Seminars in Immunology 66 (2023) 101735

Contents lists available at ScienceDirect

Seminars in Immunology
journal homepage: www.elsevier.com/locate/ysmim

Seminars in immunology special issue: Nutrition, microbiota and immunity

The unexplored microbes in health and disease
Tamar Plitt a, Jeremiah J Faith a, b, *
a
Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA
b
Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA

A R T I C L E I N F O A B S T R A C T

Keywords: Functional characterization of the microbiome’s influence on host physiology has been dominated by a few
Gut microbiota characteristic example strains that have been studied in detail. However, the extensive development of methods
Cancer for high-throughput bacterial isolation and culture over the past decade is enabling functional characterization of
In vitro
the broader microbiota that may impact human health. Characterizing the understudied majority of human
High-throughput screen
Gnotobiotics
microbes and expanding our functional understanding of the diversity of the gut microbiota could enable new
Immunology insights into diseases with unknown etiology, provide disease-predictive microbiome signatures, and advance
Culturing microbial therapeutics. We summarize high-throughput culture-dependent platforms for characterizing bacterial
Effector strains strain function and host-interactions. We elaborate on the importance of these technologies in facilitating
mechanistic studies of previously unexplored microbes, highlight new opportunities for large-scale in vitro
screens of host-relevant microbial functions, and discuss the potential translational applications for microbiome
science.

1. Introduction
Previous decades of research in bacteriology focused on a few model
organisms and pathogens with a goal of understanding the functions that
govern bacterial survival and the factors driving pathogenesis and
treatment. Despite this wealth of scientific exploration that included
complete genome sequences [1,2], gene knockout libraries [3], and
extensive transcriptional profiling [4], even the best characterized
model bacterial strains lack functional annotation of a large proportion
of their genes [5,6].
In the past 20 years, the microbiome field has transitioned much of
the bacteriology community’s focus from a few model organisms to­
wards the monumentally large challenge of characterizing all the or­
ganisms in multiple ecosystems. This change was initially spurred by
advances in genome sequencing that enabled a cost-effective means of
characterizing the members of bacterial communities using culture-
independent approaches [7,8]. Although this cataloging-of-parts phase
continues relatively unabated, with technology advances bringing
deeper sequencing to identify rarer community members with greater
resolution, this phase increased the field’s awareness of the significant
proportion of microbes that were completely unstudied. Such
discoveries led to the collecting-of-parts period that we are still in the
early to middle phase of whereby laboratories with advanced methods
for high-throughput culturing, found approaches to enrich rarer or
hard-to-culture microbes, and develop model communities of defined
bacterial strains as consortia for in vivo models or in vitro screens
[9–14]. With numerous laboratories now using reference collections of
parts [15–24], the challenge becomes how to identify the functions of so
many bacteria when each strain can potentially have a different function
from other strains in a species and the number of species alone in the
human population for gut bacteria is likely in the thousands [25].
Identification of highly penetrant microbial functions in the gut
including short-chain fatty acid (SCFA) production [26], regulatory T
cell (Treg) induction [27], Th17 cell induction [28], and bile acid
metabolism [29–33] is already producing functionally important dis­
coveries with translational implications, while fecal microbiota trans­
plantation (FMT) and defined live biotherapeutic products (LBPs)
provide the potential to identify causal influences of bacterial strains in
humans, quantify the transmissibility of strains, and translate new
functional discoveries to the clinic. There remains, however, a vast un­
explored majority of bacterial strains whose individual and collective
function is largely unknown despite years to centuries of co-habitation

* Corresponding author at: Precision Immunology Institute, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA.
E-mail address: jeremiah.faith@mssm.edu (J.J. Faith).

https://doi.org/10.1016/j.smim.2023.101735
Received 4 October 2022; Received in revised form 17 January 2023; Accepted 9 February 2023
Available online 27 February 2023
1044-5323/© 2023 Elsevier Ltd. All rights reserved.
T. Plitt and J.J. Faith
on our bodies. The cataloging-of-parts and the collecting-of-parts periods
of the microbiome field have now set the stage for the new era of
characterization-of-parts whereby we seek high-throughput scalable
platforms to identify the functions of the understudied majority of
strains in the microbiome. This phase will require studying enough
strains across the phylogenetic tree of life to identify general principles
of function across taxa and to identify exceptions that might fuel new
biological and therapeutic insights. In this review, we summarize
high-throughput approaches developed to aid in more fully exploring
the functional diversity of the human microbiome, with an emphasis on
the human gut microbiome. We focus on the implications of these
functional screens for future human translation, particularly on immune
modulation and cancer.
