New insights into Acinetobacter baumannii pathogenesis revealed by high-density pyrosequencing and transposon mutagenesis

Abstract

Acinetobacter baumannii has emerged as an important and problematic human pathogen as it is the causative agent of several types of infections including pneumonia, meningitis, septicemia, and urinary tract infections. We explored the pathogenic content of this harmful pathogen using a combination of DNA sequencing and insertional mutagenesis. The genome of this organism was sequenced using a strategy involving high-density pyrosequencing, a novel, rapid method of high-throughput sequencing. Excluding the rDNA repeats, the assembled genome is 3,976,746 base pairs (bp) and has 3830 ORFs. A significant fraction of ORFs (17.2%) are located in 28 putative alien islands, indicating that the genome has acquired a large amount of foreign DNA. Consistent with its role in pathogenesis, a remarkable number of the islands (16) contain genes implicated in virulence, indicating the organism devotes a considerable portion of its genes to pathogenesis. The largest island contains elements homologous to the Legionella/Coxiella Type IV secretion apparatus. Type IV secretion systems have been demonstrated to be important for virulence in other organisms and thus are likely to help mediate pathogenesis of A. baumannii. Insertional mutagenesis generated avirulent isolates of A. baumannii and verified that six of the islands contain virulence genes, including two novel islands containing genes that lacked homology with others in the databases. The DNA sequencing approach described in this study allows the rapid elucidation of the DNA sequence of any microbe and, when combined with genetic screens, can identify many novel genes important for microbial pathogenesis.

Figure 1.

Flowchart. Steps leading to the completion of the A. baumannii genome.

Figure 2.

Functional annotation. (A) The functional assignments of all of the called genes in A. baumannii, A. baylyi, and P. aeruginosa. (B) Graphical depiction of the gene annotation. Genes products for A. baumannii (purple), A. baylyi (blue), and P. aeruginosa (orange) are represented by COG assignment (single-letter code): (A) RNA processing and modification; (C) energy production and conversion; (D) cell division and chromosome partitioning; (E) amino acid transport and metabolism; (F) nucleotide transport and metabolism; (G) carbohydrate transport and metabolism; (H) coenzyme metabolism; (I) lipid metabolism; (J) translation, ribosomal structure, and biogenesis; (K) transcription; (L) DNA replication, recombination, and repair; (M) cell envelope biogenesis, outer membrane; (N) cell motility; (O) post-translational modification, protein turnover, chaperones; (P) inorganic ion transport and metabolism; (Q) secondary metabolites biosynthesis, transport, and catabolism; (R) general function prediction only; (S) function unknown; (T) signal transduction mechanisms; (U) intracellular trafficking, secretion, and vesicular transport; (V) defense mechanisms.

Figure 3.

Circular map of A. baumannii genome. The outermost circle shows genes color-coded by COG assignment: (gold) translation, ribosomal structure and biogenesis; (orange) RNA processing and modification; (dark orange) transcription; (maroon) DNA replication, recombination, and repair; (yellow) cell division and chromosome partitioning; (light pink) defense mechanisms; (purple) signal transduction mechanisms; (peach) cell envelope biogenesis, outer membrane; (medium purple) cell motility and secretion; (dark pink) intracellular trafficking, secretion, and vesicular transport; (light green) post-translational modification, protein turnover, chaperones; (lavender) energy production and conversion; (blue) carbohydrate transport and metabolism; (red) amino acid transport and metabolism; (green) nucleotide transport and metabolism; (light blue) coenzyme metabolism; (cyan) lipid metabolism; (dark purple) inorganic ion transport and metabolism; (sea green) secondary metabolites biosynthesis, transport, and catabolism; (light gray) general function prediction only; (ivory) function unknown; (dark gray) not in COGs. The middle circle represents the G + C percentage, colored red for regions above median GC score (38%) and blue for regions less than or equal to the median. The blue boxes indicate pAs, the yellow boxes indicate islands predicted to be involved in pathogenicity through homology and sequence composition differences, and the red and orange boxes indicate islands confirmed by the screens. The circles were drawn using the program GenomeViz (http://www.uniklinikum-giessen.de/genome/genomeviz/intro.html).

Figure 4.

Synteny map. (A) The genomes of A. baylyi (top) and A. baumannii (bottom) were compared by WebACT (http://www.webact.org/WebACT/home) and visualized by ACT (Carver et al. 2005). Red indicates similar genomic organization, whereas blue indicates inversions. (B) The genomes of six bacteria were compared for the distribution of key catabolic enzymes (from the outermost to the innermost ring): A. baumannii, A. baylyi, P. aeruginosa, N. meningitidis, E. coli K12, and B. subtilis. The island clusters were defined by Barbe et al. (2004) based on their location in the A. baylyi genome and are colored gray (−), red (I), orange (II), green (III), blue (IV), and purple (V). The end products of the pathways encoded within each of the catabolic islands are depicted. The boxes surrounding the end products are colored to match the islands from which they were derived. The circles were drawn using the program GenomeViz (http://www.uniklinikum-giessen.de/genome/genomeviz/intro.html).

Figure 5.

DNA transport machinery. Adapted from Averhoff and Friedrich (2003) with kind permission from Springer Science and Business Media. In A. baylyi and other Gram-negative bacteria, foreign DNA is delivered to and through the outer membrane transporter PilQ. ComE-bound DNA is transported to the inner membrane transporter ComA (or ComEC) via ComP, PilE and/or the type IV pilus. ComEA may assist in this delivery.

Figure 6.

Ethanol-stimulated virulence of A. baumannii. (A) Bacteria were incubated on NGM plates without (panels a,c) or with (panels b,d) 1% ethanol. A single L4 stage worm was inoculated onto lawns of E. coli OP50 (panels a,b) or A. baumannii (panels c,d) and allowed to proliferate for 4 d. (Panels a,b) The E. coli OP50 lawns are completely consumed by this time regardless of the presence of ethanol. (Panel d) The worm brood is considerably smaller on plates containing NGM + 1% ethanol and A. baumannii. (B) Bacteria and D. discoideum amoebae were incubated on SM/5 plates without (panels a,c) or with (panels b,d) 1% ethanol. (Panels a,b) In lawns containing K. aerogenes amoebae plaques form within 4 d regardless of the presence of ethanol. Amoebae are able to form plaques in A. baumannii (panel c), but plaque formation is completely inhibited when 1% ethanol is added to the media (panel d).

Figure 7.

Ethanol-stimulated virulence mutants of A. baumannii. Mutants were generated as described in Materials and Methods. (A) Six individual A. baumannii mutants were incubated on NGM (wells a–f) or NGM + 1% ethanol (wells g–j). A single L4 stage worm was inoculated onto lawns of each mutant and allowed to proliferate for 4 d. Avirulent bacterial mutants were recovered as those that allowed the worms to consume the bacterial lawns as fast or faster on NGM + 1% ethanol as on NGM alone; e.g., mutant b (b,h). (B) Six individual A. baumannii mutants were mixed with D. discoideum amoebae and incubated on SM/5 + 1% ethanol (wells a–f). These are not the same mutants as shown in A. Avirulent mutants were defined as those that allowed amoebae plaque formation; e.g., mutant d.