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* Division of Dermatology, Department of Medicine, University of California San Diego and VA San Diego Healthcare Center, San Diego;
Division of Infectious Disease, Department of Pediatrics, University of California San Diego, San Diego, California, USA;
Cellular and Molecular Microbiology Division, Medical Microbiology and Hygiene Department, University of Tubingen, Tubingen, Germany; and
Institute of Basic Medical Sciences, Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Japan
1Correspondence: Mail Code 9111B, 3350 La Jolla Village Dr., San Diego, CA 92161, USA. E-mail: rgallo{at}vapop.ucsd.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: leukocyte recruitment • cathelicidin • AMP activity • dermatitis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. The persistence of AMP activity stands in marked contrast to the human experience with design of effective pharmacologic antibiotics. In common clinical usage, the duration of effectiveness for synthetic antibiotics is very short due to the rapid evolution of various microbial strategies for resistance. Studies in animal models, however, have demonstrated that evolutionarily conserved gene-encoded antibiotics such as cathelicidins and defensins provide protection against microbial infection, while their absence results in increased risk of infectious disease (2
3
4
5)
. Clinical correlations suggest this immune function extends to humans, as patients with impaired epithelial AMP production (e.g., atopic dermatitis) are highly susceptible to secondary infection, while those with increased AMP production (e.g., psoriasis) are relatively protected (6)
.
It has been suggested that the study of AMPs may provide insight into design of more effective antibiotic therapies (1)
. To date this hope has yet to translate into reality. AMPs, such as the human cathelicidin LL-37, are highly membrane active. Their ability to kill bacteria correlates with charge and reflects an ability to disrupt the organized membrane structure of the target organism. A dependence on ionic interaction is confirmed when antimicrobial activity is evaluated in solutions containing salt. The addition of NaCl to microbial culture broth at concentrations as low as 50 mM suppresses the activity of LL-37 and many other AMPs (7
, 8)
. These results led many investigators to conclude that AMPs can only kill microbes in solutions of low ionic strength or when the peptide is expressed at very high local concentrations, observations that contradict experience in several animal models.
An alternative explanation for the function of AMPs is that these peptides have evolved from simple antibiotics to act as signals of injury. Several examples now exist that show mammalian cells respond to AMPs with specific changes in gene expression. These responses include extracellular matrix synthesis (9)
, angiogenesis (10)
, leukocyte recruitment, and cytokine release (11)
. Surface receptors such as FPRL-1 (12)
and CCR6 (13)
may mediate the host cell response to AMPs. Thus, it is possible that the association between AMP expression and infection seen in animal models and human disease may be the result of their ability to augment a cellular immune defense program rather than direct microbicidal action.
This study sought to address the apparent contradictions regarding how AMPs can function as natural antibiotics. The direct bactericidal activity of AMPs was evaluated in vitro in an animal cell culture environment. Our findings show that when select bacteria are grown in mammalian ionic conditions, they respond with an altered gene expression profile that renders them susceptible to AMPs. These observations provide insight into the true parameters of AMP activity, and suggest a novel paradigm for evaluating the function of these peptides in innate immune defense.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Antimicrobial assays
Bacteria were grown at 37°C in tryptic soy broth (TSB) (Sigma, St. Louis, MO, USA) to stationary phase, diluted 1:100 into assay medium, and grown to early log phase. Assay medium contained 20% TSB, 10% fetal bovine serum (FBS), and 150 mM NaCl, unless otherwise noted, together with 70% of additional media or media components. The additional 70% volume used to complete the assay medium included minimal essential media (MEM) (Gibco, Grand Isle, NY, USA); the buffers from MEM, which are 27 mM NaHCO3 and 1.1 mM NaH2PO4; 1 mM NaH2PO4 ± 50 mM NaHCO3. A 50 mM glycine-HCl buffer (Fisher, Tustin, CA, USA) was also used in place of 50 mM NaHCO3 in Fig. 1
c due to its similar buffering capacity. All solutions were adjusted to pH 7.4, unless otherwise noted in the figure legend. Antimicrobial peptides were added to 0.1 mL bacterial suspension containing 
1 x 106 CFU/mL in a 96-well flat bottom plate (Costar, Corning, NY, USA). Plates were incubated at 37°C for the indicated times. Inhibition of bacterial growth was determined by comparing the change in turbidity at OD600 in the presence of peptide to that in the absence of peptide. Minimal inhibitory concentration (MIC) was defined as the concentration of peptide that resulted in no detectable bacterial growth. Bactericidal and bacteristatic activity was determined by plating serial dilutions of bacteria on tryptic soy agar and enumerating colony forming units after overnight incubation at 37°C. The minimal bactericidal concentration was determined as the minimum peptide concentration resulting in no surviving colonies. Statistical significance was determined using GraphPad Prism software (v. 21., GraphPad Software, San Diego, CA, USA). Means were calculated with standard deviation, then a 2-way ANOVA was applied with a Bonferroni post-test. P values <0.001 were considered significant.
