Photorhabdus bacteria have a mutualistic relationship with nematodes, where they help the nematodes infect and kill insects. These bacteria produce bioluminescence, toxins, and other compounds that help overcome the insects' immune systems. While usually symbiotic with nematodes in insects, some Photorhabdus strains can also infect humans. The bacteria have evolved complex interactions with their hosts and produce many secondary metabolites worth studying for new drug development.

2. Photorhabdus and a Host of
Hosts
NEM 199 Seminar 0+1
Presented by,
Sujayanand, G.K.
Roll no: 9664
Division of Entomology

3.  Introduction
 Bacterial transmission in nematodes
 Bioluminescence
 Insect defence
 Pathogenicity in insects
 Toxins produced
 Leaping to humans
 conclusion

4. Introduction
 Family: Enterobacteriaceae
 Genus: Photorhabdus
 “glowing rod” – only bioluminescent terrestrial bacterium.
 Habitat:
I. gut of an infective juvenile (Symbiotic)
II. in the insect haemolymph (Parasitic)
 Photorhabdus has mutualistic relationship with
nematodes from the family Heterorhabditidae.
 Soft cuticle, soil-dwelling larvae from the orders
Coleoptera, Lepidoptera and Diptera

5.  highly motile, gram-negative, facultatively
anaerobic rods
 28◦C on blood agar, where they uniquely
produce a thin line of annular haemolysis.
Fischer-Le Saux et al. 1999
P. luminescens P. temperata P. asymbiotica
Photorhabdus

6. Phylogeny

7. Lifecycle of Heterorhabditis

8. Lifecycle of Photorhabdus

9. Bacterial transmission
 1 or 2 bacteria 100 cfu in IJ
 The nematodes feed on Photorhabdus, some bacteria
escape crushing by pharynx enter the gut of the
hermaphrodite– invade rectal gland cell (bacterial
replication).
 Colonizes IJ - develops within the adult
hermaphrodite in a process called endotokia matricida
 Lipopolysacchride biogenesis: galE and galU : role of
bacterial surface in transmission. mutation affects
virulence in insects

11. Nematode symbiont
 Photorhabdus on Luria-Bertani agar : nematode
 same bacteria cultured on lipid agar (nutrient agar
supplemented with cod liver oil and corn syrup).
 Exogenous sterols – for development -
bacteriophagous nematode Caenorhabditis
elegans, and it is likely that Heterorhabditis will
have the same requirement (Chitwood DJ. 1999.)
 Photorhabdus - iso-branched fatty acids (BCFAs),
isoC15:0 - produced - bkdABC operon.

12.  CipA and CipB have a potential role in nematode
nutrition.
 Heterorhabditis will not grow on just any strain of
Photorhabdus, rather it usually requires its specific
cognate bacterial strain

13. Signal production
 In Pt NC19, the phosphopantethienyl (PPANT)
transferase gene ngrA is required to support
nematode growth reproduction
 STs : polyketide molecules - plants in response
to stress and infection and Photorhabdus is the
only nonplant organism known to produce.
 ST is perceived by Heterorhabditis as an
indication that food (i.e. bacteria) is plentiful.
 plu4185-plu4195 involved in the production of a
polyketide molecule, anthraquinone.
 second report of Type II PKS activity in gram-
negative bacteria

14. Bioluminescence
 (a) represent a signal from bacteria to
bacteria or to the nematode, synchronizing
symbiosis;
 (b) provide a visual aposematic warning to
nocturnal scavenging animals; or even
 (c) act a lure to attract further insect prey to
the infected insect cadaver.

