Comparative analysis of iron regulated genes in mycobacteria

Iron dependent regulator, IdeR, regulates the expression of genes in response to intracellular iron levels in M. tuberculosis. Orthologs of IdeR are present in all the sequenced genomes of mycobacteria. We have used a computational approach to identify conserved IdeR regulated genes across the mycobacteria and the genes that are specific to each of the mycobacteria. Novel iron regulated genes that code for a predicted 4-hydroxy benzoyl coA hydrolase (Rv1847) and a protease dependent antibiotic regulatory system (Rv1846c, Rv0185c) are conserved across the mycobacteria. Although Mycobacterium natural-resistance-associated macrophage protein (Mramp) is present in all mycobacteria, it is, as predicted, an iron-regulated gene in only one species, M. avium subsp. paratuberculosis. We also observed an additional iron-regulated exochelin biosynthetic operon, which is present only in non-pathogenic Mycobacterium, M. smegmatis.     

PCR-linked reverse DNA hybridization using oligonucleotide-specific probes of rpoB for identification of Mycobacterium avium and Mycobacterium intracellulare

A PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes (Avp and Intp) of Mycobacterium avium and Mycobacterium intracellulare, respectively, were evaluated for the differentiation and identification of M. avium and M. intracellulare culture isolates. Among the 504 culture isolates tested by this method, 48 strains showed positive results for M. avium and 60 strains showed positive results for M. intracellulare. The other 396 culture isolates showed negative results for both M. avium and M. intracellulare. These results were consistent with those obtained from partial rpoB (306 bp) sequence analysis and biochemical tests. The negative strains obtained by this DNA hybridization method were identified as M. tuberculosis (366 strains), M. peregrinum (11 strains), M. abscessus (9 strains), M. fortuitum (8 strains), and M. flavescens (2 strains) by rpoB DNA sequence analysis. Due to the high sensitive and specific result obtained by this assay, we suggest that this PCR-linked reverse DNA hybridization method using two different specific rpoB DNA probes of M. avium and M. intracellulare would be used for the rapid and precise method for differentiation and identification of M. avium and M. intracellulare.     

Identification and comparative analysis of a genomic island in Mycobacterium avium subsp. hominissuis

Mycobacterium avium subsp. hominissuis (MAH) is an environmental bacterium causing opportunistic infections. The objective of this study was to identify flexible genome regions in MAH isolated from different sources. By comparing five complete and draft MAH genomes we identified a genomic island conferring additional flexibility to the MAH genomes. The island was absent in one of the five strains and had sizes between 16.37 and 84.85 kb in the four other strains. The genes present in the islands differed among strains and included phage- and plasmid-derived genes, integrase genes, hypothetical genes, and virulence-associated genes like mmpL or mce genes.     

Disruption of Mycobacterium avium subsp. paratuberculosis-specific genes impairs in vivo fitness

Background

Mycobacterium avium subsp. paratuberculosis (MAP) is an obligate intracellular pathogen that infects many ruminant species. The acquisition of foreign genes via horizontal gene transfer has been postulated to contribute to its pathogenesis, as these genetic elements are absent from its putative ancestor, M. avium subsp. hominissuis (MAH), an environmental organism with lesser pathogenicity. In this study, high-throughput sequencing of MAP transposon libraries were analyzed to qualitatively and quantitatively determine the contribution of individual genes to bacterial survival during infection.

Results

Out of 52384 TA dinucleotides present in the MAP K-10 genome, 12607 had a MycoMarT7 transposon in the input pool, interrupting 2443 of the 4350 genes in the MAP genome (56%). Of 96 genes situated in MAP-specific genomic islands, 82 were disrupted in the input pool, indicating that MAP-specific genomic regions are dispensable for in vitro growth (odds ratio = 0.21). Following 5 independent in vivo infections with this pool of mutants, the correlation between output pools was high for 4 of 5 (R = 0.49 to 0.61) enabling us to define genes whose disruption reproducibly reduced bacterial fitness in vivo. At three different thresholds for reduced fitness in vivo, MAP-specific genes were over-represented in the list of predicted essential genes. We also identified additional genes that were severely depleted after infection, and several of them have orthologues that are essential genes in M. tuberculosis.

Conclusions

This work indicates that the genetic elements required for the in vivo survival of MAP represent a combination of conserved mycobacterial virulence genes and MAP-specific genes acquired via horizontal gene transfer. In addition, the in vitro and in vivo essential genes identified in this study may be further characterized to offer a better understanding of MAP pathogenesis, and potentially contribute to the discovery of novel therapeutic and vaccine targets.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-415) contains supplementary material, which is available to authorized users.     