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Reverse-transcriptase PCR and quantitative real-time PCR
Approximately 1 x 108 CFU of log phase bacteria were homogenized in 1 mL of TRIzol Reagent (Gibco, Grand Island, NY, USA) in a mini BeadBeater (Biospec Products, Bartlesville, OK, USA) with 0.1 mm glass beads on max power for 5 min and RNA was extracted per TRIzol manufacturer’s directions. RT-PCR was performed on 1.5 µg RNA with Retroscript kit (Ambion, Austin, TX, USA). Briefly, first strand synthesis combined RNA with 0.4 mM dNTPs and 4 µM decamers in 16 µL at 80°C for 3 min. Reaction was iced and 2 µL 10x RT-PCR buffer, 1 µL RNase inhibitor, and 1 µL mMLV RT added followed by incubation at 42°C for 1 h, then denatured at 94°C for 10 min. Real-time (RT) quantitative PCR was performed using an Applied Biosystems 7000 Sequence Detection System (Foster City, CA, USA). Primers were designed for the target genes sigB and fliA, as well as 16 s rRNA as an endogenous control. The sequences of the sigB primers are: F-AAAGATGGTTCAACTGTTACGCTATTAG, R-GGTCATCTTGTTGCCCCATAA. The sequences of the fliA primers are: F-GCGGCATTGGGTTACTTAATG, R-CGTTCCTTGTAGGGCGTCATA. The sequences of the 16 s primers are: F-GTTATCCGGAATTATTGGGCG, R-CCGGGCTTTCACATCAGACT. 5 µL RT reaction was used in SYBR® Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) with 100 nM primers. Thermal profile: 50°C 2 min, 95°C 10 min, 40 x (94°C 15 s, 60°C 1 min). Results were analyzed using the Comparative Ct Method, as described (14)
. Product specificity was confirmed by generating dissociation curves for the reactions.
Membrane permeability
E. coli strain ML-35p, which constitutively expresses a plasmid-encoded periplasmic ß-lactamase and cytoplasmic ß-galactosidase but lacks lactose permease, was used to measure inner and outer membrane permeability. After an overnight incubation at 37°C, the culture was adjusted to 2.5–5 x 107 CFU/mL (A600=0.03) in buffer A (1 mM NaH2PO4, 20% TSB) or B (1 mM NaH2PO4, 20% TSB and 25 mM NaHCO3).
To test permeabilization of the inner membrane, formation o-nitrophenol (ONP) as the hydrolytic product of ß-galactosidase on the colorless substrate o-nitrophenyl-ß-d-galactosidase (ONPG) was determined by an increase in absorbance at 420 nm (A420). ONPG was prepared at a stock concentration of 3 mg/mL in 10 mM sodium phosphate buffer, pH 7.0 (NaPB). To study permeabilization of the outer membrane, we followed ß-lactamase activity by measuring the decrease in A390 due to the substrate nitrocefin and increase of A486 due to the red product. Nitrocefin was prepared at a stock concentration of 500 µg/mL in NaPB with 0.05 % DMSO (DMSO).
The assay mixture contained 43 µL of bacteria from stock cultured in buffer A or B, with final concentrations of 4 µM LL-37, 0.3 mg/mL ONPG or 25 µg/mL nitrocefin. Samples were incubated at 37°C in sterile 96-well microtiter plates (Corning Inc., Corning, NY, USA) and the kinetics of ß-galactosidase or ß-lactamase activity calculated by measurement of the A420 or A390/A486, respectively, with a Spectra max PLUS 384 (Molecular Devices, Sunnyvale, CA, USA). To test bacterial growth, the A600 was also monitored.
