The Use of Rap-Pcr in Studying Mycobacterium Tuberculosis Intracellular Gene During Macrophage Infection

Mycobacterium tuberculosis is the second leading cause of death from infectious agent. This study sought to detect M. tuberculosis genes; which were specifically expressed; or upregulated during intracellular infection of J774 murine macrophages; as such genes may be potential targets for novel drug action. J774 murine macrophage cell line was infected with M. tuberculosis (H37Rv strain) at 10:1 multiplicity of infection (MOI). RNA was differentially extracted from M. tuberculosis infecting J774 macrophage cell line. The control in this case was RNA from extracellular broth grown bacteria. Approximately 50 ng of RNA from intracellular derived bacteria and extracellular derived bacteria (control) were subjected to random arbitrarily primed PCR (RAP-PCR) using 50 primer combinations. Eleven differential RAP-PCR products were observed. All RAP-PCR products were cloned into pCRr2.1 and sequenced in order to determine the identity of the products. Four of the eleven products were derived from macrophage genes and another 4 products were derived from the M. tuberculosis rRNA genes (three 23S and one 16S rRNA). The 3 remaining RAP-PCR products were found to be mycobacterial genes other than ribosomal genes. The three products were genes encoding enzyme involving in a shikimate pathway; a putative carboxyphosphonoenolpyruvate phosphonomutase and a serine protease with homology to HtrA. Of the 3 mycobacterial genes other than ribosomal genes detected; none were specifically expressed during intracellular infection but bacilli

Whole genome comparison of Campylobacter jejuni human isolates using a low-cost microarray reveals extensive genetic diversity.

Campylobacter jejuni is the leading cause of bacterial food-borne diarrhoeal disease throughout the world, and yet is still a poorly understood pathogen. Whole genome microarray comparisons of 11 C. jejuni strains of diverse origin identified genes in up to 30 NCTC 11168 loci ranging from 0.7 to 18.7 kb that are either absent or highly divergent in these isolates. Many of these regions are associated with the biosynthesis of surface structures including flagella, lipo-oligosaccharide, and the newly identified capsule. Other strain-variable genes of known function include those responsible for iron acquisition, DNA restriction/modification, and sialylation. In fact, at least 21% of genes in the sequenced strain appear dispensable as they are absent or highly divergent in one or more of the isolates tested, thus defining 1300 C. jejuni core genes. Such core genes contribute mainly to metabolic, biosynthetic, cellular, and regulatory processes, but many virulence determinants are also conserved. Comparison of the capsule biosynthesis locus revealed conservation of all the genes in this region in strains with the same Penner serotype as strain NCTC 11168. By contrast, between 5 and 17 NCTC 11168 genes in this region are either absent or highly divergent in strains of a different serotype from the sequenced strain, providing further evidence that the capsule accounts for Penner serotype specificity. These studies reveal extensive genetic diversity among C. jejuni strains and pave the way toward identifying correlates of pathogenicity and developing improved epidemiological tools for this problematic pathogen.

A martyr for modernity: Qiu Jin -- feminist warrior, and revolutionary.

