Human RNase3 immune modulation by catalytic-dependent and independent modes in a macrophage-cell line infection model.

The human RNase3 is a member of the RNaseA superfamily involved in host immunity. RNase3 is expressed by leukocytes and shows broad-spectrum antimicrobial activity. Together with a direct antimicrobial action, RNase3 exhibits immunomodulatory properties. Here, we have analysed the transcriptome of macrophages exposed to the wild-type protein and a catalytic-defective mutant (RNase3-H15A). The analysis of differently expressed genes (DEGs) in treated THP1-derived macrophages highlighted a common pro-inflammatory "core-response" independent of the protein ribonucleolytic activity. Network analysis identified the epidermal growth factor receptor (EGFR) as the main central regulatory protein. Expression of selected DEGs and MAPK phosphorylation were inhibited by an anti-EGFR antibody. Structural analysis suggested that RNase3 activates the EGFR pathway by direct interaction with the receptor. Besides, we identified a subset of DEGs related to the protein ribonucleolytic activity, characteristic of virus infection response. Transcriptome analysis revealed an early pro-inflammatory response, not associated to the protein catalytic activity, followed by a late activation in a ribonucleolytic-dependent manner. Next, we demonstrated that overexpression of macrophage endogenous RNase3 protects the cells against infection by Mycobacterium aurum and the human respiratory syncytial virus. Comparison of cell infection profiles in the presence of Erlotinib, an EGFR inhibitor, revealed that the receptor activation is required for the antibacterial but not for the antiviral protein action. Moreover, the DEGs related and unrelated to the protein catalytic activity are associated to the immune response to bacterial and viral infection, respectively. We conclude that RNase3 modulates the macrophage defence against infection in both catalytic-dependent and independent manners.

Mycobacterium tuberculosis Rv3717 enhances the survival of Mycolicibacterium smegmatis by inhibiting host innate immune and caspase-dependent apoptosis.

Tuberculosis caused by Mycobacterium tuberculosis (M. tuberculosis) infection remains a serious public threat despite decades of creative endeavors. There are few reports on the roles of M. tuberculosis enzymes involved in cell envelope biosynthesis in pathogen survival and persistence. M. tuberculosis Rv3717 encodes N-acetylmuramoyl-l-alanine amidase, a cell-wall hydrolase that hydrolyzes the bond between N-acetylmuramic acid and l-alanine in cell-wall peptidoglycan. In this paper, we demonstrated the Rv3717 promoted the survival of Mycolicibacterium smegmatis(M. smegmatis) within macrophages. More importantly, we demonstrated that this effect is because MS_Rv3717 reduces the release of host pro-inflammatory cytokines such as IL-1ß, IL-6, IL-12 p40, TNF-α, and increased transcription of anti-inflammatory cytokine IL-10. At the same time, MS_Rv3717 inhibits apoptosis by inhibiting the activation of Caspase-3/9, reducing the host's elimination of M. smegmatis. Finally, from a bacterial perspective, we found Rv3717 decreased the survival of M. smegmatis under stresses such as SDS and low pH. This is the first report of the involvement of Mycobacterium cell envelope biosynthetic enzyme in host-pathogen interaction.

M.neoaurum infection increased the inhibitory function of Tregs and the death rate associated with Salmonella coinfection.

Mycobacterium neoaurum belongs to the nontuberculous mycobacteria (NTM) and is ubiquitously present in the environment. However, the changes in Treg percentages and suppressive properties in mice infected with M. neoaurum are still not elucidated. In this study, mice were intraperitoneally injected with M. neoaurum. The change in the CD4+CD25+ Treg cell percentage in the spleen was analyzed using flow cytometry. There was a significant increase in the number of CD4+CD25+ cells by week 6 postinfection, with a peak proportion of approximately 2%. The Foxp3 and IL-10 mRNA expression in CD4+CD25+ cells from the spleens of M.neoaurum-infected mice was higher than that in CD4+CD25+ cells from the spleens of noninfected controls. Proliferation suppression assay results indicated that CD4+CD25+ cells suppressed the proliferation of CD4+CD25- cells at week 6 after M.neoaurum infection, and the suppression rate reached 89.8%. However, CD4+CD25+ cells from the noninfected control group did not suppress the proliferation of CD4+CD25- cells. Based on the above results, mice were subjected to oral administration of S. Typhimurium at 6 weeks postinfection with M. neoaurum, and we found that the mortality of the M.neoaurum-S. Typhimurium infection group was higher than that of the S. Typhimurium infection group. In addition, serious pathological changes appeared in the liver and cecum of the M.neoaurum-S.Typhimurium infection group compared with those of the S. Typhimurium infection group. M. neoaurum increased Treg percentages and suppressed spleen function in mice. These results revealed the possibility that persistent M.neoaurum infection could increase the occurrence of secondary infection.

