Microbe-derived uremic solutes enhance thrombosis potential in the host
Authors: Nemet Ina , Funabashi Masanori , Li Xinmin S. , Dwidar Mohammed , Sangwan Naseer , Skye Sarah M. , Romano Kymberleigh A. , Cajka Tomas , Needham Brittany D. , Mazmanian Sarkis K. , Hajjar Adeline M. , Rey Federico E. , Fiehn Oliver , Tang W. H. Wilson , Fischbach Michael A. , Hazen Stanley L. 
Published: November 2023

p
-Cresol sulfate (
p
CS) and indoxyl sulfate (IS), gut microbiome-derived metabolites, are traditionally associated with cardiovascular disease (CVD) risks in the setting of impaired kidney function. While pharmacologic provision of
p
CS or IS can promote pro-thrombotic phenotypes, neither the microbial enzymes involved nor direct gut microbial production have been linked to CVD. Untargeted metabolomics was performed on a discovery cohort (
n
= 1,149) with relatively preserved kidney function, followed by stable isotope-dilution mass spectrometry quantification of
p
CS and IS in an independent validation cohort (
n
= 3,954). Genetic engineering of human commensals to produce
p
-cresol and indole gain-of-function and loss-of-function mutants, followed by colonization of germ-free mice, and studies on host thrombosis were performed. Systemic
p
CS and IS levels were independently associated with all-cause mortality. Both
in vitro
and within colonized germ-free mice
p
-cresol productions were recapitulated by collaboration of two organisms: a
Bacteroides
strain that converts tyrosine to 4-hydroxyphenylacetate, and a
Clostridium
strain that decarboxylates 4-hydroxyphenylacetate to
p-
cresol. We then engineered a single organism,
Bacteroides thetaiotaomicron
, to produce
p-
cresol, indole, or both metabolites. Colonizing germ-free mice with engineered strains
,
we show the gut microbial genes for
p-
cresol (
hpdBCA
) and indole (
tryptophanase
) are sufficient to confer a pro-thrombotic phenotype
in vivo
. Moreover, human fecal metagenomics analyses show that abundances of
hpdBCA
and
tryptophanase
are associated with CVD. These studies show that
p
CS and IS, two abundant microbiome-derived metabolites, play a broader potential role in CVD than was previously known. They also suggest that therapeutic targeting of gut microbial
p-
cresol- and indole-producing pathways represent rational targets for CVD.

IMPORTANCE

Alterations in gut microbial composition and function have been linked to numerous diseases. Identifying microbial pathways responsible for producing molecules that adversely impact the host is an important first step in the development of therapeutic interventions. Here, we first use large-scale clinical observations to link blood levels of defined microbial products to cardiovascular disease risks. Notably, the previously identified uremic toxins
p
-cresol sulfate and indoxyl sulfate were shown to predict 5-year mortality risks. After identifying the microbes and microbial enzymes involved in the generation of these uremic toxins, we used bioengineering technologies coupled with colonization of germ-free mice to show that the gut microbial genes that generate
p
-cresol and indole are sufficient to confer
p
-cresol sulfate and indoxyl sulfate formation, and a pro-thrombotic phenotype
in vivo
. The findings and tools developed serve as a critical step in both the study and targeting of these gut microbial pathways
in vivo
.