Differential transcriptomic and metabolic profiles of M. africanum- and M. tuberculosis-infected patients after, but not before, drug treatment.

The epidemiology of Mycobacterium tuberculosis (Mtb) and M. africanum (Maf) suggests differences in their virulence, but the host immune profile to better understand the pathogenesis of tuberculosis (TB) have not been studied. We compared the transcriptomic and metabolic profiles between Mtb- and Maf-infected TB cases to identify host biomarkers associated with lineages-specific pathogenesis and response to anti-TB chemotherapy. Venous blood samples from Mtb- and Maf-infected patients obtained before and after anti-TB treatment were analyzed for cell composition, gene expression and metabolic profiles. Prior to treatment, similar transcriptomic profiles were seen in Maf- and Mtb-infected patients. In contrast, post treatment, over 1600 genes related to immune responses and metabolic diseases were differentially expressed between the groups. Notably, the upstream regulator hepatocyte nuclear factor 4-alpha (HNF4α), which regulated 15% of these genes, was markedly enriched. Serum metabolic profiles were similar in both group pre-treatment, but the decline in pro-inflammatory metabolites post treatment were most pronounced in Mtb-infected patients. Together, the differences in both peripheral blood transcriptomic and serum metabolic profiles between Maf- and Mtb-infected patients observed over the treatment period, might be indicative of intrinsic host factors related to susceptibility to TB and/or differential efficacy of the standard anti-TB treatment on the two lineages.

M. tuberculosis Gene Expression during Transition to the "Non-Culturable" State.

We analyzed the gene expression profile under specific conditions during reversible transition of M. tuberculosis cells to the "non-culturable" (NC) state in a prolonged stationary phase. More than 500 genes were differentially regulated, while 238 genes were upregulated over all time points during NC cell formation. Approximately a quarter of these upregulated genes belong to insertion and phage sequences indicating a possible high intensity of genome modification processes taking place under transition to the NC state. Besides the high proportion of hypothetical/conserved hypothetical genes in the cohort of upregulated genes, there was a significant number of genes belonging to intermediary metabolism, respiration, information pathways, cell wall and cell processes, and genes encoding regulatory proteins. We conclude that NC cell formation is an active process involved in the regulation of many genes of different pathways. A more detailed analysis of the experimental data will help to understand the precise molecular mechanisms of dormancy/latency/persistence of M. tuberculosis in the future. The list of upregulated genes obtained in this study includes many genes found to be upregulated in other models of M. tuberculosis persistence. Thirteen upregulated genes, which are common for different models, can be considered as potential targets for the development of new anti-tuberculosis drugs directed mainly against latent tuberculosis.

Deconfounding microarray analysis - independent measurements of cell type proportions used in a regression model to resolve tissue heterogeneity bias.

OBJECTIVES: Microarray analysis requires standardized specimens and evaluation procedures to achieve acceptable results. A major limitation of this method is caused by heterogeneity in the cellular composition of tissue specimens, which frequently confounds data analysis. We introduce a linear model to deconfound gene expression data from tissue heterogeneity for genes exclusively expressed by a single cell type. METHODS: Gene expression data are deconfounded from tissue heterogeneity effects by analyzing them using an appropriate linear regression model. In our illustrating data set tissue heterogeneity is being measured using flow cytometry. Gene expression data are determined in parallel by real time quantitative polymerase chain reaction (qPCR) and microarray analyses. Verification of deconfounding is enabled using protein quantification for the respective marker genes. RESULTS: For our illustrating dataset, quantification of cell type proportions for peripheral blood mononuclear cells (PBMC) from tuberculosis patients and controls revealed differences in B cell and monocyte proportions between both study groups, and thus heterogeneity for the tissue under investigation. Gene expression analyses reflected these differences in celltype distribution. Fitting an appropriate linear model allowed us to deconfound measured transcriptome levels from tissue heterogeneity effects. In the case of monocytes, additional differential expression on the single cell level could be proposed. Protein quantification verified these deconfounded results. CONCLUSIONS: Deconfounding of transcriptome analyses for cellular heterogeneity greatly improves interpretability, and hence the validity of transcriptome profiling results.

A method for extracting RNA from dormant and germinating Bacillus subtilis strain 168 endospores.

