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1.  Targeted Killing of Streptococcus mutans by a Pheromone-Guided “Smart” Antimicrobial Peptide 
Antimicrobial Agents and Chemotherapy  2006;50(11):3651-3657.
Within the repertoire of antibiotics available to a prescribing clinician, the majority affect a broad range of microorganisms, including the normal flora. The ecological disruption resulting from antibiotic treatment frequently results in secondary infections or other negative clinical consequences. To address this problem, our laboratory has recently developed a new class of pathogen-selective molecules, called specifically (or selectively) targeted antimicrobial peptides (STAMPs), based on the fusion of a species-specific targeting peptide domain with a wide-spectrum antimicrobial peptide domain. In the current study, we focused on achieving targeted killing of Streptococcus mutans, a cavity-causing bacterium that resides in a multispecies microbial community (dental plaque). In particular, we explored the possibility of utilizing a pheromone produced by S. mutans, namely, the competence stimulating peptide (CSP), as a STAMP targeting domain to mediate S. mutans-specific delivery of an antimicrobial peptide domain. We discovered that STAMPs constructed with peptides derived from CSP were potent against S. mutans grown in liquid or biofilm states but did not affect other oral streptococci tested. Further studies showed that an 8-amino-acid region within the CSP sequence is sufficient for targeted delivery of the antimicrobial peptide domain to S. mutans. The STAMPs presented here are capable of eliminating S. mutans from multispecies biofilms without affecting closely related noncariogenic oral streptococci, indicating the potential of these molecules to be developed into “probiotic” antibiotics which could selectively eliminate pathogens while preserving the protective benefits of a healthy normal flora.
PMCID: PMC1635210  PMID: 17060534
2.  Targeted Antimicrobial Therapy Against Streptococcus mutans Establishes Protective Non-cariogenic Oral Biofilms and Reduces Subsequent Infection 
Dental biofilms are complex communities composed largely of harmless bacteria. Certain pathogenic species including Streptococcus mutans (S. mutans) can become predominant when host factors such as dietary sucrose intake imbalance the biofilm ecology. Current approaches to control S. mutans infection are not pathogen-specific and eliminate the entire oral community along with any protective benefits provided. Here, we tested the hypothesis that removal of S. mutans from the oral community through targeted antimicrobial therapy achieves protection against subsequent S. mutans colonization.
Controlled amounts of S. mutans were mixed with S. mutans-free saliva, grown into biofilms and visualized by antibody staining and cfu quantization. Two specifically-targeted antimicrobial peptides (STAMPs) against S. mutans were tested for their ability to reduce S. mutans biofilm incorporation upon treatment of the inocula. The resulting biofilms were also evaluated for their ability to resist subsequent exogenous S. mutans colonization.
S. mutans colonization was considerably reduced (9 ± 0.4 fold reduction, P=0.01) when the surface was preoccupied with saliva-derived biofilms. Furthermore, treatment with S. mutans-specific STAMPs yielded S. mutans-deficient biofilms with significant protection against further S. mutans colonization (5 minutes treatment: 38 ± 13 fold reduction P=0.01; 16 hours treatment: 96 ± 28 fold reduction P=0.07).
S. mutans infection is reduced by the presence of existing biofilms. Thus maintaining a healthy or “normal” biofilm through targeted antimicrobial therapy (such as the STAMPs) could represent an effective strategy for the treatment and prevention of S. mutans colonization in the oral cavity and caries progression.
PMCID: PMC3733586  PMID: 20737932
targeted antimicrobial therapy; antimicrobial peptide; biofilm; Streptococcus mutans; protective colonization; caries
3.  Adding Selectivity to Antimicrobial Peptides: Rational Design of a Multidomain Peptide against Pseudomonas spp. 
Currently available antimicrobials exhibit broad killing with regard to bacterial genera and species. Indiscriminate killing of microbes by these conventional antibiotics can disrupt the ecological balance of the indigenous microbial flora, often resulting in negative clinical consequences. Species-specific antimicrobials capable of precisely targeting pathogenic bacteria without damaging benign microorganisms provide a means of avoiding this problem. In this communication, we report the successful creation of the first synthetic, target-specific antimicrobial peptide, G10KHc, via addition of a rationally designed Pseudomonas-specific targeting moiety (KH) to a generally killing peptide (novispirin G10). The resulting chimeric peptide showed enhanced bactericidal activity and faster killing kinetics against Pseudomonas spp. than G10 alone. The enhanced killing activities are due to increased binding and penetration of the outer membrane of Pseudomonas sp. cells. These properties were not observed in tests of untargeted bacterial species, and this specificity allowed G10KHc to selectively eliminate Pseudomonas spp. from mixed cultures. This work lays a foundation for generating target-specific “smart” antimicrobials to complement currently available conventional antibiotics.
PMCID: PMC1426969  PMID: 16569868
4.  Systematic Approach to Optimizing Specifically Targeted Antimicrobial Peptides against Streptococcus mutans▿  
Previously we reported a novel strategy of “targeted killing” through the design of narrow-spectrum molecules known as specifically targeted antimicrobial peptides (STAMPs) (R. Eckert et al., Antimicrob. Agents Chemother. 50:3651-3657, 2006; R. Eckert et al., Antimicrob. Agents Chemother. 50:1480-1488, 2006). Construction of these molecules requires the identification and the subsequent utilization of two conjoined yet functionally independent peptide components: the targeting and killing regions. In this study, we sought to design and synthesize a large number of STAMPs targeting Streptococcus mutans, the primary etiologic agent of human dental caries, in order to identify candidate peptides with increased killing speed and selectivity compared with their unmodified precursor antimicrobial peptides (AMPs). We hypothesized that a combinatorial approach, utilizing a set number of AMP, targeting, and linker regions, would be an effective method for the identification of STAMPs with the desired level of activity. STAMPs composed of the Sm6 S. mutans binding peptide and the PL-135 AMP displayed selectivity at MICs after incubation for 18 to 24 h. A STAMP where PL-135 was replaced by the B-33 killing domain exhibited both selectivity and rapid killing within 1 min of exposure and displayed activity against multispecies biofilms grown in the presence of saliva. These results suggest that potent and selective STAMP molecules can be designed and improved via a tunable “building-block” approach.
PMCID: PMC2863653  PMID: 20211885
5.  Selective Membrane Disruption: Mode of Action of C16G2, a Specifically Targeted Antimicrobial Peptide ▿ 
The specifically targeted antimicrobial peptide (STAMP) C16G2 was developed to target the cariogenic oral pathogen Streptococcus mutans. Because the design of this peptide was novel, we sought to better understand the mechanism through which it functioned. Compared to antimicrobial peptides (AMPs) with wide spectra of activity, the STAMP C16G2 has demonstrated specificity for S. mutans in a mixed-culture environment, resulting in the complete killing of S. mutans while having minimal effect on the other streptococci. In the current study, we sought to further confirm the selectivity of C16G2 and also compare its membrane activity to that of melittin B, a classical toxic AMP, in order to determine the STAMP's mechanism of cell killing. Disruption of S. mutans cell membranes by C16G2 was demonstrated by increased SYTOX green uptake and ATP efflux from the cells similar to those of melittin B. Treatment with C16G2 also resulted in a loss of membrane potential as measured by DiSC(3)5 fluorescence. In comparison, the individual moieties of C16G2 demonstrated no specificity and limited antimicrobial activity compared to those of the STAMP C16G2. The data suggest that C16G2 has a mechanism of action similar to that of traditional AMPs and kills S. mutans through disruption of the cell membrane, allowing small molecules to leak out of the cell, which is followed by a loss of membrane potential and cell death. Interestingly, this membrane activity is rapid and potent against S. mutans, but not other noncariogenic oral streptococci.