2. Generating the microbiome strain catalog
2.1. cataloging of parts
Culture-independent sequencing transformed our ability to charac­
terize microbial consortia of the human microbiome across body sites
[34]. From the early phase of genus-focused 16S rRNA amplicon
sequencing that identified broad differences between the microbiomes
of people of different health status and treatment response [35–37], we
have moved to the metagenomics phase of characterizing species-level
differences between communities [36,38–42]. Most recently, meta­
genomics, often combined with strain genome sequencing or meta­
genomic assembly, is leading to numerous explorations of what defines a
strain, how do we track strains [43–47], how many strains exist in a
given species [48] or at a given anatomical location [49], and how
strains evolve in their native ecosystems [50–52]. Typically, species are
defined as isolates that share more than 99% of their 16S rRNA sequence
[53,54] while strains are defined as isolates of a given species that share
more than 96% of their genome content. [43,48,55,56].
The cataloging of microbiome parts is essential for providing a
structural framework to build on for functional studies. For example,
studying the functional potential of a synthetic human gut microbial
community with mostly Firmicutes and Bacteroides and a few Actino­
bacteria and Proteobacteria is more realistic to the human gut micro­
biome ecosystem than a community of only Fusobacterium and
Proteobacteria. These microbiome-part-catalogs also provide the vast
majority of functionally insightful microbiome observations directly in
humans such as which bacteria engraft in an FMT for subjects with
recurrent Clostridium difficile infection [1], ulcerative colitis [57], or
melanoma [58,59] that respond to FMT therapy. Similarly, enrichments
of taxa in specific conditions such as Crohn’s disease [42,60], colorectal
cancer [61,62], or wound healing [63] can focus the taxa selection
process for functional studies and future therapeutics – particularly now
when more opportunities are emerging for datasets to be combined to
search for consensus results across multiple human studies [41,42,60].
While the cataloging-of-parts can uncover correlations between the
relative abundance of specific species/strains of bacteria and the
onset/progression of disease, it is not sufficient to prove causality.
2.2. collecting of parts
Functionally relevant associations from statistical analyses of large
observational datasets often focus subsequent functional explorations
[64,65]. However, reductionist systems are typically required for broad
functional understanding of biological systems and components. The
rise of culture-independent cataloging of the microbiome in health and
disease was closely followed by attempts to culture gut microbial strains
in high-throughput [11], using diverse media and detection technolo­
gies [13], and with efforts to increase the culturable fraction of the gut
[9,10], skin [66], and oral microbiomes [67,68]. Lower cost genome
sequencing enabled high-throughput culturing efforts to be coupled
with genome sequencing to facilitate strain tracking and comparative
2
Seminars in Immunology 66 (2023) 101735
genomics approaches [23,43,69]. In parallel, larger collections of
defined, sequenced bacterial communities collected into larger consortia
and screening libraries also provide broad tools for functional explora­
tion [15–24]. These collections, inspired by culture-independent dis­
coveries, are now some of the key resources necessary to move from
characterizing the functional impact of individual effector strains to
understanding the individual and collective role of hundreds to thou­
sands of strains. We find particularly compelling the isolation and
exploration of functional potential of bacterial strains isolated from the
stool of frequently used fecal transplant donors, as these microbes come
from humans that have passed important FMT safety screens. The safety
data in the resulting recipients provide at least a crude representation of
the collective influence of the strains on recipient safety, and the
tracking of strains from donors to recipients provides empirical esti­
mates of the transmissibility of each strain [43].
As the microbiome field transitions from compositional descriptions
and phenotypic associations towards studies aimed at mechanistic,
functional, and interventional understanding, there arises an issue of
scale. Every individual is colonized with a unique set of bacterial strains
which collectively represent enormous functional variation. Purely in
vivo based approaches can never approach the scale and the scope
required to address this complexity and so, in vitro assays implemented
in high throughput using automation are the only realistic alternative.
To tackle questions related to functional responses across microbiota
derived strains, we can either ignore the complexity and focus on simple
systems (e.g., by using model strains such as segmented filamentous
bacteria (SFB)) or we must use a variety of tools at different scales to try
to understand the entire system. The future of human microbial thera­
peutics will therefore benefit from the parallel advancement of in vitro
screening platforms, in vivo validation and discovery, and an expansion
of the transition from FMT interventions [70] to defined bacterial strain
consortia interventions [71–74].
2.3. characterization of parts: unknown functions of the understudied
majority
Early explorations into the functional potential of human gut
microbiota strains, by necessity, focused on a limited number of strains.
