Legionnaires' Disease Mortality in Guinea Pigs Involves the p45 Mobile Genomic Element.

BACKGROUND: Legionella can cause Legionnaires' disease, a potentially fatal form of pneumonia that occurs as sporadic epidemics. Not all strains display the same propensity to cause disease in humans. Because Legionella pneumophila serogroup 1 is responsible for >85% of infections, the majority of studies have examined this serogroup, but there are 3 commonly used laboratory strains: L pneumophila serogroup 1 Philadelphia (Phil-1)-derived strains JR32 and Lp01 and 130b-derived strain AA100. METHODS: We evaluated the ability of Phil-1, JR32, Lp01, and AA100 to cause disease in guinea pigs. RESULTS: We found that, although Phil-1, JR32, and AA100 cause an acute pneumonia and death by 4 days postinfection (100%), strain Lp01 does not cause mortality (0%). We also noted that Lp01 lacks a mobile element, designated p45, whose presence correlates with virulence. Transfer of p45 into Lp01 results in recovery of the ability of this strain to cause mortality, leads to more pronounced disease, and correlates with increased interferon-γ levels in the lungs and spleens before death. CONCLUSIONS: These observations suggest a mechanism of Legionnaires' disease pathogenesis due to the presence of type IVA secretion systems that cause higher mortality due to overinduction of a proinflammatory response in the host.

Imaging tuberculosis with endogenous beta-lactamase reporter enzyme fluorescence in live mice.

The slow growth rate and genetic intractability of tubercle bacilli has hindered progress toward understanding tuberculosis, one of the most frequent causes of death worldwide. We overcame this roadblock through development of near-infrared (NIR) fluorogenic substrates for beta-lactamase, an enzyme expressed by tubercle bacilli, but not by their eukaryotic hosts, to allow real-time imaging of pulmonary infections and rapid quantification of bacteria in living animals by a strategy called reporter enzyme fluorescence (REF). This strategy has a detection limit of 6 +/- 2 x 10(2) colony-forming units (CFU) of bacteria with the NIR substrate CNIR5 in only 24 h of incubation in vitro, and as few as 10(4) CFU in the lungs of live mice. REF can also be used to differentiate infected from uninfected macrophages by using confocal microscopy and fluorescence activated cell sorting. Mycobacterium tuberculosis and the bacillus Calmette-Guérin can be tracked directly in the lungs of living mice without sacrificing the animals. Therapeutic efficacy can also be evaluated through loss of REF signal within 24 h posttreatment by using in vitro whole-bacteria assays directly in living mice. We expect that rapid quantification of bacteria within tissues of a living host and in the laboratory is potentially transformative for tuberculosis virulence studies, evaluation of therapeutics, and efficacy of vaccine candidates. This is a unique use of an endogenous bacterial enzyme probe to detect and image tubercle bacilli that demonstrates REF is likely to be useful for the study of many bacterial infections.

A Mycobacterium marinum TesA mutant defective for major cell wall-associated lipids is highly attenuated in Dictyostelium discoideum and zebrafish embryos.

Infection of the zebrafish with Mycobacterium marinum is regarded as a well-established experimental model to study the pathogenicity of Mycobacterium tuberculosis. Herein, a M. marinum transposon mutant library was screened for attenuated M. marinum phenotypes using a Dictyostelium discoideum assay. In one attenuated mutant, the transposon was located within tesA, encoding a putative type II thioesterase. Thin-layer chromatography analyses indicated that the tesA::Tn mutant failed to produce two major cell wall-associated lipids. Mass spectrometry and nuclear magnetic resonance clearly established the nature of missing lipids as phthioglycol diphthioceranates and phenolic glycolipids, respectively, indicating that TesA is required for the synthesis of both lipids. When injected into the zebrafish embryo bloodstream, the mutant was found to be highly attenuated, thus validating the performance and relevance of the Dictyostelium screen. Consistent with these in vivo findings, tesA::Tn exhibited increased permeability defects in vitro, which may explain its failure to survive in host macrophages. Unexpectedly, virulence was retained when bacteria were injected into the notochord. Histological and ultrastructural studies of the infected notochord revealed the presence of actively proliferating mycobacteria, leading to larval death. This work presents for the first time the notochord as a compartment highly susceptible to mycobacterial infection.

Hydrazide drugs that inhibit growth and proliferation of tuberculosis bacteria.