As a positive control, 2.5–5 x 107 CFU/mL of E. coli ML-35p was permeabilized with 1 µg/mL of polymyxin B (Calbiochem, La Jolla, CA, USA), 1% Triton X-100 in buffer A or B.
Nuclear membrane permeabilization in S. aureus was also examined by fluorometric measurement. 74 µL of S. aureus cultured as above were dispensed onto each well of a sterile 96-well microtiter plate (Nunc, Roskilde, Denmark). 2 µL of 640 µM LL-37 or H2O and 4 µL of 4 µM Sytox Green (Molecular Probes, Eugene, OR, USA) in 0.08% DMSO were added to each sample and the plates were incubated at 37°C in the dark. Fluorescence emission was measured with a microplate reader (Fluoroskan Ascent FL, Labsystems, Finland) at an excitation wavelength of 485 nm and an emission wavelength of 530 nm. 1 µg/mL polymyxin B, 1% Triton X-100 was used as a positive control.
Isolation and analysis of S. aureus membrane lipids
Bacteria were grown as described above for the antibacterial assay in the presence or absence of carbonate and disrupted using glass beads and a Disintegrator S (Biomatic GmbH, Rodgau, Germany). Lipids were isolated and analyzed as described recently (15)
. Briefly, polar lipids were extracted by the Bligh-Dyer procedure, vacuum dried, and dissolved in chloroform/methanol (2:1, by vol). Equal amounts of lipid extracts were spotted onto silica 60 F_254 HPTLC plates (Merck, Darmstadt, Germany) and developed with chloroform/methanol/water (65:25:4, by vol) in the first direction and chloroform/acetic acid/methanol/water (80:15:12:4, by vol) in the second direction. All lipids were visualized with molybdatophosporic acid spray (Merck) followed by charring at 120°C and treatment with ammonia vapor to improve the contrast. Phospholipids or amino group-containing lipids were selectively stained with Molybdenum Blue (Sigma) or ninhydrin spray (Merck), respectively. The lipid fatty acid composition was analyzed by gas chromatography as described recently (15)
.
Electron microscopy
Electron microscopy was performed on S. aureus Rosenbach grown to log phase in 20% TSB, 1 mM NaH2PO4, with or without addition of 25 mM NaHCO3. 32 µM LL37 or an equal volume of water was added and the bacteria were cultured 4 h. Bacteria were pelleted at 1000 x g for 5 min at 4°C, fixed in Karnovsky's Solution 24 h at 4°C, and processed for EM on a Zeiss EM 10B Transmission Electron Microscope by the VA San Diego Microscopy Core Facility.
Microarray analysis
E. coli was grown, as described earlier, in the presence or absence of NaHCO3 (buffer A or B) for 2 h. The samples for microarray analysis were prepared as described in Affymetrix Gene Chip Expression Analysis Procedure (Affymetrix, Santa Clara, CA, USA). Briefly, after centrifugation of bacteria, RNA extraction was carried out using RNeasy Mini kit (Qiagen, Germantown, MD, USA). Total RNA from four separate experiments was pooled for cDNA synthesis, RNA removed by hot-alkali treatment and cDNA was purified using a Qiaquick PCR Purification kit (Qiagen). cDNA was fragmented by DNase I treatment and terminally labeled using Enzo® BioArrayTM Terminal Labeling Kit with Biotin-ddUTP (Affymetrix, Santa Clara, CA, USA). The target was hybridized onto probe arrays. Arrays were scanned at the UCSD-VA Gene Chip Core using an Affymetrix Genechip 3000 High Resolution Scanner.
Subsequent analysis was performed using GeneSpring software (Silicon Genetics, Redwood City, CA, USA). The raw data were normalized by housekeeping genes, spiked in controls, Total Intensity, Mean Centering, Lowess correction, Chen’s Ratio Statistics, and filtered based on genes being present in at least one condition and having at least a 2-fold change.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 8
, 16)
. However, in animal model systems the expression of these gene products correlates with increased microbial resistance (4
, 17
, 18)
(Lee et al., in press). To resolve the apparent contradiction between observations in animal systems and those measured in vitro, the antimicrobial activity of the human cathelicidin peptide, LL-37, against the pathogen S. aureus was evaluated in animal tissue culture media containing 10% serum and a physiological NaCl concentration. In contrast to the bacteriologic culture media TSB, LL-37 showed significant growth inhibition of S. aureus in MEM media with 10% serum (Fig. 1a
).
