Achromobacter xylosoxidans: An Emerging Pathogen Carrying Different Elements Involved in Horizontal Genetic Transfer

In the last few years, numerous cases of multidrug-resistant Achromobacter xylosoxidans infections have been documented in immunocompromised and cystic fibrosis patients. To gain insights into the molecular mechanisms and mobile elements related to multidrug resistance in this bacterium, we studied 24 non-epidemiological A. xylosoxidans clinical isolates from Argentina. Specific primers for plasmids, transposons, insertion sequences, bla _ampC, intI1, and intI2 genes were used in PCR reactions. The obtained results showed the presence of wide host range IncP plasmids in ten isolates and a high dispersion of class 1 integrons (n?=?10) and class 2 integrons (n?=?3). Four arrays in the variable region (vr) of class 1 integrons were identified carrying different gene cassettes as the aminoglycoside resistance aac(6′)-Ib and aadA1, the trimethoprim resistance dfrA1 and dfrA16, and the β-lactamase bla _OXA-2. In only one of the class 2 integrons, a vr was amplified that includes sat2-aadA1. The bla _ampC gene was found in all isolates, confirming its ubiquitous nature. Our results show that A. xylosoxidans clinical isolates contain a rich variety of genetic elements commonly associated with resistance genes and their dissemination. This supports the hypothesis that A. xylosoxidans is becoming a reservoir of horizontal genetic transfer elements commonly involved in spreading antibiotic resistance.     

Exploring the Role of a Conserved Class A Residue in the {Omega}-Loop of KPC-2 β-Lactamase: A MECHANISM FOR CEFTAZIDIME HYDROLYSIS

Gram-negative bacteria harboring KPC-2, a class A β-lactamase, are resistant to all β-lactam antibiotics and pose a major public health threat. Arg-164 is a conserved residue in all class A β-lactamases and is located in the solvent-exposed Ω-loop of KPC-2. To probe the role of this amino acid in KPC-2, we performed site-saturation mutagenesis. When compared with wild type, 11 of 19 variants at position Arg-164 in KPC-2 conferred increased resistance to the oxyimino-cephalosporin, ceftazidime (minimum inhibitory concentration; 32→128 mg/liter) when expressed in Escherichia coli. Using the R164S variant of KPC-2 as a representative β-lactamase for more detailed analysis, we observed only a modest 25% increase in k(cat)/K(m) for ceftazidime (0.015→0.019 μm(-1) s(-1)). Employing pre-steady-state kinetics and mass spectrometry, we determined that acylation is rate-limiting for ceftazidime hydrolysis by KPC-2, whereas deacylation is rate-limiting in the R164S variant, leading to accumulation of acyl-enzyme at steady-state. CD spectroscopy revealed that a conformational change occurred in the turnover of ceftazidime by KPC-2, but not the R164S variant, providing evidence for a different form of the enzyme at steady state. Molecular models constructed to explain these findings suggest that ceftazidime adopts a unique conformation, despite preservation of Ω-loop structure. We propose that the R164S substitution in KPC-2 enhances ceftazidime resistance by proceeding through "covalent trapping" of the substrate by a deacylation impaired enzyme with a lower K(m). Future antibiotic design must consider the distinctive behavior of the Ω-loop of KPC-2.     

Immune surveillance properties of human NK cell-derived exosomes

Exosomes are nanovesicles released by normal and tumor cells, which are detectable in cell culture supernatant and human biological fluids, such as plasma. Functions of exosomes released by "normal" cells are not well understood. In fact, several studies have been carried out on exosomes derived from hematopoietic cells, but very little is known about NK cell exosomes, despite the importance of these cells in innate and adaptive immunity. In this paper, we report that resting and activated NK cells, freshly isolated from blood of healthy donors, release exosomes expressing typical protein markers of NK cells and containing killer proteins (i.e., Fas ligand and perforin molecules). These nanovesicles display cytotoxic activity against several tumor cell lines and activated, but not resting, immune cells. We also show that NK-derived exosomes undergo uptake by tumor target cells but not by resting PBMC. Exosomes purified from plasma of healthy donors express NK cell markers, including CD56(+) and perforin, and exert cytotoxic activity against different human tumor target cells and activated immune cells as well. The results of this study propose an important role of NK cell-derived exosomes in immune surveillance and homeostasis. Moreover, this study supports the use of exosomes as an almost perfect example of biomimetic nanovesicles possibly useful in future therapeutic approaches against various diseases, including tumors.     

Investigating extracellular in situ EGFR structure and conformational changes using FRET microscopy

The crystallographic structures of functional fragments of ErbBs have provided excellent insights into the geometry of growth factor binding and receptor dimerization. By placing together receptor fragments to build structural models of entire receptors, we expect to understand how these enzymes are allosterically regulated; however, several predictions from these models are inconsistent with experimental evidence from cells. The opening of this gap underlines the need to investigate intact ErbBs by combining cellular and structural studies into a full picture.     

Delayed luminescence: a novel technique to obtain new insights into water structure

Fully understanding the structure of water is a crucial point in biophysics because this liquid is essential in the operation of the engines of life. Many of its amazing anomalies seem to be tailored to support biological processes and, during about a century, several models have been developed to describe the water structuring. In particular, a theory assumes that water is a mixture of domains constituted by two distinct and inter-converting structural species, the low-density water (LDW) and the high-density water (HDW). According to this theory, by using some particular solutes or changing the water temperature, it should be possible to modify the equilibrium between the two species, changing in this way the water behavior in specific biological processes, as in governing the shape and stability of the structures of proteins. In this work, we assess the possibility of obtaining information on the structures induced in water by specific salts or by temperature by measuring the delayed luminescence (DL) of some salt solutions and of water in the super-cooled regime. Previous works have demonstrated that the delayed luminescence of a system is correlated with its dynamic ordered structures. The results show significant DL signals only when the formation of LDW domains is expected. The measurement reveals a similar activation energy for the domains both in aqueous salt solutions and super-cooled water. It is worth noting that the time trend of DL signals suggests the existence of structures unusually long-lasting in time, up to the microsecond range.