Four hydrazide drugs are shown to effectively and strongly inhibit the growth of Mycobacterium bovis BCG. The four compounds were found to be comparable to isoniazid for extent of growth inhibition. Similar to isoniazid, the four drug designs have a hydrazide functional group (-C(O)NHNH2) that replaces a former carboxyl group (-C(O)OH). Important pharmaceutical properties were determined for all drugs including Log P, polar surface area, water solubility, and violations of the Rule of 5. Values of Log P for A, B, C, D, and isoniazid were determined to be 1.08, 1.26, 1.26, 1.06, and -0.70, respectively. The polar surface area for drugs A, B, and C were calculated to be 55.12 Angstroms2, which is a value that suggests these drugs will effectively penetrate the central nervous system for targeting tuberculosis that infects that anatomical region. All drug designs and isoniazid show zero violations of the Rule of 5 indicating favorable drug bioavailability. Water solubility for all drugs varies from 1074 milligrams/liter to 16690 milligrams/liter. Growth inhibition of tuberculosis bacteria was greater than 50% for all novel drugs at concentrations of 62.5 micrograms/milliliter and higher. Cluster analysis determined that isoniazid is distinct from all new drug designs. For molecular descriptors, molecular volume is directly correlated to formula weight and polar surface area (Pearson r > 0.8800). The four novel drug designs show substantial efficacy for the clinical treatment of tuberculosis.

Identification of Mycobacterium avium pathogenicity island important for macrophage and amoeba infection.

The ability to infect macrophages is a common characteristic shared among many mycobacterial species. Mycobacterium avium, Mycobacterium tuberculosis, and Mycobacterium kansasii enter macrophages, using the complement receptors CR1, CR3, CR4, and the mannose receptor. To identify M. avium genes and host cell pathways involved in the bacterial uptake by macrophages, we screened a M. avium transposon mutant library for the inability to enter macrophages. Uptake-impaired clones were selected. Sequence of six M. avium clones identified one gene involved in glycopeptidolipid biosynthesis, one gene encoding the conserved membrane protein homologue to the M. avium subsp. paratuberculosis MAP2446c gene and four others belonging to the same region of the chromosome. Analysis of the chromosome region revealed a pathogenicity island inserted between two tRNA sequences with 58% of G+C content versus 69% in the M. avium genome. The region is unique for M. avium and is not present in M. tuberculosis or M. paratuberculosis. Although the mutants did not differ from the WT bacterium regarding the binding to macrophage cell membrane, analysis of macrophage proteins after 1 h infection revealed a deficiency in the mutant to phosphorylate certain proteins on uptake. To understand M. avium interaction with two evolutionarily distinct hosts, the mutants were evaluated for Acanthamoeba castellanii invasion. The defect in the ability of the mutants to invade both cells was highly similar, suggesting that M. avium might have evolved mechanisms that are used to enter amoebas and human macrophages.

Protection of Mycobacterium tuberculosis from reactive oxygen species conferred by the mel2 locus impacts persistence and dissemination.

Persistence of Mycobacterium tuberculosis in humans represents a major roadblock to elimination of tuberculosis. We describe identification of a locus in M. tuberculosis, mel2, that displays similarity to bacterial bioluminescent loci and plays an important role during persistence in mice. We constructed a deletion of the mel2 locus and found that the mutant displays increased susceptibility to reactive oxygen species (ROS). Upon infection of mice by aerosol the mutant grows normally until the persistent stage, where it does not persist as well as wild type. Histopathological analyses show that infection with the mel2 mutant results in reduced pathology and both CFU and histopathology indicate that dissemination of the mel2 mutant to the spleen is delayed. These data along with growth in activated macrophages and infection of Phox(-/-) and iNOS(-/-) mice and bone marrow-derived macrophages suggest that the primary mechanism by which mel2 affects pathogenesis is through its ability to confer resistance to ROS. These studies provide the first insight into the mechanism of action for this novel class of genes that are related to bioluminescence genes. The role of mel2 in resistance to ROS is important for persistence and dissemination of M. tuberculosis and suggests that homologues in other bacterial species are likely to play a role in pathogenesis.

Use of gene dosage effects for a whole-genome screen to identify Mycobacterium marinum macrophage infection loci.

