Phagocytosis influences the intracellular survival of <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis> via the endoplasmic reticulum stress response

Background

Mycobacterium smegmatis, a rapidly growing non-tuberculosis mycobacterium, is a good model for studying the pathogenesis of tuberculosis because of its genetic similarity to Mycobacterium tuberculosis (Mtb). Macrophages remove mycobacteria during an infection. Macrophage apoptosis is a host defense mechanism against intracellular bacteria. We have reported that endoplasmic reticulum (ER) stress is an important host defense mechanism against Mtb infection.

Results

In this study, we found that M. smegmatis induced strong ER stress. M. smegmatis-induced reactive oxygen species (ROS) play a critical role in the induction of ER stress-mediated apoptosis. Pretreatment with an ROS scavenger suppressed M. smegmatis-induced ER stress. Elimination of ROS decreased the ER stress response and significantly increased the intracellular survival of M. smegmatis. Interestingly, inhibition of phagocytosis significantly decreased ROS synthesis, ER stress response induction, and cytokine production.

Conclusions

Phagocytosis of M. smegmatis induces ROS production, leading to production of proinflammatory cytokines. Phagocytosis-induced ROS is associated with the M. smegmatis-mediated ER stress response in macrophages. Therefore, phagocytosis plays a critical role in the induction of ER stress-mediated apoptosis during mycobacterial infection.

     

Severe inhibition of lipooligosaccharide synthesis induces TLR2-dependent elimination of <Emphasis Type="Italic">Mycobacterium marinum</Emphasis> from THP1-derived macrophages

Background

Although mycobacterial glycolipids are among the first-line molecules involved in host–pathogen interactions, their contribution in virulence remains incomplete. Mycobacterium marinum is a waterborne pathogen of fish and other ectotherms, closely related to Mycobacterium tuberculosis. Since it causes tuberculosis-like systemic infection it is widely used as a model organism for studying the pathogenesis of tuberculosis. It is also an occasional opportunistic human pathogen. The M. marinum surface-exposed lipooligosaccharides (LOS) are immunogenic molecules that participate in the early interactions with macrophages and modulate the host immune system. Four major LOS species, designated LOS-I to LOS-IV, have been identified and characterized in M. marinum. Herein, we investigated the interactions between a panel of defined M. marinum LOS mutants that exhibited various degrees of truncation in the LOS structure, and human-derived THP-1 macrophages to address the potential of LOSs to act as pro- or avirulence factors.

Results

A moderately truncated LOS structure did not interfere with M. marinum invasion. However, a deeper shortening of the LOS structure was associated with increased entry of M. marinum into host cells and increased elimination of the bacilli by the macrophages. These effects were dependent on Toll-like receptor 2.

Conclusion

We provide the first evidence that LOSs inhibit the interaction between mycobacterial cell wall ligands and appropriate macrophage pattern recognition receptors, affecting uptake and elimination of the bacteria by host phagocytes.

     

Branched-chain amino acid aminotransferase and methionine formation in <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Background

Tuberculosis remains a major world-wide health threat which demands the discovery and characterisation of new drug targets in order to develop future antimycobacterials. The regeneration of methionine consumed during polyamine biosynthesis is an important pathway present in many microorganisms. The final step of this pathway, the conversion of ketomethiobutyrate to methionine, can be performed by aspartate, tyrosine, or branched-chain amino acid aminotransferases depending on the particular species examined.

Results

The gene encoding for branched-chain amino acid aminotransferase in Mycobacterium tuberculosis H37Rv has been cloned, expressed, and characterised. The enzyme was found to be a member of the aminotransferase IIIa subfamily, and closely related to the corresponding aminotransferase in Bacillus subtilis, but not to that found in B. anthracis or B. cereus. The amino donor preference for the formation of methionine from ketomethiobutyrate was for isoleucine, leucine, valine, glutamate, and phenylalanine. The enzyme catalysed branched-chain amino acid and ketomethiobutyrate transamination with a Km of 1.77 – 7.44 mM and a Vmax of 2.17 – 5.70 μmol/min/mg protein, and transamination of ketoglutarate with a Km of 5.79 – 6.95 mM and a Vmax of 11.82 – 14.35 μmol/min/mg protein. Aminooxy compounds were examined as potential enzyme inhibitors, with O-benzylhydroxylamine, O-t-butylhydroxylamine, carboxymethoxylamine, and O-allylhydroxylamine yielding mixed-type inhibition with Ki values of 8.20 – 21.61 μM. These same compounds were examined as antimycobacterial agents against M. tuberculosis and a lower biohazard M. marinum model system, and were found to completely prevent cell growth. O-Allylhydroxylamine was the most effective growth inhibitor with an MIC of 78 μM against M. marinum and one of 156 μM against M. tuberculosis.