RNA was extracted from dormant and germinating Bacillus subtilis 168 spores (intact spores and chemically decoated spores) by using rapid rupture followed by acid-phenol extraction. Spore germination progress was monitored by assaying colony forming ability before and after heat shock and by reading the optical density at 600 nm. The purity, yield, and composition of the extracted RNA were determined spectrophotometrically from the ratio of absorption at 260 nm to that at 280 nm; in a 2100 BioAnalyzer, giving the RNA yield/10(8) spores or cells and the distribution pattern of rRNA components. The method reported here for the extraction of RNA from dormant spores, as well as during different phases of germination and outgrowth, has proven to be fast, efficient and simple to handle. RNA of a high purity was obtained from dormant spores and during all phases of germination and growth. There was a significant increase in RNA yield during the transition from dormant spores to germination and subsequent outgrowth. Chemically decoated spores were retarded in germination and outgrowth compared with intact spores, and less RNA was extracted; however, the differences were not significant. This method for RNA isolation of dormant, germinating, and outgrowing bacterial endospores is a valuable prerequisite for gene expression studies, especially in studies on the responses of spores to hostile environmental conditions.

Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains.

A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.

Protective efficacy against tuberculosis of ESAT-6 secreted by a live Salmonella typhimurium vaccine carrier strain and expressed by naked DNA.

We have constructed a recombinant (r) attenuated Salmonella typhimurium strain which secretes ESAT-6 of Mycobacterium tuberculosis via the hemolysin secretion system of E. coli. Additionally, we have ligated ESAT-6 to different commercially available mammalian expression systems for use as naked DNA vaccines. We studied protection against M. tuberculosis induced by vaccination with each of these constructs alone or in combination in mice. Vaccination with a single dose of r S. typhimurium secreting ESAT-6 reduced numbers of tubercle bacilli in the lungs throughout the course of infection. The combined prime-boost vaccination did not considerably enhance protection.

Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens.

In 1993, the WHO declared tuberculosis a global emergency on the basis that there are 8 million new cases per year. The complete genome of the strain H37Rv of the causative microorganism, Mycobacterium tuberculosis, comprising 3924 genes has been sequenced. We compared the proteomes of two non-virulent vaccine strains of M. bovis BCG (Chicago and Copenhagen) with two virulent strains of M. tuberculosis (H37Rv and Erdman) to identify protein candidates of value for the development of vaccines, diagnostics and therapeutics. The mycobacterial strains were analysed by two-dimensional electrophoresis (2-DE) combining non-equilibrium pH gradient electrophoresis (NEPHGE) with SDS-PAGE. Distinct and characteristic proteins were identified by mass spectrometry and introduced into a dynamic 2-DE database (http://www.mpiib-berlin.mpg.de/2D-PAGE). Silver-stained 2-DE patterns of mycobacterial cell proteins or culture supernatants contained 1800 or 800 spots, respectively, from which 263 were identified. Of these, 54 belong to the culture supernatant. Sixteen and 25 proteins differing in intensity or position between M. tuberculosis H37Rv and Erdman, and H37Rv and M. bovis BCG Chicago, respectively, were identified and categorized into protein classes. It is to be hoped that the availability of the mycobacterial proteome will facilitate the design of novel measures for prevention and therapy of one of the great health threats, tuberculosis.

A dynamic two-dimensional polyacrylamide gel electrophoresis database: the mycobacterial proteome via Internet.

Proteome analysis by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and mass spectrometry, in combination with protein chemical methods, is a powerful approach for the analysis of the protein composition of complex biological samples. Data organization is imperative for efficient handling of the vast amount of information generated. Thus we have constructed a 2-D PAGE database to store and compare protein patterns of cell-associated and culture-supernatant proteins of different mycobacterial strains. In accordance with the guidelines for federated 2-DE databases, we developed a program that generates a dynamic 2-D PAGE database for the World-Wide-Web to organise and publish, via the internet, our results from proteome analysis of different Mycobacterium tuberculosis as well as Mycobacterium bovis BCG strains. The uniform resource locator for the database is http://www.mpiib-berlin.mpg.de/2D-PAGE and can be read with a Java compatible browser. The interactive hypertext markup language documents displayed are generated dynamically in each individual session from a rational data file, a 2-D gel image file and a map file describing the protein spots as polygons. The program consists of common gateway interface scripts written in PERL, minimizing the administrative workload of the database. Furthermore, the database facilitates not only interactive use, but also worldwide active participation of other scientific groups with their own data, requiring only minimal computer hardware and knowledge of information technology.

