limited antibacterial effect, whereas the inducible HBD-3 exhibited potent activity against most strains. The effect of HBD-3 on Bacteroides sp. appeared to be dependent on the presence of oxygen. Bacteroides fragilis strains isolated from blood during bacteremia or from extraintestinal infections were more resistant to HBD-3 than strains from the physiological gut flora. Thus, defensin resistance is not only species- but also strain-specific and may be clinically relevant in the host–bacteria interaction influencing mucosal translocation and systemic infection.

Mechanisms of Decreased Susceptibility to β-Defensins byTreponema denticola

Treponema denticola, a periodontal pathogen, is relatively resistant to human beta-defensins, which are small cationic antimicrobial peptides produced by a number of cells, including the gingival epithelium. Using two independent methods, we previously demonstrated that T. denticola proteases are not responsible for decreased vulnerability to defensins. In this study, we confirmed that the major outer membrane protease, dentilisin, is not responsible for T. denticola insensitivity to defensins and examined several other possible mechanisms, including reduced binding to the bacterial surface and efflux pump activity. It has been suggested that some bacteria mask their surfaces with serum proteins. T. denticola grown in a serum-free medium did not exhibit increased susceptibility to human beta-defensin 2 and 3 (hbetaD-2 and hbetaD-3, respectively), suggesting that cloaking of the outer surface with host proteins is not involved in defensin resistance. Nonetheless, we demonstrated that T. denticola binds significantly less hbetaD-2 and -3 than susceptible organisms bind, suggesting that the unusual outer membrane composition of T. denticola may discourage cationic peptide binding. Efflux pumps have been shown to mediate resistance to antibiotics and cationic peptides in other bacteria, and their role in T. denticola's relative resistance to beta-defensins was investigated. Three inhibitors of bacterial ATP-binding cassette (ABC) efflux pumps had no effect on T. denticola's susceptibility to hbetaD-2 or -3. In contrast, a proton motive force inhibitor, carbonyl cyanide 3-chlorophenylhydrazone, increased the susceptibility of T. denticola to killing by hbetaD-3, demonstrating a potential role for efflux pumps (other than ABC pumps) in resistance to this peptide. Our data suggest that the combination of decreased defensin binding and efflux of any peptide which enters the cytoplasm may explain T. denticola's relative resistance to human beta-defensins.

Neutral glycolipids of the filamentous fungus Neurospora crassa:altered expression in plant defensin-resistant mutants

To defend themselves against fungal pathogens, plants produce numerous antifungal proteins and peptides, including defensins, some of which have been proposed to interact with fungal cell surface glycosphingolipid components. Although not known as a phytopathogen, the filamentous fungus Neurospora crassa possesses numerous genes similar to those required for plant pathogenesis identified in fungal pathogens (Galagan, J. E., et al. 2003. Nature 422: 859-868), and it has been used as a model for studying plant-phytopathogen interactions targeting fungal membrane components (Thevissen, K., et al. 2003. Peptides. 24: 1705-1712). For this study, neutral glycolipid components were extracted from wild-type and plant defensin-resistant mutant strains of N. crassa. The structures of purified components were elucidated by NMR spectroscopy and mass spectrometry. Neutral glycosphingolipids of both wild-type and mutant strains were characterized as beta-glucopyranosylceramides, but those of the mutants were found with structurally altered ceramides. Although the wild type expressed a preponderance of N-2'-hydroxy-(E)-Delta3-octadecenoate as the fatty-N-acyl component attached to the long-chain base (4E,8E)-9-methyl-4,8-sphingadienine, the mutant ceramides were found with mainly N-2'-hydroxyhexadecanoate instead. In addition, the mutant strains expressed highly increased levels of a sterol glucoside identified as ergosterol-beta-glucoside. The potential implications of these findings with respect to defensin resistance in the N. crassa mutants are discussed.