Conclusion

Methionine formation from ketomethiobutyrate is catalysed by a branched-chain amino acid aminotransferase in M. tuberculosis. This enzyme can be inhibited by selected aminooxy compounds, which also have effectiveness in preventing cell growth in culture. These compounds represent a starting point for the synthesis of branched-chain aminotransferase inhibitors with higher activity and lower toxicity.

     

Role of the horizontal gene exchange in evolution of pathogenic Mycobacteria

Background

Mycobacterium tuberculosis is one of the most dangerous human pathogens, the causative agent of tuberculosis. While this pathogen is considered as extremely clonal and resistant to horizontal gene exchange, there are many facts supporting the hypothesis that on the early stages of evolution the development of pathogenicity of ancestral Mtb has started with a horizontal acquisition of virulence factors. Episodes of infections caused by non-tuberculosis Mycobacteria reported worldwide may suggest a potential for new pathogens to appear. If so, what is the role of horizontal gene transfer in this process?

Results

Availing of accessibility of complete genomes sequences of multiple pathogenic, conditionally pathogenic and saprophytic Mycobacteria, a genome comparative study was performed to investigate the distribution of genomic islands among bacteria and identify ontological links between these mobile elements. It was shown that the ancient genomic islands from M. tuberculosis still may be rooted to the pool of mobile genetic vectors distributed among Mycobacteria. A frequent exchange of genes was observed between M. marinum and several saprophytic and conditionally pathogenic species. Among them M. avium was the most promiscuous species acquiring genetic materials from diverse origins.

Conclusions

Recent activation of genetic vectors circulating among Mycobacteria potentially may lead to emergence of new pathogens from environmental and conditionally pathogenic Mycobacteria. The species which require monitoring are M. marinum and M. avium as they eagerly acquire genes from different sources and may become donors of virulence gene cassettes to other micro-organisms.

     

Evidence for positive selection on Mycobacterium tuberculosis within patients

Background

While the pathogenesis and epidemiology of tuberculosis are well studied, relatively little is known about the evolution of the infectious agent Mycobacterium tuberculosis, especially at the within-host level. The insertion sequence IS6110 is a genetic marker that is widely used to track the transmission of tuberculosis between individuals. This and other markers may also facilitate our understanding of the disease within patients.

Results

This article presents three lines of evidence supporting the action of positive selection on M. tuberculosis within patients. The arguments are based on a comparison between empirical findings from molecular epidemiology, and population genetic models of evolution. Under the hypothesis of neutrality of genotypes, 1) the mutation rate of the marker IS6110 is unusually high, 2) the time it takes for substitutions to occur within patients is too short, and 3) the amount of polymorphism within patients is too low.

Conclusions

Empirical observations are explained by the action of positive selection during infection, or alternatively by very low effective population sizes. I discuss the possible roles of antibiotic treatment, the host immune system and extrapulmonary dissemination in creating opportunities for positive selection.

     

Evaluation of the hyplex<Superscript>®</Superscript> TBC PCR test for detection of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> complex in clinical samples

Background

Tuberculosis (TB) is one of the major public health concerns worldwide. The detection of the pathogen Mycobacterium tuberculosis complex (MTBC) as early as possible has a great impact on the effective control of the spread of the disease. In our study, we evaluated the hyplex^® TBC PCR test (BAG Health Care GmbH), a novel assay using a nucleic acid amplification technique (NAAT) with reverse hybridisation and ELISA read out for the rapid detection of M. tuberculosis directly in clinical samples.

Results

A total of 581 respiratory and non-respiratory specimens from our pneumological hospital and the National TB Institute of Uzbekistan were used for the evaluation of the PCR assay. Of these, 292 were classified as TB samples and 289 as non-TB samples based on the results of the TB cultures as reference method. The PCR results were initially used to optimise the cut-off value of the hyplex^® TBC test system by means of a ROC analysis. The overall sensitivity of the assay was determined to be 83.1%. In smear-positive TB samples, the sensitivity of the hyplex^® TBC PCR test was estimated to 93.4% versus 45.1% in smear-negative samples. The specificity of the test was 99.25%. Of the two specimens (0.75%) with false-positive PCR results, one yielded a culture positive for non-tuberculous mycobacteria. Based on the assumption of a prevalence of 8% TB positives among the samples in our diagnostic TB laboratory, the positive and negative predictive values were estimated to 90.4% and 98.5%, respectively.

