Molecular characterization of the silencing complex SIR in Candida glabrata hyperadherent clinical isolates.

An important virulence factor for the fungal pathogen Candida glabrata is the ability to adhere to the host cells, which is mediated by the expression of adhesins. Epa1 is responsible for ∼95% of the in vitro adherence to epithelial cells and is the founding member of the Epa family of adhesins. The majority of EPA genes are localized close to different telomeres, which causes transcriptional repression due to subtelomeric silencing. In C. glabrata there are three Sir proteins (Sir2, Sir3 and Sir4) that are essential for subtelomeric silencing. Among a collection of 79 clinical isolates, some display a hyperadherent phenotype to epithelial cells compared to our standard laboratory strain, BG14. These isolates also express several subtelomeric EPA genes simultaneously. We cloned the SIR2, SIR3 and SIR4 genes from the hyperadherent isolates and from the BG14 and the sequenced strain CBS138 in a replicative vector to complement null mutants in each of these genes in the BG14 background. All the SIR2 and SIR4 alleles tested from selected hyper-adherent isolates were functional and efficient to silence a URA3 reporter gene inserted in a subtelomeric region. The SIR3 alleles from these isolates were also functional, except the allele from isolate MC2 (sir3-MC2), which was not functional to silence the reporter and did not complement the hyperadherent phenotype of the BG14 sir3Δ. Consistently, sir3-MC2 allele is recessive to the SIR3 allele from BG14. Sir3 and Sir4 alleles from the hyperadherent isolates contain several polymorphisms and two of them are present in all the hyperadherent isolates analyzed. Instead, the Sir3 and Sir4 alleles from the BG14 and another non-adherent isolate do not display these polymorphisms and are identical to each other. The particular combination of polymorphisms in sir3-MC2 and in SIR4-MC2 could explain in part the hyperadherent phenotype displayed by this isolate.

The GerW protein is not involved in the germination of spores of Bacillus species.

Germination of dormant spores of Bacillus species is initiated when nutrient germinants bind to germinant receptors in spores' inner membrane and this interaction triggers the release of dipicolinic acid and cations from the spore core and their replacement by water. Bacillus subtilis spores contain three functional germinant receptors encoded by the gerA, gerB, and gerK operons. The GerA germinant receptor alone triggers germination with L-valine or L-alanine, and the GerB and GerK germinant receptors together trigger germination with a mixture of L-asparagine, D-glucose, D-fructose and KCl (AGFK). Recently, it was reported that the B. subtilis gerW gene is expressed only during sporulation in developing spores, and that GerW is essential for L-alanine germination of B. subtilis spores but not for germination with AGFK. However, we now find that loss of the B. subtilis gerW gene had no significant effects on: i) rates of spore germination with L-alanine; ii) spores' levels of germination proteins including GerA germinant receptor subunits; iii) AGFK germination; iv) spore germination by germinant receptor-independent pathways; and v) outgrowth of germinated spores. Studies in Bacillus megaterium did find that gerW was expressed in the developing spore during sporulation, and in a temperature-dependent manner. However, disruption of gerW again had no effect on the germination of B. megaterium spores, whether germination was triggered via germinant receptor-dependent or germinant receptor-independent pathways.

The effects of heat activation on Bacillus spore germination, with nutrients or under high pressure, with or without various germination proteins.

Nutrient germination of spores of Bacillus species occurs through germinant receptors (GRs) in spores' inner membrane (IM) in a process stimulated by sublethal heat activation. Bacillus subtilis spores maximum germination rates via different GRs required different 75 °C heat activation times: 15 min for l-valine germination via the GerA GR and 4 h for germination with the L-asparagine-glucose-fructose-K(+) mixture via the GerB and GerK GRs, with GerK requiring the most heat activation. In some cases, optimal heat activation decreased nutrient concentrations for half-maximal germination rates. Germination of spores via various GRs by high pressure (HP) of 150 MPa exhibited heat activation requirements similar to those of nutrient germination, and the loss of the GerD protein, required for optimal GR function, did not eliminate heat activation requirements for maximal germination rates. These results are consistent with heat activation acting primarily on GRs. However, (i) heat activation had no effects on GR or GerD protein conformation, as probed by biotinylation by an external reagent; (ii) spores prepared at low and high temperatures that affect spores' IM properties exhibited large differences in heat activation requirements for nutrient germination; and (iii) spore germination by 550 MPa of HP was also affected by heat activation, but the effects were relatively GR independent. The last results are consistent with heat activation affecting spores' IM and only indirectly affecting GRs. The 150- and 550-MPa HP germinations of Bacillus amyloliquefaciens spores, a potential surrogate for Clostridium botulinum spores in HP treatments of foods, were also stimulated by heat activation.

Effects of a symbiotic on gut microbiota in Mexican patients with end-stage renal disease.

OBJECTIVES: Gut microbiota provides beneficial effects under physiological conditions, but is able to contribute to inflammatory diseases in susceptible individuals. Thus, we designed this study to test whether additional intake of symbiotic gel affects specific modifications of gut microbiota in patients with end-stage renal disease (ESRD). METHODS: Eighteen patients with ESRD diagnosis with renal replacement therapy (hemodialysis) were included in this study. They were randomly assigned to 2 treatment groups: (1) test group (nutritional counseling + symbiotic) and (2) control group (nutritional counseling + placebo). Clinical history and the evaluation of Gastrointestinal Symptom Rating Scale were performed. Gut microbiota composition was analyzed by real-time polymerase chain reaction from fecal samples. All subjects were followed for 2 months. RESULTS: Bifidobacterial counts were higher in the second samples (mean: 5.5 ± 1.72 log10 cells/g) than in first samples (4.2 ± 0.88 log 10 cells/g) in the patients of the test group (P = .0344). Also, lactobacilli counts had a little decrease in the test group (2.3 ± 0.75 to 2.0 ± 0.88 log 10 cells/g) and the control group (2.2 ± 0.90 to 1.8 ± 1.33 log 10 cells/g), between the first and the second samples. Gastrointestinal symptoms scores (scale 8-40) were reduced in the test group (start 12 [10-14] and end 9 [8-10]) compared with control group (start 11 [8-21] and end 11 [9-15]). CONCLUSIONS: Short-term symbiotic treatment in patients with ESRD can lead to the increase of Bifidobacterium counts, maintaining the intestinal microbial balance.