Download Rhizopus spp - Microbiology

Pathogenic Fungus Harbours Endosymbiotic
Bacteria For Toxin Production
Laila P. Partida-Martinez and Christian Hertweck
Nature 437, 884-888 (6 October 2005)
Michael Taveirne
Microbiology Journal Club
April 10, 2006
Rhizopus spp.
•Cosmopolitan Filamentous Soil Fungi
Kingdom: Fungi
Phylum: Zygomycota
Order: Mucorales
Family: Mucoraceae
Genus: Rhizopus
•Main Contaminant of Decaying Food
-Peach Rot
-Black Bread Mold
Rhizopus Sexual Reproduction
Sporangium
Gametangia
Hyphae
Progametangia
ProGametangia
Mature
Zygospore
Gametangia
Young
Zygospore
Rhizopus spp: The Pathogen
•Normally Infects Plant Species
•Attacks Roots of Rice Plants
-Rice Seedling Blight
-Roots Become Food for Fungus
• Rice makes up 1/5 of the calories
consumed by humans
.
• U.S.A. is third largest exporter of rice
• Only 6% of Rice is traded
internationally
Human Impact
•Pathogen in Immunosuppressed Individuals
-Zygomycosis (Mucormycosis)
•Rhinocerebral Infection Most Common
-Pulmonary, Gastrointestinal, and Cutaneous
•Inhalation of Spores is Primary Mode of Infection
•Treatment: Surgery and Anti-Fungal Medication
Burkholderia spp.
Part of the genus proteobacteria
Commonly found in soil and in groundwater
Gram negative, Motile, Obligate Aerobe
Known for its pathogenic members
mallei, pseudomallei and cepacia
Rhizoxin Toxin
Rhizoxin
•16-membered lactone ring
•Binds to the β-tubulin
•Inhibits formation of microtubule
Microtubule
•Inhibition of Mitosis and Cell Division
•Cell Death
•Possible Anti-Cancer Drug Therapy
Mitosis (metaphase)
Rhizoxin Biosynthesis
•Polyketide synthesized from acetate, methionine and serine
•Polyketides are synthesized in a similar fashion to fatty acids
•Utilizes a Polyketide Synthase Enzyme (PKS)
-Type I (Multi-domain single subunit)
-Type II (Domains on multiple subunits)
•Synthesis starts with Acetyl-CoA or Malonyl-CoA
•Elongated by Ketosynthase Domain (KS)
•Thiesterase (TS) Domain Terminates the Polyketide chain
Does Rhizopus sp.
Have PKS Genes?
• Utilized degenerate primers specific for KS domain
• Unable to find fungal KS domain
• We able to detect Bacterial Type I KS domain
• Sequencing revealed gene belong to trans AT PKS exclusively
. found in bacteria
Symbiont or Gene Transfer?
16S rDNA
KS PCR Products
Phylogenetic Affiliation
• Within Fungal Species only
one type of bacteria is found
•Bacteria belong to the genus
Burkholderia
•Within Different species of
rhizoxin producing fungi
there is closely related
Burkholderia
A True Endosymbiont
ATCC 62417
Symbiont free
ATCC 62417
ATCC 62417
Reinfected
STYO 9 fluorescent stain was used to visualize live cells
Correlating Rhizoxin Biosynthesis
With Burkholderia
• Utilized R. microsporus Strain ATCC 62417
• Treated strain with Ciprofloxacin to kill Burkholderia sp.
-Utilized KS and 16S primers for conformation
•Isolated Burkholderia spp. From ATCC 62417
• Performed HPLC to detect the presence of rhizoxin
a: Rhizoxin Reference
b: ATCC 62417
c: ATCC 62417 treated with Ciprofloxacin
d: Burkholderia spp. From ATCC 62417
Koch’s Postulate
Proving a Symbiosis
1.
The organism must be found in all animals suffering from the
disease, but not in healthy animals.
2.
The organism must be isolated from a diseased animal and
grown in pure culture.
3.
The cultured organism should cause disease when introduced
into a healthy animal.
4.
The organism must be reisolated from the experimentally
infected animal.
They were able to isolate the Burkholderia sp. And it was shown that
it could produce rhizoxin but for a limited time
Conclusions/Questions
• The fungus utilizes the rhizoxin produced by the
bacterium to feed upon the rice roots
• First example of bacterial-fungal symbiosis with a
metabolic function
• Unique because most pathogen fungi produce their own
pathogenicity factors
• The fungus benefits from the symbiosis but what benefit
does the bacteria obtain?
• How does the fungus acquire the symbiont?
Questions / Comments?