The Escherichia coli hemolysin secretion apparatus--a versatile antigen delivery system in attenuated Salmonella.

The E. coli hemolysin (HlyA) secretion apparatus represents a type I secretion system that is fully functional in Salmonella. The system which consists of the two specific membrane proteins HlyB and HlyD and the outer membrane protein TolC, recognizes on HlyA a C-terminally located signal sequence of about 60 amino acids. Fusion proteins to which this signal sequence is covalently linked at the C-terminus are also recognized by this secretion apparatus. The efficiency of secretion is dependent on the rate of folding of the reporter protein. Secretion-competent regions of a given reporter protein that is not secretable as entire protein can be screened by a recently constructed transposon TnhlyAs which allows the insertion of the secretion signal into any region of the reporter protein. The genetic information for antigens of any source ranging in size between 10 and 1000 amino acids can be easily inserted into a recently constructed secretion vector which will allow the secretion of the fused antigen(s) in attenuated Salmonella typhimurium strains and in other attenuated Enterobacteriaceae. By manipulation of the Hly secretion system the antigen can be either completely secreted into the environment, fixed on the outer membrane or arrested in the cytoplasm of the used carrier strain. By the use of appropriate attenuated Salmonella strains the antigen is delivered in isolated compartments or to the cytosolic compartment. The extracellular delivery of such antigens is also possible with the help of appropriate carrier strains. The immunological consequences of the different display of the processed antigen will be discussed in the paper by Hess et al in this volume. With a similar antigen delivery system the easy identification and molecular characterization of unknown antigens recognized by the immune system in an infection is also feasible.

Conversion of bacterial gene products to secretion-competent fusion proteins.

We describe an efficient and easy procedure that allows the generation, detection and secretion of foreign proteins by the secretion apparatus of E. coli hemolysin. The gene (or gene fragment) encoding the foreign protein (or protein domain) is inserted in-frame into a residual portion of the hemolysin gene (hlyA), encoding the HlyA secretion signal (HlyAs). Generally, the expressed fusion is efficiently secreted into the culture supernatant of the producing strain. The new approach allows the direct generation of fusion proteins from genomic DNA fragments. The successful use of this method is demonstrated by cloning of random chromosomal DNA fragments from Salmonella typhimurium.

Extracellular PagC-HlyAs fusion protein for the generation and identification of Salmonella-specific antibodies.

The outer membrane protein, PagC, of Salmonella typhimurium was converted into a secreted protein by linking the 61-amino-acid long, C-terminal signal sequence of the E. coli hemolysin protein (HlyAs) to the mature PagC peptide. This PagC-HlyAs fusion protein was expressed and efficiently secreted into the culture supernatant by E. coli upon complementation with the hemolysin secretion proteins HlyB and HlyD. Polyclonal antibodies raised against this fusion protein not only recognized PagC in the membrane fraction of all salmonellae by Western blotting, but also reacted with proteins of smaller size in other gram-negative bacteria tested. A monoclonal antibody against the PagC-HlyAs fusion protein recognized only PagC in membrane fractions. The antibody-binding domain was determined using synthetic peptides derived from specific PagC domains. Sera from Salmonella-infected human patients and from a rabbit infected with S. typhimurium did not react with PagC in immunoblots, suggesting that PagC may not be recognized as a major antigen by the humoral immune system.

SlyA, a regulatory protein from Salmonella typhimurium, induces a haemolytic and pore-forming protein in Escherichia coli.

