Distribution of the putative type VI secretion system core genes in Klebsiella spp.

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Abstract

The type VI secretion system (t6ss) is a recently characterized secretion system which appears to be involved in bacterial pathogenesis as a potential nano-syringe for the translocation of effector proteins into the eukaryotic host cell cytoplasm.

Until now no evidence was provided for the presence of t6ss in the genomes of the sequenced representatives of Klebsiella spp., including the human opportunistic pathogen Klebsiella pneumoniae. However, in a previous study by Lawlor et al. (2005), were revealed two insertion mutants in hypothetical proteins of K. pneumoniae with decreased ability to infect mouse spleen. Interestingly, these two putative proteins appear to be homologues with two characterized t6ss core proteins of Yersinia pestis.

In order to investigate the presence of genes encoding for putative t6ss core components and putative effectors in Klebsiella spp., we have undertaken an in silico genome mining in three fully and one partially sequenced strains of K. pneumoniae, as well as a strain of the Klebsiella variicola. Moreover, we have investigated the phylogenetic relatedness of three core proteins of the Klebsiella t6ss with their orthologues of various bacteria species. Our analysis evidenced three distinguishable, conserved syntenies in Klebsiella spp. genomes that contain the recognised as putative t6ss genes. The results of our work taken together with the results on the functional analysis of insertion mutants, strongly suggest the existence of an organised t6ss mechanism that likely accounts of the host–pathogen interaction.

Research highlights

▶ The chromosomes of Klebsiella spp. comprise 2–3 conserved loci of T6SS genes. ▶ These T6SS loci coincide with a reported virulence locus. ▶ The T6SS loci of Klebsiella strains exhibit more than 95% homology and they are phylogenetically highly related to Yersinia T6SS. ▶ The Klebsiella T6SS loci are acquired horizontally and maintained through evolution.

Introduction

The type VI secretion system (hereafter t6ss) is a recently characterized secretion system that appears to constitute an injectisome (phage-tail-like), which has the potential to translocate bacterial effector proteins directly into the eukaryotic host cell cytoplasm (Bingle et al., 2008, Cascales, 2008, Filloux et al., 2008, Shrivastava and Mande, 2008), analogous to the t3ss and t4ss machineries. The t6ss at first drew attention as a conserved family of pathogenicity islands or as an atypical t4ss gene locus in Gram-negative bacteria, before being identified as protein-encoding secretory machinery required for virulence in the Vibrio choleraeDictyostelium discoideum model system (Pukatzki et al., 2006).

The t6ss of V. cholerae and Pseudomonas aeruginosa have been shown to be involved in export of Hcp (Haemolysin-Coregulated Protein) and the valine-glycine repeat (Vgr) proteins. These proteins are proposed effectors that seem associated with cytotoxicity in some in vitro models, but their exact function is still under investigation (Mougous et al., 2006, Pukatzki et al., 2006).

A large number of sequenced bacterial genomes contain genes that were predicted to code for t6ss components, mostly within the class of Proteobacteria, but also within the Planctomycetes and Acidobacteria (Tseng et al., 2009, Boyer et al., 2009). T6ss clusters are usually found within pathogenicity islands for example, P. aeruginosa-HSI, enteroaggregative Escherichia coli-pheU, Salmonella typhimurium-SCI (Salmonella centrisome island), Francisella tularensis-FPI (Francisella pathogenicity island), Agrobacterium tumefaciens (Wu et al., 2008), Pectobacterium atrosepticum (Liu et al., 2008) and Xanthomonas oryzae (Tseng et al., 2009) or on chromosomal locations that show a bias towards virulence or survival in the host. Interestingly, it has been shown that the t6ss is required for efficient root colonization by the nitrogen-fixing plant mutualists Mesorhizobium loti and Rhizobium leguminosarum (Bingle et al., 2008) but also to determine host specificity of Pisum sativum (Bladergroen et al., 2003).