PMCID: PMC3122425  PMID: 21518845
6.  Identification of a Novel Antimicrobial Peptide from Human Hepatitis B Virus Core Protein Arginine-Rich Domain (ARD) 
PLoS Pathogens  2013;9(6):e1003425.
The rise of multidrug-resistant (MDR) pathogens causes an increasing challenge to public health. Antimicrobial peptides are considered a possible solution to this problem. HBV core protein (HBc) contains an arginine-rich domain (ARD) at its C-terminus, which consists of 16 arginine residues separated into four clusters (ARD I to IV). In this study, we demonstrated that the peptide containing the full-length ARD I–IV (HBc147-183) has a broad-spectrum antimicrobial activity at micro-molar concentrations, including some MDR and colistin (polymyxin E)-resistant Acinetobacter baumannii. Furthermore, confocal fluorescence microscopy and SYTOX Green uptake assay indicated that this peptide killed Gram-negative and Gram-positive bacteria by membrane permeabilization or DNA binding. In addition, peptide ARD II–IV (HBc153-176) and ARD I–III (HBc147-167) were found to be necessary and sufficient for the activity against P. aeruginosa and K. peumoniae. The antimicrobial activity of HBc ARD peptides can be attenuated by the addition of LPS. HBc ARD peptide was shown to be capable of direct binding to the Lipid A of lipopolysaccharide (LPS) in several in vitro binding assays. Peptide ARD I–IV (HBc147-183) had no detectable cytotoxicity in various tissue culture systems and a mouse animal model. In the mouse model by intraperitoneal (i.p.) inoculation with Staphylococcus aureus, timely treatment by i.p. injection with ARD peptide resulted in 100-fold reduction of bacteria load in blood, liver and spleen, as well as 100% protection of inoculated animals from death. If peptide was injected when bacterial load in the blood reached its peak, the protection rate dropped to 40%. Similar results were observed in K. peumoniae using an IVIS imaging system. The finding of anti-microbial HBc ARD is discussed in the context of commensal gut microbiota, development of intrahepatic anti-viral immunity and establishment of chronic infection with HBV. Our current results suggested that HBc ARD could be a new promising antimicrobial peptide.
Author Summary
Antibiotics-resistant pathogens have been a major problem to our public health. Recently, in our studies of human hepatitis B virus (HBV), we accidentally discovered potent and broad spectrum antimicrobial peptides from HBV core protein (HBc) arginine-rich domain (ARD). The peptides are mainly composed of SPRRR repeats and are effective against both Gram-positive and Gram-negative bacteria, as well as fungi. We found different bactericidal mechanisms of the ARD peptides, which involved LPS binding, DNA binding and membrane permeabilization in various tested bacteria, such as P. aeruginosa, K. pneumoniae, E. coli and S. aureus. We also found that this ARD peptide was effective for colistin-resistant A. baumannii. The peptides exhibited no hemolysis activity to human red blood cells and no cytotoxicity to human hepatoma cells and kidney cells. Furthermore, the ARD peptide was shown to be safe and protective in the animal model. Recently, intestinal flora was found to influence the development of immunity. We discussed here the potential involvement of the antimicrobial activity of HBc ARD in the establishment of HBV chronic infection in the newborns. We proposed here that the HBc ARD peptides could serve as an alternative to the conventional antibiotics in clinical medicine.
PMCID: PMC3681751  PMID: 23785287
7.  Novel Riboswitch Ligand Analogs as Selective Inhibitors of Guanine-Related Metabolic Pathways 
PLoS Pathogens  2010;6(4):e1000865.
Riboswitches are regulatory elements modulating gene expression in response to specific metabolite binding. It has been recently reported that riboswitch agonists may exhibit antimicrobial properties by binding to the riboswitch domain. Guanine riboswitches are involved in the regulation of transport and biosynthesis of purine metabolites, which are critical for the nucleotides cellular pool. Upon guanine binding, these riboswitches stabilize a 5′-untranslated mRNA structure that causes transcription attenuation of the downstream open reading frame. In principle, any agonistic compound targeting a guanine riboswitch could cause gene repression even when the cell is starved for guanine. Antibiotics binding to riboswitches provide novel antimicrobial compounds that can be rationally designed from riboswitch crystal structures. Using this, we have identified a pyrimidine compound (PC1) binding guanine riboswitches that shows bactericidal activity against a subgroup of bacterial species including well-known nosocomial pathogens. This selective bacterial killing is only achieved when guaA, a gene coding for a GMP synthetase, is under the control of the riboswitch. Among the bacterial strains tested, several clinical strains exhibiting multiple drug resistance were inhibited suggesting that PC1 targets a different metabolic pathway. As a proof of principle, we have used a mouse model to show a direct correlation between the administration of PC1 and the reduction of Staphylococcus aureus infection in mammary glands. This work establishes the possibility of using existing structural knowledge to design novel guanine riboswitch-targeting antibiotics as powerful and selective antimicrobial compounds. Particularly, the finding of this new guanine riboswitch target is crucial as community-acquired bacterial infections have recently started to emerge.
Author Summary
During the last 30 years, bacterial resistance to antibiotics has become a major problem. This situation is partly because today's antibiotics are mainly based on a limited selection of chemical scaffolds, which makes it easier for bacterial pathogens to quickly develop resistance against new drug derivatives. This recurrent problem of multiple drug resistance implies a constant need to search for novel microbial targets and to modulate their activity using artificial molecules. Riboswitches are newly discovered gene regulatory elements that represent attractive targets for antimicrobial drugs. Riboswitches are RNA structures located in untranslated regions of messenger RNAs that regulate the expression of genes involved in the transport and metabolism of small metabolites. We have identified a new antibiotic specifically targeting riboswitches found in a subgroup of bacteria including Staphylococcus aureus and Clostridium difficile, which are nosocomial pathogens responsible for a significant mortality rate in hospitals, and increased health care costs. The riboswitch controls the expression of guaA that appears essential for virulence in the mammalian host. A murine model was used as a proof of principle to show that such an antibiotic could inhibit the growth of S. aureus in a mammal. Our work provides new insights into the discovery and design of novel antimicrobial agents against bacterial pathogens.
PMCID: PMC2858708  PMID: 20421948
8.  Single molecule resolution of the antimicrobial action of quantum dot-labeled sushi peptide on live bacteria 
BMC Biology  2009;7:22.
Antimicrobial peptides are found in all kingdoms of life. During the evolution of multicellular organisms, antimicrobial peptides were established as key elements of innate immunity. Most antimicrobial peptides are thought to work by disrupting the integrity of cell membranes, causing pathogen death. As antimicrobial peptides target the membrane structure, pathogens can only acquire resistance by a fundamental change in membrane composition. Hence, the evolution of pathogen resistance has been a slow process. Therefore antimicrobial peptides are valuable alternatives to classical antibiotics against which multiple drug-resistant bacteria have emerged. For potential therapeutic applications as antibiotics a thorough knowledge of their mechanism of action is essential. Despite the increasingly comprehensive understanding of the biochemical properties of these peptides, the actual mechanism by which antimicrobial peptides lyse microbes is controversial.
