Enzymes with phospholipase C activity in Mycobacterium tuberculosis have been recently described. The three genes encoding these proteins, plcA, plcB, and plcC, are located at position 2351 of the genomic map of M. tuberculosis H37Rv and are arranged in tandem. We have previously described the presence of variations in the restriction fragment length polymorphism patterns of the plcA and plcB genes in M. tuberculosis clinical isolates. In the present work we investigated the origin of this polymorphism by sequence analysis of the phospholipase-encoding regions of 11 polymorphic M. tuberculosis clinical isolates. To do so, a long-PCR assay was used to amplify a 5,131-bp fragment that contains the plcA and plcB genes and part of the plcC gene. In the M. tuberculosis strains studied the production of an amplicon ϳ1,400 bp larger than anticipated was observed. Sequence analysis of the PCR products indicated the presence of a foreign sequence that corresponded to an IS6110 element. We observed insertion elements in the plcA, plcB, and plcC genes. One site in plcB had the highest incidence of transposition (5 out of 11 strains). In two strains the insertion element was found in plcA in the same nucleotide position. In all the cases, IS6110 was transposed in the same direction. The high level of transposition in the phospholipase region can lead to the excision of fragments of genomic DNA by recombination of neighboring IS6110 elements, as demonstrated by finding the deletion, in two strains, of a 2,837-bp fragment that included plcA and most of plcB. This can explain the negative results obtained by some authors when detecting the mtp40 sequence (plcA) by PCR. Given the high polymorphism in this region, the use of the mtp40 sequence as a genetic marker for M. tuberculosis sensu stricto is very restricted.The mtp40 gene was first described as a 402-bp open reading frame (ORF) encoding a 13.8-kDa specific protein of Mycobacterium tuberculosis (21). This gene was cloned in a 3.1-kbp BamHI fragment, and, after sequencing the whole insert, Leão et al. noted the presence of an ORF of 1,170 bp and the beginning of another (15). Johansen et al. (13) completely sequenced the second ORF, and they also demonstrated in vitro that these genes encode phospholipase C activities. They named the ORFs mpcA and mpcB. The sequence called mtp40 actually constitutes only a part of the mpcA gene. After the whole M. tuberculosis H37Rv genome was sequenced, two more phospholipase genes were decribed: an ORF beside mpcB and another related sequence at position 1755 of the genome, located beside an IS6110 element (4). From this point on these genes were designated plc (phospholipase C) genes; the three ORFs arranged in tandem were called plcA, plcB, and plcC, and the fragment at position 1755 in M. tuberculosis H37Rv was called plcD. We use this nomenclature in the present work.Since there have been conflicting results concerning the presence of the mtp40 sequence in the M. tuberculosis complex, we previously studied its distribution within a c...

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Knowledge of Entamoeba histolytica biology in the last 17 years has been acquired largely as a consequence of this parasite's axenic cultivation in TPS-1 or TYI-S-33 media. Unfortunately, there are often low yields in these media, due to variability of their main components, Panmede and yeast extract. We describe a medium, PEHPS, of which the main components are extracts of ox liver, and ox and swine pancreas (EHP). 5 strains of E. histolytica and 2 of E. invadens were quickly and easily adapted to PEHPS and serially cultivated for 3 years. Yields progressively rose initially, and then became stable. Depending on the strain, average yields in the last 6 months of this study were 1.3 to 3.1 x 10(5) amoebae/ml for E. histolytica and 5.5 to 5.7 x 10(5) for E. invadens. All the 18 EHP batches tested supported vigorous amoebal growth. PEHPS had 2 additional advantages: (a) it was stable at 4 degrees C or 25 degrees C for 9 months, and at -10 degrees C for at least 2 years, and (b) it supported amoebal growth with inocula as low as one trophozoite/ml. PEHPS avoids the variability shown by TPS-1 and TYI-S-33, and could therefore be a good alternative for axenic amoebal cultivation.

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We developed an in vitro tissue-culture model to analyze the process involved in mycobacterial spread through lung epithelial cell monolayers. A549 cells were infected with low numbers of viable Mycobacterium tuberculosis bacilli expressing the gfp gene. Subsequent addition of a soft agarose overlay prevented the dispersal of the bacilli from the initial points of attachment. By fluorescence microscopy the bacteria were observed to infect and grow within the primary target cells; this was followed by lysis of the infected cells and subsequent infection of adjacent cells. This process repeated itself until an area of clearing (plaque formation) was observed. The addition of amikacin after initial infection did not prevent intracellular growth; however, subsequent plaque formation was not observed. Plaque formation was also observed after infection with Mycobacterium bovis BCG bacilli, but the plaques were smaller than those formed after infection with M. tuberculosis. These observations reinforce the possibility that cell-to-cell spreading of M. tuberculosis bacilli, particularly early in the course of infection within lung macrophages, pneumocytes, and other cells, may be an important component in the infectious process.

We developed an in vitro tissue-culture model to analyze the process involved in mycobacterial spread through lung epithelial cell monolayers. A549 cells were infected with low numbers of viable Mycobacterium tuberculosis bacilli expressing the gfp gene. Subsequent addition of a soft agarose overlay prevented the dispersal of the bacilli from the initial points of attachment. By fluorescence microscopy the bacteria were observed to infect and grow within the primary target cells; this was followed by lysis of the infected cells and subsequent infection of adjacent cells. This process repeated itself until an area of clearing (plaque formation) was observed. The addition of amikacin after initial infection did not prevent intracellular growth; however, subsequent plaque formation was not observed. Plaque formation was also observed after infection with Mycobacterium bovis BCG bacilli, but the plaques were smaller than those formed after infection with M. tuberculosis. These observations reinforce the possibility that cell-to-cell spreading of M. tuberculosis bacilli, particularly early in the course of infection within lung macrophages, pneumocytes, and other cells, may be an important component in the infectious process. ß

Mycobacterium tuberculosis is a serious worldwide health threat, killing almost 2 million people per year. Alternative antimycobacterial drugs are urgently needed; studies have shown that medicinal plants traditionally used to treat respiratory diseases are a potential source of compounds to treat tuberculosis. This paper studied the antimycobacterial activity of 28 extracts from four different plant species that have been used in traditional Mexican medicine to treat tuberculosis. Bark and leaf crude extracts of Juglans regia L., Juglans mollis Engelm., Carya illinoensis (Wangenh) K. Koch and Bocconia frutescens showed in vitro anti-M. tuberculosis activity. Hexane bark extracts from C. illinoensis, J. mollis and J. regia were the most active with a minimal inhibitory concentration (MIC) of 31, 50 and 100 microg/mL, respectively. Ethanol bark extracts from C. illinoensis and J. mollis showed activity at 100 and 125 microg/mL, respectively. Leaf extracts had the lowest activity. Methanol and hexane leaves extracts from B. frutescens had a MIC of 125 microg/mL. None of the aqueous extracts showed antimycobacterial activity.

Entamoeba histolytica phospholipase A and lysophospholipase activities from a vesicular subcellular fraction (P30) were analyzed. The products, obtained using specific substrates labeled with 14C or 3H, indicated the presence of phospholipase A1 and A2 as well as lysophospholipase L1 activities. The enzymes detected could participate in phospholipid metabolism and the alkaline phospholipase A2 may contribute to E. histolytica cytopathogenicity.