A Cr(VI)-reducing Microbacterium sp. strain SUCR140 enhances growth and yield of Zea mays in Cr(VI) amended soil through reduced chromium toxicity and improves colonization of arbuscular mycorrhizal fungi.

Pot culture experiments were conducted in a glasshouse to evaluate the effects of four efficient Cr(VI)-reducing bacterial strains (SUCR44, SUCR140, SUCR186, and SUCR188) isolated from rhizospheric soil, and four arbuscular mycorrhizal fungi (AMF-Glomus mosseae, G. aggregatum, G. fasciculatum, and G. intraradices) alone or in combination, on Zea mays in artificially Cr(VI)-amended soil. Presence of a strain of Microbacterium sp. SUCR140 reduced the chromate toxicity resulting in improved growth and yields of plants compared to control. The bioavailability of Cr(VI) in soil and its uptake by the plant reduced significantly in SUCR140-treated plants; the effects of AMF, however, either alone or in presence of SUCR140 were not significant. On the other hand, presence of AMF significantly restricted the transport of chromium from root to the aerial parts of plants. The populations of AMF chlamydospores in soil and its root colonization improved in presence of SUCR140. This study demonstrates the usefulness of an efficient Cr(VI)-reducing bacterial strain SUCR140 in improving yields probably through reducing toxicity to plants by lowering bioavailability and uptake of Cr(VI) and improving nutrient availability through increased mycorrhizal colonization which also restricted the transport of chromium to the aerial parts.

Environmental mycobacteria: a threat to human health?

In many cases, bacterial pathogens are close relatives to nonpathogens. Pathogens seem to be limited lineages within nonpathogenic bacteria. Nonpathogenic isolates are generally more diverse and widespread in the environment and it is generally considered that environmental bacteria do not pose a risk to human health as clinical isolates do; this may not be the case with mycobacteria, but environmental mycobacteria have not been well studied. It is documented that several environmental mycobacteria constitute a source for human infections. Diverse mycobacterial environmental isolates are rarely involved in human disease. Environmental mycobacteria may have a role in degradation of different compounds. Environmental mycobacteria have had a long interaction with humans, maybe as long as the human species, and may have contributed to human evolution.

Evaluation of signal peptide prediction algorithms for identification of mycobacterial signal peptides using sequence data from proteomic methods.

Secreted proteins play an important part in the pathogenicity of Mycobacterium tuberculosis, and are the primary source of vaccine and diagnostic candidates. A majority of these proteins are exported via the signal peptidase I-dependent pathway, and have a signal peptide that is cleaved off during the secretion process. Sequence similarities within signal peptides have spurred the development of several algorithms for predicting their presence as well as the respective cleavage sites. For proteins exported via this pathway, algorithms exist for eukaryotes, and for Gram-negative and Gram-positive bacteria. However, the unique structure of the mycobacterial membrane raises the question of whether the existing algorithms are suitable for predicting signal peptides within mycobacterial proteins. In this work, we have evaluated the performance of nine signal peptide prediction algorithms on a positive validation set, consisting of 57 proteins with a verified signal peptide and cleavage site, and a negative set, consisting of 61 proteins that have an N-terminal sequence that confirms the annotated translational start site. We found the hidden Markov model of SignalP v3.0 to be the best-performing algorithm for predicting the presence of a signal peptide in mycobacterial proteins. It predicted no false positives or false negatives, and predicted a correct cleavage site for 45 of the 57 proteins in the positive set. Based on these results, we used the hidden Markov model of SignalP v3.0 to analyse the 10 available annotated proteomes of mycobacterial species, including annotations of M. tuberculosis H37Rv from the Wellcome Trust Sanger Institute and the J. Craig Venter Institute (JCVI). When excluding proteins with transmembrane regions among the proteins predicted to harbour a signal peptide, we found between 7.8 and 10.5% of the proteins in the proteomes to be putative secreted proteins. Interestingly, we observed a consistent difference in the percentage of predicted proteins between the Sanger Institute and JCVI. We have determined the most valuable algorithm for predicting signal peptidase I-processed proteins of M. tuberculosis, and used this algorithm to estimate the number of mycobacterial proteins with the potential to be exported via this pathway.