Here we investigate how Sushi 1, an antimicrobial peptide derived from the horseshoe crab (Carcinoscorpius rotundicauda), induces lysis of Gram-negative bacteria. To follow the entire process of antimicrobial action, we performed a variety of experiments including transmission electron microscopy and fluorescence correlation spectroscopy as well as single molecule tracking of quantum dot-labeled antimicrobial peptides on live bacteria. Since in vitro measurements do not necessarily correlate with the in vivo action of a peptide we developed a novel fluorescent live bacteria lysis assay. Using fully functional nanoparticle-labeled Sushi 1, we observed the process of antimicrobial action at the single-molecule level.
Recently the hypothesis that many antimicrobial peptides act on internal targets to kill the bacterium has been discussed. Here, we demonstrate that the target sites of Sushi 1 are outer and inner membranes and are not cytosolic. Further, our findings suggest four successive steps of the bactericidal process: 1) Binding, mediated mainly by charged residues in the peptide; 2) Peptide association, as peptide concentration increases evidenced by a change in diffusive behavior; 3) Membrane disruption, during which lipopolysaccharide is not released; and 4) Lysis, by leakage of cytosolic content through large membrane defects.
PMCID: PMC2685782  PMID: 19432949
9.  Discerning the Complexity of Community Interactions Using a Drosophila Model of Polymicrobial Infections 
PLoS Pathogens  2008;4(10):e1000184.
A number of human infections are characterized by the presence of more than one bacterial species and are defined as polymicrobial diseases. Methods for the analysis of the complex biological interactions in mixed infections with a large number of microorganisms are limited and do not effectively determine the contribution of each bacterial species to the pathogenesis of the polymicrobial community. We have developed a novel Drosophila melanogaster infection model to study microbe–microbe interactions and polymicrobe–host interactions. Using this infection model, we examined the interaction of 40 oropharyngeal isolates with Pseudomonas aeruginosa. We observe three classes of microorganisms, one of which acts synergistically with the principal pathogen, while being avirulent or even beneficial on its own. This synergy involves microbe–microbe interactions that result in the modulation of P. aeruginosa virulence factor gene expression within infected Drosophila. The host innate immune response to these natural-route polymicrobial infections is complex and characterized by additive, suppressive, and synergistic transcriptional activation of antimicrobial peptide genes. The polymicrobial infection model was used to differentiate the bacterial flora in cystic fibrosis (CF) sputum, revealing that a large proportion of the organisms in CF airways has the ability to influence the outcome of an infection when in combination with the principal CF pathogen P. aeruginosa.
Author Summary
Bacterial infections often involve more than one species. The lung disease of cystic fibrosis (CF) patients provides examples of polymicrobial infections whereby diverse and dynamic microbial communities are a characteristic of CF airways. The significance of microbe–microbe interactions and the interplay of the communities with the host have not been thoroughly investigated. We describe a novel Drosophila model to discern the biological interactions between microbes within microbial communities, as well as the interactions between the communities and the innate immune system. Using fly survival as a readout of relevant interactions, we show that mixed infections may additively or synergistically enhance the pathogenicity of a microbial community. The polymicrobial infection model was used to differentiate the bacterial flora in CF sputum, revealing that a large proportion of the organisms in CF airways has the ability to influence the outcome of an infection when in combination with the principal CF pathogen Pseudomonas aeruginosa. We show that during the synergistic-type mixed infections, P. aeruginosa virulence gene expression is altered within live Drosophila compared to mono-species infections. The immune response to microbial communities takes many forms and can include synergistic activation of antimicrobial peptide gene expression. We postulate that the biological interactions exposed using this model may contribute to the transition from chronic stable infections to acute pulmonary exacerbation infections in CF.
PMCID: PMC2566602  PMID: 18949036
10.  Clinical Efficacy of a Specifically Targeted Antimicrobial Peptide Mouth Rinse: Targeted Elimination of Streptococcus mutans and Prevention of Demineralization 
Caries Research  2011;45(5):415-428.
Streptococcus mutans, the major etiological agent of dental caries, has a measurable impact on domestic and global health care costs. Though persistent in the oral cavity despite conventional oral hygiene, S. mutans can be excluded from intact oral biofilms through competitive exclusion by other microorganisms. This suggests that therapies capable of selectively eliminating S. mutans while limiting the damage to the normal oral flora might be effective long-term interventions to fight cariogenesis. To meet this challenge, we designed C16G2, a novel synthetic specifically targeted antimicrobial peptide with specificity for S. mutans. C16G2 consists of a S. mutans-selective ‘targeting region’ comprised of a fragment from S. mutans competence stimulation peptide (CSP) conjoined to a ‘killing region’ consisting of a broad-spectrum antimicrobial peptide (G2). In vitro studies have indicated that C16G2 has robust efficacy and selectivity for S. mutans, and not other oral bacteria, and affects targeted bacteria within seconds of contact.
In the present study, we evaluated C16G2 for clinical utility in vitro, followed by a pilot efficacy study to examine the impact of a 0.04% (w/v) C16G2 rinse in an intra-oral remineralization/demineralization model.
Results and Conclusions
C16G2 rinse usage was associated with reductions in plaque and salivary S. mutans, lactic acid production, and enamel demineralization. The impact on total plaque bacteria was minimal. These results suggest that C16G2 is effective against S. mutans in vivo and should be evaluated further in the clinic.
PMCID: PMC3169368  PMID: 21860239
Antimicrobial; Antimicrobial peptide; Caries; Demineralization; Dental plaque; Lactic acid; Mouth rinse; Oral therapeutic; Selective antibiotic; Selective therapeutic; Specifically targeted antimicrobial peptide; Streptococcus mutans; Targeted antimicrobial
11.  Enhancement of Antimicrobial Activity against Pseudomonas aeruginosa by Coadministration of G10KHc and Tobramycin▿  
Antimicrobial Agents and Chemotherapy  2006;50(11):3833-3838.
Pseudomonas aeruginosa is a common opportunistic human pathogen that is associated with life-threatening acute infections and chronic airway colonization during cystic fibrosis. Previously, we converted the wide-spectrum antimicrobial peptide novispirin G10 into a selectively-targeted antimicrobial peptide (STAMP), G10KHc. Compared to novispirin G10, the STAMP had an enhanced ability to kill Pseudomonas mendocina. In this study, we explored the activity of G10KHc against P. aeruginosa. G10KHc was found to be highly active (as active as tobramycin) against P. aeruginosa clinical isolates. Most interestingly, we observed a synergistic-like enhancement in killing activity when biofilms and planktonic cultures of P. aeruginosa were cotreated with G10KHc and tobramycin. The data indicate that the mechanism of enhanced activity may involve increased tobramycin uptake due to G10KHc-mediated cell membrane disruption. These results suggest that G10KHc may be useful against P. aeruginosa during acute and chronic infection states, especially when it is coadministered with tobramycin.
