Mazen Saleh

Bioinformatic comparison of bacterial secretomes.

The rapid increasing number of completed bacterial genomes provides a good opportunity to compare their proteomes. This study was undertaken to specifically compare and contrast their secretomes—the fraction of the proteome with predicted N-terminal signal sequences, both type I and type II. A total of 176 theoretical bacterial proteomes were examined using the ExProt program. Compared with the Gram-positives, the Gram-negative bacteria were found, on average, to contain a larger number of potential Sec-dependent sequences. In the Gram-negative bacteria but not in the others, there was a positive correlation between proteome size and secretome size, while there was no correlation between secretome size and pathogenicity. Within the Gram-negative bacteria, intracellular pathogens were found to have the smallest secretomes. However, the secretomes of certain bacteria did not fit into the observed pattern. Specifically, the secretome of Borrelia burgdoferi has an unusually large number of putative lipoproteins, and the signal peptides of mycoplasmas show closer sequence similarity to those of the Gram-negative bacteria. Our analysis also suggests that even for a theoretical minimal genome of 300 open reading frames, a fraction of this gene pool (up to a maximum of 20%) may code for proteins with Sec-dependent signal sequences.

Proteomics of Desulfovibrio desulfuricans and X-ray absorption spectroscopy to investigate mercury methylation in the presence of selenium

The effects of mercury added as Hg(2+) and selenium as selenite to cultures of the sulfate reducing bacterium Desulfovibrio desulfuricans were investigated under controlled laboratory conditions. There was no significant difference in the growth curves in comparison to control except in the 0.5 μM Hg-6.3 μM Se combined system in which Hg methylation was significantly reduced. A significant decrease in the production of methylmercury indicates a disruption of the methylation process due to the presence of the relatively high concentrations of Se in the system, suggesting a modification of the biological pathway. The results of detailed 2D gel electrophoresis in combination with mass spectrometry confirmed that the Hg methylation process should certainly be influenced when the protein Dde_1198 protein-glutamate O-methyltransferase was totally suppressed in a culture containing 0.5 μM Hg and 6.3 μM Se. Since this protein plays an important role in the methylation process, its suppression in the presence of Se brings a possible explanation for the antagonism between Se and Hg in natural systems. The experiment involving the determination of Hg and Se in membrane proteins separated by 1D gel thin-layer isoelectric focusing revealed that when both elements were present in a culture, the concentration of Hg in the separated proteins was significantly lower in comparison to those without added Se to the culture and vice versa. Finally, near-edge X-ray absorption spectroscopy and extended X-ray absorption fine structure were used to corroborate the presence of a very inert solid HgSe in the cell membrane obtained from the culture containing 0.5 μM Hg and 6.3 μM Se. This confirms the protective effect of Se against Hg assimilation at the molecular level and reinforces the findings of our research group in numerous field and laboratory studies.

Indicators from archaeal secretomes

Just as in the Eukarya and the Bacteria, members of the Archaea need to export proteins beyond the cell membrane. This would be required to fulfill a variety of essential functions such as nutrient acquisition and biotransformations, maintenance of extracellular structures and more. Apart from the Eukarya and the Bacteria however, members of the Archaea share a number of unique characteristics. Does this uniqueness extend to the protein secretion system? It was the objective of this study to answer this question. To overcome the limited experimental information on secreted proteins in Archaea, this study was carried out by subjecting the available archaeal genomes, which represent halophiles, thermophiles, and extreme thermophiles, to bioinformatics analysis. Specifically, to examine the properties of the secretomes of the Archaea using the ExProt program. A total of 24 genomes were analyzed. Secretomes were found to fall in the range of 6% of total ORFs (Methanopyrus kandleri) to 19% (Halobacterium sp. NRC-1). Methanosarcina acetivorans has the highest fraction of lipoproteins (at 89) and the lowest (at 1) were members of the Thermoplasma, Pyrobaculum aerophilum, and Nanoarchaeum equitans. Based on the Tat consensus sequence, contribution of these secreted proteins to the secretomes were negligible, making up 8 proteins out of a total of 7105 predicted exported proteins. Amino acid composition, an attribute of signal peptides not used as a selection criteria by ExProt, of predicted archaeal signal peptides show that in the haloarchaea secretomes, the frequency of the amino acid Lys is much lower than that seen in bacterial signal peptides, but is compensated for by a higher frequency of Arg. It also showed that higher frequencies for Thr, Val, and Gly contribute to the hydrophobic character in haloarchaeal signal peptides, unlike bacterial signal peptides in which the hydrophobic character is dominated by Leu and Ile.

