Francesca Borsetti

The bacterial response to the chalcogen metalloids Se and Te.

Microbial metabolism of inorganics has been the subject of interest since the 1970s when it was recognized that bacteria are involved in the transformation of metal compounds in the environment. This area of research is generally referred to as bioinorganic chemistry or microbial biogeochemistry. Here, we overview the way the chalcogen metalloids Se and Te interact with bacteria. As a topic of considerable interest for basic and applied research, bacterial processing of tellurium and selenium oxyanions has been reviewed a few times over the past 15 years. Oddly, this is the first time these compounds have been considered together and their similarities and differences highlighted. Another aspect touched on for the first time by this review is the bacterial response in cell-cell or cell-surface aggregates (biofilms) against the metalloid oxyanions. Finally, in this review we have attempted to rationalize the considerable amount of literature available on bacterial resistance to the toxic metalloids tellurite and selenite.

Identification of TLR4 as the Receptor That Recognizes Shiga Toxins in Human Neutrophils

Hemolytic uremic syndrome (HUS) caused by intestinal Shiga toxin–producing Escherichia coli infections is a worldwide health problem, as dramatically exemplified by the German outbreak occurred in summer 2011 and by a constant burden of cases in children. Shiga toxins (Stx) play a pivotal role in HUS by triggering endothelial damage in kidney and brain through globotriaosylceramide (Gb3Cer) receptor targeting. Moreover, Stx interact with human neutrophils, as experimentally demonstrated in vitro and as observed in patients with HUS. A neutrophil-protective role on endothelial damage (sequestration of circulating toxins) and a causative role in toxin delivery from the gut to the kidney (piggyback transport) have been suggested in different studies. However, the receptor that recognizes Stx in human neutrophils, which do not express Gb3Cer, has not been identified. In this study, by competition and functional experiments with appropriate agonists and antagonists (LPS, anti-TLR4 Abs, respectively), we have identified TLR4 as the receptor that specifically recognizes Stx1 and Stx2 in human neutrophils. Accordingly, these treatments displaced both toxin variants from neutrophils and, upon challenge with Stx1 or Stx2, neutrophils displayed the same pattern of cytokine expression as in response to LPS (assessed by quantitative RT-PCR, ELISA, or multiplexed Luminex-based immunoassays). Moreover, data were supported by adequate controls excluding any potential interference of contaminating LPS in Stx-binding and activation of neutrophils. The identification of the Stx-receptor on neutrophils provides additional elements to foster the understanding of the pathophysiology of HUS and could have an important effect on the development of therapeutic strategies.

Effects of the Metalloid Oxyanion Tellurite (TeO32−) on Growth Characteristics of the Phototrophic Bacterium Rhodobacter capsulatus

ABSTRACT This work examines the effects of potassium tellurite (K2TeO3) on the cell viability of the facultative phototroph Rhodobacter capsulatus. There was a growth mode-dependent response in which cultures anaerobically grown in the light tolerate the presence of up to 250 to 300 μg of tellurite (TeO32−) per ml, while dark-grown aerobic cells were inhibited at tellurite levels as low as 2 μg/ml. The tellurite sensitivity of aerobic cultures was evident only for growth on minimal salt medium, whereas it was not seen during growth on complex medium. Notably, through the use of flow cytometry, we show that the cell membrane integrity was strongly affected by tellurite during the early growth phase (≤50% viable cells); however, at the end of the growth period and in parallel with massive tellurite intracellular accumulation as elemental Te0 crystallites, recovery of cytoplasmic membrane integrity was apparent (≥90% viable cells), which was supported by the development of a significant membrane potential (Δψ = 120 mV). These data are taken as evidence that in anaerobic aquatic habitats, the facultative phototroph R. capsulatus might act as a natural scavenger of the highly soluble and toxic oxyanion tellurite.

Reduction of potassium tellurite to elemental tellurium and its effect on the plasma membrane redox components of the facultative phototroph Rhodobacter capsulatus.

Summary. Anaerobically light-grown cells of Rhodobacter capsulatus B100 are highly resistant to the toxic oxyanion tellurite (TeO32−; minimal inhibitory concentration, 250 μg/ml). This study examines, for the first time, some structural and biochemical features of cells and plasma membrane fragments of this facultative phototroph grown in the presence of 50μg of K2TeO3 per ml. Through the use of transmission microscopy and X-ray microanalysis we show that several “needlelike” shaped granules of elemental tellurium are accumulated into the cytosol near the intracytoplasmic membrane system. Flash-spectroscopy, oxygen consumption measurements, and difference spectra analysis indicated that membrane vesicles (chromatophores) isolated from tellurite-grown cells are able to catalyze both photosynthetic and respiratory electron transport activities, although they are characterized by a low c-type cytochrome content (mostly soluble cytochrome c2). This feature is paralleled by a low cytochrome c oxidase activity and with an NADH-dependent respiration which is catalyzed by a pathway leading to a quinol oxidase (Qox) inhibited by high (millimolar) concentrations of cyanide (CN−). Conversely, membranes from R. capsulatus B100 cells grown in the absence of tellurite are characterized by a branched respiratory chain in which the cytochrome c oxidase pathway (blocked by CN− in the micromolar range) accounts for 35–40% of the total NADH-dependent oxygen consumption, while the remaining activity is catalyzed by the quinol oxidase pathway. These data have been interpreted to show that tellurite resistance of R. capsulatus B100 is characterized by the presence of a modified plasma-membrane-associated electron transport system.