PMCID: PMC1635211  PMID: 16940063
12.  Targeted antimicrobial activity of a specific IgG–SMAP28 conjugate against Porphyromonas gingivalis in a mixed culture 
Antimicrobial peptides coupled to a ligand, receptor or antibody for a specific pathogenic bacteria could be used to develop narrow-spectrum pharmaceuticals with ‘targeted’ antimicrobial activity void of adverse reactions often associated with the use of broad-spectrum antibiotics. To assess the feasibility of this approach, in this study sheep myeloid antimicrobial peptide (SMAP) 28 was linked to affinity- and protein G-purified rabbit immunoglobulin G (IgG) antibodies specific to the outer surface of Porphyromonas gingivalis strain 381. The selective activity of the P. gingivalis IgG–SMAP28 conjugate was then assessed by adding it to an artificially generated microbial community containing P. gingivalis, Aggregatibacter actinomycetemcomitans and Peptostreptococcus micros. The specificity of the P. gingivalis IgG–SMAP28 conjugate in this mixed culture was concentration-dependent. The conjugate at 50 μg protein/mL lacked specificity and killed P. gingivalis, A. actinomycetemcomitans and P. micros. The conjugate at 20 μg protein/mL was more specific and killed P. gingivalis. This is an initial step to develop a selective antimicrobial agent that can eliminate a specific periodontal pathogen, such as P. gingivalis, from patients with periodontal disease without harming the normal commensal flora.
PMCID: PMC3169388  PMID: 18778918
Porphyromonas gingivalis; Aggregatibacter actinomycetemcomitans; Peptostreptococcus micros; Cathelicidins; Targeted antimicrobial activity; SMAP28
13.  Integrative genome-scale metabolic analysis of Vibrio vulnificus for drug targeting and discovery 
Chromosome 1 of Vibrio vulnificus tends to contain larger portion of essential or housekeeping genes on the basis of the genomic analysis and gene knockout experiments performed in this study, while its chromosome 2 seems to have originated and evolved from a plasmid.The genome-scale metabolic network model of V. vulnificus was reconstructed based on databases and literature, and was used to identify 193 essential metabolites.Five essential metabolites finally selected after the filtering process are 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine (AHHMP), D-glutamate (DGLU), 2,3-dihydrodipicolinate (DHDP), 1-deoxy-D-xylulose 5-phosphate (DX5P), and 4-aminobenzoate (PABA), which were predicted to be essential in V. vulnificus, absent in human, and are consumed by multiple reactions.Chemical analogs of the five essential metabolites were screened and a hit compound showing the minimal inhibitory concentration (MIC) of 2 μg/ml and the minimal bactericidal concentration (MBC) of 4 μg/ml against V. vulnificus was identified.
Discovering new antimicrobial targets and consequently new antimicrobials is important as drug resistance of pathogenic microorganisms is becoming an increasingly serious problem in human healthcare management (Fischbach and Walsh, 2009). There clearly exists a gap between genomic studies and drug discovery as the accumulation of knowledge on pathogens at genome level has not successfully transformed into the development of effective drugs (Mills, 2006; Payne et al, 2007). In this study, we dissected the genome of a microbial pathogen in detail, and subsequently developed a systems biological strategy of employing genome-scale metabolic modeling and simulation together with metabolite essentiality analysis for effective drug targeting and discovery. This strategy was used for identifying new drug targets in an opportunistic pathogen Vibrio vulnificus CMCP6 as a model.
V. vulnificus is a Gram-negative halophilic bacterium that is found in estuarine waters, brackish ponds, or coastal areas, and its Biotype 1 is an opportunistic human pathogen that can attack immune-compromised patients, and causes primary septicemia, necrotized wound infections, and gastroenteritis. We previously found that many metabolic genes were specifically induced in vivo, suggesting that specific metabolic pathways are essential for in vivo survival and virulence of this pathogen (Kim et al, 2003; Lee et al, 2007). These results motivated us to carry out systems biological analysis of the genome and the metabolic network for new drug target discovery.
V. vulnificus CMCP6 has two chromosomes. We first re-sequenced genomic regions assembled in low quality and low depth, and subsequently re-annotated the whole genome of V. vulnificus. Horizontal gene transfer was suspected to be responsible for the diversification of each chromosome of V. vulnificus, and the presence of metabolic genes was more biased to chromosome 1 than chromosome 2. Further studies on V. vulnificus genome revealed that chromosome 2 is more prone to diversification for better adaptation to the environment than its chromosome 1, while chromosome 1 tends to expand their genetic repertoire while maintaining the core genes at a constant level.
Next, a genome-scale metabolic network VvuMBEL943 was reconstructed based on literature, databases and experiments for systematic studies on the metabolism of this pathogen and prediction of drug targets. The VvuMBEL943 model is composed of 943 reactions and 765 metabolites, and covers 673 genes. The model was validated by comparing its simulated cell growth phenotype obtained by constraints-based flux analysis with the V. vulnificus-specific experimental data previously reported in the literature. In this study, constraints-based flux analysis is an optimization-based simulation method that calculates intracellular fluxes under the specific genetic and environmental condition (Kim et al, 2008). As a result, 17 growth phenotypes were correctly predicted out of 18 cases, which demonstrate the validity of VvuMBEL943.
The main objective of constructing VvuMBEL943 in this study is to predict potential drug targets by system-wide analysis of the metabolic network for the effective treatment of V. vulnificus. To achieve this goal, a set of drug target candidates was predicted by taking a metabolite-centric approach. Metabolite essentiality analysis is a concept recently introduced for the study of cellular robustness to complement conventional reaction or gene-centric approach (Kim et al, 2007b). Metabolite essentiality analysis observes changes in flux distribution by removing each metabolite from the in silico metabolic network. Hence, metabolite essentiality predicts essential metabolites whose absence causes cell death. By selecting essential metabolites, it is possible to directly screen only their structural analogs, which substantially reduces the number of chemical compounds to screen from the chemical compound library. As a result of implementing this approach, 193 metabolites were initially identified to be essential to the cell. These essential metabolites were then further filtered based on the predetermined criteria, mainly organism specificity and multiple connectivity associated with each metabolite, in order to reduce the number of initial target candidates towards identifying the most effective ones.
Five essential metabolites finally selected are 2-amino-4-hydroxy-6-hydroxymethyl-7,8-dihydropteridine (AHHMP), D-glutamate (DGLU), 2,3-dihydrodipicolinate (DHDP), 1-deoxy-D-xylulose 5-phosphate (DX5P), and 4-aminobenzoate (PABA). Enzymes that consume these essential metabolites were experimentally verified to be essential, which indeed demonstrates the essentiality of these five metabolites. On the basis of the structural information of these five essential metabolites, whole-cell screening assay was performed using their analogs for possible antibacterial discovery. We screened 352 chemical analogs of the essential metabolites selected from the chemical compound library, and found a hit compound 24837, which shows the minimal inhibitory concentration (MIC) of 2 μg/ml and minimal bactericidal concentration (MBC) of 4 μg/ml, showing good antibacterial activity without further structural modification. Although this study demonstrates a proof-of-concept, the approaches and their rationale taken here should serve as a general strategy for discovering novel antibiotics and drugs based on systems-level analysis of metabolic networks.
Although the genomes of many microbial pathogens have been studied to help identify effective drug targets and novel drugs, such efforts have not yet reached full fruition. In this study, we report a systems biological approach that efficiently utilizes genomic information for drug targeting and discovery, and apply this approach to the opportunistic pathogen Vibrio vulnificus CMCP6. First, we partially re-sequenced and fully re-annotated the V. vulnificus CMCP6 genome, and accordingly reconstructed its genome-scale metabolic network, VvuMBEL943. The validated network model was employed to systematically predict drug targets using the concept of metabolite essentiality, along with additional filtering criteria. Target genes encoding enzymes that interact with the five essential metabolites finally selected were experimentally validated. These five essential metabolites are critical to the survival of the cell, and hence were used to guide the cost-effective selection of chemical analogs, which were then screened for antimicrobial activity in a whole-cell assay. This approach is expected to help fill the existing gap between genomics and drug discovery.