Bacterial RNA induces myocyte cellular dysfunction through the activation of PKR.

Severe sepsis and the ensuing septic shock are serious life threatening conditions. These diseases are triggered by the hosts over exuberant systemic response to the infecting pathogen. Several surveillance mechanisms have evolved to discriminate self from foreign RNA and accordingly trigger effective cellular responses to target the pathogenic threats. The RNA-dependent protein kinase (PKR) is a key component of the cytoplasmic RNA sensors involved in the recognition of viral double-stranded RNA (dsRNA). Here, we identify bacterial RNA as a distinct pathogenic pattern recognized by PKR. Our results indicate that natural RNA derived from bacteria directly binds to and activates PKR. We further show that bacterial RNA induces human cardiac myocyte apoptosis and identify the requirement for PKR in mediating this response. In addition to bacterial immunity, the results presented here may also have implications in cardiac pathophysiology.

Septic sera induces apoptosis and DNA fragmentation factor 40 activation in fibroblasts

Sepsis, the systemic response to infection, is the leading cause of death in the intensive care units worldwide. Septic patients can succumb through the development of early refractory hypotension or late multiple organ dysfunction. Misregulation of apoptosis during sepsis may contribute to cellular dysfunction and multiple organ dysfunction. Utilizing a tissue culture model which mimics the human disease, we demonstrate that the addition of sera derived from septic patients induces apoptosis in human fibroblast cells. Addition of septic sera to 2fTGH cells induced apoptosis by activating caspase 8, caspase 3 and DNA fragmentation factor 40 (DFF 40). Interestingly, the addition of septic sera to cells which lack STAT1 (U3A cells) did not activate DFF 40. U3A cells were also shown to be resistant to septic serum induced apoptosis. These data suggest that DFF 40 mediated apoptosis plays a significant role in mediating sepsis induced cellular dysfunction.

Depleting proteins from the growth medium of Mycoplasma capricolum unmasks bacterium-derived enzymatic activities

Mycoplasma constitutes a unique group of bacteria best characterized as lacking peptidoglycan and having one of the smallest genomes of all free-living prokaryotes. Members of this group also represent important pathogens of humans, animals, and plants. Our understanding of the interaction between these pathogens and their hosts is limited, partly due to our inadequate knowledge of the secreted enzymes and virulence factors of these pathogens. Analysis of secreted proteins of mycoplasma has been hampered by their fastidious growth requirements where protein-rich growth supplements are required. Simple ultrafiltration of the complete medium through a 10kDa cut-off membrane successfully removed virtually all of the polypeptides in the medium and supported the growth of Mycoplasma capricolum (type California kid). This modification (AM medium) exposed the activities of a number of enzymes produced by this bacterium during growth including; acid and alkaline phosphatase, gelatinase, and beta-lactamase activities. We also show that the spent culture medium contained hemolysin activity.

Smoking: Disease or Therapy

During the past 20 years Canada and several other state members of the WHO have been using regular sales tax increases as part of a policy to curb tobacco use. The WHO recent report (2015) on tobacco use classifies smoking as a disease. This report shows that the effect of tax increase on tobacco use is marginal and is difficult to isolate from the effects of other tobacco use measures such as restrictions on marketing, sales to minors, and public education. It is proposed that the unreasonable increase in tobacco taxes introduces additional stress on the smokers and further compromises their health. It is further proposed that tobacco use is treated as a form of selftherapy rather than a disease.