Induction of β-Defensin Resistance in the Oral AnaerobePorphyromonas gingivalis

Induction of resistance of oral anaerobes to the effects of human beta-defensin 1 (hbetaD-1) to hbetaD-4 was investigated by pretreating cells with either sublethal levels of defensins or environmental factors, followed by a challenge with lethal levels of defensins. Cultures of Porphyromonas gingivalis were (i) pretreated with defensins at 1 ng/ml, (ii) heated to 42 degrees C (heat stress), (iii) exposed to normal atmosphere (oxidative stress), or (iv) exposed to 1 mM hydrogen peroxide (peroxide stress). Samples (10 microl) were distributed among the wells of sterile 384-well plates containing hbetaD-1 to -4 (100 microg/ml). Plates were incubated at 37 degrees C for 36 h in an anaerobe chamber. Growth inhibition was determined by a system that measures the total nucleic acid of a sample with a DNA binding dye. The MICs of the four defensins for P. gingivalis were 3 to 12 microg/ml. We found that sublethal levels of the defensins and heat and peroxide stress, but not oxidative stress, induced resistance to 100 microg of defensin per ml in P. gingivalis. Resistance induced by sublethal levels of hbetaD-2 lasted 90 min, and the resistance induced by each defensin was effective against the other three. Multiple strains exposed to hbetaD-2 all evidenced resistance induction. Defensin resistance is vital to the pathogenic potential of several human pathogens. This is the first report describing the induction of defensin resistance in the oral periodontal pathogen P. gingivalis. Such resistance may have an effect on the ability of oral pathogens to persist in the mouth and to withstand innate human immunity.

Staphylococcus aureus resists human defensins by production of staphylokinase, a novel bacterial evasion mechanism.

Alpha-defensins are peptides secreted by polymorphonuclear cells and provide antimicrobial protection mediated by disruption of the integrity of bacterial cell walls. Staphylokinase is an exoprotein produced by Staphylococcus aureus, which activates host plasminogen. In this study, we analyzed the impact of interaction between alpha-defensins and staphylokinase on staphylococcal growth. We observed that staphylokinase induced extracellular release of alpha-defensins from polymorphonuclear cells. Moreover, a direct binding between alpha-defensins and staphylokinase was shown to result in a complex formation. The biological consequence of this interaction was an almost complete inhibition of the bactericidal effect of alpha-defensins. Notably, staphylokinase with blocked plasminogen binding site still retained its ability to neutralize the bactericidal effect of alpha-defensins. In contrast, a single mutation of a staphylokinase molecule at position 74, substituting lysine for alanine, resulted in a 50% reduction of its alpha-defensin-neutralizing properties. The bactericidal properties of alpha-defensins were tested in 19 S. aureus strains in vitro and in a murine model of S. aureus arthritis. Staphylococcal strains producing staphylokinase were protected against the bactericidal effect of alpha-defensins. When staphylokinase was added to staphylokinase-negative S. aureus cultures, it almost totally abrogated the effect of alpha-defensins. Finally, human neutrophil peptide 2 injected intra-articularly along with bacteria alleviated joint destruction. In this study, we report a new property of staphylokinase, its ability to induce secretion of defensins, to complex bind them and to neutralize their bactericidal effect. Staphylokinase production may therefore be responsible in vivo for defensin resistance during S. aureus infections.

MprF-mediated biosynthesis of lysylphosphatidylglycerol, an important determinant in staphylococcal defensin resistance

Frequently bacteria are exposed to membrane-damaging cationic antimicrobial molecules (CAMs) produced by the host’s immune system (defensins, cathelicidins) or by competing microorganisms (bacteriocins). Staphylococcus aureus achieves CAM resistance by modifying anionic phosphatidylglycerol with positively charged L-lysine, resulting in repulsion of the peptides. Inactivation of the novel S. aureus gene, mprF, which is found in many bacterial pathogens, has resulted in the loss of lysylphosphatidylglycerol (L-PG), increased inactivation by CAM-containing neutrophils, and attenuated virulence. We demonstrate here that expression of mprF is sufficient to confer L-PG production in Escherichia coli, which indicates that MprF represents the L-PG synthase. L-PG biosynthesis was studied in vitro and found to be dependent on phosphatidylglycerol and lysyl-tRNA, two putative substrate molecules. Further addition of cadaverin, a competitive inhibitor of the lysyl-tRNA synthetases, or of RNase A abolished L-PG biosynthesis, thereby confirming the involvement of lysyl-tRNA. This study forms the basis for further detailed analyses of L-PG biosynthesis and its role in bacterial infections.

MP061 MprF-mediated defensin resistance in Staphylococcus aureus

MP061 MprF-mediated defensin resistance in Staphylococcus aureus

Staphylococcus aureus strains lacking D-alanine modifications of teichoic acids are highly susceptible to human neutrophil killing and are virulence attenuated in mice.

Staphylococcus aureus is resistant to alpha-defensins, antimicrobial peptides that play an important role in oxygen-independent killing of human neutrophils. The dlt operon mediates d-alanine incorporation into teichoic acids in the staphylococcal cell envelope and is a determinant of defensin resistance. By using S. aureus wild-type (WT) and Dlt- bacteria, the relative contributions of oxygen-dependent and -independent antimicrobial phagocyte components were analyzed. The Dlt- strain was efficiently killed by human neutrophils even in the absence of a functional respiratory burst, whereas the killing of the WT organism was strongly diminished when the respiratory burst was inhibited. Human monocytes, which do not produce defensins, inactivated the WT and Dlt- bacteria with similar efficiencies. In addition, mice injected with the Dlt- strain had significantly lower rates of sepsis and septic arthritis and fewer bacteria in the kidneys, compared with mice infected with the WT strain.