PMCID: PMC3049409  PMID: 21245845
drug discovery; drug targeting; genome analysis; metabolic network; Vibrio vulnificus
14.  Achieving Probiotic Effects via Modulating Oral Microbial Ecology 
Advances in dental research  2009;21(1):53-56.
Unlike many pathogens are foreign invaders, oral “pathogens” such as Streptococcus mutans are part of the “normal” oral microbial flora. While they express certain pathogenic properties, the balance of synergistic and antagonistic interactions determines whether these çommensal pathogens cause damage or not. Recognition of these microbial ecology based pathogeneses argues for new strategies for disease treatment and prevention.
Probiotics, potentially beneficial live bacteria or yeasts, have been used to combat dental caries. This includes the application of S. mutans types that cannot produce acids or other bacteria that interfere with the pathogenic effects of S. mutans. While these approaches show therapeutic effects against S. mutans experimentally, the conversion into commercial products remains a challenge, due to safety and shelf life issues. New high-tech approaches, such as quorum sensing interference of pathogenic bacteria or targeted antimicrobial therapies, offer novel ways to achieve probiotic effects against dental caries.
PMCID: PMC2777612  PMID: 19710082
15.  Implication of the Mosquito Midgut Microbiota in the Defense against Malaria Parasites 
PLoS Pathogens  2009;5(5):e1000423.
Malaria-transmitting mosquitoes are continuously exposed to microbes, including their midgut microbiota. This naturally acquired microbial flora can modulate the mosquito's vectorial capacity by inhibiting the development of Plasmodium and other human pathogens through an unknown mechanism. We have undertaken a comprehensive functional genomic approach to elucidate the molecular interplay between the bacterial co-infection and the development of the human malaria parasite Plasmodium falciparum in its natural vector Anopheles gambiae. Global transcription profiling of septic and aseptic mosquitoes identified a significant subset of immune genes that were mostly up-regulated by the mosquito's microbial flora, including several anti-Plasmodium factors. Microbe-free aseptic mosquitoes displayed an increased susceptibility to Plasmodium infection while co-feeding mosquitoes with bacteria and P. falciparum gametocytes resulted in lower than normal infection levels. Infection analyses suggest the bacteria-mediated anti-Plasmodium effect is mediated by the mosquitoes' antimicrobial immune responses, plausibly through activation of basal immunity. We show that the microbiota can modulate the anti-Plasmodium effects of some immune genes. In sum, the microbiota plays an essential role in modulating the mosquito's capacity to sustain Plasmodium infection.
Author Summary
The Anopheles gambiae mosquito that transmits the malaria-causing parasite Plasmodium has an intestinal bacterial flora, or microbiota, which comprises a variety of species. Elimination of this microbiota with antibiotic treatment will render the Anopheles mosquito more susceptible to Plasmodium infection. In this study we show that these bacteria can inhibit the infection of the mosquito with the human malaria parasite Plasmodium falciparum through a mechanism that involves the mosquito's immune system. Our study suggests that the microbial flora of mosquitoes is stimulating a basal immune activity, which comprises several factors with known anti-Plasmodium activity. The same immune factors that are needed to control the mosquito's microbiota are also defending against the malaria parasite Plasmodium. This complex interplay among the mosquito's microbiota, the innate immune system, and the Plasmodium parasite may have significant implications for the transmission of malaria in the field where the bacterial exposure of mosquitoes may differ greatly between ecological niches.
PMCID: PMC2673032  PMID: 19424427
16.  Prevalence of Local Immune Response against Oral Infection in a Drosophila/Pseudomonas Infection Model 
PLoS Pathogens  2006;2(6):e56.
Pathogens have developed multiple strategies that allow them to exploit host resources and resist the immune response. To study how Drosophila flies deal with infectious diseases in a natural context, we investigated the interactions between Drosophila and a newly identified entomopathogen, Pseudomonas entomophila. Flies orally infected with P. entomophila rapidly succumb despite the induction of both local and systemic immune responses, indicating that this bacterium has developed specific strategies to escape the fly immune response. Using a combined genetic approach on both host and pathogen, we showed that P. entomophila virulence is multi-factorial with a clear differentiation between factors that trigger the immune response and those that promote pathogenicity. We demonstrate that AprA, an abundant secreted metalloprotease produced by P. entomophila, is an important virulence factor. Inactivation of aprA attenuated both the capacity to persist in the host and pathogenicity. Interestingly, aprA mutants were able to survive to wild-type levels in immune-deficient Relish flies, indicating that the protease plays an important role in protection against the Drosophila immune response. Our study also reveals that the major contribution to the fly defense against P. entomophila is provided by the local, rather than the systemic immune response. More precisely, our data points to an important role for the antimicrobial peptide Diptericin against orally infectious Gram-negative bacteria, emphasizing the critical role of local antimicrobial peptide expression against food-borne pathogens.
Normal feeding and digestion involves the ingestion of many microorganisms. Many are innocuous, some are commensal, and others may be pathogenic. Eukaryotes have thus evolved complex mechanisms to detect, control, and if necessary, eliminate intestinal microbes. Insects are no exception, and the fruit fly, Drosophila, employs a physical barrier within the intestinal lumen and the peritrophic matrix, and an innate immune response which exhibits similarities to the mammalian counterpart. Pseudomonas entomophila was identified as a novel entomopathogenic bacterium that can infect and colonize the gut of Drosophila. In this paper, Liehl et al. describe one specific secreted virulence factor of P. entomophila, the zinc metalloprotease, AprA, which they demonstrate to be required for defense against the host gut epithelial immune response. AprA defends P. entomophila against the Drosophila antimicrobial peptides, produced by the gut innate immune response. P. entomophila aprA mutants are attenuated for virulence in wild-type Drosophila but are equally infective as wild-type bacteria in immune-deficient mutant flies that do not express these antimicrobial peptides. Although secreted proteases have previously been described as a potentially important defense against host immune proteins, this is one of the rare examples of an in vivo demonstration of such a specific role against insect antimicrobial peptides.
PMCID: PMC1475658  PMID: 16789834
17.  The Oct1 homolog Nubbin is a repressor of NF-κB-dependent immune gene expression that increases the tolerance to gut microbiota 
BMC Biology  2013;11:99.
Innate immune responses are evolutionarily conserved processes that provide crucial protection against invading organisms. Gene activation by potent NF-κB transcription factors is essential both in mammals and Drosophila during infection and stress challenges. If not strictly controlled, this potent defense system can activate autoimmune and inflammatory stress reactions, with deleterious consequences for the organism. Negative regulation to prevent gene activation in healthy organisms, in the presence of the commensal gut flora, is however not well understood.