We recently identified two loci, mel1 and mel2, that affect macrophage infection by Mycobacterium marinum. The ability of these loci to confer enhanced infection in trans is presumably due to gene dosage effects since their presence on plasmids increases expression from five- to eightfold. Reasoning that this phenomenon would allow identification of other mycobacterial genes involved in macrophage infection, we conducted a screen of an M. marinum DNA library that provides 2.6-fold coverage of the entire genome for clones that affect macrophage infection. Our preliminary screen identified 76 plasmids that carry loci affecting macrophage infection. We eliminated plasmids that do not confer the expected phenotype when retransformed (70%), that have identical physical maps (5%), or that carry either of the mel1 or mel2 loci (14%) from further consideration. Four loci that confer enhanced infection (mel) and four that confer repressed infection (mrl) of macrophages were identified, and two of each group were chosen for detailed analysis. Saturating transposon mutagenesis was used to identify the loci responsible, and M. marinum mutants were constructed in the genes involved. We expect these genes to provide insight into how mycobacteria parasitize macrophages, an important component of innate immunity.

Four hydrazide compounds that inhibit the growth of mycobacterium tuberculosis.

Over one-third of the world's population has been exposed to Mycobacterium tuberculosis (TB). New drug designs are necessitated by the appearance of multi-drug resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB). This work presents four hydrazide compounds that inhibit Mycobacterium tuberculosis growth at potency comparable to isoniazid. The hydrazide drugs A, B, C, and D were synthesized utilizing microwave excitation methodologies. Hydrazide agents are produced from parent carboxyl compounds. All reactions were accomplished in dry conditions. Using suitable molecular scaffolds the final hydrazides possessed the desired properties in Log P, polar surface area, molecular weight, etc. All four hydrazide compounds induced at least 60% inhibition of TB at concentrations at less than 31.5 microgram/mL. Measured as relative survival, all four hydrazide compounds induced greater than 95% death of TB bacteria at concentrations less than 31.5 microgram/mL. Drugs A, B, C, D exhibited zero violations of the Rule of 5, indicating favorable bioavailability. Although isoniazid, A, B, C, and D were determined to have a polar surface area less than 70 Angstroms2, the values of Log P (a measurement of lipophilicity) showed a broad range of -1.463 (drug C) to 4.46 (drug A). The numerical values of polar surface area suggests that all drugs would have greater than 50% intestinal absorption. For all drugs the number of amine (-NH) and hydroxyl (-OH) groups remains constant at three, with number of oxygens and nitrogens varying only three to four. ANOSIM (analysis of similarity) indicated that isoniazid, A, B, C, and D are highly similar. Correlation of molecular properties for all five drugs is greater than r = 0.9500. Drugs A, B, C, and D are members of two homologous series of anti-tuberculosis agents. All hydrazides effectively inhibited TB at 31.5 micrograms/mL and lower concentrations.

The Mycobacterium marinum mel2 locus displays similarity to bacterial bioluminescence systems and plays a role in defense against reactive oxygen and nitrogen species.

BACKGROUND: Mycobacteria have developed a number of pathways that provide partial protection against both reactive oxygen species (ROS) and reactive nitrogen species (RNS). We recently identified a locus in Mycobacterium marinum, mel2, that plays a role during infection of macrophages. The molecular mechanism of mel2 action is not well understood. RESULTS: To better understand the role of the M. marinum mel2 locus, we examined these genes for conserved motifs in silico. Striking similarities were observed between the mel2 locus and loci that encode bioluminescence in other bacterial species. Since bioluminescence systems can play a role in resistance to oxidative stress, we postulated that the mel2 locus might be important for mycobacterial resistance to ROS and RNS. We found that an M. marinum mutant in the first gene in this putative operon, melF, confers increased susceptibility to both ROS and RNS. This mutant is more susceptible to ROS and RNS together than either reactive species alone. CONCLUSION: These observations support a role for the M. marinum mel2 locus in resistance to oxidative stress and provide additional evidence that bioluminescence systems may have evolved from oxidative defense mechanisms.

Antibacterial activity of dipeptide constructs of acetylsalicylic acid and nicotinic acid.

