Genetic and Phenotypic Differences Between Legionella Pneumophila Strains

Journal of Clinical Microbiology. Apr, 2002  |  Pubmed ID: 11923356

Legionnaires' disease is a potentially lethal pneumonia that is primarily due to infection by the species Legionella pneumophila, although more than 40 other species are known. Certain L. pneumophila subgroups, particularly serogroup 1, are associated with the majority of the epidemics. The genetic bases for these differences in virulence have not been determined. Three strains, AA100, JR32, and Lp01, have been used in many molecular pathogenesis studies of L. pneumophila. We found genetic differences between these strains by PCR and Southern analyses that may be related to their ability to cause disease. We also examined the distribution of these genetic loci in clinical and environmental isolates of Legionella and found a correlation between the presence of two of these loci, rtxA and lvh, and the ability to cause disease in humans. Examination of the interactions of these strains with host cells suggested that they differ in important phenotypic characteristics including adherence, entry, and intracellular replication. Furthermore, in the mouse model of infection they display differing levels of replication in lungs. These studies emphasize the importance of further investigation into the genetic makeup of these strains, which is likely to lead to the identification of additional factors involved in Legionella pathogenesis.

Role of the Legionella Pneumophila RtxA Gene in Amoebae

Microbiology (Reading, England). Jun, 2002  |  Pubmed ID: 12055287

Legionella pneumophila infects humans, causing Legionnaires' disease, from aerosols generated by domestic and environmental water sources. In aquatic environments L. pneumophila is thought to replicate primarily in protozoa. A 'repeats in structural toxin' (RTX) gene, rtxA, from L. pneumophila was identified recently that plays a role in entry and replication in human macrophages and also has the ability to infect mice. However, the role of this gene in the interaction of L. pneumophila with environmental protozoa and its distribution in different Legionella species has not been examined. Southern analyses demonstrated that rtxA is present in all L. pneumophila isolates tested and correlates with species that have been shown to cause disease in humans. To evaluate the importance of rtxA in the interaction with protozoa a series of studies was carried out in an environmental host for L. pneumophila, Acanthamoeba castellanii. The L. pneumophila rtxA gene plays a role in both adherence and entry into A. castellanii similar to that observed in human monocytic cells. Furthermore, it was found that rtxA is involved in intracellular survival and trafficking. In addition to demonstrating involvement of rtxA in the interaction of L. pneumophila with host cells, these data support a role for this gene both during disease in humans and in environmental reservoirs.

Bifunctional Constructs of Aspirin and Ibuprofen (non-steroidal Anti-inflammatory Drugs; NSAIDs) That Express Antibacterial and Alkylation Activities

Biotechnology and Applied Biochemistry. Jun, 2003  |  Pubmed ID: 12533193

Ibuprofen and aspirin are two common non-steroidal anti-inflammatory drugs (NSAIDs). Both NSAIDs have a carbonyl carbon [-C(O)-], which was utilized to attach a nitrogen mustard (N-mustard) ester group or a tripeptide group. The tripeptide consisted of a L-Gly-D-Ala-D-Ala sequence, where D-Ala-D-Ala is the reactive site for antibacterial activity and L-Gly serves as a linker to the NSAID carrier drug. The aspirin tripeptide and N-mustard show significant antibacterial activity at >or=5.0 x 10(-5) M against penicillin-susceptible or -resistant Escherichia coli. The partition coefficients (log Kow)log P of aspirin and ibuprofen tripeptide drugs were -1.05 and 2.23, respectively. The NSAIDs served as carrier drugs of the N-mustard group which expressed alkylation activity directed towards the nucleophilic primary amine of p -chloroaniline. Hydrolysis of the N-mustard agents yielded the parent structure of aspirin (or ibuprofen) and an N-mustard moiety, 2-[bis(2-chloroethyl)amino]ethanol. The (log Kow)log P for the N-mustard structures of aspirin and ibuprofen were 2.61 and 5.63, respectively. The (log Kow)log P value of 2-[bis(2-chloroethyl)amino]ethanol was 0.56. Fluorescamine was utilized to determine unreacted p -chloroaniline at known time intervals, which permitted calculation of rate constants and rate equations. The aspirin N-mustard agent expressed strong antibacterial activity against a penicillin-resistant bacteria and first-order alkylation kinetics. The ibuprofen N-mustard and 2-[bis(2-chloroethyl)amino]ethanol followed second-order alkylation kinetics. All N-mustard and tripeptide compounds showed zero violations of the Rule of 5. Values of TPSA (molecular polar surface area), C log P and molecular dipoles were calculated.

