Plasticity of Repetitive DNA Sequences within a Bacterial (Type IV) Secretion System Component

DNA rearrangement permits bacteria to regulate gene content and expression. In Helicobacter pylori, cagY, which contains an extraordinary number of direct DNA repeats, encodes a surface-exposed subunit of a (type IV) bacterial secretory system. Examining potential DNA rearrangements involving the cagY repeats indicated that recombination events invariably yield in-frame open reading frames, producing alternatively expressed genes. In individual hosts, H. pylori cell populations include strains that produce CagY proteins that differ in size, due to the predicted in-frame deletions or duplications, and elicit minimal or no host antibody recognition. Using repetitive DNA, H. pylori rearrangements in a host-exposed subunit of a conserved bacterial secretion system may permit a novel form of antigenic evasion

Long non-coding RNA GAS5 and miR-126-3p as molecular biomarkers of response to sorafenib in human cancer cells.

lncRNAs and microRNAs are implicated in several biological functions and their dysregulation is frequently found in cancer. To better understand the molecular mechanism of the multikinase inhibitor sorafenib, we profiled the expression of a panel of ncRNAs in a sorafenib-treated hepatocellular carcinoma (HCC) cell line. Among the most modulated, we found the dysregulation of the lncRNAs GAS5, HOTTIP and HOXA-AS2 and the miR-126-3p in HCC, renal and breast carcinoma cell lines. The diagnostic performance of GAS5 and miR-126-3p was verified in solid and liquid biopsies from HCC patients. miR-126-3p was decreased in HCC tissues respect to their correspondent peri-tumoral tissues. The levels of circulating miR-126-3p and GAS5 were significantly higher and lower respectively, in HCC patients respect to healthy subjects. This study highlighted that GAS5 and miR-126-3p were involved in the response to sorafenib of different cancer cell types and they were good diagnostic biomarkers of HCC in liquid biopsies

Long non-coding RNA GAS5 and miR-126-3p as molecular biomarkers of response to sorafenib in human cancer cells.

lncRNAs and microRNAs are implicated in several biological functions and their dysregulation is frequently found in cancer. To better understand the molecular mechanism of the multikinase inhibitor sorafenib, we profiled the expression of a panel of ncRNAs in a sorafenib-treated hepatocellular carcinoma (HCC) cell line. Among the most modulated, we found the dysregulation of the lncRNAs GAS5, HOTTIP and HOXA-AS2 and the miR-126-3p in HCC, renal and breast carcinoma cell lines. The diagnostic performance of GAS5 and miR-126-3p was verified in solid and liquid biopsies from HCC patients. miR-126-3p was decreased in HCC tissues respect to their correspondent peri-tumoral tissues. The levels of circulating miR-126-3p and GAS5 were significantly higher and lower respectively, in HCC patients respect to healthy subjects. This study highlighted that GAS5 and miR-126-3p were involved in the response to sorafenib of different cancer cell types and they were good diagnostic biomarkers of HCC in liquid biopsies

Indole-containing arene-ruthenium complexes with broad spectrum activity against antibiotic-resistant bacteria

Antimicrobial resistant (AMR) bacteria are emerging and spreading globally, threatening our ability to treat common infectious diseases. The development of new classes of antibiotics able to kill or inhibit the growth of such AMR bacteria through novel mechanisms of action is therefore urgently needed. Here, a new family of indole-containing arene ruthenium organometallic compounds are screened against several bacterial species and drug resistant strains. The most active complex [(p-cym)Ru(O-cyclohexyl-1H-indole-2-carbothioate)Cl] (3) shows growth inhibition and bactericidal activity against different organisms (Acinetobacter baumannii, Mycobacterium abscessus, Mycobacterium tuberculosis, Staphylococcus aureus, Salmonella enterica serovar typhi and Escherichia coli), demonstrating broad-spectrum inhibitory activity. Importantly, this compound series exhibits low toxicity against human cells. Owing to the novelty of the antibiotic family, their moderate cytotoxicity, and their inhibitory activity against Gram positive, Gram negative and acid-fast, antibiotic resistant microorganisms, this series shows significant promise for further development