A chromosomal fragment from Salmonella typhimurium, when cloned in Escherichia coli, generates a haemolytic phenotype. This fragment carries two genes, termed slyA and slyB. The expression of slyA is sufficient for the haemolytic phenotype. The haemolytic activity of E. coli carrying multiple copies of slyA is found mainly in the cytoplasm, with some in the periplasm of cells grown to stationary phase, but overexpression of SlyB, a 15 kDa lipoprotein probably located in the outer membrane, may lead to enhanced, albeit unspecific, release of the haemolytic activity into the medium. Polyclonal antibodies raised against a purified SlyA-HlyA fusion protein identified the overexpressed monomeric 17 kDa SlyA protein mainly in the cytoplasm of E. coli grown to stationary phase, although smaller amounts were also found in the periplasm and even in the culture supernatant. However, the anti-SlyA antibodies reacted with the SlyA protein in a periplasmic fraction that did not contain the haemolytic activity. Conversely, the periplasmic fraction exhibiting haemolytic activity did not contain the 17 kDa SlyA protein. Furthermore, S. typhimurium transformed with multiple copies of the slyA gene did not show a haemolytic phenotype when grown in rich culture media, although the SlyA protein was expressed in amounts similar to those in the recombinant E. coli strain. These results indicate that SlyA is not itself a cytolysin but rather induces in E. coli (but not in S. typhimurium) the synthesis of an uncharacterised, haemolytically active protein which forms pores with a diameter of about 2.6 nm in an artificial lipid bilayer. The SlyA protein thus seems to represent a regulation factor in Salmonella, as is also suggested by the similarity of the SlyA protein to some other bacterial regulatory proteins. slyA- and slyB-related genes were also obtained by PCR from E. coli, Shigella sp. and Citrobacter diversus but not from several other gram-negative bacteria tested.

Salmonella strain secreting active listeriolysin changes its intracellular localization.

We describe the construction of an attenuated Salmonella dublin aroA strain which secretes via the Escherichia coli hemolysin secretion machinery an active hybrid cytolysin consisting of listeriolysin from Listeria monocytogenes and the C-terminal secretion signal of E. coli hemolysin. This hemolytic S. dublin strain is partially released into the cytoplasm of the host cell following uptake by J774 macrophage cells, whereas the nonhemolytic control S. dublin aroA strain remains in the phagosome.

Synthesis and secretion of bacterial antigens by attenuated Salmonella via the Escherichia coli hemolysin secretion system.

We describe a plasmid system which allows the secretion of foreign antigens in attenuated Salmonella aroA strains by the secretion apparatus of E. coli hemolysin. The gene (or gene fragment) encoding the antigen is inserted in frame into a residual position of the hlyA gene, encoding the HlyA secretion signal (HlyAs). Generally, the fused gene is efficiently expressed and the synthesized antigen is in part secreted into the culture supernatant and in part exposed on the surface of the producing Salmonella strain. The successful use of this approach is demonstrated with two antigens of Salmonella typhimurium, PagC and SlyA, both of which are potent virulence factors but produced only in small amounts under in vitro culture conditions and two virulence proteins of Listeria monocytogenes, p60 and listeriolysin. Interestingly the listeriolysin fusion protein proved to be cytolytically active and allowed, when expressed in Salmonella, the escape of these bacteria into the cytoplasm of infected macrophages.

A topological model for the haemolysin translocator protein HlyD.

A topological model for HlyD is proposed that is based on results obtained with gene fusions of lacZ and phoA to hlyD. Active HlyD-LacZ fusion proteins were only generated when lacZ was fused to hlyD within the first 180 bp (60 amino acids). HlyD-PhoA proteins exhibiting alkaline phosphatase (AP) activity were obtained when phoA was inserted into hlyD between nucleotides 262 (behind amino acid position 87) and 1405 (behind amino acid position 468, only 10 amino acids away from the C-terminus of HlyD). Active insertions of phoA into the middle region of hlyD were not observed on in vivo transposition but such fusions exhibiting AP activity could be constructed by in vitro techniques. A fusion protein that carried the PhoA part close to the C-terminal end of HlyD proved to be the most stable HlyD-PhoA fusion protein. In contrast to the other, rather unstable, HlyD-PhoA+ fusions, no proteolytic degradation product of this HlyD-PhoA protein was observed and nearly all the alkaline phosphatase activity was membrane bound. Protease accessibility and cell fractionation experiments indicated that the alkaline phosphatase moiety of this fusion protein was located in the periplasm as for all other HlyD-PhoA+ proteins. These data and computer-assisted predictions suggest a topological model for HlyD with the N-terminal 60 amino acids located in the cytoplasm, a single transmembrane segment from amino acids 60 to 80 and a large periplasmic region extending from amino acid 80 to the C-terminus.(ABSTRACT TRUNCATED AT 250 WORDS)