Comparative genomics of the emerging human pathogen Photorhabdus asymbiotica with the insect pathogen Photorhabdus luminescens

Abstract

Background: The Gram-negative bacterium Photorhabdus asymbiotica (Pa) has been recovered from human infections in both North America and Australia. Recently, Pa has been shown to have a nematode vector that can also infect insects, like its sister species the insect pathogen P. luminescens (Pl). To understand the relationship between pathogenicity to insects and humans in Photorhabdus we have sequenced the complete genome of Pa strain ATCC43949 from North America. This strain (formerly referred to as Xenorhabdus luminescens strain 2) was isolated in 1977 from the blood of an 80 year old female patient with endocarditis, in Maryland, USA. Here we compare the complete genome of Pa ATCC43949 with that of the previously sequenced insect pathogen P. luminescens strain TT01 which was isolated from its entomopathogenic nematode vector collected from soil in Trinidad and Tobago.

Results: We found that the human pathogen Pa had a smaller genome (5,064,808 bp) than that of the insect pathogen Pl (5,688,987 bp) but that each pathogen carries approximately one megabase of DNA that is unique to each strain. The reduced size of the Pa genome is associated with a smaller diversity in insecticidal genes such as those encoding the Toxin complexes (Tc's), Makes caterpillars floppy (Mcf) toxins and the Photorhabdus Virulence Cassettes (PVCs). The Pa genome, however, also shows the addition of a plasmid related to pMT1 from Yersinia pestis and several novel pathogenicity islands including a novel Type Three Secretion System (TTSS) encoding island. Together these data suggest that Pa may show virulence against man via the acquisition of the pMT1-like plasmid and specific effectors, such as SopB, that promote its persistence inside human macrophages. Interestingly the loss of insecticidal genes in Pa is not reflected by a loss of pathogenicity towards insects.

Conclusion: Our results suggest that North American isolates of Pa have acquired virulence against man via the acquisition of a plasmid and specific virulence factors with similarity to those shown to play roles in pathogenicity against humans in other bacteria.

Figure 1

Strains of P. asymbiotica, either from the USA or Australia, are the only Photorhabdus that carry plasmids. Restriction enzyme (Bgl2) analysis of Pa plasmids confirms the similarity of plasmids in different USA (lanes 1–4) and Australian (lanes 5–7) isolates. Strains are from North America: 1, ATCC43950 (San Antonio, Texas); 2, ATCC43951 (San Antonio, Texas); 3; ATCC43952 (San Antonio, Texas); 4, ATCC43949 (Maryland) and Australia: 5, Beaudesert (Queensland), 6, Murwillumbah (New South Wales) and 7, Gladstone (Queensland). See Gerrard et al. 2004 reference [6] for more details of Australian strains and their collection.

Figure 2

(A) Predicted open reading frames in the pPAU1 circular plasmid sequenced from Pa ATCC43949 presented in a linear diagram. Most of the open reading frames on the plasmid predict transposases with high similarity to those found in Y. pestis (see Table 1) (B) Two-dimensional SDS-PAGE gel of Pa supernatants grown at 30°C (left panel) and 37°C (right panel). Proteins that are differentially expressed are circled and labelled S1-S12. Analysis of proteins S1-S12 by tryptic digest and comparison to a database of predicted tryptic digest fragments from predicted Pa proteins identified S1 as YP1042-like and S4 as Ail-like. The fully annotated sequence of pPAU1 is GenBank accession number AC FM162592.

Figure 3

Schematic circular diagram of the single 5,064,808 bp chromosome of Pa ATCC43949. The circles show (from outside to inside): 1, DNA coordinates (black); 2, CDSs colour coded as to function (black, pathogenicity/adaptation; dark grey, essential metabolism; red, DNA replication, transcription and restriction modification; green, transmembrane/outer membrane; cyan and magenta, degradation of large and small molecules respectively; yellow, intermediary metabolism; light green, hypothetical; light blue, regulators, orange, conserved hypothetical; brown, pseudogenes; pink, transposons and phage); 3, GC skew and 4, GC deviation. The fully annotated sequence of Pa ATCC43949 is GenBank accession number AC FM162591.

Figure 4

Venn diagrams showing the results of a BLASTCLUST analysis of orthologous CDS between Pa and Pl. (A) Numbers of orthologs with 75% identity and (B) numbers of orthologs at 95% identity.

Figure 5

Alignment of the genomes of Pl TT01 and Pa ATCC43949 using the ARTEMIS comparison tool. Note the broad linear alignment of the two genomes with the presence of several large inversions. The breakpoints of these chromosomal rearrangements are flanked by numerous transposons and directly repeated sequences.

Figure 6

Diagram comparing the tcd islands of Pa ATCC43949 with those from two different strains of Pl: TT01 and W14. The tcd island of Pa appears to represent a conserved 'core' of genes found in all strains whilst both Pl strains contain additional copies of tcdA-like, tcdB-like and tccC-like genes inserted adjacent to this core. Note also that the tcd island of Pl W14 has gained several copies of pdl genes that encode lipases thought to be responsible for the release of mature W14 Tc complexes into the bacterial supernatant.

Figure 7

Diagram comparing the tca islands of Pa ATCC43949 with those from Pl TT01 and Pl W14. Note the presence of all three A, B and C elements (tcaA, tcaB and tcaC) in the tca island of Pl W14 which are required for full oral insecticidal activity in the bacterial supernatant and that the tcaA and tcaB genes in both Pa ATCC43949 and Pl TT01 have been either deleted or truncated.

Figure 8

Diagram comparing the tcb-island of Pa ATCC43949 with those from Pl TT01 and Pl W14. Note that Pl W14 has an intact island with a complete copy of the tcbA gene, whereas tcbA is either deleted or largely truncated from the two other strains (see Figure 7).

Figure 9

Diagram comparing the genomic context of mcf1 and mcf2-encoding islands in Pa ATCC43949, Pl TT01 and Pseudomonas fluorescens. Genes similar to mcf1 are found in all Photorhabdus strains and also in the plant-associated bacterium P. fluorescens where the mcf1-like toxin encoding gene is called fitD. Note that fitD in P. fluorescens and mcf2 in Pl TT01 are encoded adjacent to ABC transporters, suggesting that type 1 secretion may be responsible for release of the Mcf-like toxin from the bacterial cell.

Figure 10

The genome of Pa ATCC43949 carries an additional Type Three Secretion System encoding island (T3SS2) that is not present in Pl TT01. This T3SS2 island carries two blocks of genes similar to those found in clinical isolates of Vibrio parahaemolyticus, the first similar to vpa1343-1345 and the second similar to vpa1361-1350.

Figure 11

Difference in the behaviour of GFP labelled Pa ATCC43949 bacteria when exposed to insect hemocytes from Manduca sexta (left panel) and mouse-derived macrophage cell lines (right panel). Note that Pa cells merely adhere to the surface of the insect phagocytes whereas in the presence of macrophages the bacteria grow as filaments that protrude from and invade the mouse phagocytes. Phagocytes are labelled with a TRITC-phalloidin conjugate to visualize their actin cytoskeletons.

Figure 12

Growth of Pl TT01 and Pa ATCC43949 LB medium in the presence or absence of human blood serum at 30°C and/or 37°C. Note that Pa ATCC43949 experiences a long lag phase when grown at 37°C in LB which is abolished by the addition of human serum. The asterisks denote that bacterial cells grew as filaments at 37°C in a fashion similar to their growth in the presence of mouse macrophages (see Figure 11).