Identification of a Gene That Affects the Efficiency of Host Cell Infection by Legionella Pneumophila in a Temperature-dependent Fashion

Infection and Immunity. Nov, 2003  |  Pubmed ID: 14573644

The ability to infect host cells is critical for the survival and replication of intracellular pathogens in humans. We previously found that many genes involved in the ability of Legionella pneumophila to infect macrophages are not expressed efficiently under standard laboratory growth conditions. We have developed an approach using expression of L. pneumophila genes from an exogenous constitutive promoter on a low-copy-number vector that allows identification of genes involved in host cell infection. Through the use of this strategy, we found that expression of a gene, lvhB2, enhances the efficiency of L. pneumophila infection of mammalian cells. The putative protein encoded by lvhB2 has similarity to structural pilin subunits of type IV secretion systems. We confirmed that this gene plays a role in host cell infection by the construction of an in-frame deletion in the L. pneumophila lvhB2 gene and complementation of this mutant with the wild-type gene. The lvhB2 mutant does not display a very obvious defect in interactions with host cells when the bacteria are grown at 37 degrees C, but it has an approximately 100-fold effect on entry and intracellular replication when grown at 30 degrees C. These data suggest that lvhB2 plays an important role in the efficiency of host cell infection by L. pneumophila grown at lower temperatures.

Identification of Two Mycobacterium Marinum Loci That Affect Interactions with Macrophages

Infection and Immunity. Dec, 2004  |  Pubmed ID: 15557611

Mycobacterium marinum is closely related to Mycobacterium tuberculosis, the cause of tuberculosis in humans. M. marinum has become an important model system for the study of the molecular mechanisms involved in causing tuberculosis in humans. Through molecular genetic analysis of the differences between pathogenic and nonpathogenic mycobacteria, we identified two loci that affect the ability of M. marinum to infect macrophages, designated mel(1) and mel(2). In silico analyses of the 11 putative genes in these loci suggest that mel(1) encodes secreted proteins that include a putative membrane protein and two putative transglutaminases, whereas mel(2) is involved in secondary metabolism or biosynthesis of fatty acids. Interestingly, mel(2) is unique to M. marinum and the M. tuberculosis complex and not present in any other sequenced mycobacterial species. M. marinum mutants with mutations in mel(1) and mel(2), constructed by allelic exchange, are defective in the ability to infect both murine and fish macrophage cell lines. These data suggest that the genes in mel(1) and mel(2) are important for the ability of M. marinum to infect host cells.

Molecular Properties and Antibacterial Activity of the Methyl and Ethyl Ester Derivatives of Ampicillin

Physiological Chemistry and Physics and Medical NMR. 2004  |  Pubmed ID: 16268120

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.