Development of a method to investigate the hydrolysis of xenobiotic esters by a Mycobacterium smegmatis homogenate

One of the main problems in combating tuberculosis is caused by a poor penetration of drugs into the mycobacterial cells. A prodrug approach via activation inside mycobacterial cells is a possible strategy to overcome this hurdle and achieve efficient drug uptake. Esters are attractive candidates for such a strategy and we and others communicated previously the activity of esters of weak organic acids against mycobacteria. However very little is known about ester hydrolysis by mycobacteria and no biological model is available to study the activation of prodrugs by these microorganisms. To begin filling this gap, we have embarked in a project to develop an in vitro method to study prodrug activation by mycobacteria using Mycobacterium smegmatis homogenates. Model ester substrates were ethyl nicotinate and ethyl benzoate whose hydrolysis was monitored and characterized kinetically. Our studies showed that in M. smegmatis most esterase activity is associated with the soluble fraction (cytosol) and is preserved by storage at 5 °C or at room temperature for one hour, or by storage at − 80 °C up to one year. In the range of homogenate concentrations studied (5-80% in buffer), k_obs varied linearly with homogenate concentration for both substrates. We also found that the homogenates showed Michaelis-Menten kinetics behavior with both prodrugs. Since ethyl benzoate is a good substrate for the mycobacterial esterases, this compound can be used to standardize the esterasic activity of homogenates, allowing results of incubations of prodrugs with homogenates from different batches to be readily compared.     

Conformational analyses of mycothiol, a critical intracellular glycothiol in Mycobacteria

Intracellular thiols are essential biomolecules, which play several critical roles in living organisms including controlling intracellular redox potential and acting as cofactors for several vital detoxification enzymes including S-transferases and formaldehyde dehydrogenases. The tripeptide gamma-L-glutamyl-L-cysteinylglycine, more commonly known as glutathione, is well known as the major intracellular thiol in eukaryotes and in some bacteria. However, glutathione is absent in the Actinomycetales bacteria such as Mycobacteria and Streptomyces and is believed to be replaced by 1-D-myo-inosityl-2-(N-acetyl-L-cysteinyl)amido-2-deoxy-alpha-D-glucopyranoside, mycothiol, in these organisms. Although much is known about the chemistry and biochemistry of glutathione, currently much less is known concerning mycothiol and its properties. The structure of mycothiol is composed of a glycoside linkage between myo-inositol and D-glucosamine with an N-acetyl-L-cysteine linked to the 2'-amino group of the d-glucosamine moiety. Mycothiol is currently of intense interest due to its essential role in the cellular physiology of Mycobacteria, such as Mycobacterium tuberculosis, and its possible role in antimycobacterial drug resistance. A detailed investigation of its chemistry is therefore essential in ameliorating our knowledge of this key glycothiol, and in shedding additional light on its biochemical role in these pathogenic organisms. This report presents a detailed conformational analysis of mycothiol utilizing a variety of force fields and stochastic search protocols. Cluster analyses of energetically low lying conformations have indicated the presence of several key conformations that are populated in the gas phase and with implicit water solvation. These conformations are compared to recent NMR studies on a derivative of mycothiol. This information should be an important contribution to our basic understanding of the chemistry of this glycothiol and critical in the design of novel inhibitors of pathogen enzymes that require it.     

Detection and persistence of fecal Bacteroidales as water quality indicators in unchlorinated drinking water

The results of this study support the use of fecal Bacteroidales qPCR as a rapid method to complement traditional, culture-dependent, water quality indicators in systems where drinking water is supplied without chlorination or other forms of disinfection. A SYBR-green based, quantitative PCR assay was developed to determine the concentration of fecal Bacteroidales 16S rRNA gene copies. The persistence of a Bacteroides vulgatus pure culture and fecal Bacteroidales from a wastewater inoculum was determined in unchlorinated drinking water at 10 °C. B. vulgatus 16S rRNA gene copies persisted throughout the experimental period (200 days) in sterile drinking water but decayed faster in natural drinking water, indicating that the natural microbiota accelerated decay. In a simulated fecal contamination of unchlorinated drinking water, the decay of fecal Bacteroidales 16S rRNA gene copies was considerably faster than the pure culture but similar to that of Escherichia coli from the same wastewater inoculum.     

