Photorhabdus Host of Hosts

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

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

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

 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

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

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.

 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

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

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.

 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

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

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).

 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

 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

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)

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.

Photorhabdus toxins with the
insect immune system
Tcd pathogenicity island

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).

PO cascade inhibition by stilbene

Nodule formation
Dose response curve

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

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

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

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).

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

“Makes Caterpillars Floppy” Toxins

Photorhabdus
virulence cassettes (PVCs).

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).

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)

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.

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

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)

Behaviour of GFP labelled Pa
ATCC43949 bacteria

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.

 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.

Photorhabdus Host of Hosts

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