To identify the factor in animal cell culture media responsible for increasing antimicrobial activity, the components of MEM were systematically tested. All media tested contained 20% TSB, 150 mM NaCl, and 10% FBS at pH 7.4. 20% TSB was chosen because it allowed robust bacterial growth in the absence of AMPs. The concentration of TSB did not affect the ability of LL-37 to kill S. aureus, as no difference in killing was observed when the assay was performed in MEM containing 0, 5, 10, or 20% TSB (data not shown). The presence of only the salts from MEM, CaCl2, KCl, and MgSO4 was not sufficient to enhance antimicrobial activity, nor were phenol red, amino acids or vitamins present in this medium (data not shown). Addition of the buffering components of MEM (27 mM NaHCO3 and 1.1 mM NaH2PO4,) was the only treatment that showed effects similar to that of complete MEM (Fig. 1b
). Further testing identified the carbonate anion as the active molecule (Fig. 1b
). As a control, similar testing was performed using solutions buffered with either NaHCO3 or glycine-HCl. No increase in antimicrobial activity was seen at any pH in glycine-based buffers (Fig. 1c
), indicating the unique carbonate effect was not simply a reflection of pH stabilization. We also examined whether environmental CO2 could diffuse into the media to potentiate killing. In the absence of carbonate, overnight growth and assay in a 5% CO2 environment did not result in significantly enhanced antimicrobial activity.
Carbonate enhances the antimicrobial activity of a structurally diverse panel of peptides and renders diverse bacteria susceptible
To determine whether the observed carbonate-induced increase in antimicrobial activity applied to peptides other than LL-37, a structurally diverse panel of AMPs was tested (Fig. 1d
). The human and murine cathelicidins LL-37 and mouse cathelin-related antimicrobial peptide (mCRAMP) are both 
-helical, the porcine cathelicidin PR-39 is a proline/arginine-rich linear peptide, and murine -defensin cryptdin-4 (19)
and human ß defensin 2 (hBD-2) (20)
have ß sheet structures. None of the peptides tested had significant activity in the presence of NaCl and serum without carbonate (MIC>64 µM), but in the presence of NaHCO3 all showed significant inhibition of bacterial growth (i.e., LL-37 MIC=4 µM). Of the panel tested here, only the anionic human defense peptide dermcidin did not show increased antimicrobial activity in the presence of carbonate.
Next, to compare the influence of carbonate on S. aureus with enhanced resistance to pharmacologic antibiotics, we tested two MRSA isolates, ATCC 81025 and 81056, and found both demonstrated an increase in susceptibility to LL-37 (MIC>32 µM in the absence of carbonate, and >98% inhibition of 81056 and 100% inhibition of 81025 at 8 µM with carbonate). A similar AMP-enhancing effect was seen with two important Gram-negative human pathogens. Enteroinvasive O29 E. coli were completely inhibited by 16 µM mCRAMP in the presence of carbonate but were resistant to cathelicidin without it. Similarly, Salmonella dublin Lane was inhibited 50% without carbonate, while totally inhibited in its presence (Fig. 1e
).
Carbonate acts on bacteria to render them susceptible
It has been reported that carbonate can affect the -helical structure of LL-37 (21)
. This opens the possibility that the potentiating effects of carbonate could reflect structural changes in AMPs to somehow increase their potency, although the diversity of peptides tested seemed to diminish this possibility. To test whether the effects observed were due to action on the peptide or on the bacteria, we studied the ability of LL-37 to kill S. aureus as a function of the time of exposure to NaHCO3 (Fig. 2
). Bacteria were grown to log phase in the presence or absence of NaHCO3, then washed free of media and resuspended in fresh media with or without NaHCO3 and with or without LL-37. S. aureus grown initially without NaHCO3 were not inhibited by LL-37 even if NaHCO3 was added at the time of LL-37 exposure. In contrast, bacteria grown initially in the presence of NaHCO3 were killed by LL-37 even if carbonate was removed prior to LL-37 addition. Separate experiments demonstrated that bacteria must grow for at least 2 h in carbonate-containing media to show increased susceptibility to LL-37 (data not shown).