15.  lux operon that contains all of the genes
required for the production of light from fatty
acids and molecular O2, i.e. luxCDABE
 fatty acid reductase encoded by luxC and
luxD
 FMN supplies electron

17. Symbiotic Vs Pathogenicity
 a gene with homology to hexA from Erwinia :
represses symbiosis factors in P2 - key player
in the regulatory network controlling
pathogenicity and mutualism
 Bacterial toxins & hydrolytic enzymes:
septicemia
Joyce & Clarke

18. Secondary metabolites

19. Pathogenicity
TTSS: effector protein, LopT, homologous to YopT from
Yersinia →inhibit phagocytosis (Brugirard-Ricaud et al., 2004; 2005).
ST: inhibits the activity of PO, an enzyme required for melanin
production , involved in stabilization of the nodule.
Tc and the Mcf toxins - target and induce apoptosis in, insect
immune cells a novel BH3-domain-mediated effect (Dowling et al.,
2004 and Waterfield et al., 2005).

20.  Epithelial cells: local antimicrobial peptides
(AMPs) and reactive oxygen species (ROS)
 fat body and haemocytes major role in innate
immunity
 Recognize invading pathogens, insects use
pattern recognition proteins (PRPs)
- hemolin,
- immulectin-2 (IML-2) and
- peptidoglycan recognition protein (PGRP)
 PRPs bind to conserved pathogen associated
molecular pattern (PAMP) in Manduca.
Humoral response

21.  IML-2 is crucial in protecting insects against
Gram-negative bacteria and binds to serine
proteases in plasma, which participate in
activating pro-PO (PPO) to active PO
 (RNAi)-mediated knockdown of any of these
genes results in increased susceptibility to
Photorhabdus (Eleftherianos, et al. 2006)
 silencing of IML-2 prevented normal activation
of PO, which was linked to impaired ability of
the insect to encapsulate the bacteria (Steiner,
2004)
Humoral response

22. Humoral response
 detection of Photorhabdus by PRPs: ↑
transcription levels of AMPs (attacin, cecropin,
lebocin, lysozyme and moricin) are high in the
Manduca fat body.
 older Manduca larvae are less able to induce the
transcription of PRP and AMP mRNA - host age
or developmental stage in bacterial immune
challenge. (Eleftherianos, et al. 2008)

23. Cellular response
 One type of cytokine involved in the
haemocyte spreading process is plasmatocyte
spreading peptide (PSP).
 Photorhabdus can inhibit phospholipase A2,
which catalyzes the first step of eicosanoid
biosynthesis and is important for haemocyte
nodulation
 Makes caterpillars floppy 1 (Mcf1) is a toxin
that destroys both insect hemocytes and the
midgut by apoptosis.

24. Photorhabdus toxins with the
insect immune system
Tcd pathogenicity island

25. Phagocytosis evasion
 TccC3 causes ADP-ribosylation of actin and
TccC5 causes ADP-ribosylation of the Rho
GTPases RhoA and Rac, resulting in their
activation (Lang et al., 2010).
 Both TccC3 and TccC5 enter the cytosol via
TcdA1 where they act together to disrupt the
actin cytoskeleton.
 TccC3 and TccC5 are active against both
lepidopteran and human cells.
 Photox – toxin inhibits the polymerization of
actin filaments - effects the cytoskeleton of the
target cell (Visschedyk et al., 2010).

27. PO cascade inhibition by stilbene

28. Nodule formation
Dose response curve

29. Monoxenic infection
 ST: broad-spectrum antibiotic - strong
antibacterial and antifungal properties
 Proteinaceous antibacterial molecules
 S- type pyocins: toxic proteins capable of degrading
macromolecules in target cells. E.g. Lumicin
 R-type pyocins: larger, modified bacteriophage tail structures
 highly toxic Tcd complex is expressed by Pl
W14 early in infection in low amounts and
that the highly expressed Tca is produced
only after death

30. Toxin Complexes (Tcs)
 The toxin complexes (Tcs) are encoded by the
PAI I (pathogenity island I)
 four such complexes, namely Tca, Tcb, Tcc
and Tcd,
 tca and tcd encode for orally active toxins,
responsible for the majority of the insecticidal
activity in Manduca secta

