H-NOX-MEDIATED NITRIC OXIDE

SENSING MODULATES SYMBIOTIC

COLONIZATION BY VIBRIO

FISCHERI

Yanling Wang et. Al.

Presented by Lucas Man

Vibrio fischeri and Euprymna scolopes

• Hawaiian bobtail squid and its bioluminescent

symbiont

• V. fischeri colonizes the light organ

• Bioluminescence provides defense through

counterillumination

Pic source: Discover Magazine; V. fischeri genome project

Vibrio fischeri and Euprymna scolopes

• Colonization of the light organ involves:

• Secretion of mucus into seawater by E. scolopes

• V. fischeri migrating and aggregating in mucus before

traveling into pores and through ducts to reach crypts in

light organ

• Mucus shed by E. scolopes contains high levels

of nitric oxide

Pic source: Cell Microbiol

Bonus paper: “NO means „yes‟ in the squid-vibrio symbiosis:

nitric oxide (NO) during the initial stages of a

beneficial association” – SK. Davidson et. al. Cell Microbiol.

• Found active NO

synthase and NO-filled

vesicles in secreted

mucus

• Also discovered that NO

synthase activity drops

once colonization of the

light organ is complete Pic source: Cell Microbiol

Nitric Oxide (NO)

• In high concentrations, it can serve as a

antimicrobial

• It can also serve as a signaling molecule

• Key biological messenger in vertebrates

• H-NOX: heme NO/oxygen-binding protein

• High sequence identity with soluble guanylate cyclase

(sGC), a eukaryote NO receptor

• Found in V. fischeri

Hypothesis

• NO is a symbiotic signal

• H-NOX can sense host-derived NO

• H-NOX can regulate V. fischeri genes

Pic source: PDB

H-NOX protein

Experiment

• Step 1: Determine if H-NOXvf binds NO with high affinity

• Method: Spectroscopic characterization of H-NOXvf complexes with

NO, CO and O2

Fig. 1

• Results:

• Stable complexes with NO and CO

but not O2

• UV peak positions closely match

those of sGC

Experiment• Step 2: Determine genes that might be regulated by NO in

the presence of H-NOXvf

• Method:

• Compare transcriptional profile of wild-type cells to hnoX-insertion

mutant cells (YLW1 cells)

• Cells were cultured in 4 experimental groups: wild-type, wild in the

presence of NO (released from DEA-NONOate), mutant, mutant in the

presence of NO; 3 replicates in each experimental group

• Extract total RNA from cultures

Wild-type Wild-type

+NO

Mutant Mutant

+NO

Experiment

• Results:

• Bacterial defensive responses to

NO not dependent on H-NOX

• In wild-type cells, NO down-

regulated hemin-use genes, in

mutant cells these genes were

unaffected by NO exposure

• Many of the genes that were

down-regulated contained a motif

similar to the FUR regulon

Fig. 2

Experiment

• Step 3: Test growth rates of wild-type and mutant

cells with hemin as primary iron source • Hypothesis: mutant cells will grow more rapidly than wild-type

cells in a medium in which hemin is the primary iron source

when exposed to NO

• Method:

• Cultured wild-type and mutant cells in medium with only hemin

as iron source

• Treated one-half of each culture with NO twice

• Also cultured mutant and wild-type cells either carrying a

plasmid vector or the plasmid vector containing the H-NOX gene

Experiment

• Results:

• Mutant cells grew faster on

hemin when NO is present

• When H-NOX gene is added

with the plasmid vector to

mutant cells, growth with NO

matches wild-type cells

• Supports hypothesis that H-

NOX mediates hemin-use in

presence of NO

Fig. 3

Experiment

• Step 4: compare colonization competence of wild-

type and mutant cells

• Method: grew juvenile squids in seawater either

containing mutant or wild-type V. fischeri

• Also grew juvenile squids in a mixed culture containing

1:1 ratio of mutant and wild-type cells to compare

competition

• Used onset of luminescence as marker for colonization

Experiment

• Results:

• H-NOX mutant actually more

proficient in initiation of

colonization

• H-NOX mutant had 10-fold

higher colonization efficiency

(density of cells required to

colonize ½ of a juvenile squid

cohort)

Fig. 4

Experiment

• Results:

• Mutant cells outcompeted the wildtype by 16-fold after

24 hours, advantage dropped to 3-fold by 48 hours

• Wild-type cells had an advantage over mutant in the

presence of added ironFig. 4

• NO synthase inhibitor

reduces mutant advantage

• Vector insertion of HNOX

gene removes mutant

advantage

Conclusions

• H-NOX senses NO, represses V. fisheri’s ability to use

hemin, suppresses rapid growth

• Why? What could be the advantage?

• Possibility: high intracellular concentration of iron can generate

toxic levels of hydroxyl radicals

• Host already generates a high level of oxidants, hemin accumulation

could lead to toxic levels

• H-NOXvf-NO could prime V. fischeri for oxidative stress and protect the

symbiont until oxidants have been reduced

• Oxidative stress could cause harmful mutations

• Could explain why competitive advantage of the mutant decreased after

the first 24 hours (Figure 4)

Conclusions

• The take-home message:

• H-NOX in V. fischeri is an NO sensor that influences the

expression of genes associated with hemin acquisition

(and possibly others as well)

• Pathway is still unknown, it is possible that H-NOXvf-NO targets

the FUR regulon

• H-NOX and NO sensing plays a role in symbiotic

colonization by V. fischeri

• Host-symbiont biomolecular cross-talk

Questions?