Whole-Genome Sequence of the Microcin E492-Producing Strain Klebsiella pneumoniae RYC492

Marcoleta, Andrés E.; Gutiérrez-Cortez, Sergio; Maturana, Daniel; Monasterio, Octavio; Lagos, Rosalba

doi:10.1128/genomea.00178-13

Cited by 8 publications

(10 citation statements)

References 13 publications

(13 reference statements)

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“… References K. pneumoniae subsp. pneumoniae 1084 Liver abscess Complete 2 8 and 14 NC_018522 [ 21 , 22 ] K. pneumoniae NTUH-K2044 Liver abscess Complete 2 3 and 22 NC_012731 [ 23 ] K. pneumoniae JHCK1 Meningitis Draft 2 9 and 15 NZ_ANGH02000012 [ 24 , 25 ] K. pneumoniae RYC492 Stool Draft 1 11 NZ_APGM01000001 [ 26 , 27 ] K. pneumoniae WGLW2 Sputum Draft 1 9 NZ_JH930419 NR K. pneumoniae WGLW5 Mouse Draft 2 6 and 19 NZ_JH930428 NR NR no reference, WGLW2 and WGLW5 genomes were submitted by Broad Institute, BioProjects PRJNA181874, PRJNA169454 and PRJNA181876, PRJNA169456 respectively, available in NCBI. a CRISPR/Cas system was detected with the CRISPRFinder software.…”

Section: Resultsmentioning

confidence: 99%

Survey of clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR/Cas) systems in multiple sequenced strains of Klebsiella pneumoniae

et al. 2015

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BackgroundIn recent years the emergence of multidrug resistant Klebsiella pneumoniae strains has been an increasingly common event. This opportunistic species is one of the five main bacterial pathogens that cause hospital infections worldwide and multidrug resistance has been associated with the presence of high molecular weight plasmids. Plasmids are generally acquired through horizontal transfer and therefore is possible that systems that prevent the entry of foreign genetic material are inactive or absent. One of these systems is CRISPR/Cas. However, little is known regarding the clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR/Cas) system in K. pneumoniae. The adaptive immune system CRISPR/Cas has been shown to limit the entry of foreign genetic elements into bacterial organisms and in some bacteria it has been shown to be involved in regulation of virulence genes. Thus in this work we used bioinformatics tools to determine the presence or absence of CRISPR/Cas systems in available K. pneumoniae genomes.ResultsThe complete CRISPR/Cas system was identified in two out of the eight complete K. pneumoniae genomes sequences and in four out of the 44 available draft genomes sequences. The cas genes in these strains comprises eight cas genes similar to those found in Escherichiacoli, suggesting they belong to the type I-E group, although their arrangement is slightly different. As for the CRISPR sequences, the average lengths of the direct repeats and spacers were 29 and 33 bp, respectively. BLAST searches demonstrated that 38 of the 116 spacer sequences (33%) are significantly similar to either plasmid, phage or genome sequences, while the remaining 78 sequences (67%) showed no significant similarity to other sequences. The region where the CRISPR/Cas systems were located is the same in all the Klebsiella genomes containing it, it has a syntenic architecture, and is located among genes encoding for proteins likely involved in metabolism and resistance to antibiotics.ConclusionsThe CRISPR/Cas system is not widely distributed in K. pneumoniae genomes, those present most likely belong to type I-E with few differences from the arrangement of the cse3 gene and most of the spacers have not been are not described yet. Given that the CRISPR/Cas system is scarcely distributed among K. pneumoniae genomes it is not clear whether it is involved in either immunity against foreign genetic material or virulence. We consider that this study represents a first step to understand the role of CRISPR/Cas in K. pneumoniae.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1285-7) contains supplementary material, which is available to authorized users.

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Section: Resultsmentioning

confidence: 99%

Survey of clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR/Cas) systems in multiple sequenced strains of Klebsiella pneumoniae

et al. 2015

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“…In K. pneumoniae , several virulence and mobility factors are encoded in PAIs, including fimbrial assembly proteins, yersiniabactin siderophore synthesis and uptake determinants, colibactin toxin synthesis proteins, a virulence-related phospholipase D, and conjugative transfer-related proteins ( Koczura and Kaznowski, 2003 ; Lin et al, 2008 ; van Aartsen, 2008 ; Putze et al, 2009 ; Chen et al, 2010 ; Lery et al, 2014 ). Among other putative virulence-related factors of K. pneumoniae likely encoded in PAIs are the determinants required for MccE492 and salmochelin siderophore production ( Marcoleta et al, 2013a ; Lai et al, 2014 ).…”

Section: Introductionmentioning

confidence: 99%

“…This 13-kbp segment showed to be sufficient to allow the heterologous production and export of MccE492 with the same biochemical properties than the MccE492 purified from Kp RYC492. However, the genomic context of the MccE492 production cluster in its former host remained unexplored until the recent sequencing, assembly, and annotation of the Kp RYC492 genome, where a preliminary inspection indicated that the MccE492 cluster could be located inside a GI ( Marcoleta et al, 2013a ). In this work, we performed bioinformatics and experimental analyses indicating that the MccE492 cluster actually forms part of an unstable 23-kbp GI (named GIE492) with hallmark features of this kind of genetic elements.…”