Identification of Mycobacterium Avium Genes That Affect Invasion of the Intestinal Epithelium

Infection and Immunity. Jul, 2005  |  Pubmed ID: 15972512

Invasion of intestinal mucosa of the host by Mycobacterium avium is a critical step in pathogenesis and likely involves several different bacterial proteins, lipids, glycoproteins, and/or glycolipids. Through the screening of an M. avium genomic library in Mycobacterium smegmatis, we have identified a number of M. avium genes that are associated with increased invasion of mucosal epithelial cells. In order to further investigate these genes, we cloned six of them into a plasmid downstream of a strong mycobacterial promoter (L5 mycobacterial phage promoter), resulting in constitutive expression. Bacteria were then evaluated for increased expression and examined for invasion of HT-29 intestinal epithelial cells. The genes identified encode proteins that are similar to (i) M. tuberculosis coenzyme A carboxylase, (ii) M. tuberculosis membrane proteins of unknown function, (iii) M. tuberculosis FadE20, (iv) a Mycobacterium paratuberculosis surface protein, and (v) M. tuberculosis cyclopropane fatty acyl-phopholipid synthase. The constitutive expression of these genes confers to M. avium the ability to invade HT-29 intestinal epithelial cells with a severalfold increase in efficiency compared to both the wild-type M. avium and M. avium containing the vector alone. Using the murine intestinal ligated loop model, it was observed that the constitutive expression of M. avium proteins has a modest impact on the ability to enter the intestinal mucosa when compared with the wild-type control, suggesting that under in vivo conditions these genes are expressed at higher levels. Evaluation of the expression of these invasion-related genes indicated that under conditions similar to the intestinal lumen environment, the genes identified are upregulated. These data suggest that invasion of the intestinal mucosa is an event that requires the participation of several bacterial factors and the expression of the genes that encode them is less observed under standard laboratory growth conditions.

Identification of Mycobacterium Marinum Macrophage Infection Mutants

Microbial Pathogenesis. Apr, 2006  |  Pubmed ID: 16451826

Mycobacterium marinum is an important pathogen of humans, amphibians and fish. Most pathogenic mycobacteria, including M. marinum, infect, survive and replicate primarily intracellularly within macrophages. We constructed a transposon mutant library in M. marinum using Tn5367 delivered by phage transduction in the shuttle phasmid phAE94. We screened 529 clones from the transposon library directly in macrophage infection assays. All clones were screened for their ability to initially infect macrophages as well as survive and replicate intracellularly. We identified 19 mutants that fit within three classes: class I) defective for growth in association with macrophages (42%), class II) defective for macrophage infection (21%) and class III) defective for infection of and growth in association with macrophages (37%). Although 14 of the macrophage infection mutants (Mim) carry insertions in genes that have not been previously identified, five are associated with virulence of mycobacteria in animal models. These observations confirm the utility of mutant screens directly in association with macrophages to identify new virulence determinants in mycobacteria. We complemented four of the Mim mutants with their M. tuberculosis homologue, demonstrating that secondary mutations are not responsible for the observed defect in macrophage infection. The genes we identified provide insight into the molecular mechanisms of macrophage infection by M. marinum.

A Mycobacterium Marinum Mel2 Mutant is Defective for Growth in Macrophages That Produce Reactive Oxygen and Reactive Nitrogen Species

Infection and Immunity. Jan, 2007  |  Pubmed ID: 17030568

Macrophages produce reactive oxygen species (ROS) and reactive nitrogen species (RNS) in response to bacterial infections. Mycobacteria are relatively resistant to ROS, but RNS inhibit growth of, and possibly even kill, mycobacteria in activated macrophages. We recently constructed a Mycobacterium marinum mel2 locus mutant, which is known to affect macrophage infection. We found previously that the mel2 locus confers resistance to ROS and RNS in laboratory medium, suggesting that this locus might play a similar role during growth in macrophages. Since J774A.1 murine macrophages produce high levels of ROS and RNS upon activation with gamma interferon (IFN-gamma), we examined the effects of IFN-gamma on ROS and RNS production by these cells as well as the effects on growth of M. marinum in these cells. We found that an M. marinum mutant with mutation of the first gene in the mel2 locus, melF, is defective for growth in IFN-gamma-plus-lipopolysaccharide-treated J774A.1 cells and that this defect is abrogated by the presence of either inhibitors of nitric oxide synthase or ROS scavengers. Furthermore, the M. marinum melF mutant displays a defect at late stages in the mouse footpad model of infection. These phenotypic characteristics could be complemented fully by the entire mel2 locus but only partially by the presence of melF alone, supporting data suggesting that this insertion mutation has polar effects on downstream genes in the mel2 locus. These observations demonstrate that the M. marinum mel2 locus plays a role in resistance to ROS and RNS produced by activated macrophages.