Tellurite uptake by cells of the facultative phototroph Rhodobacter capsulatus is a ΔpH-dependent process

The uptake by light‐grown cells of Rhodobacter capsulatus of the highly toxic metalloid oxyanion tellurite (TeO3 2−) was examined. We show that tellurite is rapidly taken up by illuminated cells in a process which is inhibited by the protonophore carbonyl cyanide‐p‐trifluoromethoxyphenyl‐hydrazone (FCCP) and by the K+/H+ exchanger nigericin. Notably, the light‐driven membrane potential (Δψ) is enhanced by K2TeO3≥200 μM. Further, tellurite uptake is largely insensitive to valinomycin, strongly repressed by the sulfhydryl reagent N‐ethylethylmaleimide (NEM) and competitively inhibited by phosphate. We conclude that tellurite is transported into cells by a ΔpH‐dependent, non‐electrogenic process which is likely to involve the phosphate transporter (PiT family).

Cyclooxygenase-2 Silencing for the Treatment of Colitis: A Combined In Vivo Strategy Based on RNA Interference and Engineered Escherichia Coli

Nonpathogenic-invasive Escherichia coli (InvColi) bacteria are suitable for genetic transfer into mammalian cells and may act as a vehicle for RNA Interference (RNAi) in vivo. Cyclooxygenase-2 (COX-2) is overexpressed in ulcerative colitis (UC) and Crohns disease (CD), two inflammatory conditions of the colon and small intestine grouped as inflammatory bowel disease (IBD). We engineered InvColi strains for anti-COX-2 RNAi (InvColi(shCOX2)), aiming to investigate the in vivo feasibility of a novel COX-2 silencing strategy in a murine model of colitis induced by dextran sulfate sodium (DSS). Enema administrations of InvColi(shCOX2) in DSS-treated mice led to COX-2 downregulation, colonic mucosa preservation, reduced colitis disease activity index (DAI) and increased mice survival. Moreover, DSS/InvColi(shCOX2)-treated mice showed lower levels of circulating pro-inflammatory cytokines and a reduced colitis-associated shift of gut microbiota. Considering its effectiveness and safety, we propose our InvColi(shCOX2) strategy as a promising tool for molecular therapy in intestinal inflammatory diseases.

Metals and Metalloids in Photosynthetic Bacteria: Interactions, Resistance and Putative Homeostasis Revealed by Genome Analysis

Microbial metabolism of metals and metalloids has been a topic of interest since the early seventies when it was recognized that bacteria are involved in the transformation of metal compounds in the environment. For this reason, bacterial processing of inorganic compounds has been reviewed several times over the past decade. However, this is the first time that the metal(loid)s metabolism of photosynthetic bacteria has been considered in detail as compared to non-phototrophs. Another aspect touched on for the first time in this chapter is the analysis of genomes of representative phototrophs in an attempt to reveal common or unique features of the interactions of these bacteria with metal(loid)s. This work not only identified new genes linked to metal resistance, but also contributed to unify the nomenclature used among the genomes of different photosynthetic species. Based on our analysis, similarities and differences can be used more efficiently in future work as new ‘preys’ that have been either hypothesized or described generically in the past have also been uncovered.

Manganese is a Deinococcus radiodurans growth limiting factor in rich culture medium

To understand the effects triggered by Mn2+ on Deinococcus radiodurans, the proteome patterns associated with different growth phases were investigated. In particular, under physiological conditions we tested the growth rate and the biomass yield of D. radiodurans cultured in rich medium supplemented or not with MnCl2. The addition of 2.5-5.0 µM MnCl2 to the medium neither altered the growth rate nor the lag phase, but significantly increased the biomass yield. When higher MnCl2 concentrations were used (10-250 µM), biomass was again found to be positively affected, although we did observe a concentration-dependent lag phase increase. The in vivo concentration of Mn2+ was determined in cells grown in rich medium supplemented or not with 5 µM MnCl2. By atomic absorption spectroscopy, we estimated 0.2 and 0.75 mM Mn2+ concentrations in cells grown in control and enriched medium, respectively. We qualitatively confirmed this observation using a fluorescent turn-on sensor designed to selectively detect Mn2+in vivo. Finally, we investigated the proteome composition of cells grown for 15 or 19 h in medium to which 5 µM MnCl2 was added, and we compared these proteomes with those of cells grown in the control medium. The presence of 5 µM MnCl2 in the culture medium was found to alter the pI of some proteins, suggesting that manganese affects post-translational modifications. Further, we observed that Mn2+ represses enzymes linked to nucleotide recycling, and triggers overexpression of proteases and enzymes linked to the metabolism of amino acids.