When there is little to compare to, any significant phenotypic pertur­
bation causally linked to a specific bacterial strain is of interest [75] and
many observations ask the simplest question – what is difference be­
tween a germ-free or antibiotic treated mouse and a mouse colonized
with a complex diverse microbiota? Since the goal for a clinical inter­
vention will never be to transition someone between a germ-free and
colonized state or to provide an antibiotic cocktail to deplete the
microbiome for life, practical implementation of microbiome functional
discoveries requires identifying individual strains or groups of strains
that perturb host physiology. The earliest examples of such experiments
identified a few sample strains or consortia that could perturb a host
phenotype, for instance, through the induction of Th17 cells [28], Tregs
[76], or colitis [77] (Fig. 1A). In microbiomes beyond the gut, similar
experiments identified skin microbiome strains that induce IL-17A+
CD8+ T cells [78] and oral microbes that induce Th1 cells [79]. The
challenge of these initial forays is that broad conclusions can only be
based on highly significant narrow explorations, and there are far too
few examples to determine how unique the results are relative to all
possible experiments with all possible strains.
As modest-sized functional explorations with synthetic defined mi­
crobial communities became possible by the advances in gnotobiotic
caging systems and anaerobic culturing, we were able to better under­
stand the context for earlier findings and their potential use in humans
(Fig. 1B). For example, among gut lamina propria Th17 cells, it appears
that potent Th17 inducing strains [28] are rare strains that are not
harbored by many people or animals [15,80]. In contrast for Tregs, the
microbiome is clearly fundamental to their induction in the lamina
propria [27], and there are a multitude of individual strains and groups
T. Plitt and J.J. Faith Seminars in Immunology 66 (2023) 101735

Fig. 1. Scalable functional characterization of gut microbiome strains. In this illustrative example, the therapeutic aim is to identify bacterial strains that induce high
proliferation in mammalian cells and whose removal or suppression could reduce the risk of colorectal cancer. (A) Early functional explorations of the gut microbiota
focused on a few sample bacterial strains that influenced host physiology. The green dot corresponds to a unique bacteria strain that is present in a healthy donor and
induced low colonic carcinoma cell proliferation. The orange dot corresponds to another unique bacterial strain that is present in a CRC donor and is found to induce
high colonic carcinoma cell proliferation. Both of these bacterial strains belong to the Proteobacteria phylum. Here we might conclude that Proteobacteria in people
with CRC are uniquely capable of inducing colonic carcinoma cell proliferation. (B) Synthetic defined microbial communities enabled exploration of colonic car­
cinoma cell proliferation potential of different bacterial species across multiple taxa per phylum and disease state. Within each disease state, there exist strains that
have a wide range of colonic carcinoma cell proliferative properties. Here we might conclude that multiple phyla contain strains that can induce high colonic
carcinoma cell proliferation, but they are uniquely common in CRC patients. (C) Building on recent expansions of high-throughput culturing, functional screens are
now being performed on the population scale with hundreds of bacterial strains, often with multiple strains per species. On the population scale, we have sufficient
strains in each of our categories of interest (taxonomy and disease status) to see the underlying distributions driving our earlier observations. In this population scale
example, Actinobacteria universally induce high colonic carcinoma cell proliferation and could serve as targets for therapeutic removal or suppression to ultimately
reduced CRC risk, while only a fraction of the Proteobacteria induce high colonic carcinoma cell proliferation. While earlier sample- and consortium-scale exper­
iments showed enrichment of high proliferation-inducing strains in CRC, this effect is explained by CRC enrichment with Actinobacteria and Bacteroides. (D) A
challenge and opportunity for the field moving forward is to develop relevant in vitro and in vivo systems that are the most relevant for advancing microbiome
science towards clinical interventions using the functionally characterized strains. Abbreviation: IBD, inflammatory bowel disease; CRC, colorectal cancer; T1D, type
1 diabetes; FMT, fecal microbiota transplantation.

3
T. Plitt and J.J. Faith
of strains able to induce different Treg subsets [76,81,82] through a
variety of mechanisms [26,30].
Empirical observations of strains that repeatedly engraft in fecal
transplants, fractionated fecal transplants, and defined LBPs in subjects
whose disease status is improved by a microbial therapeutic are likely
the shortest route to developing minimal bacterial consortia to improve
human health. In parallel, there will always be a desire to understand the
mechanisms and general principles driving such successes so that we can
determine the cause of treatment failures, improve future microbial
therapeutics, and envision future applications that are not visible until
we build the basic foundations necessary to see a little further. To begin
assembling this foundation as a scientific community will require
identifying new in vitro, ex vivo, and in silico methods to bring addi­
tional scale beyond the animal experiments that have been foundational
to date and the human observations that are our motivation for explo­
ration [70,83]. The microbiome field’s extensive cataloging-of-parts and
growing collection-of-parts are the raw resources that were necessary to
open this next era of characterization-of-parts that will facilitate our
understanding of the functional attributes of each strain in a broader
context of populations of hundreds of strains (Fig. 1C).
