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.

Gonzalo Nuñez,Octavio Monasterio,Andrés E Marcoleta,Rosalba Lagos,Camilo Berríos-Pastén

doi:10.3389/fmicb.2016.00849

Abstract

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.

Highlights

Klebsiella pneumoniae is a Gram-negative bacterium normally considered as an opportunistic pathogen causative of nosocomial infections (Podschun and Ullmann, 1998; Brisse et al, 2006)
In order to gain information about the genomic context of the MccE492 gene cluster, we examined the sequence of the KpRYC492 chromosome and noticed that it is located inside a region with hallmark features of GIs
It is recognized as a main force directing the arising of new strains with further metabolic capabilities and able to adapt to changing environments. This is true for pathogenic bacteria, where a large set of virulence-related genes are encoded in mobile elements that can be transferred among different strains and species coexisting in a particular habitat

Summary

Introduction

Klebsiella pneumoniae is a Gram-negative bacterium normally considered as an opportunistic pathogen causative of nosocomial infections (Podschun and Ullmann, 1998; Brisse et al, 2006). In the last two decades an increasing number of community-acquired invasive K. pneumoniae infections have emerged, mainly in the form of pyogenic liver abscesses that are often accompanied by severe metastatic infections such as endophthalmitis, meningitis, and necrotizing fasciitis (Chang and Chou, 1995; Siu et al, 2012; Struve et al, 2015). Those cases were restricted to Southeast Asia but currently an increasing number of cases are being reported globally. Genomic analysis of the hypervirulent strains revealed that their virulence is mainly determined by a specific gene profile acquired horizontally (Holt et al, 2015)

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Results

Conclusion