Two dipeptide drugs are synthesized utilizing an acetylsalicylic acid or nicotinic acid molecule for the framework. A D-alanine-D-alanine dipeptide moiety is attached to the carbonyl carbon of acetylsalicylic acid (I) and nicotinic acid (II). Dipeptide derivatives (I) and (II) showed significant reduction of Escherichia coli (E. coli) bacterial growth and colony-forming units. A mixture of (I) and (II) induced growth inhibition of 8%, 17.5%, 28%, and 42.5% at concentrations of 100, 200, 300, and 400 microg/mL, respectively. Ampicillin demonstrated much less growth inhibition of this penicillin-resistant E. coli bacteria. Derivatives (I) and (II) showed significant reduction of colony-forming units at concentrations higher than 200 microg/mL, whereas ampicillin showed no significant affect on colony-forming units. Both (I) and (II) produced no violations of the Rule of 5, indicating favorable characteristics for bioavailability. Molecular properties were determined and showed (I) to have a Log Kow of -0.22 with aqueous solubility of 683.8 mg/L, whereas (II) had a Log Kow of -1.00 and aqueous solubility of 6859 mg/L. A mixture of the parent compounds acetyl salicylic acid and nicotinic acid demonstrated some antibacterial activity.

Derivatives of cephalothin that inhibit ampicillin resistant Escherichia coli.

Two derivatives of cephalothin, compound I and II, were synthesized and demonstrated strong growth inhibition of ampicillin resistant Escherichia coli (E. coli). Compound I is the propyl ester of the parent cephalothin antibiotic, while compound II is the butyl ester derivative. The ester substituent replaces the former carboxyl group of cephalothin. Compounds I and II are stable at room temperature and have increased lipophilicity compared to cephalothin due to the presence of the ester substituent. The MIC50 of I and II were determined to be 55 microg/mL and 30 microg/mL, respectively. Cephalothin showed less than 20% growth inhibition of E. coli at all concentrations based on assay of colony forming units. Compounds I and II showed greater than 50% growth inhibition of E. coli at all concentrations greater than 50 microg/mL (more than 65% at 400 microg/mL). Pharmacological properties such as octanol/water partition Log P and polar surface area were determined. Values for polar surface area suggested 35% of I or II present in the intestinal system would be absorbed. The Log P values for I and II are 2.061 and 2.62, respectively, which indicate I and II will penetrate the blood-brain barrier more effectively than cephalothin. The lipophilic substituent constant (pi) for I and II are 1.592 and 1.95, respectively, which indicates the ester substituents contribute a strong lipophilic trait. Properties of molar refractivity, parachor, and molar volume, which describe van-der-Waals interactions, are also determined.

The Caenorhabditis elegans p38 MAPK Gene plays a key role in protection from mycobacteria.

Mitogen-activated protein kinases (MAPK) are critical mediators of cellular responses to pathogens and are activated in response to infection, but investigation is difficult in multi-cell hosts due to developmental lethality of mutations. Mycobacterium marinum (Mm) is an established model for tuberculosis, a disease afflicting nearly one-third of the world's population. We found that Mm-infected Caenorhabditis elegans display >80% mortality, but nonpathogenic M. smegmatis cause <15% mortality. C. elegans display pathological changes when infected with Mm, whereas Mm mutants produce lower mortality, suggesting that C. elegans is a promising virulence model for detailed genetic analysis. C. elegans MAPK mutants are hypersusceptible to mycobacterial infection; however, the C. elegans TOL-like, TGF-ß and insulin-like pathway genes do not play important roles in susceptibility. We show that pathogenic mycobacteria inhibit MAPK-mediated protection through the MAPK phosphatase gene and demonstrate that C. elegans provide a genetically tractable pathogenicity model of both the host and pathogen.

Application of Fluorescent Protein Expressing Strains to Evaluation of Anti-Tuberculosis Therapeutic Efficacy In Vitro and In Vivo.

The slow growth of Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), hinders development of new diagnostics, therapeutics and vaccines. Using non-invasive real-time imaging technologies to monitor the disease process in live animals would facilitate TB research in all areas. We developed fluorescent protein (FP) expressing Mycobacterium bovis BCG strains for in vivo imaging, which can be used to track bacterial location, and to quantify bacterial load in live animals. We selected an optimal FP for in vivo imaging, by first cloning six FPs: tdTomato, mCherry, mPlum, mKate, Katushka and mKeima, into mycobacteria under either a mycobacterial Hsp60 or L5 promoter, and compared their fluorescent signals in vitro and in vivo. Fluorescence from each FP-expressing strain was measured with a multimode reader using the optimal excitation and emission wavelengths for the FP. After normalizing bacterial numbers with optical density, the strain expressing L5-tdTomato displayed the highest fluorescence. We used the tdTomato-labeled M. bovis BCG to obtain real-time images of pulmonary infections in living mice and rapidly determined the number of bacteria present. Further comparison between L5-tdTomato and Hsp60-tdTomato revealed that L5-tdTomato carried four-fold more tdTomato gene copies than Hsp60-tdTomato, which eventually led to higher protein expression of tdTomato. Evaluating anti-TB efficacy of rifampicin and isoniazid therapy in vitro and in vivo using the L5-tdTomato strain demonstrated that this strain can be used to identify anti-TB therapeutic efficacy as quickly as 24 h post-treatment. These M. bovis BCG reporter strains represent a valuable new tool for evaluation of therapeutics, vaccines and virulence.