Conclusions

The hyplex^® TBC PCR test is an accurate NAAT assay for a rapid and reliable detection of M. tuberculosis in various respiratory and non-respiratory specimens. Compared to many other conventional NAAT assays, the hyplex^® TBC PCR test is in a low price segment which makes it an attractive option for developing and emerging countries with high TB burdens.

     

Molecular Identification of <Emphasis Type="Italic">Malassezia</Emphasis> Species in Patients with <Emphasis Type="Italic">Malassezia</Emphasis> folliculitis in Sfax,Tunisia

Aim

Malassezia folliculitis is caused by the invasion of hair follicles by large numbers of Malassezia cells. Several Malassezia researches still use cultures, morphology and biochemical techniques. The aim of this study was to identify Malassezia species isolated from patients diagnosed with folliculitis, at the Parasitology and Mycology Laboratory of Sfax University Hospital, and to explore the genetic diversity of Malassezia by using PCR-RFLP and PCR-sequencing targeting the rDNA region of the Malassezia genome.

Patients and Methods

Specimens were taken from 27 patients with Malassezia folliculitis. For the molecular identification, PCR amplification of the 26S rDNAD1/D2 region was carried out using the Malup and Maldown primers and three restriction enzymes (BanI, MspI and HeaII) for RFLP analysis. The nucleotide sequences of each isolate were compared to those in the NCBI GenBank by using BLASTIN algorithm.

Results

Three species of Malassezia yeasts were identified among the 31 Malassezia strains isolated: M. globosa (83.9%), M. sympodialis (12. 9%) and M. furfur (3.2%). The sequence analysis of M. globosa showed six genotypes.

Conclusion

There is a high genotypic variability of M. globosa colonizing patients with folliculitis.

     

Genetically diverse mice are novel and valuable models of age-associated susceptibility to <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Background

Tuberculosis, the disease due to Mycobacterium tuberculosis, is an important cause of morbidity and mortality in the elderly. Use of mouse models may accelerate insight into the disease and tests of therapies since mice age thirty times faster than humans. However, the majority of TB research relies on inbred mouse strains, and these results might not extrapolate well to the genetically diverse human population. We report here the first tests of M. tuberculosis infection in genetically heterogeneous aging mice, testing if old mice benefit from rapamycin.

Findings

We find that genetically diverse aging mice are much more susceptible than young mice to M. tuberculosis, as are aging human beings. We also find that rapamycin boosts immune responses during primary infection but fails to increase survival.

Conclusions

Genetically diverse mouse models provide a valuable resource to study how age influences responses and susceptibility to pathogens and to test interventions. Additionally, surrogate markers such as immune measures may not predict whether interventions improve survival.

     

Impacts of peat bulk density,ash deposition and rainwater chemistry on establishment of peatland mosses

Background and aims

Peatland moss communities play an important role in ecosystem function. Drivers such as fire and atmospheric pollution have the capacity to influence mosses via multiple pathways. Here, we investigate physical and chemical processes which may influence establishment and growth of three key moss species in peatlands.

Methods

A controlled factorial experiment investigated the effects of different peat bulk density, ash deposition and rainwater chemistry treatments on the growth of Sphagnum capillifolium, S. fallax and Campylopus introflexus.

Results

Higher peat bulk density limited growth of both Sphagnum species. S. capillifolium and C. introflexus responded positively to ash deposition. Less polluted rain limited growth of C. introflexus. Biomass was well correlated with percentage cover in all three species.

Conclusions

Peat bulk density increases caused by fire or drainage can limit Sphagnum establishment and growth, potentially threatening peatland function. Ash inputs may have direct benefits for some Sphagnum species, but are also likely to increase competition from other bryophytes and vascular plants which may offset positive effects. Rainwater pollution may similarly increase competition to Sphagnum, and could enhance positive effects of ash addition on C. introflexus growth. Finally, cover can provide a useful approximation of biomass where destructive sampling is undesirable.

     

Three-dimensional models of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> proteins Rv1555, Rv1554 and their docking analyses with sildenafil,tadalafil, vardenafil drugs,suggest interference with quinol binding likely to affect protein’s function

Background

Earlier based on bioinformatics analyses, we had predicted the Mycobacterium tuberculosis (M.tb) proteins; Rv1555 and Rv1554, among the potential new tuberculosis drug targets. According to the ‘TB-drugome’ the Rv1555 protein is ‘druggable’ with sildenafil (Viagra), tadalafil (Cialis) and vardenafil (Levitra) drugs. In the present work, we intended to understand via computer modeling studies, how the above drugs are likely to inhibit the M.tb protein’s function.