Here we provide examples from this newly emerging literature in the
microbiome field of scientists exploring dozens to hundreds of strains
with in vitro, ex vivo, and in silico approaches. For example, larger
explorations, facilitated by ex vivo screens that are less limited by the
scale of mouse experimentation, suggest that high proportions of Th17
cells in gnotobiotic mice can be obtained by single potent effector strains
with highly penetrant phenotype like SFB [28] and similarly high Th17
cells can be obtained by altering microbiome composition so that it is
composed of only moderate Th17-inducing strains and no low
Th17-inducing strains [84]. We anticipate this emerging trend will
continue to expand as new assays are imagined and more laboratories
have access to large cultured communities of strains.
Ultimately, it will be important to weigh the utility of in vitro, ex
vivo, in vivo, human observational, and human interventional ap­
proaches in an unbiased manner to identify the best usage of resources
and time to maximize the human relevance of the microbiome field. A
continuous learning and cycling between all of these approaches
(Fig. 1D) is likely necessary for us to reach the point of identifying
minimal therapeutic sets of strains, strain metabolites, or strain proteins
that cause, prevent, or treat human disease.
2.4. Metabolic potential
Screens to understand the metabolic potential of gut microbiota
strains and communities represent an area ripe for discovery with major
advantages over other types of host/microbe screens. First, the meta­
bolic potential of a bacterial strain in vitro and in vivo is likely similar
when given the same substrates [22]. Second, there are numerous
human relevant microbiota influenced metabolites to explore from
SCFAs [85] to drug metabolism [86,87]. In addition, these approaches
can be performed with either native microbes or in a cloned expression
library to facilitate identifying causal genes, metabolites, and enzymes
[88,89]. However, challenges and opportunities to the broader appli­
cation of microbial strain metabolic screens to therapeutics include the
difficulty of identifying metabolites, and the challenge of comparing
results across metabolomics measurement platforms. Additionally, the
substantial cross-feeding and metabolic redundancy that occurs in
multi-species communities can limit the predictability of single strain in
vitro screens for metabolic processes and compounds for more common
chemicals in the gut like bile salts and SCFAs.
The gut microbiota plays a considerable role in host metabolism,
producing active catecholamines [90], regulating serotonin biosynthesis
[91], and generating butyrate [92]. In fact, evidence suggests that a
significant proportion of microbial metabolites can translocate from gut
lumen to the systemic circulation influencing distant organs and the
central nervous system (CNS) [93]. For instance, studies focused on
4
Seminars in Immunology 66 (2023) 101735
characterizing the role of the gut microbiota on the gut-brain axis
signaling found that metabolites produced by certain strains of Escher­
ichia coli activated c-Fos in the arcuate nucleus (ARC) and stimulated the
firing rate of proopiomelanocortin neurons in the ARC, which play a role
in appetite control and energy expenditure [94,95]. A more recent study
utilized high-coverage metabolomics to describe microbiota-derived
metabolites implicated in the interorgan transport of gut microbial
metabolites and gut-brain axis [96].
In this review, we will emphasize the role of microbial metabolites in
cancer. Microbial metabolites can have a protective role against carci­
nogenesis by contributing to inflammatory tone or influencing the bal­
ance between proliferation and apoptosis in tissues [97]. Several
microbial metabolites generated from dietary fiber and fats have an
established effect on cancer [97,98]. Understanding the effects of these
metabolites on cancer growth and spread offers the potential for ther­
apeutic interventions – either by direct transfer of strains or adminis­
tration of bacterial products.
The intestinal fermentation of dietary fiber releases SCFAs, princi­
pally acetic, propionic, and butyric acids, which help maintain the
integrity of intestinal epithelium [99] and are otherwise unavailable
from diet. These SCFAs have been studied in the context of intratumoral
inflammation and demonstrated to have anti-inflammatory effects on
myeloid cells [100] and colonic Tregs [101–103]. Butyrate, in partic­
ular, is the principal energy source for colonic epithelial cells and has
been shown to have tumor suppressive functions [103]. Butyrate can
inhibit histone deacetylase activity in colonocytes and immune cells,
downregulating proinflammatory cytokines and inducing apoptosis in
colorectal cancer (CRC) cells [103–106]. The effects of SCFAs on colonic
epithelial cells can also be calibrated by the receptors they bind, such as
G protein-coupled receptors (GPCRs) [107–109]. Gpr109, for instance,
is expressed on colonic epithelial cells and intestinal myeloid cells and is
important for Treg generation [109]. Loss of this receptor increases
susceptibility to colitis associated colorectal cancer (caCRC) [109].
