ppGpp and cytotoxicity diversity in Shiga toxin-producing Escherichia coli (STEC) isolates.

Shiga toxin-producing Escherichia coli (STEC) is a known food pathogen, which main reservoir is the intestine of ruminants. The abundance of different STEC lineages in nature reflect a heterogeneity that is characterised by the differential expression of certain genotypic characteristics, which in turn are influenced by the environmental conditions to which the microorganism is exposed. Bacterial homeostasis and stress response are under the control of the alarmone guanosine tetraphosphate (ppGpp), which intrinsic levels varies across the E. coli species. In the present study, 50 STEC isolates from healthy sheep were evaluated regarding their ppGpp content, cytotoxicity and other relevant genetic and phenotypic characteristics. We found that the level of ppGpp and cytotoxicity varied considerably among the examined strains. Isolates that harboured the stx2 gene were the least cytotoxic and presented the highest levels of ppGpp. All stx2 isolates belonged to phylogroup A, while strains that carried stx1 or both stx1 and stx2 genes pertained to phylogroup B1. All but two stx2 isolates belonged to the stx2b subtype. Strains that belonged to phylogroup B1 displayed on average low levels of ppGpp and high cytotoxicity. Overall, there was a negative correlation between cytotoxicity and ppGpp.

ppGpp and cytotoxicity diversity in Shiga toxin-producing Escherichia coli (STEC) isolates

Shiga toxin-producing Escherichia coli (STEC) is a known food pathogen, which main reservoir is the intestine of ruminants. The abundance of different STEC lineages in nature reflect a heterogeneity that is characterised by the differential expression of certain genotypic characteristics, which in turn are influenced by the environmental conditions to which the microorganism is exposed. Bacterial homeostasis and stress response are under the control of the alarmone guanosine tetraphosphate (ppGpp), which intrinsic levels varies across the E. coli species. In the present study, 50 STEC isolates from healthy sheep were evaluated regarding their ppGpp content, cytotoxicity and other relevant genetic and phenotypic characteristics. We found that the level of ppGpp and cytotoxicity varied considerably among the examined strains. Isolates that harboured the stx2 gene were the least cytotoxic and presented the highest levels of ppGpp. All stx2 isolates belonged to phylogroup A, while strains that carried stx1 or both stx1 and stx2 genes pertained to phylogroup B1. All but two stx2 isolates belonged to the stx2b subtype. Strains that belonged to phylogroup B1 displayed on average low levels of ppGpp and high cytotoxicity. Overall, there was a negative correlation between cytotoxicity and ppGpp.

The Pst system of Streptococcus mutans is important for phosphate transport and adhesion to abiotic surfaces.

The Pst system is a high-affinity inorganic phosphate transporter found in many bacterial species. Streptococcus mutans, the etiological agent of tooth decay, carries a single copy of the pst operon composed of six cistrons (pstS, pstC1, pstC, pstB, smu.1134 and phoU). Here, we show that deletion of pstS, encoding the phosphate-binding protein, reduces phosphate uptake and impairs cell growth, which can be restored upon enrichment of the medium with high concentrations of inorganic phosphate. The relevance of Pst for growth was also demonstrated in the wild-type strain treated with an anti-PstS antibody. Nevertheless, a reduced ability to bind to saliva-coated surfaces was observed, along with the reduction of extracellular polysaccharide production, although no difference on pH acidification was observed between mutant and wild-type strains. Taken together, the present data indicate that the S. mutans Pst system participates in phosphate uptake, cell growth and expression of virulence-associated traits.

In vitro antimicrobial effect of bacteriophages on human dentin infected with Enterococcus faecalis ATCC 29212.