Section: Introductionmentioning

confidence: 99%

Klebsiella pneumoniae Asparagine tDNAs Are Integration Hotspots for Different Genomic Islands Encoding Microcin E492 Production Determinants and Other Putative Virulence Factors Present in Hypervirulent Strains

et al. 2016

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Due to the developing of multi-resistant and invasive hypervirulent strains, Klebsiella pneumoniae has become one of the most urgent bacterial pathogen threats in the last years. Genomic comparison of a growing number of sequenced isolates has allowed the identification of putative virulence factors, proposed to be acquirable mainly through horizontal gene transfer. In particular, those related with synthesizing the antibacterial peptide microcin E492 (MccE492) and salmochelin siderophores were found to be highly prevalent among hypervirulent strains. The determinants for the production of both molecules were first reported as part of a 13-kbp segment of K. pneumoniae RYC492 chromosome, and were cloned and characterized in E. coli. However, the genomic context of this segment in K. pneumoniae remained uncharacterized. In this work, we provided experimental and bioinformatics evidence indicating that the MccE492 cluster is part of a highly conserved 23-kbp genomic island (GI) named GIE492, that was integrated in a specific asparagine-tRNA gene (asn-tDNA) and was found in a high proportion of isolates from liver abscesses sampled around the world. This element resulted to be unstable and its excision frequency increased after treating bacteria with mitomycin C and upon the overexpression of the island-encoded integrase. Besides the MccE492 genetic cluster, it invariably included an integrase-coding gene, at least seven protein-coding genes of unknown function, and a putative transfer origin that possibly allows this GI to be mobilized through conjugation. In addition, we analyzed the asn-tDNA loci of all the available K. pneumoniae assembled chromosomes to evaluate them as GI-integration sites. Remarkably, 73% of the strains harbored at least one GI integrated in one of the four asn-tDNA present in this species, confirming them as integration hotspots. Each of these tDNAs was occupied with different frequencies, although they were 100% identical. Also, we identified a total of 47 asn-tDNA-associated GIs that were classified into 12 groups of homology differing in theencoded functionalities but sharing with GIE492 a conserved recombination module and potentially its mobility features. Most of these GIs encoded factors with proven or potential role in pathogenesis, constituting a major reservoir of virulence factors in this species.

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“…The steps for MccE92 synthesis, post-translational modification, processing, export and uptake from target cells are shown in Figure 1A (Thomas et al, 2004;Strahsburger et al, 2005;Lagos et al, 2009;Marcoleta et al, 2013a). The gene cluster encoding for active MccE492 is located in the genomic island GI-E492 (Marcoleta et al, 2013b(Marcoleta et al, , 2016, which was found to be highly prevalent among liver abscessassociated strains of K. pneumoniae (Marcoleta et al, 2016(Marcoleta et al, , 2018Lam et al, 2018). A striking characteristic of MccE492 is that it forms amyloid fibers both in vivo and in vitro ( Figure 1A; Bieler et al, 2005;Arranz et al, 2012;Marcoleta et al, 2013a;Aguilera et al, 2016).…”

Section: Introductionmentioning

confidence: 99%

Exploiting Zebrafish Xenografts for Testing the in vivo Antitumorigenic Activity of Microcin E492 Against Human Colorectal Cancer Cells

et al. 2020

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One of the approaches to address cancer treatment is to develop new drugs not only to obtain compounds with less side effects, but also to have a broader set of alternatives to tackle the resistant forms of this pathology. In this regard, growing evidence supports the use of bacteria-derived peptides such as bacteriocins, which have emerged as promising anti-cancer molecules. In addition to test the activity of these molecules on cancer cells in culture, their in vivo antitumorigenic properties must be validated in animal models. Although the standard approach for such assays employs experiments in nude mice, at the initial stages of testing, the use of high-throughput animal models would permit rapid proof-of-concept experiments, screening a high number of compounds, and thus increasing the possibilities of finding new anti-cancer molecules. A validated and promising alternative animal model are zebrafish larvae harboring xenografts of human cancer cells. Here, we addressed the anti-cancer properties of the antibacterial peptide microcin E492 (MccE492), a bacteriocin produced by Klebsiella pneumoniae, showing that this peptide has a marked cytotoxic effect on human colorectal cancer cells in vitro. Furthermore, we developed a zebrafish xenograft model using these cells to test the antitumor effect of MccE492 in vivo, demonstrating that intratumor injection of this peptide significantly reduced the tumor cell mass. Our results provide, for the first time, evidence of the in vivo antitumoral properties of a bacteriocin tested in an animal model. This evidence strongly supports the potential of this bacteriocin for the development of novel anti-cancer therapies.

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Whole-Genome Sequence of the Microcin E492-Producing Strain Klebsiella pneumoniae RYC492

Cited by 8 publications

References 13 publications

Survey of clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR/Cas) systems in multiple sequenced strains of Klebsiella pneumoniae

Survey of clustered regularly interspaced short palindromic repeats and their associated Cas proteins (CRISPR/Cas) systems in multiple sequenced strains of Klebsiella pneumoniae

Klebsiella pneumoniae Asparagine tDNAs Are Integration Hotspots for Different Genomic Islands Encoding Microcin E492 Production Determinants and Other Putative Virulence Factors Present in Hypervirulent Strains

Exploiting Zebrafish Xenografts for Testing the in vivo Antitumorigenic Activity of Microcin E492 Against Human Colorectal Cancer Cells

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