Molecular properties and antibacterial activity of the methyl and ethyl ester derivatives of ampicillin.

Ampicillin is a beta-lactam antibiotic that is effective against gram-negative bacteria. Ampicillin has a single carboxyl group (-C(O)OH) within its structure which is suitable for forming ester compounds. Diazomethane and diazoethane were utilized to react with ampicillin to form the methyl and ethyl esters, respectively. The ester derivatives of ampicillin were solubilized together (mole ratio 1:1) in LB media and penicillin resistant Escherichia coli added to measure antibacterial activity. Growth inhibition of bacteria was monitored by optical density after a known time period and with known specific concentrations of the ampicillin esters present. Significant growth inhibition of penicillin resistant bacteria occurred at concentrations of the combined methyl and ethyl ampicillin esters from less than 50 microgram/mL to more than 150 microgram/mL. Molecular properties of the ester compounds were determined. The two ester derivatives showed values of Log BB, Log P, polar surface area, intestinal absorption, and solubility suitable for clinical application. The two ester compounds showed zero violations of the Rule of 5 indicating good bioavailability. The two ester derivatives showed greater intestinal absorbance and greater penetration of the blood brain barrier than the parent ampicillin. Favorable druglikeness was determined for both ester derivatives.

Real-time bioluminescence imaging of mixed mycobacterial infections.

Molecular analysis of infectious processes in bacteria normally involves construction of isogenic mutants that can then be compared to wild type in an animal model. Pathogenesis and antimicrobial studies are complicated by variability between animals and the need to sacrifice individual animals at specific time points. Live animal imaging allows real-time analysis of infections without the need to sacrifice animals, allowing quantitative data to be collected at multiple time points in all organs simultaneously. However, imaging has not previously allowed simultaneous imaging of both mutant and wild type strains of mycobacteria in the same animal. We address this problem by using both firefly (Photinus pyralis) and click beetle (Pyrophorus plagiophthalamus) red luciferases, which emit distinct bioluminescent spectra, allowing simultaneous imaging of two different mycobacterial strains during infection. We also demonstrate that these same bioluminescence reporters can be used to evaluate therapeutic efficacy in real-time, greatly facilitating our ability to screen novel antibiotics as they are developed. Due to the slow growth rate of mycobacteria, novel imaging technologies are a pressing need, since they can they can impact the rate of development of new therapeutics as well as improving our understanding of virulence mechanisms and the evaluation of novel vaccine candidates.

Antibacterial Derivatives of Ciprofloxacin to Inhibit Growth of Necrotizing Fasciitis Associated Penicillin Resistant Escherichia coli.

Escherichia coli (E. coli) is associated with necrotizing fasciitis (type I) and can induce enough damage to tissue causing hypoxia. Three ester derivatives of the broad-spectrum antibiotic ciprofloxacin were placed into bacteria culture simultaneously with the parent ciprofloxacin (drug 1) to ascertain the level of antibacterial activity. The n-propyl (drug 2), n-pentyl (drug 3), and n-octyl (drug 4) esters of ciprofloxacin were synthesized under mixed phase conditions and by microwave excitation. The formation of ester derivatives of ciprofloxacin modified important molecular properties such as Log P and polar surface area which improves tissue penetration, yet preserved strong antibacterial activity. The Log P values for drugs 1, 2, 3, and 4 became -0.701, 0.437, 1.50, and 3.02, respectively. The polar surface areas for drugs 1, 2, 3, and 4 were determined to be 74.6 Angstroms(2), 63.6 Angstroms(2), 63.6 Angstroms(2), and 63.6 Angstroms(2), respectively. These values of Log P and polar surface area improved tissue penetration, as indicated by the determination of dermal permeability coefficient (K p ) and subsequently into the superficial fascial layer. All drugs induced greater than 60% bacterial cell death at concentrations less than 1.0 micrograms/milliliter. The ester derivatives of ciprofloxacin showed strong antibacterial activity toward penicillin resistant E. coli.