Evaluation of a rapid and inexpensive liquid culture system for the detection of Mycobacterium avium subsp. paratuberculosis in bovine faeces

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent of paratuberculosis, a chronic granulomatous enteric disease of ruminants. MAP detection by faecal culture provides the definitive diagnosis of the infection. Automated liquid systems for MAP culture are more sensitive and rapid than culture on solid media, but they are expensive and require specialised equipment. In this study, a non-automated culture method using a modified Middlebrook 7H9 liquid medium (7H9+) was compared with Herrold's solid medium (HEYM) and direct real-time PCR on dairy cattle faeces. MAP growth in 7H9+ was monitored weekly by real-time PCR until the 12th week post-inoculation. The analytical sensitivity of the three methods was evaluated using faecal samples from a healthy cow spiked with ten-fold dilutions of MAP organisms (10⁴-10^− 1) and naturally MAP-infected faeces serially diluted 1 to 10 in negative faecal samples. The limits of detection of the solid culture and direct real-time PCR were 10² and 10³ MAP/g, respectively. In comparison, the 7H9+ culture revealed as few as 1 MAP/g. A marked reduction in time to detection of the pathogen, compared with HEYM culture, was obtained. In addition, the three methods were applied to environmental faecal samples collected from a high- and a low-prevalence herd. The culture in 7H9+ showed to be the most sensitive test in the low-prevalence herd and provided faster results than HEYM. In the high-prevalence herd the three methods showed the same sensitivity and the real-time PCR had the shortest turnaround time. In conclusion, the use of 7H9+ for MAP-detection from cattle faeces maximizes diagnostic sensitivity and reduces turnaround time and, therefore, could replace culture in solid medium. Hence, we propose a two-step protocol for the assessment of MAP faecal excretion based on: 1) direct real-time PCR on all samples; and 2) inoculation of negative samples into 7H9+ and analysis after 3 and, if necessary, 6 weeks by real-time PCR.     

Companion animal parasitology: a clinical perspective

In recent years there have been many changes to the ways that clinical veterinary science is conducted and nowhere is this more evident than in companion animal practice. Veterinarians working with pet dogs and cats are facing new challenges associated with the emergence and re-emergence of parasitic diseases. Some, such as Neospora caninum, have been recently recognised; others like Giardia and Cryptosporidium have been reported with increasing frequency, in part as a result of laboratory tests with improved sensitivity and specificity. In many regions, the emergence of parasitic diseases has been a consequence of pet travel and exotic diseases pose a unique diagnostic challenge for the veterinarian, as the index of suspicion for these conditions may be absent. The ranges of certain vector-borne diseases such as babesiosis, hepatozoonosis, ehrlichiosis, leishmaniasis and dirofilariasis are extending due to ecological and climatic changes and enhanced by animals with subclinical infection returning home from endemic areas. In companion animal practice, veterinarians have the additional responsibility of providing accurate information about the zoonotic transmission of parasite infections from pets, especially to those most vulnerable such as children, the elderly and the immunocompromised. Effective education is vital to allay public concerns and promote responsible pet ownership.     

Role of MAP kinase activation in Nramp1 mRNA stability in RAW264.7 macrophages expressing Nramp1(Gly169)

Nramp1 (natural resistance-associated macrophage protein 1) is a phagosomal iron transport molecule. In addition to its anti-microbial activity, Nramp1 exerts a wide range of pleiotropic effects, including increased stability of Nramp1 mRNA and a variety of other mRNA species. Previously, we showed that the increased stability of Nramp1 mRNA is regulated by an oxidant-generated signaling pathway that requires PKC. In the current study, we show that inhibition of ERK1,2 and p38 MAP kinase activities decreases Nramp1 mRNA stability in Mycobacterium avium infected RAW264.7 cells expressing Nramp1(Gly169) but not in RAW264.7-Nramp1(Asp169) cells. Phosphorylation of ERK1,2 and p38 MAP kinases, which could be inhibited by the anti-oxidant BHA and a protein kinase C inhibitor, was higher in M. avium infected RAW264.7-Nramp1(Gly169) cells than in RAW26.47-Nramp1(Asp169) cells. These results suggest that generation of oxidants by Nramp1 iron transport activates MAP kinase signaling cascades that result in stabilization of Nramp1 mRNA.     

High resolution melting PCR to differentiate Mycobacterium avium subsp. paratuberculosis"cattle type" and "sheep type"

In this study, we describe a novel HRM-PCR assay that clearly differentiates the two main types of Mycobacterium avium subsp. paratuberculosis (cattle and sheep) based on the polymorphic variation of a previously described tandem repeat. This modern genotyping technique has several advantages over alternative methods, including cost, ease of use and rapidity.     