Propionate activation of Gpr43, expressed in hematopoietic tissues and
in the colon, reduces murine pro-B BaF3 cell proliferation in a
dose-dependent manner [110]. Compared to normal colon tissues,
Gpr43 expression is significantly reduced or lost in colorectal adeno­
carcinoma tissues and their corresponding lymph node metastatic ade­
nocarcinomas [106]. Restored Gpr43 expression in human colonic
adenocarcinoma cells led to increased apoptotic cell death upon SCFA
treatment [106].
However, conflicting effects of SCFA in cancer prevention have also
been documented. Two mouse studies reported opposing effects of
butyrate on tumorigenesis. For instance, butyrate had tumor-promoting
effects in a mouse model of intestinal tumorigenesis driven by mutations
in the DNA mismatch repair gene, Msh2, and the tumor suppressor gene,
Apc [111]. On the other hand, butyrate ultimately suppressed tumori­
genesis in the AOM/DSS model of colitis-associated cancer by reducing
cellular proliferation and increasing apoptosis [92]. The latter effects
were explained by the fact that neoplastic colonocytes favor glycolysis
for cellular energy, leading to a decreased butyrate metabolism,
increased nuclear accumulation, and subsequent inhibition of histone
deacetylases. These studies highlight the wide range of effects that a
single microbial metabolite can elicit in tumor models, hindering the
translation of microbiome and diet data into practical clinical in­
terventions in the context of cancer. Thus, expanding research on the
uncharacterized taxa and metabolites will allow us to identify metabo­
lites with more specific or complementary effects.
Beyond SCFAs, high-fat diet (HFD) and obesity are also thought to be
involved in the pathogenesis of specific cancers by altering the gut
microbiota and inducing inflammation [83,98–103,112,114–117]. In
fact, obesity is now regarded as an inflammatory state [97,118] and
inflammation and HFD are considered primary risk factors for hepato­
cellular carcinoma (HCC) progression [119]. Studies in obese mice
treated with an antibiotic cocktail that reduces the number of
commensal intestinal bacteria showed a reduced risk of developing HCC
T. Plitt and J.J. Faith
by depleting the microbiome and the subsequent secretion of
pro-carcinogenic and proinflammatory metabolites and toxins [120].
Another mechanism by which HFD influences cancer risk is through
the secretion of bile acids [97,121]. Primary bile acids are synthesized
from cholesterol in the liver and secreted from the gall bladder into
duodenum to solubilize and digest fats. Bacteria in the distant small
intestine and colon can metabolize primary bile acids into various sec­
ondary bile acids, which are then absorbed in the colon or returned to
the liver. Secondary bile acids have been shown to exert immunoregu­
latory functions and have been implicated in the development and
progression of liver cancers [121,122]. For instance, in 2018, Ma et al.
showed that production of secondary bile acids by Clostridium species
contributed to tumor progression in various mouse models of primary or
metastatic liver cancer [122]. The pro-tumorigenic effect was due to the
secondary bile acid-driven downregulation of CXCL16 in liver sinusoidal
endothelial cells, resulting in a reduced recruitment of natural killer T
(NKT) cells in the liver [122]. In another study, enrichment of certain
Clostridium species in response to a HFD led to increased levels of the
secondary bile acid, deoxycholic acid (DCA), a gut bacterial metabolite
known to cause DNA damage. Accumulation of DCA in the liver led to an
accelerated progression of carcinogen-induced liver cancer [113]. In
addition to diet, an earlier study by Yoshimoto found that genetic sus­
ceptibility to obesity can also increase DCA-mediated activation of
mitogenic and proinflammatory response program in hepatic stellate
cells (HSCs), potentiating liver cancer in mice [120].
Given that the gut microbiota generate, modify, or influence these
host-relevant metabolites, numerous opportunities exist to broadly
characterize the metabolic capacities of bacterial strains in a high-
throughput manner. Recently, Han et.al., demonstrated a metab­
olomics pipeline to identify and characterize microbiota-dependent
metabolites (MDM) [123]. In a scalable in vitro platform, the team
grew 178 individual human gut microorganisms, spanning 130 species
and 6 phyla, in rich complex culture media and used mass-spectrometry
to generate a reference dataset of metabolomic profiles for each strain.