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To determine whether altered susceptibility to AMPs correlates with changes in membrane permeability, S. aureus and E. coli membrane permeability was examined directly. S. aureus cultured in carbonate exhibited an increase in permeability to Sytox green in response to LL-37 (Fig. 3
a). This effect was also observed in E. coli by measuring ß-galactosidase release in response to Polymyxin B (Fig. 3b
) or LL-37 (Fig. 3c
). These data suggested that differences in the bacterial cell membrane or in membrane repair and synthesis mechanisms may make them more susceptible to permeabilization by cationic compounds. One important mechanism of bacterial AMP resistance reflects modification of cell wall lipids with cationic substitutions to decrease surface negative charge (15)
. Examination of membrane charge by binding of cytochrome c (22)
and direct analysis of fatty acid and lipid composition of S. aureus showed no significant changes in response to growth in carbonate-containing media (data not shown). However, transmission electron microscopy of S. aureus treated with NaHCO3 did show thinning of the cell wall (Fig. 4
a, c). Subsequent addition of LL-37 produced severe membrane blebbing, disintegration of the cell wall and widespread bacterial lysis (Fig. 4d
).
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Carbonate alters gene expression of key regulatory factors
Our data demonstrated that carbonate enhancement of bacterial AMP susceptibility occurred without alterations in membrane charge, but with potential structural alterations in the cell wall. The potential effects of carbonate on bacterial genes not previously associated with AMP sensitivity was explored by global transcriptional profiling. DNA microarray analysis of E. coli O29 cultured in the presence or absence of NaHCO3 identified changes in the expression of several candidate genes (Table 1
). Notably, > 30 flagellar genes, which have been implicated in virulence in Salmonella and stress response (23)
, were decreased >2-fold by culture in carbonate in E. coli O29. In fact, of the 10 genes with the largest decrease in expression shown in Table 1
, five were flagellar genes. A large decrease in expression was also observed for barA (24)
, which encodes a kinase that controls various virulence factors through the global regulator OmpR (25)
.
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For confirmatory analysis we chose the fliA gene, which encodes a sigma factor that regulates many flagellar genes and can influence bacterial virulence and AMP sensitivity (23)
. Comparison of fliA to sigma factors in S. aureus revealed sequence similarities to the alternative sigma factor, sigB. SigB contributes to S. aureus virulence and is involved in a broad range of stress responses, including control of cell wall thickness and integrity (26
, 27)
. Using quantitative RT-PCR, we verified a 5-fold decrease in E. coli fliA expression and a 10-fold decrease in S. aureus sigB upon growth in carbonate-containing media (Fig. 5
a, b). Thus, one important effect of carbonate upon the bacteria appears to be suppression of global regulatory genes involved in coordinating potential resistance phenotypes.
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To directly test the hypothesis that a decrease in the expression of sigB in S. aureus could contribute to the increase sensitivity to LL-37 observed when cultured in carbonate, we evaluated S. aureus 
sigB mutant (NKSB) for growth inhibition in response to LL-37. We found the sigB mutant NKSB to be more susceptible to LL-37 during early growth periods from 2 to 8 h post-inoculum (Fig. 5c
). Calculation of MIC in this period showed MIC of 16 µM for NKSB compared with wild-type parent strain MIC of >64 µM. However, unlike S. aureus grown in carbonate-containing buffers, the sigB mutant increase in sensitivity was transient.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Many of these molecules are thought to be integral mediators of host barrier defense, including the cathelicidins and defensins, whose family members have been described in most human epithelia (29)
. AMPs are either constitutively expressed at locations where initial interaction with potential invading microbes occurs or induced upon recognition of injury. The in vivo significance of several of these peptides has been demonstrated in animal models, yet many of these molecules appear to be inactive, or minimally active, when antimicrobial activity is directly evaluated in vitro using physiologic salt concentrations or serum (7)
(21)
. This has lead to a debate as to the true relevance of these so-called "natural antibiotics" to immune defense. Here we demonstrate that bacterial susceptibility is significantly enhanced in the mammalian ionic environment in which AMPs have evolved their defense role.