Qui Jin, at one level, was an oriental twentieth-century Judith, the mythical Jewish widow from Bethulia who cut off the head of Holofernes, the Assyrian general besieging the city, thus saving the Israelites from destruction. Qui Jin was, as Judith was, a self-reliant heroine who when others seemed 'helpless and demoralized undertook to save them single-handedly', or in her case virtually single-handedly. This, of course, was both her making and her unmaking. In Chinese terms the story of Qui Jin, like the story of Judith if less famous, less publicised, more recent, is the story of an icon at once central and at the same time marginal to tradition. She contradicted the most cherished customs on Confucian Chinese culture. She was a radical force who thrust her way to the centre of the concentric circles of customs surrounding this culture and was pushed back to the margins by conservatism. Nevertheless Qui Jin was not without success. She challenged a long-established mythology of exclusively masterful patriarchy - and created a counter myth of purposeful patriotic feminism. She was a counter-cultural icon who changed perceptions of Chinese femininity. She gave courage, confidence and purpose to those women who came after her and absorbed her ambitions for modern Chinese womanhood. For them she was a modern national heroine and a personification of a modern nation of equal men and women. For Qui Jin the body was an instrument of female revolution to be trained, strengthened and prepared for confrontation. As a revolutionary militant she was a failure; as a revolutionary talisman she was a success. For the Chinese women of the 1911 Revolution hers was an exemplary emancipatory story: subscribe, struggle, sacrifice. Patriotism through feminism is the purpose. Her heroism was firmly outside the historic patriarchal order. Her adulation is thus all the more remarkable because of the profound traditions she rejected, the controversial mannerisms she adopted, the uncompromising attitudes she embraced. She eschewed motherhood, abandoned marriage, dismissed femininity, and yet won acclaim in the most traditional of cultures. Qui Jin was hardly a cynosure of universal acclaim but she was admired, respected and emulated by radical Chinese women and men seeking a new society accommodating women. Her modern feminism struggled to overcome an ancient patriarchy. Here was her appeal. She exuded no moral ambiguity. Consequently, if she was demonized by the conventional; she was deified by the radical - and inspired them as the contemplated and attempted to construct the future. There is a point, of course, that should not be overlooked. Qui Jin, in fact, is not divorced from occidental culture and political iconography. Qui Jin is closely associated with the attitudes, aspirations and fantasies of modern Western feminism. As Margarita Stocker observes, a 'romantic heroine, angry feminist, radical, activist is one example of a pervasive figure', in modern Western cultural mythology 'a figure we may sum up as the Woman with a Gun'. Force, that potent means to power, is available to the gun user irrespective of age of sex, with a resulting 'crucial alteration in the sexual politics of violence'. The Woman with a Gun can now be emphatically heroic - without duplicity, without deceitfulness, without subterfuge. Moral ambiguity in action has been abandoned. She becomes an unambiguous potent force - an armed woman faces an armed man on equal terms - physically, psychologically, morally. Equality offers the legal right and responsibility to kill in the name of patriotism. Modern culture has just caught up with Qui Jin.

The early evolution of modern sport in Latin America: a mainly English middle-class inspiration.

In any consideration of cultural diffusion, what matters is not only what happens to a cultural form when it arrives, but that it arrives. In the twentieth century, Latin America, like too many other areas of the world, has been a place of diplomatic turmoil, social inequality, political paranoia, capitalist exploitation and class conflict. However, despite all this, and through all this, it may be stated factually and without sentimentality, that it has also been a place where people have survived and thrived, worked, loved and played. Modern sport has brought to their play both unhappy moments of disillusion and disappointment and marvellous opportunities for illusion and pleasure. Modern sport, with its beauty spots and warts, is the reality and while there are things to criticize, there are also many things to applaud. Along with others, the English middle class played a not insignificant part in the arrival of modern sport in Latin America.

Extraction of RNA from Intracellular Mycobacterium tuberculosis : Methods, Considerations, and Applications.

Pathogenicity in Mycobacterium tuberculosis may be thought of as a multifactorial process with both pathogen and host-response effector molecules contributing to the process of infection, leading either to immunopathology and disease or control of infection and long-term persistence. Little is known about this at a genetic level, but it is becoming recognized that bacterial virulence constitutes the correct temporal and spatial regulation of many genes that may be necessary for a particular phase in infection in response to specific environmental cues.

Detection of mRNA transcripts and active transcription in persistent Mycobacterium tuberculosis induced by exposure to rifampin or pyrazinamide.

Mycobacterium tuberculosis can persist in an altered physiological state for many years after initial infection, and it may reactivate to cause active disease. An analogous persistent state, possibly consisting of several different subpopulations of bacteria, may arise during chemotherapy; this state is thought to be responsible for the prolonged period required for effective chemotherapy. Using two models of drug-induced persistence, we show that both microaerophilic stationary-phase M. tuberculosis treated with a high dose of rifampin in vitro and pyrazinamide-induced persistent bacteria in mice are nonculturable yet still contain 16S rRNA and mRNA transcripts. Also, the in vitro persistent, plate culture-negative bacteria incorporate radioactive uridine into their RNA in the presence of rifampin and can rapidly up-regulate gene transcription after the replacement of the drug with fresh medium and in response to heat shock. Our results show that persistent M. tuberculosis has transcriptional activity. This finding provides a molecular basis for the rational design of drugs targeted at persistent bacteria.

Evaluation of reverse transcription-PCR and a bacteriophage-based assay for rapid phenotypic detection of rifampin resistance in clinical isolates of Mycobacterium tuberculosis.