Numerical and genetic analysis of polycyclic aromatic hydrocarbon-degrading mycobacteria.

Ability to degrade high molecular weight polycyclic aromatic hydrocarbons (PAHs) has been found in diverse species of fast-growing mycobacteria. This study included several PAH-degrading mycobacteria from heavily contaminated sites and an uncontaminated humus soil in the Natural Park, Schwäbische Alb, Germany. The numerical analysis with a total of 131 tests showed that isolates from humus soil and contaminated sites had similar substrate utilization patterns for primary alcohols from ethanol to pentanol, 1,4-butanediol, benzyl alcohol, hexadecane, ethyl acetate, fluoranthene, phenanthrene, and pyrene as the sole carbon and energy (C/E) sources. Significant differences between the two subgroups isolated from humus soil and contaminated sites were observed in the utilization of polyalcoholic sugars, including adonitol, D: -arabitol, L: -arabitol, erythritol, inositol, rhamnose, sorbitol, and xylitol. Among isolates from humus soil, strain PYR100 showed high similarity in 16S rDNA sequence with M. vanbaalenii strain PYR-1 (=DSM 7251, 100%) and M. austroafricanum ATCC 33464 (99.9%). In addition to the numerical analysis, the 16S-23S intergenic spacer sequence was useful for discriminating between the closely related strains PYR100 and PYR-1 (98% similarity). The patterns of the variable V2 and V3 regions in the ribosomal RNA gene corresponding to Escherichia coli positions 179 to 197 and 1006 to 1023, respectively, were useful for dividing fast-growing and thermosensitive PAH-degrading mycobacteria into ten subgroups consistent with the phylogenetic positions.

The molecular biology of recombination in Mycobacteria: what do we know and how can we use it?

Recombination is a ubiquitous genetic process which results in the exchange of DNA between two substrates. Homologous recombination occurs between DNA species with identical sequence whereas illegitimate recombination can occur between DNA with very little or no homology. Site-specific recombination is often used by temperate phages to stably integrate into bacterial chromosomes. Characterisation of the mechanisms of recombination in mycobacteria has mainly focussed on RecA-dependent homologous recombination and phage-directed site-specific recombination. In contrast the high frequency of illegitimate recombination in slow-growing mycobacteria has not been explained. The role of DNA repair in dormancy and infection have not yet been fully established, but early work suggests that RecA-mediated pathways are not required for virulence. All three recombination mechanisms have been utilised in developing genetic techniques for the analysis of the biology and pathogenesis of mycobacteria. A recently developed method for studying essential genes will generate further insights into the biology of these important organisms.

Preventing drug access to targets: cell surface permeability barriers and active efflux in bacteria.

Bacteria, being unicellular, are constantly exposed to toxic compounds in their environment. Gram-negative bacteria and mycobacteria are unusually successful in surviving in the presence of toxic compounds because they combine two mechanisms of resistance. They produce effective permeability barriers, comprising the outer membrane and the mycolate-containing cell wall, on the cell surface. Further, they actively pump out drug molecules that trickle through the barrier, often utilizing multidrug efflux pumps. In Gram-negative bacteria, multidrug pumps of exceptionally wide specificity frequently interact with outer membrane channels and accessory proteins, forming multisubunit complexes that extrude drug molecules directly into the medium, bypassing the outer membrane barrier.

[Physiology of xerophytic microorganisms that grow under very dry conditions]. / Fiziologiia kserofitynykh mikroorganizmov, rastushchikh v ochen' sukhikh usloviiakh.

The biology of xerophytic microorganisms surviving and growing under conditions imitating Martian ones was studied, as well as the zone of tolerance of several microorganisms towards the activity of water. The xerophytic nature of microorganisms is suggested to be evaluated by means of quantitative determination, using gas chromatography, of carbon dioxide which is evolved when microorganisms are cultivated on media with different values of aw.