This screen demonstrated that the phylogenetic relationship between
strains can predict some of the metabolic potential of each strain, with a
number of exceptions where significant differences exist between
metabolomic profiles of highly phylogenetically related strains. Scaling
this type of platform across multiple growth media and larger numbers
of strains will continue to advance our knowledge of the shared and
unique metabolite potential of strains across taxonomy and human
disease. And ultimately, it may allow us to use comparative genomics to
associate genomic features with metabolic outputs [123]. In related
work, Wong et al. used a strain-dropout strategy to show that there is
functional redundancy within the bile acid 7a-dehydroxylation niche as
long as Clostridium scindens or Clostridium hylemonae is present in the
community [124]. However, dropping out either strain led to variations
in the bile acid metabolism outside the niche. These studies redefine
how we think about strain-level variation in the microbiome.
Gut microorganisms are functionally differentiated by their ability to
metabolize specific structurally distinct carbon sources or glycans.
Synthetic glycans (SGs) from diverse, naturally occurring mono­
saccharides, or natural glycans that selectively drive the gut micro­
biota’s generation of beneficial metabolites or the relative abundance of
specific strains provide one potential therapeutic strategy [125]. Using a
bead-based approach combined with 16S rRNA sequencing, Patnode et.
al., reported strain-specific and glycan-specific binding phenotypes
among 160 Bacteroides and Parabacteroides strains [126]. In a more
recent screen of carbohydrate utilization of 354 members across 29
species of the Bacteroidetes phylum [127], Pudlo et al. found strongly
enriched glycan utilization abilities within species demonstrating a
vertical evolution functional imprint on gut microbial strains. However,
similar to studies by Han et al., a clear mosaicism existed at the level of
individual strains suggesting that horizontal gene transfer or perhaps
convergent evolution provide important contributions to the metabolic
capacity of individual strains.
5
Seminars in Immunology 66 (2023) 101735
Combining in vitro metabolic screens with in vivo validation studies
already has multiple proof-of-principle demonstrations and appears to
be an area that is primed for growth in the next decade. For near term
human relevance, a key will be identifying rare metabolites from gut
microbial strains that have important beneficial health properties and
are absent in people with a specific disease. Moving to applications
beyond singularly potent metabolite generation or drug metabolism will
require understanding the complex problem of what determines the
average metabolic output of mixtures of strains with highly overlapping
metabolic potential (e.g., short chain fatty acids or secondary bile salt)
when given diverse inputs from human diet and host metabolites.
2.5. Immunogenic potential
2.5.1. cell mediated immunity
Given the interconnectivity of the cells in the human immune sys­
tem, developing scalable models of immune function is a challenge.
With numerous important roles identified for the microbiota in the
shaping of our immune system, it is exciting that large-scale applications
are beginning to emerge that take advantage of in vitro, ex vivo, and in
silico platforms as well as tools from genomics and DNA synthesis.
Mounting evidence indicates that the microbiome may influence a
myriad of immune mediated diseases including inflammatory bowel
disease (IBD), rheumatoid arthritis, type 2 diabetes, allergic diseases,
and cancers [128]. These data are further supported by studies
demonstrating that the gut microbiota directly modulates an antigen
specific host immune response [129–131]. For example, Perez-Munoz
et al. colonized germ-free mice with a strain of Bacteroides thetaiotao­
micron and were able to isolate an antigen specific T cell towards the
strain as well as identify the protein epitope that was shared across
species which was largely predictive of the cross reactivity of the T cell
[132]. Yang et al., demonstrated that intestinal Th17 cells in
SFB-colonized mice express T-cell antigen receptor (TCR) specific for
SFB [133]. Similarly, tolerance to the pathobiont, Helicobacter hepaticus
(Hh), was shown to be mediated by Hh-specific Tregs [134]. Treg in­
duction by a range of individual or groups of microbes have been re­
ported by multiple groups [27,81,135]. Others have shown the
specificity of IL17A+ CD8 + T cell response to the skin commensal
Staphylococcus epidermidis lasts for months and is not associated with
inflammation but rather induced to calibrate barrier immunity and
provide heterologous protection against invasive pathogens [78].