New rapid phenotypic assays for the detection of rifampin resistance in Mycobacterium tuberculosis have recently been described, but most of these require liquid cultures, which reduces the utility of many tests in terms of turnaround times. In the United Kingdom, over 90% of rifampin-resistant isolates are also resistant to isoniazid, so rifampin resistance can be used as a sensitive marker for multidrug-resistant tuberculosis. In this study, two new rapid phenotypic assays were compared to the standard resistance ratio method on 91 clinical isolates of M. tuberculosis. One, the phage amplified biologically (PhaB) assay, has been described previously and is based on the inability of susceptible isolates of M. tuberculosis to support the replication of bacteriophage D29 in the presence of inhibitory doses of rifampin. The other employed reverse transcription (RT)-PCR to demonstrate a reduction in inducible dnaK mRNA levels in susceptible isolates treated with rifampin. After incubation for 18 h with 4 microg of rifampin per ml, the PhaB assay showed concordance with the resistance ratio method for 46 of 46 (100%) susceptible and 31 of 31 (100%) resistant isolates, while RT-PCR showed concordance for 46 of 48 (96%) susceptible and 35 of 36 (97%) resistant isolates. We believe these assays provide a reliable rapid means of susceptibility testing with a total turnaround time of only 48 h, although the PhaB assay is better in terms of its lower technical demand and cost and its applicability to tuberculosis susceptibility testing in developing countries.

Mycobacterium tuberculosis expresses a novel pH-dependent divalent cation transporter belonging to the Nramp family.

Mammalian natural resistance-associated macrophage protein (Nramp) homologues are important determinants of susceptibility to infection by diverse intracellular pathogens including mycobacteria. Eukaryotic Nramp homologues transport divalent cations such as Fe(2+), Mn(2+), Zn(2+), and Cu(2+). Mycobacterium tuberculosis and Mycobacterium bovis (bacillus Calmette-Guérin [BCG]) also encode an Nramp homologue (Mramp). RNA encoding Mramp induces approximately 20-fold increases in (65)Zn(2+) and (55)Fe(2+) uptake when injected into Xenopus laevis oocytes. Transport is dependent on acidic extracellular pH and is maximal between pH 5.5 and 6.5. Mramp-mediated (65)Zn(2+) and (55)Fe(2+) transport is abolished by an excess of Mn(2+) and Cu(2+), confirming that Mramp interacts with a broad range of divalent transition metal cations. Using semiquantitative reverse transcription PCR, we show that Mramp mRNA levels in M. tuberculosis are upregulated in response to increases in ambient Fe(2+) and Cu(2+) between <1 and 5 microM concentrations and that this upregulation occurs in parallel with mRNA for y39, a putative metal-transporting P-type ATPase. Using a quantitative ratiometric PCR technique, we demonstrate a fourfold decrease in Mramp/y39 mRNA ratios from organisms grown in 5-70 microM Cu(2+). M. bovis BCG cultured axenically and within THP-1 cells also expresses mRNA encoding Mramp. Mramp exemplifies a novel prokaryotic class of metal ion transporter. Within phagosomes, Mramp and Nramp1 may compete for the same divalent cations, with implications for intracellular survival of mycobacteria.

Regulation of hmp gene transcription in Mycobacterium tuberculosis: effects of oxygen limitation and nitrosative and oxidative stress.

The Mycobacterium tuberculosis hmp gene encodes a protein which is homologous to flavohemoglobin in Escherichia coli. Northern blotting analysis demonstrated that hmp transcription increased when a microaerophilic culture became oxygen limited as it entered stationary phase at 20 days. There was a fivefold increase of the hmp transcripts during early stationary phase compared with the value which was observed in the exponential growth phase. This induction of hmp transcription was not due to changes in the mRNA stability since the half-life of hmp mRNA was very short in a 20-day microaerophilic culture. No induction of hmp mRNA was observed during entry into stationary phase when the culture was continuously aerated. hmp transcription was induced after a short exposure of a late-exponential-phase culture to anaerobic conditions. These data indicate that oxygen limitation is the trigger for hmp gene transcription. In addition, when a microaerophilic culture entered into the stationary phase at 20 days, transcription of hmp increased to a small extent after exposure to S-nitrosoglutathione (a nitric oxide [NO] releaser) and sodium nitroprusside (an NO+ donor) and decreased after exposure to paraquat (a superoxide generator) and H2O2. In log phase (4 days) and late stationary phase (40 days), the transcription of hmp was unaffected by nitrosative and oxidative stress. Three primer extension products were observed. The -10 region is 100% identical to that of promoter T3 in mycobacteria and shows a strong similarity to the -10 sequence of hmp and rpoS promoters in E. coli. These observations of hmp mRNA induction in response to O2 limitation and nitrosative stress suggest that the hmp gene of M. tuberculosis may have a role in protection of the organism from NO killing under microaerophilic conditions.