31. Gut active toxins
 Tc produced by Photorhabdus is a high
molecular weight gut active toxin that is lethal to
Lepidoptera, Coleoptera, Hymenoptera and
Dictyoptera when injected into the hemolymph
or orally ingested.
 Tca: M. sexta, having a LD50 of 875 ng/cm2 of
artificial diet and causing significant weight
reduction at 40 ng/cm2

32. Transgenics
 Oral toxicity to insects has been achieved by
cloning Tca (tcdA) from P. luminescens strain
W14 into Arabidopsis to create a transgenic
plant capable of killing first instar M. sexta (Liu,
et al., 2003).
 Expression of tcdA in recombinant E. coli leads
to oral toxicity at high expression levels, but
components B and C (encoded by tcdB and
tccC) are needed to reconstitute full oral
toxicity (Waterfield, et al., 2005).

33. Photorhabdus Insect Related (Pir)
Toxins
 Photorhabdus insect related (Pir) toxins act as
binary proteins.
 PirA shows little similarity to known proteins
 PirB shows high homology with the N-terminal
region of the pore-forming domain of the
Cry2A insecticidal toxin, suggestive of the
existence of a similar motif in these binary
proteins

34. “Makes Caterpillars Floppy” Toxins

35. Photorhabdus
virulence cassettes (PVCs).

36. Photorhabdus Genome
 P. luminescence subspecies laumondii strain
TT01 genome was sequenced (Duchaud et
al., 2003).
 Single circular chromosome – 5,688,987 bp
with an average GC content
- Pa 42.2%
- Pl 42.8%
 2 paralogs plu4092 and plu4436 encode
proteins : JH of Leptinotarsa decemlineata
 For rapid elimination of insect polyphenols e.g.
plu4258 adjacent to glutathione transferase
(plu4259).

37. P. asymbiotica
 Pa was first isolated only from clinical infections,
with 14 cases reported USA and Australia.
 Contact with soil as the primary route of infection.
 most strains of P. luminescens and P. temperata do
not grow at temperatures >32°C, whilst P.
asymbiotica can grow at 37°C.
 Pa comprises a single circular chromosome of
5,064,808 bp.
 Acquired a plasmid related to pMT1 from Yersinia
pestis, the causative agent of the bubonic
plague(Wilkinson, et al., 2009)

38. BLASTCLUST analysis
@ 95% identity only 770 predicted proteins are similar
between the two genomes but this number increases to 2,823
predicted proteins at the 75% identity level.

39. Variation in tca island of Pa
Pa tca-island, all of tcaA and most of tcaB are deleted but a new tccC homologue has
been acquired

40. TTSS variation
 lopT homolog is absent from the equivalent
TTSS island, contain a gene previously termed
lopU (pau01043) that is similar to the ExoU
effector from Pseudomonas aeruginosa (Brugirard-
Ricaud et al., 2004; J Bacteriol, 186(13):4376-4381).
 ExoU has phospholipase activity that disrupts
epithelial and macrophage cell lines (Finck-
Barbancon et al., 1997; Mol Microbiol 25(3):547-557)

41. Behaviour of GFP labelled Pa
ATCC43949 bacteria

42. Conclusion
 Photorhabdus live in a mutualistic relationship
with insect pathogenic nematodes of the genus
Heterorhabditis. This symbiosis is highly
evolved and is highly strain specific.
 Photorhabdus and its interaction with the
immune system.
 The large repertoire of secondary metabolite
gene clusters suggests these bacteria
represent an excellent source of novel drug
candidates.

43.  P. asymbiotica is capable of causing infections
in humans, providing an excellent model of an
emerging human pathogen.
 The ability to extend this fully functional
bacterial lux system to eukaryotic cells opens
many opportunities for continuous bioprocess
monitoring, high-throughput screening
systems, and in vivo sensor and diagnostic
applications.