The specificity of the mucosal immune response to the gut com­
mensals has profound implications for our understanding of tissue-
specific immunity and pathologies. In accordance with this idea, Spin­
dler et al. recently profiled antigen-specific responses directed towards
individual strains in the setting of a multi-strain defined community and
found lamina propria and mesenteric lymph node T cells specific for
bacterial strains at the species level, with the ability of Th17 cells to
discriminate between some strains of the same species [84]. This spec­
ificity of T cell responses enabled ex vivo recall assays to demonstrate
the in vivo Th17 contribution of individuals strains in a multi-strain
community. In addition, knowing the Th17 inducing potential of these
strains allowed for the generation of custom defined strain subsets that
collectively induced predictable numbers of Th17 cells in gnotobiotic
mice.
In an important recent application of a scalable in vitro pipeline
designed to understand interactions between T cells and gut microbes,
Nagashima et al. colonized germ-free mice with 97 bacterial strains to
educate their immune system with the multi-strain consortia [69].
Two-weeks later, T cells from the large intestine were co-cultured ex
vivo with each of the 97 strains to identify the specificity of the T cells
towards each strain. Although the global T cell pool was clearly being
driven by antigen-specific T cells towards certain strains, it was also
found that many T cells recognized multiple strains of bacteria. Using a
beautiful combination of TCR-sequencing to identify relevant T cells,
DNA synthesis, a T cell reporter line to generate 92 T cell clonotypes,
T. Plitt and J.J. Faith
and comparative genomics across the large strain library, the authors
identified abundant TCR clonotypes that were polyspecific for 18 Fir­
micutes in the strain collection. In addition, the authors discovered that
these 18 strains shared a conserved substrate binding protein from an
ABC transport system that turned out to be a T cell epitope. In many
ways, these observations parallel prior work with small numbers of
strains that identified a specific T cell epitope to B. thetaiotaomicron and
the shared reactivity of an antigen specific T cell towards other members
of the Bacteroidetes [132]. However, by taking advantage of more
scalable platforms and larger communities of bacterial strains, we have
the opportunity to pursue more interactions and to have a better un­
derstanding of how such observations might extrapolate to humans
colonized with large numbers of microbes in an ecosystem that may gain
or lose strains over time.
High-throughput screening platforms have also been developed for
identifying interactions between microbes and innate immune cells. For
example, a recent screen of a synthetic plant root bacterial community
identified strains that suppress MAMP-triggered immunity induced by
flg2 (a 22-amino acid epitope in bacterial flagellin). This immune sup­
pression influenced the ability of different bacteria to colonize the root
and could be manipulated with subsets of the 35 strains identified to
have suppressive effects in a monocolonization screen [136]. Another
recent study explored the interactions between gut microbial strains and
toll like receptors (TLRs). TLRs are a major sub-family of pattern
recognition receptors (PRRs) that are widely expressed by a variety of
cells. In this study, 277 commensal bacterial strains from healthy in­
dividuals and individuals with IBD were co-cultured with myeloid cells
or dendritic cells to quantify the cytokine response to each strain [23].
The cytokine responses to these 277 gut bacterial strains were found to
be as strong as responses toward enteric pathogens and varied across
phyla, down to the strain level [23]. Similar to the population scale
metabolomics screen [123] and the carbohydrate utilization profiles
from a large screening library [127], a great deal of phylogenetically
enriched patterns could be identified in the innate immune responses
towards gut bacterial strains, while numerous exceptions to these phy­
logenic patterns highlight how strain diversity and horizontal gene
transfer allows for a great diversity of responses. Moreover, Spindler
et al. found that myeloid cells differentially rely on toll-like receptor 2
(TLR2) or TLR4 activation to sense particular taxa, an observation that
predicted in vivo function. Finally, in vitro co-culture assays have also
been successfully employed to explore the functional potential of strains
from the cervicovaginal microbiome on the cervicovaginal epithelial
barrier [137] and represent one of the more generalizable and scalable
systems for exploring other microbiomes.
2.6. Humoral immunity
IgA responses have been demonstrated to be influenced by the gut
microbiome [138–141]. Yang et al. evaluated fecal IgA responses to
commensal bacteria and found Bacteroides ovatus to be the most prom­
inent IgA inducer in the colon of gnotobiotic mice [139]. Further
characterization of 19 unique B. ovatus strains revealed strain-specific
IgA responses. Transplanting low-IgA mice with a multiplex cocktail
of high-IgA inducing strains, but not individual ones, led to an increase
of fecal IgA. Follow up studies found the bulk secretory IgA antibody
repertoire tailored to recognize the “self” gut bacteria with a minor
fraction of IgAs displaying cross-reactivity [24]. At the same time,
Rollenske et al. identified a range of antigen-specific monoclonal IgAs
with a diverse immunoglobulin gene repertoire targeting defined bac­
terial antigens [138]. The distinct functional effects of these IgAs were
highly dependent on the antigen recognized.