We show that the Drosophila homolog of mammalian Oct1/POU2F1 transcription factor, called Nubbin (Nub), is a repressor of NF-κB/Relish-driven antimicrobial peptide gene expression in flies. In nub1 mutants, which lack Nub-PD protein, excessive expression of antimicrobial peptide genes occurs in the absence of infection, leading to a significant reduction of the numbers of cultivatable gut commensal bacteria. This aberrant immune gene expression was effectively blocked by expression of Nub from a transgene. We have identified an upstream regulatory region, containing a cluster of octamer sites, which is required for repression of antimicrobial peptide gene expression in healthy flies. Chromatin immunoprecipitation experiments demonstrated that Nub binds to octamer-containing promoter fragments of several immune genes. Gene expression profiling revealed that Drosophila Nub negatively regulates many genes that are involved in immune and stress responses, while it is a positive regulator of genes involved in differentiation and metabolism.
This study demonstrates that a large number of genes that are activated by NF-κB/Relish in response to infection are normally repressed by the evolutionarily conserved Oct/POU transcription factor Nub. This prevents uncontrolled gene activation and supports the existence of a normal gut flora. We suggest that Nub protein plays an ancient role, shared with mammalian Oct/POU transcription factors, to moderate responses to immune challenge, thereby increasing the tolerance to biotic stress.
PMCID: PMC3849502  PMID: 24010524
Antimicrobial peptides; Drosophila; Gene regulation; Host-pathogen interaction; Immune signaling; Innate immunity; NF-kappaB; Oct /POU transcription factors; Stress response
Lactobacillus species maintain the vaginal ecosystem in a healthy condition by production of antimicrobial substances. Depletion of lactobacilli in the vagina results in bacterial vaginosis (BV), where the normal flora is replaced by several bacterial pathogens, usually Gardnerella vaginalis and obligate anaerobes. BV may cause complications such as premature labor, low birth weight and increased risk of HIV acquisition. The currently recommended antibiotic treatments for BV are not always effective and often lead to reoccurrence of the infection. In many cases, this is due to the antibiotic-resistant forms of the pathogens. Therefore, there is an interest in the development of treatments using antimicrobials derived primarily from Lactobacillus spp., such as ribosomally produced antimicrobial peptides (bacteriocins) and lactic acid. These substances effectively inhibit pathogenic bacteria, are safe and do not pose any threat to healthy vaginal Lactobacillus spp. It may be possible to find an effective treatment against BV while reducing the infection’s reoccurrence and the treatment-related complications through hurdle technology. This would be achieved by combining antimicrobials produced by Lactobacillus spp. with different natural antimicrobials obtained from plants or other non-pathogenic organisms.
PMCID: PMC2908489  PMID: 20657710
Bacterial vaginosis; Bacteriocins; Lactic acid; Lactobacilli; Natural antimicrobials
19.  The Antimicrobial Peptide Histatin-5 Causes a Spatially Restricted Disruption on the Candida albicans Surface, Allowing Rapid Entry of the Peptide into the Cytoplasm 
PLoS Pathogens  2008;4(10):e1000190.
Antimicrobial peptides play an important role in host defense against microbial pathogens. Their high cationic charge and strong amphipathic structure allow them to bind to the anionic microbial cell membrane and disrupt the membrane bilayer by forming pores or channels. In contrast to the classical pore-forming peptides, studies on histatin-5 (Hst-5) have suggested that the peptide is transported into the cytoplasm of Candida albicans in a non-lytic manner, and cytoplasmic Hst-5 exerts its candicidal activities on various intracellular targets, consistent with its weak amphipathic structure. To understand how Hst-5 is internalized, we investigated the localization of FITC-conjugated Hst-5. We find that Hst-5 is internalized into the vacuole through receptor-mediated endocytosis at low extracellular Hst-5 concentrations, whereas under higher physiological concentrations, Hst-5 is translocated into the cytoplasm through a mechanism that requires a high cationic charge on Hst-5. At intermediate concentrations, two cell populations with distinct Hst-5 localizations were observed. By cell sorting, we show that cells with vacuolar localization of Hst-5 survived, while none of the cells with cytoplasmic Hst-5 formed colonies. Surprisingly, extracellular Hst-5, upon cell surface binding, induces a perturbation on the cell surface, as visualized by an immediate and rapid internalization of Hst-5 and propidium iodide or rhodamine B into the cytoplasm from the site using time-lapse microscopy, and a concurrent rapid expansion of the vacuole. Thus, the formation of a spatially restricted site in the plasma membrane causes the initial injury to C. albicans and offers a mechanism for its internalization into the cytoplasm. Our study suggests that, unlike classical channel-forming antimicrobial peptides, action of Hst-5 requires an energized membrane and causes localized disruptions on the plasma membrane of the yeast. This mechanism of cell membrane disruption may provide species-specific killing with minimal damage to microflora and the host and may be used by many other antimicrobial peptides.
Author Summary
In most healthy individuals, the yeast Candida albicans is found within the oral cavity as part of the normal microflora. Though under immunocompromising conditions, this benign microbe can become an opportunistic pathogen causing oral candidiasis (i.e. thrush). Although antifungal drugs are typically efficacious, the paucity of drugs and their increasing usage has led to rising drug resistance. Thus, researchers have begun to look at alternative therapeutics, such as the candidacidal salivary peptide histatin-5. To date, little is known about the initial binding and subsequent internalization that facilitates Hst-5's killing activity. Thus, our study attempted to determine how Hst-5 is internalized into C. albicans. It was thought that Hst-5 is transported into the cytoplasm without disruption of the plasma membrane. However, our study finds that Hst-5, under physiological concentrations, disrupts the plasma membrane and is rapidly translocated into the cytoplasm, leading to cell death. Interestingly, the internalization of Hst-5 is initiated from a single spatially restricted site on the plasma membrane rather than multiple breaches on the cell surface. This novel mechanism of membrane disruption provides new insights into how Hst-5 and other antimicrobial peptides may be acting on pathogenic microorganisms.
PMCID: PMC2568956  PMID: 18974864
20.  Modelling within-Host Spatiotemporal Dynamics of Invasive Bacterial Disease 
PLoS Biology  2008;6(4):e74.
Mechanistic determinants of bacterial growth, death, and spread within mammalian hosts cannot be fully resolved studying a single bacterial population. They are also currently poorly understood. Here, we report on the application of sophisticated experimental approaches to map spatiotemporal population dynamics of bacteria during an infection. We analyzed heterogeneous traits of simultaneous infections with tagged Salmonella enterica populations (wild-type isogenic tagged strains [WITS]) in wild-type and gene-targeted mice. WITS are phenotypically identical but can be distinguished and enumerated by quantitative PCR, making it possible, using probabilistic models, to estimate bacterial death rate based on the disappearance of strains through time. This multidisciplinary approach allowed us to establish the timing, relative occurrence, and immune control of key infection parameters in a true host–pathogen combination. Our analyses support a model in which shortly after infection, concomitant death and rapid bacterial replication lead to the establishment of independent bacterial subpopulations in different organs, a process controlled by host antimicrobial mechanisms. Later, decreased microbial mortality leads to an exponential increase in the number of bacteria that spread locally, with subsequent mixing of bacteria between organs via bacteraemia and further stochastic selection. This approach provides us with an unprecedented outlook on the pathogenesis of S. enterica infections, illustrating the complex spatial and stochastic effects that drive an infectious disease. The application of the novel method that we present in appropriate and diverse host–pathogen combinations, together with modelling of the data that result, will facilitate a comprehensive view of the spatial and stochastic nature of within-host dynamics.
