Comparative analysis of <i>Klebsiella pneumoniae</i> genomes identifies a phospholipase D family protein as a novel virulence factor

International audienceBACKGROUND: Klebsiella pneumoniae strains are pathogenic to animals and humans, in which they are both a frequent cause of nosocomial infections and a re-emerging cause of severe community-acquired infections. K. pneumoniae isolates of the capsular serotype K2 are among the most virulent. In order to identify novel putative virulence factors that may account for the severity of K2 infections, the genome sequence of the K2 reference strain Kp52.145 was determined and compared to two K1 and K2 strains of low virulence and to the reference strains MGH 78578 and NTUH-K2044. RESULTS: In addition to diverse functions related to host colonization and virulence encoded in genomic regions common to the four strains, four genomic islands specific for Kp52.145 were identified. These regions encoded genes for the synthesis of colibactin toxin, a putative cytotoxin outer membrane protein, secretion systems, nucleases and eukaryotic-like proteins. In addition, an insertion within a type VI secretion system locus included sel1 domain containing proteins and a phospholipase D family protein (PLD1). The pld1 mutant was avirulent in a pneumonia model in mouse. The pld1 mRNA was expressed in vivo and the pld1 gene was associated with K. pneumoniae isolates from severe infections. Analysis of lipid composition of a defective E. coli strain complemented with pld1 suggests an involvement of PLD1 in cardiolipin metabolism. CONCLUSIONS: Determination of the complete genome of the K2 reference strain identified several genomic islands comprising putative elements of pathogenicity. The role of PLD1 in pathogenesis was demonstrated for the first time and suggests that lipid metabolism is a novel virulence mechanism of K. pneumoniae

Evidence that a laminin-like insect protein mediates early events in the interaction of a Phytoparasite with its vector's salivary gland.

Phytomonas species are plant parasites of the family Trypanosomatidae, which are transmitted by phytophagous insects. Some Phytomonas species cause major agricultural damages. The hemipteran Oncopeltus fasciatus is natural and experimental host for several species of trypanosomatids, including Phytomonas spp. The invasion of the insect vectors' salivary glands is one of the most important events for the life cycle of Phytomonas species. In the present study, we show the binding of Phytomonas serpens at the external face of O. fasciatus salivary glands by means of scanning electron microscopy and the in vitro interaction of living parasites with total proteins from the salivary glands in ligand blotting assays. This binding occurs primarily through an interaction with a 130 kDa salivary gland protein. The mass spectrometry of the trypsin-digest of this protein matched 23% of human laminin-5 β3 chain precursor sequence by 16 digested peptides. A protein sequence search through the transcriptome of O. fasciatus embryo showed a partial sequence with 51% similarity to human laminin β3 subunit. Anti-human laminin-5 β3 chain polyclonal antibodies recognized the 130 kDa protein by immunoblotting. The association of parasites with the salivary glands was strongly inhibited by human laminin-5, by the purified 130 kDa insect protein, and by polyclonal antibodies raised against the human laminin-5 β3 chain. This is the first report demonstrating that a laminin-like molecule from the salivary gland of O. fasciatus acts as a receptor for Phytomonas binding. The results presented in this investigation are important findings that will support further studies that aim at developing new approaches to prevent the transmission of Phytomonas species from insects to plants and vice-versa

Matching of the amino acid sequences of the 130 kDa protein of <i>Oncopeltus fasciatus</i> salivary glands with the human laminin-5 β3 chain amino acid sequence.

<p>(<b>A</b>). Protein spots cut from the gel were treated with porcine trypsin and the peptides were spotted onto a MALDI-TOF sample plate (Voyager- DE, Applied Biosystem, CA, USA). Peptide mass fingerprints were analyzed using Protein Prospector MS-Fit interface (<a href="http://prospector.ucsf.edu" target="_blank">http://prospector.ucsf.edu</a>). (<b>B</b>). Underlined letters represent the amino acid sequences of the salivary gland protein that matched the mass spectrometry data to protein sequences in the NCBI database. The underlined sequences indicate the peptides of the salivary gland protein that were matched with the amino acid sequence of the precursor of the human laminin-5 β3 chain.</p

2D SDS-PAGE and corresponding ligand blotting that show the binding of live parasites to the 130 kDa polypeptide spot present in the total protein extract of <i>Oncopeltus faciatus</i> salivary glands.