The Mycobacterium Marinum Mel2 Locus Displays Similarity to Bacterial Bioluminescence Systems and Plays a Role in Defense Against Reactive Oxygen and Nitrogen Species

BMC Microbiology. 2007  |  Pubmed ID: 17239244

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.

Derivatives of Cephalothin That Inhibit Ampicillin Resistant Escherichia Coli

Medicinal Chemistry (Shāriqah (United Arab Emirates)). Jan, 2007  |  Pubmed ID: 17266623

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.

Antibacterial Activity of Dipeptide Constructs of Acetylsalicylic Acid and Nicotinic Acid

Drug Delivery. Feb, 2007  |  Pubmed ID: 17364875

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.

Identification of Mycobacterium Avium Pathogenicity Island Important for Macrophage and Amoeba Infection

Proceedings of the National Academy of Sciences of the United States of America. Jun, 2007  |  Pubmed ID: 17578930

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.

Virulent Mycobacteria and the Many Aspects of Macrophage Uptake

Future Microbiology. Oct, 2007  |  Pubmed ID: 17927465

Use of Gene Dosage Effects for a Whole-genome Screen to Identify Mycobacterium Marinum Macrophage Infection Loci

Infection and Immunity. Jul, 2008  |  Pubmed ID: 18443095

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.

Pseudomonas Aeruginosa PAO1 Virulence Factors and Poplar Tree Response in the Rhizosphere

Microbial Biotechnology. Jan, 2008  |  Pubmed ID: 21261818

Whole-transcriptome analysis was used here for the first time in the rhizosphere to discern the genes involved in the pathogenic response of Pseudomonas aeruginosa PAO1 as well as to discern the response of the poplar tree. Differential gene expression shows that 185 genes of the bacterium and 753 genes of the poplar tree were induced in the rhizosphere. Using the P. aeruginosa transcriptome analysis, isogenic knockout mutants, and two novel plant assays (poplar and barley), seven novel PAO1 virulence genes were identified (PA1385, PA2146, PA2462, PA2463, PA2663, PA4150 and PA4295). The uncharacterized putative haemolysin repressor, PA2463, upon inactivation, resulted in greater poplar virulence and elevated haemolysis while this mutant remained competitive in the rhizosphere. In addition, disruption of the haemolysin gene itself (PA2462) reduced the haemolytic activity of P. aeruginosa, caused less cytotoxicity and reduced barley virulence, as expected. Inactivating PA1385, a putative glycosyl transferase, reduced both poplar and barley virulence. Furthermore, disrupting PA2663, a putative membrane protein, reduced biofilm formation by 20-fold. Inactivation of PA3476 (rhlI) increased virulence with barley as well as haemolytic activity and cytotoxicity, so quorum sensing is important in plant pathogenesis. Hence, this strategy is capable of elucidating virulence genes for an important pathogen.

Four Hydrazide Compounds That Inhibit the Growth of Mycobacterium Tuberculosis

Physiological Chemistry and Physics and Medical NMR. 2008  |  Pubmed ID: 20070040

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.