Use of Non-mammalian Models to Assess the Virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosa (P. aeruginosa) is the archetypical opportunistic pathogen that accounts for significant numbers of cases of hospital-acquired infections. It adapts to diverse environments in part because it may “modify” its genome through horizontal gene transfer (HGT). Caenorhabditis elegans (C. elegans) and Acanthamoeba spp. (A. castellanii and A. polyphaga) can be employed as in vivo surrogates to investigate the virulence associated with P. aeruginosa clinical isolates and with the presence of genomic islands, genetic elements acquired through HGT. A PA14 based P. aeruginosa pathogenicity island (PAPI)-2 minus mutant exhibited only a minor reduction in virulence as measured by C. elegans survival using the slow-killing assay (SKA) model of virulence but significant attenuation of virulence was found as assessed by the expansion of A. castellanii but not A. polyphaga film on non-nutrient agar. A C. elegans highthroughput assay (HTA) was also established as model of infection and validated against the SKA. The HTA was employed to screen a panel of P. aeruginosa mutants created by deletion of tRNA-associated genomic islands. Few mutants (5 out of 21) showed attenuated virulence toward C. elegans in HTA many of which were confirmed to be less virulent toward the nematode using SKA. Screening of clinical isolates linked the strains isolated from blood culture of a patient with lower survival of C. elegans in HTA compared with strains isolated from sputum. Moreover, type III secretion system effector (T3SS) exoU-positive strains and strains developing pigments during growth were more virulent toward C. elegans in HTA than T3SS effector exoS-positive strains and pigment-less strains, respectively. Since pigment development was also linked to exoU and this gene has been associated with genomic islands integrated at tRNALys10, it was inferred that these genomic islands may bear functions affecting C. elegans survival possibly enhancing iron scavenging

Deletion of TnAbaR23 results in both expected and unexpected antibiogram changes in a multidrug-resistant acinetobacter baumannii strain

Since the 2006 discovery of the Acinetobacter baumannii strain AYE AbaR1 resistance island, similar elements have been reported in numerous members of this species. As AbaR1 is distantly related to Tn7, we have renamed it TnAbaR1. TnAbaR transposons are known to carry multiple antibiotic resistance- and efflux-associated genes, although none have been experimentally studied en bloc. We deleted the TnAbaR transposon in A. baumannii A424, which we have designated TnAbaR23, and characterized independent deletion mutants DCO163 and DCO174. The NotI pulsed-field gel electrophoresis (PFGE) profile of strain DCO174 was consistent with targeted deletion of TnAbaR23 alone, but strain DCO163 apparently harbored a second large genomic deletion. Nevertheless, “subtractive amplification” targeting 52 TnAbaR and/or resistance-associated loci yielded identical results for both mutants and highlighted genes lost relative to strain A424. PCR mapping and genome sequencing revealed the entire 48.3-kb sequence of TnAbaR23. Consistent with TnAbaR23 carrying two copies of sul1, both mutants exhibited markedly increased susceptibility to sulfamethoxazole. In contrast, loss of tetAR(A) resulted in only a minor and variable increase in tetracycline susceptibility. Despite not exhibiting a growth handicap, strain DCO163 was more susceptible than strain DCO174 to 9 of 10 antibiotics associated with mutant-to-mutant variation in susceptibility, suggesting impairment of an undefined resistance-associated function. Remarkably, despite all three strains sharing identical gyrA and parC sequences, the ciprofloxacin MIC of DCO174 was >8-fold that of DCO163 and A424, suggesting a possible paradoxical role for TnAbaR23 in promoting sensitivity to ciprofloxacin. This study highlights the importance of experimental scrutiny and challenges the assumption that resistance phenotypes can reliably be predicted from genotypes alone

Acinetobacter Insertion Sequence ISAba11 Belongs to a Novel Family That Encodes Transposases with a Signature HHEK Motif

Experimental and in silico PCR analysis targeting ISAba11 and TnAbaR islands in 196 epidemiologically unrelated Acinetobacter strains representative of ≥19 species were performed. The first two Acinetobacter baumannii ISAba11 elements identified had been found to map to the same site on TnAbaR transposons. However, no further evidence of physical linkage between the two elements was demonstrated. Indeed, examination of 25 definite or putative insertion sites suggested limited sequence specificity. Importantly, an aacC1-tagged version of ISAba11 was shown to actively transpose in A. baumannii. Similarity searches identified nine iso-ISAba11 elements in Acinetobacter and one in Enhydrobacter and single representatives of four distant homologs in bacteria belonging to the phyla “Cyanobacteria” and Proteobacteria. Phylogenetic, sequence, and structural analyses of ISAba11 and/or its associated transposase (TnpISAba11) suggested that these elements be assigned to a new family. All five homologs encode transposases with a shared extended signature comprising 16 invariant residues within the N2, N3, and C1 regions, four of which constituted the cardinal ISAba11 family HHEK motif that is substituted for the YREK DNA binding motif conserved in the IS4 family. Additionally, ISAba11 family members were associated with either no flanking direct repeat (DR) or an ISAba11-typical 5-bp DR and possessed variable-length terminal inverted repeats that exhibited extensive intrafamily sequence identity. Given the limited pairwise identity among TnpISAba11 homologs and the observed restricted distribution of ISAba11, we propose that substantial gaps persist in the evolutionary record of ISAba11 and that this element represents a recent though potentially highly significant entrant into the A. baumannii gene pool