Carbonate is a ubiquitous molecule in many microenvironments of the body, including blood, sweat, and gastrointestinal, urogenital, and respiratory tracts. Secretions containing carbonate at the concentrations evaluated in this study are generated from or bathe AMP-expressing tissues such as skin, gut, airway epithelia, and neutrophils (30
31
32)
. Our findings show that when grown in the presence of carbonate, both Gram-positive and Gram-negative bacteria may show dramatically increased AMP sensitivity. For example, the major human pathogen S. aureus has been described as resistant to mammalian AMPs, with an MIC to many cathelicidins and defensins of >32 µM (8)
. In mouse studies, the concentration of cathelicidin in wounds has been estimated as only between 3 and 10 µM (33)
. Our data now show that in the presence of carbonate at physiologic concentrations S. aureus is suppressed by the murine cathelicidin mCRAMP at 8 µM, within the effective concentration range found in vivo.
The AMP-enhancing effect of carbonate is due to alterations in bacterial susceptibility. Carbonate enhances activity of a broad range of antimicrobials including polymyxin and peptides with -helical, ß-sheet, and linear structures. Carbonate does not need to be present in the media at the same time as the AMP to produce increased bacterial susceptibility, suggesting its effects are independent of any potential changes in the structural conformation of AMPs as previously reported. Culture of S. aureus and E. coli in the presence of carbonate increased the ability of LL-37 and polymyxin B to produce membrane permeabilization, indicating the anion has a deleterious effect on the bacteria’s ability to resist external stresses that threaten its membrane integrity.
Bacterial growth in the presence of carbonate appears to produce significant changes in gene expression that correspond to the enhanced AMP sensitivity phenotype. In the Gram-negative E. coli, no difference in growth rate was observed upon carbonate treatment, but a global alteration in gene expression was seen with more than 300 gene transcripts altered >2-fold. Among the genes with significantly decreased expression was the global regulator fliA, as confirmed by quantitative RT-PCR analysis. Analysis of the Gram-positive S. aureus response to carbonate also showed marked changes in transcript abundance, including genes involved in virulence, stress response, and cell wall maintenance. Sharing homology to fliA, the S. aureus global regulatory gene sigB was also significantly suppressed when the Gram-positive pathogen was grown in the presence of carbonate.
In earlier investigations, deletion of sigB restored methicillin susceptibility in resistant strains of S. aureus (34)
, while overexpression of sigB increased cell wall thickness as well as resistance to cell wall antibiotics (27)
. Ultrastructural analysis of S. aureus grown in the presence of carbonate shows a decrease in cell wall thickness that corresponded to the enhanced susceptibility to AMP mediated lysis. Since SigB mutants of S. aureus are known to have decreased cell wall thickness, this suggests that functional phenotypic changes observed in our study in response to carbonate may be partly linked to sigB. However, the deletion of sigB did not confer the same level of susceptibility as culture of the parental strain in carbonate-containing media. Moreover, carbonate further enhanced the susceptibility of sigB-deficient bacteria to LL-37 (data not shown). This indicates that additional effects beyond suppression of sigB expression are involved in the AMP-enhancing properties of carbonate. Our data indicate that investigation of families of genes that are involved in the control of cell wall synthesis and repair may be fruitful in understanding microbial resistance systems and how native antimicrobial defense systems can function effectively.
The discovery that carbonate enhances microbial susceptibility to AMPs is important from many perspectives. First, it reconciles the apparent paradox described between the demonstrated in vivo innate immune function of AMPs and their lack of in vitro antimicrobial activity. The presence of carbonate in mammalian tissues may dictate alterations in bacterial gene expression and cell wall integrity that render them more susceptible to innate immune defense molecules. This phenomenon may also represent a mechanism to maintain susceptible microbial populations. AMPs are evolutionarily conserved, predicting their presence in the environment for millennia, yet bacteria have only developed incomplete resistance. The low level of carbonate in external reservoirs for microbial growth ensures that in these settings no survival advantage exists for the further development of AMP resistance.
These observations also provide an alternate paradigm for antimicrobial discovery and suggest that analysis of microbial susceptibility to compounds should be performed with organisms grown in an environment corresponding to the mammalian host for which they represent a potential pathogen. Potential new classes of effective antimicrobials may be discovered by this approach. Compounds that may have otherwise been deemed ineffective during in vitro screening may be effective in the animal. This approach would represent a major change in the continuing struggle for development of effective antibiotics in the setting of rapidly evolving antibiotic resistance, and would exploit the immune defense strategy evolved by natural antibiotic peptides.
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ACKNOWLEDGMENTS |
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Received for publication May 27, 2005. Accepted for publication October 5, 2005.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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