Indole and 7-hydroxyindole Diminish Pseudomonas Aeruginosa Virulence

Microbial Biotechnology. Jan, 2009  |  Pubmed ID: 21261883

Indole is an extracellular biofilm signal for Escherichia coli, and many bacterial oxygenases readily convert indole to various oxidized compounds including 7-hydroxyindole (7HI). Here we investigate the impact of indole and 7HI on Pseudomonas aeruginosa PAO1 virulence and quorum sensing (QS)-regulated phenotypes; this strain does not synthesize these compounds but degrades them rapidly. Indole and 7HI both altered extensively gene expression in a manner opposite that of acylhomoserine lactones; the most repressed genes encode the mexGHI-opmD multidrug efflux pump and genes involved in the synthesis of QS-regulated virulence factors including pyocyanin (phz operon), 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) signal (pqs operon), pyochelin (pch operon) and pyoverdine (pvd operon). Corroborating these microarray results, indole and 7HI decreased production of pyocyanin, rhamnolipid, PQS and pyoverdine and enhanced antibiotic resistance. In addition, indole affected the utilization of carbon, nitrogen and phosphorus, and 7HI abolished swarming motility. Furthermore, 7HI reduced pulmonary colonization of P. aeruginosa in guinea pigs and increased clearance in lungs. Hence, indole-related compounds have potential as a novel antivirulence approach for the recalcitrant pathogen P. aeruginosa.

Protection of Mycobacterium Tuberculosis from Reactive Oxygen Species Conferred by the Mel2 Locus Impacts Persistence and Dissemination

Infection and Immunity. Jun, 2009  |  Pubmed ID: 19349422

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.

Mycobacterium Tuberculosis Interferes with the Response to Infection by Inducing the Host EphA2 Receptor

The Journal of Infectious Diseases. Jun, 2009  |  Pubmed ID: 19426113

Mycobacterium tuberculosis is an unusual pathogen, persisting for years in infected persons despite an immune response. Erythropoietin-producing hepatoma (Eph) receptors are critical for tissue organization. One hallmark of tuberculosis is the presence of granulomas consisting of organized immune cells. The importance of granuloma structure makes it likely that Eph receptors play a role in immunity to tuberculosis.

Efficacy of Using Sutures Treated with Povidone-iodine or Chlorhexidine for Preventing Growth of Staphylococcus and Escherichia Coli

Plastic and Reconstructive Surgery. Jul, 2009  |  Pubmed ID: 19568081

Application of Optical Imaging to Study of Extrapulmonary Spread by Tuberculosis

Tuberculosis (Edinburgh, Scotland). Dec, 2009  |  Pubmed ID: 20006298

The incidence of extrapulmonary tuberculosis is increasing, possibly due to the high frequency of co-infection with HIV. Extrapulmonary infections complicate diagnosis, have higher mortality rates and are more difficult to treat. Insight into the mechanisms involved in extrapulmonary spread of tuberculosis is critical to improving management. We set out to better understand extrapulmonary spread kinetics in mice and guinea pigs as well as the effects of infectious dose. We found that extrapulmonary spread occurs at a discrete time point when infected by low-dose aerosol, but at high-dose aerosol it occurs within the first 24h. The ability to follow tuberculosis in real-time during infection would allow us to better address the mechanisms involved. We found that mycobacteria can be optically imaged after pulmonary infection in the mouse lung, suggesting that this technology could be applied to study of extrapulmonary spread of tuberculosis.

Design of Ciprofloxacin Derivatives That Inhibit Growth of Methicillin Resistant Staphylococcus Aureus (MRSA) and Methicillin Susceptible Staphylococcus Aureus (MSSA)

Medicinal Chemistry (Shāriqah (United Arab Emirates)). Mar, 2010  |  Pubmed ID: 20367615