Author Summary
Global patterns and mechanistic determinants of bacterial spread in mammalian organisms are difficult to obtain through numerical and topographical mapping of a single bacterial population. Appreciation of the true pathogenetic events during infections needs to be based on the understanding of the fine interactions that control the infection dynamics of individual subpopulations in the same host. We have used molecular techniques to tag individually otherwise identical subpopulations of bacteria. We have used these bacteria, called wild-type isogenic tagged strains (WITS), in simultaneous infections in the same animal to gather insights into the patterns of spread of individual subpopulations of bacteria in the tissues and interactions between bacteria and phagocytes. Combining numerical fluctuation in the WITS populations with mathematical modelling and statistical analysis, we have gathered data on the relative occurrence of bacterial growth and death in different phases of the disease process. Our analyses support a model in which shortly after infection, concomitant death and rapid bacterial replication lead to the establishment of independent bacterial subpopulations in different organs. Later, decreased microbial mortality leads to an exponential increase in the number of bacteria that spread locally, with subsequent mixing of bacteria between organs. The work illustrates the importance of unravelling heterogeneous traits of infections to reconstruct and understand the true nature of the global disease process.
Genetically identical bacterial strains reveal the population dynamics and interactions of subpopulations of bacteria with the host's immune system in vivo during infection.
PMCID: PMC2288627  PMID: 18399718
21.  Histidine-Rich Glycoprotein Protects from Systemic Candida Infection 
PLoS Pathogens  2008;4(8):e1000116.
Fungi, such as Candida spp., are commonly found on the skin and at mucosal surfaces. Yet, they rarely cause invasive infections in immunocompetent individuals, an observation reflecting the ability of our innate immune system to control potentially invasive microbes found at biological boundaries. Antimicrobial proteins and peptides are becoming increasingly recognized as important effectors of innate immunity. This is illustrated further by the present investigation, demonstrating a novel antifungal role of histidine-rich glycoprotein (HRG), an abundant and multimodular plasma protein. HRG bound to Candida cells, and induced breaks in the cell walls of the organisms. Correspondingly, HRG preferentially lysed ergosterol-containing liposomes but not cholesterol-containing ones, indicating a specificity for fungal versus other types of eukaryotic membranes. Both antifungal and membrane-rupturing activities of HRG were enhanced at low pH, and mapped to the histidine-rich region of the protein. Ex vivo, HRG-containing plasma as well as fibrin clots exerted antifungal effects. In vivo, Hrg−/− mice were susceptible to infection by C. albicans, in contrast to wild-type mice, which were highly resistant to infection. The results demonstrate a key and previously unknown antifungal role of HRG in innate immunity.
Author Summary
It has been estimated that humans contain about 1 kg of microbes, an observation that reflects our coexistence with colonizing microbes such as bacteria and fungi. The fungal species Candida is present as a commensal at mucosal surfaces and on skin. Although it may cause life-threatening infections, such as sepsis, particularly in immunocompromised individuals, it seldom causes disease in normal individuals. In order to control our microbial flora, humans as well as virtually all life forms are armoured with various proteins and peptides that comprise integral parts of our innate immune system. Here we describe a new component in this system; histidine-rich glycoprotein (HRG), an abundant plasma protein. We show, using a combination of microbiological, biochemical, and biophysical methods, that HRG exerts a potent antifungal activity, which is mediated via a histidine-rich region of the protein, and targets ergosterol-rich membrane structures such as those of Candida. HRG killed Candida both in plasma as well as when incorporated into fibrin clots. In mouse infection models, HRG was protective against systemic infection by Candida, indicating a novel antifungal role of HRG in innate immunity.
PMCID: PMC2537934  PMID: 18797515
22.  Antimicrobial activity of innate immune molecules against Streptococcus pneumoniae, Moraxella catarrhalis and nontypeable Haemophilus influenzae 
Despite its direct connection to the nasopharynx which harbors otitis media pathogens as part of its normal flora, the middle ear cavity is kept free of these bacteria by as yet unknown mechanisms. Respiratory mucosal epithelia, including those of the middle ear and eustachian tube, secrete antimicrobial effectors including lysozyme, lactoferrin and β defensins-1 and -2. To elucidate the role of these innate immune molecules in the normal defense and maintenance of sterility of respiratory mucosa such as that of the middle ear, we assessed their effect on the respiratory pathogens nontypeable Haemophilus influenzae (NTHi) 12, Moraxella catarrhalis 035E, and Streptococcus pneumoniae 3, and 6B.
Two assay methods, the radial assay and the liquid broth assay, were employed for testing the antimicrobial activity of the molecules. This was done in order to minimize the possibility that the observed effects were artifacts of any single assay system employed. Also, transmission electron microscopy (TEM) was employed to evaluate the effect of antimicrobial innate immune molecules on OM pathogens. For the statistical analysis of the data, Student's t-test was performed.
Results of the radial diffusion assay showed that β defensin-2 was active against all four OM pathogens tested, while treatment with β defensin-1 appeared to only affect M. catarrhalis. The radial assay results also showed that lysozyme was quite effective against S. pneumoniae 3 and 6B and was partially bacteriostatic/bactericidal against M. catarrhalis. Lysozyme however, appeared not to affect the growth of NTHi. Thus, lysozyme seems to have a more pronounced impact on the growth of the Gram-positive S. pneumoniae as compared to that of Gram-negative pathogens. Lactoferrin on the other hand, enhanced the growth of the bacteria tested. The results of the radial assays were confirmed using liquid broth assays for antimicrobial activity, and showed that lysozyme and β defensin-2 could act synergistically against S. pneumoniae 6B. Moreover, in the liquid broth assay, β defensin-1 showed a modest inhibitory effect on the growth of S. pneumoniae 6B. As assessed by ultrastructural analysis, lysozyme and β defensin-2, and to a much lesser extent, β defensin-1, appeared to be able to cause damage to the bacterial membranes.
Here we report that lysozyme and the β defensins can inhibit the growth of clinical isolates of otitis media pathogens – namely NTHi strain 12, S. pneumoniae strains 3 and 6B and M. catarrhalis strain 035E – and cause ultrastructural damage to these pathogens. Moreover, we demonstrate that lysozyme and β defensin-2 can act synergistically against S. pneumoniae. These findings are consistent with the concept that secreted antimicrobial peptides and other components of innate immunity constitute the first line of defense protecting host mucosal surfaces, including the tubotympanal (eustachian tube and middle ear cavity) mucosa, against pathogens.
PMCID: PMC428576  PMID: 15125783
23.  The Circadian Clock Protein Timeless Regulates Phagocytosis of Bacteria in Drosophila 
PLoS Pathogens  2012;8(1):e1002445.