Results

The three-dimensional computer models for M.tb proteins; Rv1555 and Rv1554 constructed on the template of equivalent membrane anchor subunits of the homologous E.coli quinol fumarate reductase respiratory protein complex, followed by drug docking analyses, suggested that the binding of above drugs interferes with quinol binding sites. Also, we experimentally observed the in-vitro growth inhibition of E.coli bacteria containing the homologous M.tb protein sequences with sildenafil and tadalafil drugs.

Conclusions

The predicted binding sites of the drugs is likely to affect the above M.tb proteins function as quinol binding is known to be essential for electron transfer function during anaerobic respiration in the homologous E.coli protein complex. Therefore, sildenafil and related drugs currently used in the treatment of male erectile dysfunction targeting the human phosphodiesterase 5 enzyme may be evaluated for their plausible role as repurposed drugs to treat human tuberculosis.

     

Exosomes derived from tumor cells genetically modified to express <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> antigen: a novel vaccine for cancer therapy

Objectives

To examine the potential of exosomes derived from the tumor cells, which had been genetically modified to express a Mycobacterium tuberculosis antigen, as a cancer vaccine aimed at overcoming the weak immunogenicity of tumor antigens.

Results

We transfected B16 melanoma cells with a plasmid encoding the M. tuberculosis antigen, early secretory antigenic target-6 (ESAT-6). The secreted exosomes bearing both tumor-associated antigens and the pathogenic antigen (or their epitopes) were collected. When the exosomes were injected into foot pads of mice, they significantly (p < 0.05) evoked cellular immunity against both ESAT-6, and B16 tumor cells. Intra-tumoral injection of the exosomes significantly suppressed (p < 0.001) tumor growth in syngeneic B16 tumor-bearing mice, while the exosomes derived from the non-transfected B16 cells showed no effect on tumor growth, although both exosomes should have similar tumor antigens.

Conclusions

Exosomes bearing both tumor antigens and the M. tuberculosis antigen (or their epitopes) have a high potential as a candidate for cancer vaccine to overcome the immune escape by tumor cells.

     

Unexpected high circulation of <Emphasis Type="Italic">Plasmodium vivax</Emphasis> in asymptomatic children from Kédougou,southeastern Senegal

Background

Malaria in Senegal is due essentially to infections by Plasmodium falciparum and, to a lesser extent to Plasmodium malariae and Plasmodium ovale. By the use of molecular methods, detection of Plasmodium vivax has been recently reported in the region of Kedougou, raising the question of appraisal of its potential prevalence in this setting.

Methods

A retrospective serological study was carried out using 188 samples taken from 2010 to 2011 in a longitudinal school survey during which 48 asymptomatic children (9–11 years) were recruited. Four collections of samples collected during two successive dry and rainy seasons were analysed for antibody responses to P. vivax and P. falciparum. Recombinant P. falciparum and P. vivax MSP1 antigens and total P. falciparum schizont lysate from African 07/03 strain (adapted to culture) were used for ELISA. Nested PCR amplification was used for molecular detection of P. vivax.

Results

A surprising high prevalence of IgG responses against P. vivax MSP1 was evidenced with 53% of positive samples and 58% of the individuals that were found positive to this antigen. There was 77% of responders to P. falciparum outlined by 63% of positive samples. Prevalence of responders did not differ as function of seasons. Levels of antibodies to P. falciparum fluctuated with significant increasing between dry and rainy season (P < 0.05), contrary to responses to P. vivax. There was a significant reciprocal relationship (P < 10^?3) between antibody responses to the different antigens, but with weak coefficient of correlation (Rho around 0.3) underlining a variable profile at the individual level. Clear molecular signature was found in positive IgG to P. vivax msp1 samples by PCR.

Conclusion

This cross-sectional longitudinal study highlights the unexpected high circulation of P. vivax in this endemic area. Sero-immunology and molecular methods are powerful additive tools to identify endemic sites where relevant control measures have to be settled and monitored.

     

Development of a Dot-Blot Assay for the Detection of Mould-Specific IgE in the Belgian Population

Background

Data on mould sensitization in the general population are scarce and mostly on Aspergillus fumigatus, Alternaria alternata and Cladosporium herbarum.

Objectives

To validate a dot-blot assay for the detection of specific IgE and evaluate the prevalence of mould sensitization in a healthy population.

Methods

The dot-blot assay was validated against the CAP test. Sensitization rate to ten common indoor and outdoor mould species in 344 serum samples was calculated. For each serum with more than one reactivity, the “major sensitization” defined as the strongest response against a single mould species was calculated.