Three derivatives of ciprofloxacin (compound B, C, and D) were constructed utilizing microwave synthesis methodology (compound D) or diazoalkane reaction in nonaqueous solvent (compounds B and C). The final structures of the derivatives featured an ester group in place of the original carboxyl group of the ciprofloxacin. These ester groups contained aliphatic single carbon (compound B), two carbon length (compound C), or three carbon length propyl ester group (compound D). The ester groups strongly affected the molecular properties of the parent ciprofloxacin. As the size of the ester group increased the formula weight, molar volume, and number of rotatable bonds increased. The Log P for these compounds were -0.701, -0.441, -0.065, 0.437 for ciprofloxacin, B, C, and compound D, respectively. Numerical values of dermal permeability coefficient (Kp) increased rapidly as length of the ester carbon chain increased. The immediate consequence of Kp increase is an increased skin penetration rate based on dose and time span of administration. Polar surface area for ciprofloxacin is 74.569 Angstroms(2), but decreases to 63.575 Angstroms(2) for all three derivatives. All three derivatives of ciprofloxacin showed zero violations of the Rule of 5, indicating these drugs would have favorable bioavailability. Compounds A, B, C, and D were placed into tissue culture with methicillin resistant and susceptible Staphylococcus aureus (MRSA and MSSA, respectively) to determine levels of bacterial growth inhibition. All compounds induced greater than 60 % inhibition of MSSA at concentrations as low as 15.63 micrograms/milliliter. All four compounds induced greater than 80 % inhibition of MRSA at concentratins as low as 15.63 micrograms/milliliter. Development of novel drug designs will benefit the clinical treatment of dangerous infections of MSSA and MRSA.

Imaging Tuberculosis with Endogenous Beta-lactamase Reporter Enzyme Fluorescence in Live Mice

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2010  |  Pubmed ID: 20566877

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

Molecular Microbiology. May, 2011  |  Pubmed ID: 21375593

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.

Whole-body Imaging of Infection Using Fluorescence

Current Protocols in Microbiology. May, 2011  |  Pubmed ID: 21538304

Optical imaging is emerging as a powerful tool to study physiological, neurological, oncological, cell biological, molecular, developmental, immunological, and infectious processes. This unit describes the use of fluorescent reporters for biological organisms, components, or events. We describe the application of fluorescence imaging to examination of infectious processes, in particular subcutaneous and pulmonary bacterial infections, but the same approaches are applicable to nearly any infectious route. The strategies described use mycobacterial infections as an example, but nearly identical systems can be used for Pseudomonas, Legionella, Salmonella, Escherichia, Borrelia, and Staphylococus, suggesting that the approaches are generally applicable to nearly any infectious agent. Two strategies for fluorescence imaging are described: the first method uses reporter enzyme fluorescence (REF), and the second uses fluorescent proteins for fluorescence imaging. Methods are described in detail to facilitate successful application of these emerging technologies to nearly any experimental system.

Whole-body Imaging of Infection Using Bioluminescence

Current Protocols in Microbiology. May, 2011  |  Pubmed ID: 21538305

Bioluminescence imaging is a powerful technique to visualize and monitor biological processes in numerous systems. This unit describes two strategies for bioluminescence imaging that can be used to study bacterial infection in mice. One method is to express a luciferase gene in the bacteria; the second method is to use bacteria that express both a luciferase and β-lactamase along with a substrate containing caged luciferin, which is released by β-lactamase hydrolysis and reacts with luciferase to generate light. For both strategies, bioluminescent signals are imaged using an IVIS live animal imaging system (Caliper Life Sciences). The bioluminescence images are analyzed to localize bioluminescent bacteria, quantify signal, and determine the wavelengths of the signals produced. The correlation of bacterial numbers with signal intensity in vivo can be determined, allowing a quantitative measure of bacterial numbers in mice in real time. Methods are described in detail to facilitate successful application of these emerging technologies in nearly any experimental system.

Hydrazide Drugs That Inhibit Growth and Proliferation of Tuberculosis Bacteria

Physiological Chemistry and Physics and Medical NMR. 2011  |  Pubmed ID: 21970157

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.

Random Inducible Controlled Expression (RICE) for Identification of Mycobacterial Virulence Genes

Tuberculosis (Edinburgh, Scotland). Dec, 2011  |  Pubmed ID: 22079212

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.