This study assessed the effect of bacteriophages on the viability of Enterococcus faecalis. Human dental roots were inoculated with a suspension of E. faecalis at three different multiplicities of infection - 0.1, 1.0 and 10.0. The phage lysate was able to significantly inhibit bacteria growth when incubated at the multiplicities of infection of 1.0, 10.0 and 0.1. The dental roots were also inoculated with bacteria for 6 days to allow bacterial penetration into the teeth tubules. Addition of the phage lysate to the roots following the 6-day incubation period led to a substantial reduction in bacteria viability. Phage therapy may be an important alternative for the treatment of root canal infections refractory to conventional endodontic therapy.

Phagocytosis as a potential mechanism for microbial defense of mouse placental trophoblast cells.

Trophoblast giant cells are active phagocytes during implantation and post-implantation. Phagocytosis decreases during placental maturation as the phagocytic function of nutrition is gradually replaced by the direct uptake of nutrients by the labyrinth zone trophoblast. We hypothesize that, after placental maturation, trophoblast cells maintain phagocytic functions for purposes other than nutrition. This study employs histological techniques to examine the ability of trophoblast cells to phagocytose microorganisms (yeast or bacteria)--in vivo in females receiving thioglycolate to activate macrophages and in vitro in the presence of phagocytic promoters such as interferon-gamma and complement component C3. Placental trophoblast cells from the second half of gestation show basal phagocytosis that can be dramatically up-regulated by these promoters when microorganisms are inoculated into pregnant animals or introduced into culture systems. Stimulated trophoblast cells phagocytosed organisms more rapidly and in greater numbers than non-stimulated trophoblast exposed to the same numbers of organisms. Taken together, our results indicate that trophoblast cells do not lose their ability to phagocytose during the placentation process, which may imply that trophoblast cells participate in embryonic and fetal innate immune defense through elimination of microorganisms present at the maternal-fetal interface.

Differentially expressed genes in the liver of thioacetamide treated rats

Thioacetamide is a hepatotoxic and hepatocarcinogenic compound that affects liver metabolism, inhibits mRNA transport and induces enlargement of the nucleolus. To investigate the effect of thioacetamide at the molecular level, differential display RT-PCR was conducted. Analysis of nineteen differentially expressed genes demonstrated that ten cDNAs have their expression inhibited while the other nine were positively affected by thioacetamide. Two of the cDNAs were homologous to known genes-TAP and ankyrin-binding glycoprotein-1, two corresponded to repetitive sequences and seven were homologous to expressed sequence tags. The differential expression of some of the isolated cDNAs was confirmed by northern hybridization. It is proposed that since the product of TAP is involved in mRNA transport, thioacetamide inhibition of TAP expression might, at least partially, explain the thioacetamide-induced swelling of the nucleolus.

Transcriptional analysis of the pst operon of Escherichia coli.

The pst operon of Escherichia coli, which encodes the phosphate-specific transport system, is composed of five genes, pstS, pstC, pstA, pstB and phoU, whose transcription is induced by phosphate starvation. A phosphate-regulated promoter located upstream of the most proximal gene ( pstS) controls the transcription of the entire operon. Though the full-length pst mRNA could be detected by an improved RT-PCR protocol, Northern analysis using several pst-specific probes failed to reveal this transcript. Instead, smaller but distinct pst mRNA species were evident. Primer-extension experiments localized the 5' ends of pst mRNAs within the operon. The data suggest that the full-length mRNA is rapidly processed post-transcriptionally.

Resistance to antiretroviral drugs in patients with primary HIV-1 infection. Investigators of the Quebec Primary Infection Study.

The widespread use of antiretroviral agents (ARVs) and the growing occurrence of HIV strains resistant to these drugs have given rise to serious concerns regarding the transmission of resistant viruses to newly infected persons. Plasma viral RNA from 80 individuals newly infected between 1997 and 1999 was genotyped by automated sequencing to analyze the profile of viruses resistant to nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTIs and NNRTIs) and to protease inhibitors (PIs). The prevalence of mutations that conferred primary resistance to PIs (L10I, D30Y, V82A, L90M) was 15% of the cohort. RT genotypic variants, associated with high-level resistance to ARVs, were observed in 21% of individuals, including NRTI, NNRTI and multidrug (MDR) resistance in 6, 5, and 10% of cases, respectively. The phenotypic susceptibility of viral isolates to ARVs was also assayed and showed transmission of high-level resistance to ZDV, 3TC, and PIs in those individuals with MDR. The transmission of drug-resistant HIV genotypic variants is a serious problem that merits further attention by public health officials, virologists, and clinicians.