Until now, little is known about the presence of t6ss in the genomes of the sequenced representatives of Klebsiella spp. including the human opportunistic pathogen K. pneumoniae. This microorganism produces several virulence factors, including antiphagocytic capsular polysaccharides (CPS), (Cortes et al., 2002), lipopolysaccharides (Shankar-Sinha et al., 2004), and iron acquisition systems (Nassif and Sansonetti, 1986), however their presence seems not sufficient for K. pneumoniae-induced colonisation and infection of the host. Evidences for new virulence loci have been reported by Lawlor et al. (2005) who had undertaken a large scale screening of about 4800 K. pneumoniae transposon insertion mutants in the mouse model. A total of 106 independent mutants failed to be recovered from either the lungs or spleens of infected mice. Two of these insertions that displayed decreased ability to infect mouse spleen are in hypothetical ORF of K. pneumoniae genome and appear to be homologue with the Yersinia pestis protein (YPO1467) (Lawlor et al., 2005). In our database search, the pre-mentioned Yersinia protein is annotated as t6ss core component and seems to by highly homologue (65.5%) with the putative K. pneumoniae protein.

In this study we present the results of a comparative in silico analysis of putative t6ss-related genes found in the genome of the three fully sequenced K. pneumoniae strains (K. pneumoniae 342, K. pneumoniae NTUH-K2044 & K. pneumoniae subsp. pneumoniae MGH 78578), one partially sequenced strain (K. pneumoniae subsp. rhinoscleromatis ATCC 13884) and the Klebsiella variicola strain At-22. Our analysis allowed us to predict the presence of three different genetic loci potentially coding for core proteins of a t6ss. We also present a phylogenetic analysis based on the protein sequences of the putative ATPases, ClpV/B, IcmF and VgrG homologues, and the respective phylogenetic trees, which provide a basis for understanding the phylogenetic roots of t6ss in pathogenic K. pneumoniae. Our results, together with the functional analysis of the insertion mutants suggest that a t6ss mechanism may assists colonisation and infection of the host by Klebsiella.

Section snippets

Materials and methods

Complete annotated and draft sequenced Klebsiella spp. genomes were downloaded from the NCBI Genome database. Protein and nucleotide sequences from P. aeruginosa HSI-I and HSI-III PAO1 strain and E. coli strain APEC O1, were used for BLASTP, BLASTN and reverse BLAST against the Klebsiella genomes in the NCBI and KEGG data bases. Only proteins showing the highest E-value (E-value of <e10) were retained. Clusters containing at least five genes encoding proteins with similarity to known t6ss core

Results

The opportunistic pathogen P. aeruginosa (that chronically infects the lungs of 98% of cystic fibrosis patients) encodes three IcmF-associated homologous protein (IAHP) gene clusters in its genome. These clusters designated HSIs, encode components of t6ss, which mediate cytotoxicity in phagocytic cells and are required for the extracellular secretion of four proteins lacking canonical hydrophobic amino-terminal signal sequences in V. cholerae.

In a genome-wide in silico analysis, a list of the

Discussion

Our genomic mining and data analysis evidenced three distinguishable, conserved syntenies in Klebsiella spp. genomes that contain the recognised putative t6ss genes. Accumulating data show that multiple copies of the t6ss locus, is a frequent phenomenon in proteobacteria, where this secretion system appears to be confined. In our work we have included both phytosymbiotic and human pathogenic strains that exhibit an overall genetic identity between 66% and 98%, with the phytosymbiotic Kp342 to

Conclusion

Our study provides a basis for focused investigations on this newly discovered and poorly understood secretion system in Klebsiella spp. Evidence on the functionality of the genetic loci described here, have been provided previously by Lawlor et al. (2005), but the proper expression of the t6ss core proteins and their role in different Klebsiella strains are currently under study in our laboratory.

Acknowledgements

We are grateful to Dr. Manolis D. Ladoukakis (University of Crete) for critical reading of this manuscript and useful suggestions on phylogenetic studies.

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