Survival of bacterial infection is the result of complex host-pathogen interactions. An often-overlooked aspect of these interactions is the circadian state of the host. Previously, we demonstrated that Drosophila mutants lacking the circadian regulatory proteins Timeless (Tim) and Period (Per) are sensitive to infection by S. pneumoniae. Sensitivity to infection can be mediated either by changes in resistance (control of microbial load) or tolerance (endurance of the pathogenic effects of infection). Here we show that Tim regulates resistance against both S. pneumoniae and S. marcescens. We set out to characterize and identify the underlying mechanism of resistance that is circadian-regulated. Using S. pneumoniae, we found that resistance oscillates daily in adult wild-type flies and that these oscillations are absent in Tim mutants. Drosophila have at least three main resistance mechanisms to kill high levels of bacteria in their hemolymph: melanization, antimicrobial peptides, and phagocytosis. We found that melanization is not circadian-regulated. We further found that basal levels of AMP gene expression exhibit time-of-day oscillations but that these are Tim-independent; moreover, infection-induced AMP gene expression is not circadian-regulated. We then show that phagocytosis is circadian-regulated. Wild-type flies exhibit up-regulated phagocytic activity at night; Tim mutants have normal phagocytic activity during the day but lack this night-time peak. Tim appears to regulate an upstream event in phagocytosis, such as bacterial recognition or activation of phagocytic hemocytes. Interestingly, inhibition of phagocytosis in wild type flies results in survival kinetics similar to Tim mutants after infection with S. pneumoniae. Taken together, these results suggest that loss of circadian oscillation of a specific immune function (phagocytosis) can have significant effects on long-term survival of infection.
Author Summary
Circadian rhythms, or daily oscillations in physiological functions, are nearly ubiquitous among animals. Though immune parameters are known to vary with time of day, circadian regulation of the immune system is not well understood. Studies of circadian biology and innate immunity are well established in the genetically tractable model organism, the fruit fly. We use this model to investigate the mechanisms underlying circadian-regulated immunity. We and others have found that flies infected at different times of day exhibit different mean survival times. There are three main components of fly immune defense against bacterial pathogens: antimicrobial peptide synthesis, reactive oxygen species generation, and phagocytosis, or the ingestion of microbes by host immune cells. Of these three, we found that only the phagocytic activity of host immune cells is circadian-regulated and peaks at night, during the rest phase. This night-time peak of phagocytosis correlates with the time of day of maximal resistance to infection by the bacteria Streptococcus pneumoniae. Mutants that lack circadian regulation also lack this night-time peak in phagocytosis. Inhibition of phagocytosis sensitized wild type flies to infection but did not further sensitize circadian mutants. These results suggest that this night-time peak in immune system function has significant effects on survival of infection.
PMCID: PMC3257305  PMID: 22253593
24.  Rudra Interrupts Receptor Signaling Complexes to Negatively Regulate the IMD Pathway 
PLoS Pathogens  2008;4(8):e1000120.
Insects rely primarily on innate immune responses to fight pathogens. In Drosophila, antimicrobial peptides are key contributors to host defense. Antimicrobial peptide gene expression is regulated by the IMD and Toll pathways. Bacterial peptidoglycans trigger these pathways, through recognition by peptidoglycan recognition proteins (PGRPs). DAP-type peptidoglycan triggers the IMD pathway via PGRP-LC and PGRP-LE, while lysine-type peptidoglycan is an agonist for the Toll pathway through PGRP-SA and PGRP-SD. Recent work has shown that the intensity and duration of the immune responses initiating with these receptors is tightly regulated at multiple levels, by a series of negative regulators. Through two-hybrid screening with PGRP-LC, we identified Rudra, a new regulator of the IMD pathway, and demonstrate that it is a critical feedback inhibitor of peptidoglycan receptor signaling. Following stimulation of the IMD pathway, rudra expression was rapidly induced. In cells, RNAi targeting of rudra caused a marked up-regulation of antimicrobial peptide gene expression. rudra mutant flies also hyper-activated antimicrobial peptide genes and were more resistant to infection with the insect pathogen Erwinia carotovora carotovora. Molecularly, Rudra was found to bind and interfere with both PGRP-LC and PGRP-LE, disrupting their signaling complex. These results show that Rudra is a critical component in a negative feedback loop, whereby immune-induced gene expression rapidly produces a potent inhibitor that binds and inhibits pattern recognition receptors.
Author Summary
The innate immune system controls the immediate response to infection. Innate immunity relies on germline encoded receptors, receptors that are present at birth, to recognize germs and trigger a protective response. Invertebrates (i.e., insects) rely on innate immunity to survive in microbial-rich environments, such as rotting fruit. However, uncontrolled innate immune responses are dangerous, leading to severe pathologies like sepsis, inflammatory bowel diseases, and lupus. Therefore, the intensity and duration of the innate immune response is kept in-check by multiple regulatory mechanisms. Here, we have identified a new feedback regulator of the Drosophila (the fruit fly) immune response, which we call Rudra. Using various approaches, we show that in the absence of Rudra the innate immune system is hyper-activated. This elevated immune response leads to better protection against bacterial infection. On the other hand, when present in excess, Rudra prevents the activation of the immune response. Furthermore, we show that Rudra turns off the immune response by binding to the receptors that are responsible for detecting bacteria, thereby preventing downstream responses.
PMCID: PMC2483946  PMID: 18688280
25.  Elucidation of the RamA Regulon in Klebsiella pneumoniae Reveals a Role in LPS Regulation 
PLoS Pathogens  2015;11(1):e1004627.
Klebsiella pneumoniae is a significant human pathogen, in part due to high rates of multidrug resistance. RamA is an intrinsic regulator in K. pneumoniae established to be important for the bacterial response to antimicrobial challenge; however, little is known about its possible wider regulatory role in this organism during infection. In this work, we demonstrate that RamA is a global transcriptional regulator that significantly perturbs the transcriptional landscape of K. pneumoniae, resulting in altered microbe-drug or microbe-host response. This is largely due to the direct regulation of 68 genes associated with a myriad of cellular functions. Importantly, RamA directly binds and activates the lpxC, lpxL-2 and lpxO genes associated with lipid A biosynthesis, thus resulting in modifications within the lipid A moiety of the lipopolysaccharide. RamA-mediated alterations decrease susceptibility to colistin E, polymyxin B and human cationic antimicrobial peptide LL-37. Increased RamA levels reduce K. pneumoniae adhesion and uptake into macrophages, which is supported by in vivo infection studies, that demonstrate increased systemic dissemination of ramA overexpressing K. pneumoniae. These data establish that RamA-mediated regulation directly perturbs microbial surface properties, including lipid A biosynthesis, which facilitate evasion from the innate host response. This highlights RamA as a global regulator that confers pathoadaptive phenotypes with implications for our understanding of the pathogenesis of Enterobacter, Salmonella and Citrobacter spp. that express orthologous RamA proteins.
Author Summary
Bacteria can rapidly evolve under antibiotic pressure to develop resistance, which occurs when target genes mutate, or when resistance-encoding genes are transferred. Alternatively, microbes can simply alter the levels of intrinsic proteins that allow the organism to “buy” time to resist antibiotic pressure. Klebsiella pneumoniae is a pathogen that causes significant blood stream or respiratory infections, but more importantly is a bacterium that is increasingly being reported as multidrug resistant. Our data demonstrate that RamA can trigger changes on the bacterial surface that allow Klebsiella to survive both antibiotic challenge, degradation by host immune peptides and resist phagocytosis. We demonstrate that the molecular basis of increased survival of ramA overexpressing K. pneumoniae, against host-derived factors is associated with RamA-driven alterations of the lipid A moiety of Klebsiella LPS. This modification is likely to be linked to Klebsiella’s ability to resist the host response so that it remains undetected by the immune system. The relevance of our work extends beyond RamA in Klebsiella as other pathogens such as Enterobacter spp and Salmonella spp. also produce this protein. Thus our overarching conclusion is that the intrinsic regulator, RamA perturbs host-microbe and microbe-drug interactions.
PMCID: PMC4310594  PMID: 25633080

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