The effect of thioacetamide on the activity and expression of cytosolic rat liver glutathione-S-transferase.

The effect of thioacetamide (TA), an hepatotoxic and hepatocarcinogenic compound, on the expression and activity of the cytosolic enzyme glutathione-S-transferase (GST) was studied in rat liver. Four h following the administration of 14C-labeled thioacetamide (50 mg/Kg), several subunits of GST were found to be radioactively labeled. A single sublethal dose of TA (250 mg/Kg) decreased by three-fold the expression of class alpha GST at 24-48 h of treatment, but did not significantly affect the transcription of class mu GST. The activity of the enzyme toward 1-chloro-2,4-dinitrobenzene was mildly inhibited (66% of the control) by a 24 h TA treatment and gradually increased thereafter. It is proposed that the covalent binding of TA or its derivative to the GST subunits does not affect the activity of the enzyme. Nevertheless, GST activity inhibition is due to the deleterious effect of TA on GST transcription.

Prevalence of HIV-1 resistant to antiretroviral drugs in 81 individuals newly infected by sexual contact or injecting drug use. Investigators of the Quebec Primary Infection Study.

OBJECTIVE: Prolonged treatment with antiretroviral drugs results in the selection of HIV-1 variants with mutations conferring resistance to nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTI and NNRTI) or to protease inhibitors (PI). There is serious concern about transmission of resistant viruses to newly infected persons. This study monitored the prevalence of resistant viruses in individuals undergoing primary HIV infection. DESIGN: Resistance testing was performed on 81 individuals infected between 1997 and 1999 by injecting drug use (n =21), sexual (n = 56), or unknown (n = 4) transmission. METHODS: Automated sequencing was used to genotype the reverse transcriptase (RT) and protease regions of virus isolated from patients' plasma. The phenotypic susceptibility of stimulated peripheral blood mononuclear cells to antiretroviral drugs was assayed. Line probe assays detected quasispecies variations in wild-type and mutated RT codons. RESULTS: A high prevalence of PI and RT genotypic variants, associated with high-level resistance to antiretroviral drugs, was observed in individuals newly infected by injecting drug use (PI = 24%, RT = 24%) or sexual transmission (PI = 12%, RT = 22%). The PI mutations, L101, V82A, and L90M, were found in 10.5, 3 and 4% of cases, respectively; whereas for RT, primary mutations at positions T215Y (zidovudine), M184V (lamivudine), T69D/A (zalcitabine), and K103N (multi-NNRTI) were present in 8, 5, 4, and 4% of subjects, respectively. Resistance to NRTI was demonstrated by phenotypic, genotypic, and line probe analyses. Transmission of multidrug (NRTI/NNRTI/PI) resistance in eight subjects (9.9%) was confirmed by showing that source partners possessed viruses of similar genotype. CONCLUSIONS: The transmission of drug-resistant HIV is a serious problem that merits further attention by public health officials as well as virologists and clinicians.

The integration host factor (IHF) affects the expression of the phosphate-binding protein and of alkaline phosphatase in Escherichia coli.

The genes encoding alkaline phosphatase (phoA) and the inducible inorganic phosphate transport system Pst (pstS,C,A,B,U) belong to the PHO regulon. Mutants of Escherichia coli lacking the global regulatory protein integration host factor (IHF) show an increased level of alkaline phosphatase and a decreased level of Pst. IHF binds weakly but specifically to a DNA fragment containing the promoter region of the pst operon but does not bind to a fragment that includes the promoter region of phoA. It is proposed that IHF is a positive regulator of the pst operon and as such controls indirectly the expression of phoA.