Staphylococcus aureus resistance to human defensins and evasion of neutrophil killing via the novel virulence factor MprF is based on modification of membrane lipids with l-lysine.

Defensins, antimicrobial peptides of the innate immune system, protect human mucosal epithelia and skin against microbial infections and are produced in large amounts by neutrophils. The bacterial pathogen Staphylococcus aureus is insensitive to defensins by virtue of an unknown resistance mechanism. We describe a novel staphylococcal gene, mprF, which determines resistance to several host defense peptides such as defensins and protegrins. An mprF mutant strain was killed considerably faster by human neutrophils and exhibited attenuated virulence in mice, indicating a key role for defensin resistance in the pathogenicity of S. aureus. Analysis of membrane lipids demonstrated that the mprF mutant no longer modifies phosphatidylglycerol with l-lysine. As this unusual modification leads to a reduced negative charge of the membrane surface, MprF-mediated peptide resistance is most likely based on repulsion of the cationic peptides. Accordingly, inactivation of mprF led to increased binding of antimicrobial peptides by the bacteria. MprF has no similarity with genes of known function, but related genes were identified in the genomes of several pathogens including Mycobacterium tuberculosis, Pseudomonas aeruginosa, and Enterococcus faecalis. MprF thus constitutes a novel virulence factor, which may be of general relevance for bacterial pathogens and represents a new target for attacking multidrug resistant bacteria.

PhoP/PhoQ: macrophage‐specific modulators of Salmonella virulence?

The regulation of gene expression by the two-component regulatory system PhoP/PhoQ is necessary for Salmonella typhimurium survival within macrophages, defensin resistance, acid resistance, and murine typhoid fever pathogenesis. Salmonella experience multiple environments during mammalian infection and survival requires tightly regulated gene expression. After phagocytosis by macrophages, signal transduction by PhoQ results in the transcription of phoP-activated genes (pags) encoding proteins essential to bacterial survival and virulence. One such gene, pagC, encodes an envelope protein with amino acid similarity to an epithelial cell invasion protein of Yersina enterocolitica, Ail, and a bacteriophage lambda outer membrane protein, Lom. The PhoP and PhoQ proteins can also repress the synthesis of proteins, encoded by phoP repressed genes (prgs), when pags are maximally expressed. If prgs encode receptors for toxic compounds, prg repression may protect the cell within macrophages when pag expression is most necessary. At least one prg locus, prgH, is required for full S. typhimurium mouse virulence. Within the macrophage, different environments may stimulate a switch from pag to prg expression that is necessary to Salmonella survival. prg expression may also be necessary for surviving nonmacrophage environments. Study of the PhoP regulon should lead to the discovery of new virulence factors, increase knowledge of how gene regulation is essential to bacterial virulence, and perhaps lead to the development of better vaccines for typhoid fever.

Characterization of defensin resistance phenotypes associated with mutations in the phoP virulence regulon of Salmonella typhimurium

The defensin sensitivities of Salmonella typhimurium strains with mutations in the phoP/phoQ two-component virulence regulon were tested by using purified defensins NP-1 and NP-2. Strains with mutations in either gene of the regulatory pair (phoP [transcriptional activator] or phoQ [membrane sensor kinase]) had increased sensitivities to defensin. The predicted periplasmic domain of the PhoQ protein contained a markedly anionic domain that could interact with cationic proteins and that could be responsible for resistance to defensin. Because insertion mutations in phoP are polar on phoQ, we constructed strains that expressed the PhoQ protein in the absence of PhoP to test whether resistance to defensin requires only the phoQ gene product. We found that resistance to defensin requires the function of both components of this regulatory system, because strains expressing PhoQ without PhoP were still markedly sensitive to defensins. This implied that a pag (phoP-activated gene) product is responsible for defensin resistance. We also tested for the ability of defensins NP-1, NP-5, and HNP-1 to activate pag expression and found that these peptides have no effect. Defensin resistance is not the only virulence characteristic controlled by the PhoP-PhoQ regulon because mutations in pagC, as well as ones in the phoP locus that resulted in constitutive pag activation (phenotype PhoPc), had no effect on defensin resistance, even though they rendered the organism avirulent and deficient in survival within macrophages. The virulence defect conferred by mutations in the phoP-phoQ two-component regulatory system is not completely explained by alterations in resistance to cationic proteins and involves the control of other proteins necessary for S. typhimurium survival within macrophages.