Structure determination of lipopeptides from Mycobacterium avium subspecies paratuberculosis and identification of antigenic lipopeptide probes

Mycobacterium avium subspecies paratuberculosis (MAP) causes chronic illnesses mostly in ruminants. MAP infection of intestinal tissue triggers a fatal inflammatory disorder, Johne's disease (paratuberculosis). Development of fast and reliable diagnostic methods for Johne's disease in clinically suspected ruminants requires the discovery of MAP-specific antigens that induce immune responses. Despite a longtime interest in finding such antigens that can detect serum antibody responses with high sensitivity, the antigens currently used for a diagnosis of the MAP infections are the crude extracts from the whole cell. We performed the serum antibody response assay-guided purification of the ethanol extract from MAP isolated from an infected cow. With the results of extensive fractionations and in vitro assays, we identified that arachidyl-d-Phe-N-Me-l-Val-l-Ile-l-Phe-l-Ala-OH (named lipopeptide IIß, 3) exhibited the highest antibody binding activity in serum of a MAP-infected cattle compared with the other lipopeptides isolated from MAP. The absolute chemistry of 3 was determined unequivocally via our high-performance liquid chromatography (HPLC)–amino acid databases. α-Amino lipopeptide IIß and its fluorescent probes were synthesized and evaluated in serum antibody binding activity assays. Lipopeptide IIß-(2S)-NH₂ (9) and its dansyl and fluorescein isothiocyanate (FITC) probes (10 and 11) exhibited antibody-mediated binding activity; thus, such MAP-specific lipopeptide probes can be potential biomarkers for the development of rapid and accurate diagnosis of Johne's disease.     

Tandem screening of toxic compounds on GFP-labeled bacteria and cancer cells in microtiter plates

A 96-well fluorescence-based assay has been developed for the rapid screening of potential cytotoxic and bacteriocidal compounds. The assay is based on detection of green fluorescent protein (GFP) in HeLa human carcinoma cells as well as gram negative (Escherichia coli) and gram positive bacteria (Mycobacterium avium). Addition of a toxic compound to the GFP marked cells resulted in the loss of the GFP fluorescence which was readily detected by fluorometry. Thirty-nine distinct naphthoquinone derivatives were screened and several of these compounds were found to be toxic to all cell types. Apart from differences in overall toxicity, two general types of toxic compounds were detected, those that exhibited toxicity to two or all three of the cell types and those that were primarily toxic to the HeLa cells. Our results demonstrate that the parallel screening of both eukaryotic and prokaryotic cells is not only feasible and reproducible but also cost effective.     

Dietary glycerol and adult access to water: effects on fecundity and longevity in the almond moth

The quality of food eaten by larval insects will affect traits such as gamete production, fat reserves, muscle bulk and body size in the adult. Moreover, larvae also depend on high moisture content in the diet for survival. The almond moth (Ephestia cautella) (W.) (Lepidoptera; Pyralidae) does not feed as an adult although it continues to drink water. We tested the idea that an almond moth could compensate for a low-water diet as a larva by increasing its water intake as an adult. We reared larvae on two different food sources with different moisture regimes; standard laboratory diet with glycerol (relatively wet) and standard diet without glycerol (relatively dry). Half the adult moths from each treatment were given water to drink before their first and only mating. Our results show that wet larval diets (i.e. containing glycerol) significantly decreased fecundity (total number of eggs laid and the proportion of hatched larvae), whilst it significantly increased male and female longevity. The interaction effect of water access for adult males and females was significant, independent of the glycerol in the larval diet. Longevity in females that were not presented with water as adults was slightly higher if mated with a male that had had access to water, suggesting a mating donation of water. However, females that received water as adults showed a decreased longevity if mated with a male who had also had access to water as an adult, indicating a negative effect of water if received by both males and females. In addition, when the larval diet included glycerol, increased number of eggs laid decreased female longevity, whilst an absence of glycerol in the larval diet resulted in low female longevity that was unlinked with fecundity. Glycerol is used in many artificial insect diets and the fact that it shows a strong effect on key life-history traits (reproductive output and longevity in this species), merits careful re-examination of its effects on these important traits in other laboratory models. We also discuss the possibility that larval diet can affect female reproductive decisions.     