<p>(<b>A</b>) 2D SDS-PAGE of total protein extract from salivary glands stained with Coomassie Blue. The arrow indicates spots at the 130 kDa region. The numbers on the horizontal axis refer to the pH gradient (pI) and the numbers on the vertical axis refer to the molecular mass standard expressed in kilodaltons. (<b>B</b>) Ligand blotting showing one spot at the 130 kDa region. The spot on the PVDF membrane indicates the binding of biotinylated live parasites to the 130 kDa protein. For further details see the Methods.</p

Scanning electron microscopy of <i>Oncopeltus fasciatus</i> salivary glands infected with <i>Phytomonas serpens</i>.

<p>The parasites were injected laterally into the thorax of the insects and the salivary glands were explanted at different time points after injection. (<b>A</b>) Outer surface of the salivary gland showing a high number of parasites between two salivary gland lobes and a few parasites attached to the gland, 48 h post-infection. Scale bar: 10 µm. (<b>B–C</b>) Large numbers of parasites bound to the salivary gland, 72 h post-infection. Scale bars: 100 µm (<b>B</b>) and 50 µm (<b>C</b>). SG: salivary gland; P: parasite; SGL1, SGL2 and SGL3: salivary gland lobes. The asterisk (*) indicates the salivary duct. For further details see the Methods.</p

Comparison of laminin subunit β through pairwise alignment of β3 laminin of <i>Homo sapiens</i> (LamB3Hs, gi|119613854), β1-like of the turkey <i>Meleagris gallopavo</i> (LamB1Mg, gi|326911240) and β1-like of <i>Oncopeltus fasciatus</i> (LamB1Of).

<p>The alignment of β3 laminin of <i>H. sapiens</i> and β1-like of <i>M. gallopavo</i> with the partial sequence of β1-like identified in the transcriptome of <i>O. fasciatus</i> embryo <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048170#pone.0048170-EwenCampen1" target="_blank">[28]</a> shows a highly conserved region at the domain VI among these molecules. Black shaded residues are identical or similar amino acids present in all three sequences. Grey shaded residues are identical or similar amino acids present in two of the three sequences. The consensus sequence is represented under alignment lines. In the red rectangles are highlighted the regions with higher similarity between all three sequences. The alignments were performed using GENEDOC software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048170#pone.0048170-Nicholas1" target="_blank">[90]</a>.</p

Inhibition of the <i>in vitro</i> interaction between <i>Phytomonas serpens</i> and salivary glands explanted from <i>Oncopeltus fasciatus</i>.

<p>The parasites were pre-incubated for 30 min in the presence of human laminin-5 (Lam-5) (<b>A</b>) or in the presence of the purified 130 kDa salivary gland protein (p130) (<b>B</b>). In a parallel system, the salivary glands were pre-incubated in the presence of anti-human laminin-5 β3 chain antibodies (anti-β3 antibodies) (<b>C</b>). In the control systems, the parasites and salivary glands were pre-incubated in the absence of the proteins and antibodies, respectively. The proteins and antibodies were used at the indicated concentrations or dilutions. Each bar represents the mean ± standard error of at least three independent experiments. The <i>P</i> values are indicated on the panels.</p

Immunoblotting of the 130 kDa salivary gland protein probed with anti-human laminin-5 β3 chain antibodies.

<p>Total salivary gland proteins were extracted and separated by 10% SDS-PAGE (<b>a</b>). The 130 kDa band was cut and purified from the gel and the purity of the product was evaluated by 10% SDS-PAGE stained with silver nitrate (<b>b</b>). The purified 130 kDa protein was probed with anti-human laminin-5 β3 chain antibodies (<b>c</b>). The arrow shows the position of the 130 kDa protein on SDS-PAGE and the stained band by immunoblotting.</p