Identification and analysis of genomic islands in Burkholderia cenocepacia AU 1054 with emphasis on pathogenicity islands

Abstract

Background: Genomic islands (GIs) are genomic regions that reveal evidence of horizontal DNA transfer. They can code for many functions and may augment a bacterium's adaptation to its host or environment. GIs have been identified in strain J2315 of Burkholderia cenocepacia, whereas in strain AU 1054 there has been no published works on such regions according to our text mining and keyword search in Medline.

Results: In this study, we identified 21 GIs in AU 1054 by combining two computational tools. Feature analyses suggested that the predictions are highly reliable and hence illustrated the advantage of joint predictions by two independent methods. Based on putative virulence factors, four GIs were further identified as pathogenicity islands (PAIs). Through experiments of gene deletion mutants in live bacteria, two putative PAIs were confirmed, and the virulence factors involved were identified as lipA and copR. The importance of the genes lipA (from PAI 1) and copR (from PAI 2) for bacterial invasion and replication indicates that they are required for the invasive properties of B. cenocepacia and may function as virulence determinants for bacterial pathogenesis and host infection.

Conclusions: This approach of in silico prediction of GIs and subsequent identification of potential virulence factors in the putative island regions with final validation using wet experiments could be used as an effective strategy to rapidly discover novel virulence factors in other bacterial species and strains.

Keywords: B. cenocepacia AU1054; Genomic Island; Pathogenicity Island; Virulence factor.

Fig. 1

Cumulative GC profile of chromosome II of B. cenocepacia AU 1054. All GIs tend to be ascending straight lines (rend colour), indicating they are compositionally homogeneous and AT-richer than the core region

Fig. 2

Blast search result of AU 1054 PAIs against genomes in the same species of B. cenocepacia. a, (b), (c), and (d) Correspond to PAI 1, PAI 2, PAI 3 and PAI 4, respectively. In the four figures only those segments with e-values less than 1e-20 are regarded as effective match. The other six stains are arranged according to match length of their homologous to PAIs in AU1054. That is to say, if a strain has the largest homologous match length, it will be assigned most adjacent with AU 1054. Note that confirmed or putative VFs are marked on the bar of AU 1054 as blue box

Fig. 3

Survival in and adherence to A549 cells of B. cenocepacia strains. a Growth of WT AU1054 versus mutant strains AU1054∆lipA and AU1054∆copR cultured in LB. The optical density at OD₆₀₀ was measured hourly over 14 h. b Intracellular survival of WT AU1054 and derivative mutant strains in A549 cells. Bacterial infections were performed with MOI of 10, and bacterial survival was represented as recovery rate of CFUs at 24 h relative to that at 2 h. c Bacterial adherence assays with different AU1054 strains in A549 cells. Bacterial infections were performed with an MOI of 50. Adherence values were calculated by determining the percentages of bacterial CFUs after adhesion relative to that of original CFUs added for infection (*P < 0.05; **P < 0.01; ***P < 0.001; ns: no significant difference)