Results

Intra- and inter-assay variations were both below 20%, and the positivity threshold of the test was of 0.418 kU/L for A. fumigatus. Correlation with CAP results was strong. The overall prevalence of sensitization was 32.8%, and the commonest sensitizations were against A. alternaria, A. flavus and A. niger (around 15%). The most frequent “major reactivities” were against A. niger and A. alternata (20–30%). In 25.1% of the samples, “major reactivities” were directed against a group of moulds commonly found indoor (Penicillium spp., Aspergillus versicolor, Cladosporium sphaerospermum and Cladosporium cladosporioides).

Conclusions

The dot-blot assay was validated for the detection of mould-specific IgE. In the general population, sensitization to indoor species was common and accounted for 25% of overall mould sensitizations.

     

The prevalence and genomic characteristics of hepatitis E virus in murine rodents and house shrews from several regions in China

Background

Urban rodents and house shrews are closely correlated in terms of location with humans and can transmit many pathogens to them. Hepatitis E has been confirmed to be a zoonotic disease. However, the zoonotic potential of rat HEV is still unclear. The aim of this study was to determine the prevalence and genomic characteristics of hepatitis E virus (HEV) in rodents and house shrews.

Results

We collected a total of 788 animals from four provinces in China. From the 614 collected murine rodents, 20.19% of the liver tissue samples and 45.76% of the fecal samples were positive for HEV. From the 174 house shrews (Suncus murinus), 5.17% fecal samples and 0.57% liver tissue samples were positive for HEV. All of the HEV sequences obtained in this study belonged to Orthohepevirus C1. However, we observed a lower percentage of identity in the ORF3 region upon comparing the amino acid sequences between Rattus norvegicus and Rattus losea. HEV derived from house shrews shared a high percentage of identity with rat HEV. Notably, the first near full-length of the HEV genome from Rattus losea is described in our study, and we also report the first near full-length rat HEV genomes in Rattus norvegicus from China.

Conclusion

HEV is prevalent among the three common species of murine rodents (Rattus. norvegicus, Rattus. tanezumi, and Rattus. losea) in China. HEV sequences detected from house shrews were similar to rat HEV sequences. The high identity of HEV from murine rodents and house shrews suggested that HEV can spread among different animal species.

     

Five new species of dictyostelid social amoebae (Amoebozoa) from Thailand

Background

Dictyostelid cellular slime molds (dictyostelids) are common inhabitants of the soil and leaf litter layer of fields and forests, along with animal dung, where they feed mostly on bacteria. However, reports on the species diversity of dictyostelids in South Asia, particularly Thailand, are limited. The research reported in this paper was carried out to increase our knowledge of the species diversity of this group of organisms in northern Thailand.

Results

Forty soil samples were collected at four localities in northern Thailand to assess the species richness of dictyostelids. These samples yielded five dictyostelid isolates that were not morphologically consistent with any described species. Based on molecular signatures, all five of these isolates were assigned to the family Cavenderiaceae, genus Cavenderia. All five share a number of morphological similarities with other known species from this family. The new taxa differ from previously described species primarily in the size and complexity of their fruiting bodies (sorocarps). This paper describes these new species (Cavenderia aureostabilis, C. bhumiboliana, C. protodigitata, C. pseudoaureostipes, and C. subdiscoidea) based on a combination of morphological characteristics and their phylogenetic positions.

Conclusions

At least 15 taxa of dictyostelids were obtained from the four localities in northern Thailand, which indicates the high level of species diversity in this region. Five species were found to be new to science. These belong to the family Cavenderiaceae, genus Cavenderia, and were described based on both morphology and phylogeny.

     

Differential Immune Responses and Protective Effects in Avirulent Mycobacterial Strains Vaccinated BALB/c Mice

Screening live mycobacterial vaccine candidates is the important strategy to develop new vaccines against adult tuberculosis (TB). In this study, the immunogenicity and protective efficacy of several avirulent mycobacterial strains including Mycobacterium smegmatis, M. vaccae, M. terrae, M. phlei, M. trivial, and M. tuberculosis H37Ra were compared with M. bovis BCG in BALB/c mice. Our results demonstrated that differential immune responses were induced in different mycobacterial species vaccinated mice. As BCG-vaccinated mice did, M. terrae immunization resulted in Th1-type responses in the lung, as well as splenocytes secreting IFN-γ against a highly conserved mycobacterial antigen Ag85A. M. smegmatis also induced the same splenocytes secreting IFN-γ as BCG and M. terrae did. In addition, M. terrae and M. smegmatis-immunized mice predominantly increased expression of IL-10 and TGF-β in the lung. Most importantly, mice vaccinated with H37Ra and M. vaccae could provide the same protection in the lung against virulent M. tuberculosis challenge as BCG. The result may have important implications in developing adult TB vaccine.