Molecular analysis of the Mycobacterium tuberculosis lux-like mel2 operon.

Using a high throughput genetic strategy, designated Random Inducible Controlled Expression (RICE), we identified the six gene mel2 locus in Mtb and M. marinum. Interestingly, three of the genes present in mel2 have similarities to bioluminescence genes. Similar to other bacterial bioluminescence systems, mel2 facilitates detoxification of reactive oxygen species (ROS). Through the use of thin layer chromatography (TLC) we demonstrate enhanced production of the cell wall virulence lipid, pthiocerol dimycoserosate (PDIM), in a Mtb mel2 mutant relative to the wild type strain in the presence of both H2O2 and diamide oxidative stresses. Furthermore, propionate toxicity assays revealed increased accumulation of triacylglycerol (TAG) in the mel2 mutant relative to wild type. These observations provide the first evidence that mel2 plays a critical role in Mtb lipid biosynthesis.

Small molecule hydrazide agents to inhibit growth and proliferation of mycobacterium tuberculosis.

Four novel drug designs for the treatment of Mycobacterium tuberculosis are analyzed and shown to prevent the growth and proliferation of this dangerous bacteria. All four agents, designated A, B, C, and D, are hydrazide type compounds, where D has three hydrazide functional groups. Agents B and C have a halogenated aromatic ring substituent, while A contains a pyridine ring. Pharmaceutical properties such as Log P, polar surface area, and violations of the Rule of 5 are determined for all agents. The Polar surface area for these four agents ranged from 55.121 A2 to 165.363 A2 and Log P values for A, B, C, and D were determined at -0.916, 0.95, 0.974, and -4.921, respectively. Drug designs A, B, and C show zero violations of the Rule of 5, where D exhibits only one violation, which are outcomes describing favorable bioavailability. Values of polar surface area for A, B, and C affirm an intestinal absorption of greater than 60% as well as the potential for crossing the blood brain barrier for targeting bacterial meningitis of the central nervous system. Interaction with Mycobacterium tuberculosis was monitored over a 14 day interval with agents at known concentration. Agents A, B, C, and D elicited more than 60% inhibition of bacterial growth by day 14 at concentrations of as little as 30 micrograms/ milliliter. All agents reduced bacteria survival to less than 60% by day 7 of culture. The inhibition of bacterial growth induced by agents A, B, C, and D was comparable to that of isoniazid. K-means cluster analysis of descriptors determined isoniazid most similar to agents A, B, and C. Other characteristics of these small hydrazide compounds render supportive evidence for an efficacious clinical application.

Using luciferase to image bacterial infections in mice.

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load. This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Random inducible controlled expression (RICE) for identification of mycobacterial virulence genes.

We have developed Random Inducible Controlled Expression (RICE), a high throughput genetic approach to identify regulated virulence pathways in pathogenic mycobacteria. RICE allows expression of bacterial genes under conditions where they are normally off, e.g. under laboratory growth conditions, via the use of an inducible or constitutive promoter as well as gene dosage effects due to the presence of the gene on a plasmid. Mycobacterial genomic DNA can be digested to yield random fragments for cloning into a suicide expression vector downstream of a mycobacterial promoter or with their own promoter on a replicating plasmid increasing expression by gene dosage effects. The plasmid DNA is normally amplified in Escherichia coli and delivered into mycobacteria to select for recombinants or plasmid transformants. The resulting library is then directly screened for enhanced host cell interactions in functional assays that evaluate the efficiency of adherence, entry and replication inside host cells. This approach has resulted in identification of several virulence factors from pathogenic mycobacteria. Our analysis of one such locus identified by RICE, the mycobacterial enhanced entry locus (mel2), found that the genes present facilitate bacterial persistence inside the host by protecting the pathogen against oxidative damage. Thus, we have developed a genetic strategy that offers several advantages: (i) it allows identification of bacterial genetic elements that have a direct role during host-pathogen interactions (ii) it can be used to identify virulence factors in a broad range of pathogens and (iii) it can reveal genes that are only induced at specific stages of infection.