Endemic cattle diseases: comparative epidemiology and governance

Cattle are infected by a community of endemic pathogens with different epidemiological properties that invoke different managerial and governmental responses. We present characteristics of pathogens that influence their ability to persist in the UK, and describe a qualitative framework of factors that influence the political response to a livestock disease. We develop simple transmission models for three pathogens (bovine viral diarrhoea virus, bovine herpesvirus and Mycobacterium avium spp. paratuberculosis) using observed cattle movements, and compare the outcomes to an extensive dataset. The results demonstrate that the epidemiology of the three pathogens is determined by different aspects of within- and between-farm processes, which has economic, legal and political implications for control. We consider how these pathogens, and Mycobacterium bovis (the agent of bovine tuberculosis), may be classified by the process by which they persist and by their political profile. We further consider the dynamic interaction of these classifications with pathogen prevalence and with the action taken by the government.     

Functional analyses in the hemipteran Oncopeltus fasciatus reveal conserved and derived aspects of appendage patterning in insects

The conservation of expression of appendage patterning genes, particularly Distal-less, has been shown in a wide taxonomic sampling of animals. However, the functional significance of this expression has been tested in only a few organisms. Here we report functional analyses of orthologues of the genes Distal-less, dachshund, and homothorax in the appendages of the milkweed bug Oncopeltus fasciatus (Hemiptera). This hemimetabolous insect has typical legs but highly derived mouthparts. Distal-less, dachshund, and homothorax are conserved in their individual expression patterns and functions in the legs of Oncopeltus, but their functions in other appendages are in some cases divergent. We find that specification of antennal identity does not require wild-type Distal-less activity in Oncopeltus as it does in Drosophila. Additionally, the mouthparts of Oncopeltus show novel patterns of gene expression and function, relative to other insects. Expression of Distal-less in the maxillary stylets of Oncopeltus does not seem necessary for proper development of this appendage, while dachshund and homothorax are crucial for formation of the mandibular and maxillary stylets. These data are used to evaluate hypotheses for the evolution of hemipteran mouthparts and the evolution of developmental mechanisms in insect appendages in general.     

Visualization of Mycobacterium avium in Crohn's tissue by oil-immersion microscopy

Studies seeking Mycobacterium avium subsp. paratuberculosis in Crohn's disease by PCR have generated inconsistent findings. As an alternative, microscopy offers a number of advantages, including direct visualization of organisms in tissue. Experimental infections have demonstrated that M. avium organisms can be seen by both acid-fast staining and species-specific in situ hybridization, but because they are smaller than M. tuberculosis, oil-immersion microscopy (×1000 magnification) is needed. We performed a blinded search for M. avium in paraffin-embedded surgical resections from Crohn's and control subjects at two centres. Specimens were coded and subjected to acid-fast staining and ribosomal RNA in situ hybridization for M. avium rRNA. Agreement between these two methods was good (42/52 patients, κ = 0.60) and similar results were observed for patients from two centers. Together, both methods provided positive results in 10 of 17 Crohn's subjects (59%, 95% CI: 36–78), contrasting with only 5 of 35 control subjects (Odds ratio for Crohn's vs. controls = 8.6, p = 0.002). M. avium organisms had an intracellular localization within inflammatory lesions, but were often observed as lone organisms outside of granulomas. Using two assays in two settings, presence of M. avium organisms was strongly associated with Crohn's disease.     

Cost of oviposition site selection in a water strider Aquarius paludum insularis: Egg mortality increases with oviposition depth

Females generally avoid selecting sites for oviposition which have a high predation risk to increase offspring survival. Previous studies have focused on costs to ovipositing females. However, although offspring may also incur costs by being oviposited at low predation risk sites, no studies have focused on costs to offspring. Such costs to offspring were examined by using Aquarius paludum insularis, females of which avoid eggs parasitism by ovipositing at deep sites. Deep sites are safe from egg parasitism but may be unsuitable for hatching due to environmental factors. We examined the costs to offspring at deep sites by comparing the hatching rate, the duration to hatching and the proportion of drowned larvae between eggs that were set at three levels of water depth (0 cm, 25 cm and 50 cm depth). While the hatching rate at 50 cm was lower than that at 0 cm, the rate at 25 cm did not differ from that at 0 cm. Duration to hatching and the proportion of drowned larvae did not differ between the three depths. It is suggested that the declining survival rate of A. paludum eggs was due to increased water pressure at greater depth. Such a cost may exist in other species and such an observation may aid in understanding oviposition site selection.