The relation between ppGpp and the PHO regulon in Escherichia coli.

Starving of Escherichia coli cells for inorganic orthophosphate (Pi) results in the accumulation of spoT-dependent ppGpp and induces the expression of genes of the PHO regulon. In a delta relA delta spoT strain that is unable to accumulate ppGpp the expression of two genes (phoA and pstS) that belong to the PHO regulon is impaired, even in constitutive mutants. The transcription of phoA and of pstS is not affected in the ppGpp0 strain, and therefore this impairment is due to a post-transcriptional defect. Conversely, overexpression of ppGpp inhibits transcription of these two PHO regulon genes. In phoB mutants, the accumulation of ppGpp during Pi starvation is diminished, suggesting that PhoB or one of the PHO products is involved in the control of ppGpp accumulation. We propose that the presence of ppGpp in the cell, but not its accumulation as a result of the starvation stress, is important for the expression of the PHO genes.

Resistance to (-)-2',3'-dideoxy-3'-thiacytidine (3TC) in HIV-1 isolated from paediatric patients.

We conducted detailed virological evaluations of 16 HIV-1-infected paediatric patients treated with 3TC (lamivudine) monotherapy. High-level phenotypic resistance against this compound (up to 2,500-fold) was seen in virtually all cases, usually within 8-12 weeks of initiation of therapy. This was concomitant with the appearance of the M184V mutation in viral reverse transcriptase, previously shown to be responsiblefor such resistance. Viral burden fell in virtually all cases after commencement of therapy, and remained below baseline in each instance studied, despite a rebound effect and the appearance of drug resistance. Viral isolates from some patients underwent a switch from a non-syncytium-inducing (NSI) to a syncytium-inducing (SI) phenotype during the course of the study, although no relationship was apparent between dose of drug employed, time to development of drug resistance or time of appearance of SI phenotype.

Guanosine 3',5'-bispyrophosphate (ppGpp) synthesis in cells of Escherichia coli starved for Pi.

Cells of Escherichia coli which enter a phase of starvation for Pi induce the synthesis of the nucleotide guanosine 3',5'-bispyrophosphate (ppGpp). This induction is relA independent but depends on the spoT gene product. A mutant unable to produce ppGpp is impaired in the expression of two genes which belong to the pho regulon, a defect which is dependent on the product of spoT. We suggest that ppGpp is essential for the proper induction of the genes which belong to the pho regulon.

HIV resistance to anti-viral drugs.

The use of zidovudine (ZDV) and other forms of nucleoside therapy, including dideoxyinosine (ddI), to treat HIV-infected individuals has led to both longer survival and improved quality of life. However, ZDV-resistant variants of HIV-1 can be isolated from patients undergoing prolonged therapy with this drug. HIV drug resistance against ZDV, ddI and other nucleosides is attributable to a series of point mutations within the pol gene of HIV-1 that encodes the viral enzyme, reverse transcriptase (RT). This is not surprising, since the virus is known to replicate at high rates in infected individuals; moreover the RT which mediates transcription of proviral DNA from viral genomic RNA is known to be highly error-prone. Thus, mutants of HIV-1, which possess a drug resistance phenotype and genotype, may be expected to emerge under the selective pressure of long-term anti-viral chemotherapy. HIV drug resistance occurs most commonly in individuals with low CD4 counts, who have progressed to more serious forms of disease. Moreover, viruses obtained from patients with AIDS generally display higher levels of resistance, relative to pre-treatment isolates, than do viruses from patients with more limited illness. Although observations of drug resistance can be correlated with disease progression and a weakened immune system, it is still unclear whether a cause and effect relationship exists. Current clinical research is designed to answer this question while testing the notion that combinations of nucleosides and immuno-stimulatory drugs may provide important clinical benefits.