In the context of disease, multiple groups found taxa specific IgA
responses in relation to perturbations in the gut microbiota. For
instance, Palm et al. examined IgA coating patterns in mice with a
transmissible colitogenic microbiota and found high IgA coating spe­
cifically marked known disease-driving members of the intestinal
6
Seminars in Immunology 66 (2023) 101735
microbiota [142]. Colonization of GF mice with bacteria from human
IBD patients that were highly coated with IgA conferred dramatic sus­
ceptibility to colitis. In addition, Alexander et al. characterized antibody
reactivity in IBD patients using microbiota antigen arrays directed at
recombinant bacterial flagellins [143]. Compared to healthy controls, a
subset of patients with Crohn’s disease displayed elevated serum IgG
response to multiple flagellins. The multi-flagellin reactive patients also
exhibited elevated flagellin-specific T effector memory (TEM) cells, a
reduced ratio of flagellin-reactive Treg to effector T cells, and a high
frequency of disease complications. Understanding the specificity of
antibodies towards gut microbial strains is an area that is well suited for
high-throughput functional screening experiments. The availability of
strain- and species-specific hybridomas along with the numerous
antibody-based in vitro assays currently in the literature facilitate
high-throughput screens.
In addition to IgA, intestinal IgG and IgM antibodies have also been
reported to react to the gut commensal antigens [144]. While these
antibodies are not as well-characterized, they share similar specificities
to IgA. Homeostatic commensal IgG antibodies, specifically IgG2b and
IgG3, commonly bind gram-negative antigens (i.e., Proteobacteria).
IgG2b and IgG3 are transmitted to neonates in breast milk and maternal
transmission of these IgG antibodies coordinates with IgA to limit
mucosal T cell responses, and modulates neonatal intestinal ILC3 and
macrophage differentiation [146]. Zeng et al., identified murein lipo­
protein (MLP) as a major antigen that induced systemic IgG in both mice
and humans [145]. Administration of anti-MLP conferred protection
against Salmonella infection.
3. Oncogenic potential
Perturbations in the composition and metabolic output of the gut
microbiota are associated with diseases ranging from autoimmune and
metabolic diseases to cancer. The mechanisms by which microbes
contribute to carcinogenesis fall into three broad categories: 1) pro­
moting gastrointestinal (GI) inflammation, 2) inducing genotoxicity
within intestinal epithelial cells, and 3) promoting cell proliferation of
intestinal epithelial cells [97].
3.1. Inflammation
Although mucosal barriers maintain symbiotic relationship between
the gut microbiota and the host, they are susceptible to constant envi­
ronmental insults. Cancer and inflammatory disorders arise when these
mucosal surface barriers are breached, and microbes pass into the
lamina propria, overturning immune tolerance and inciting mucosal
inflammation [97,147–149]. Upon mucosal barrier breach, proin­
flammatory pathways become activated serving as a critical component
of tumor progression. Inflammatory factors in the tumor microenvi­
ronment, such as reactive oxygen species (ROS) and reactive nitrogen
species (RNS), further contribute to tumor growth and metastasis [150].
Subsequent tumor engagement of PRRs results in further cell prolifera­
tion by activating cell survival signaling through NF-kB, a master
regulator in cancer-associated inflammation [151–153]. For instance,
CRC-associated Fusobacterium nucleatum has been shown to activate
NF-kB signaling within the tumor microenvironment through various
mechanisms, such as PRR engagement [154–156] or FadA binding of
E-cadherin on epithelial cells [157]. NF-kB regulates the transcription of
over 100 proinflammatory, as well as anti-apoptotic, genes. Nod-like
receptors (NLR), which are cytosolic PRRs, have also been linked to
cancer as mice deficient in NLRs display an enhanced susceptibility to
caCRC [158–161]. For instance, colons of NOD2-/- mice express signif­
icantly higher levels of inflammatory genes and activation of inflam­
matory pathways [158].
Beyond the impact on the innate immune system, mucosal barrier
breach also has deleterious consequences on the adaptive immune arm.
The inappropriate interaction between intestinal epithelium, gut
T. Plitt and J.J. Faith
microbiota, and the immune cells is the major contributor to inflam­
matory pathogenesis of the gut. Numerous studies have implicated the
role of the IL23/IL17 axis [162,163], TNFa [152,164,165], T follicular
helper cells, [166] and STAT3 [167] in tumor growth and progression.
As discussed above, several in vitro and ex vivo screening platforms
have been developed for identifying interactions between hundreds of
microbes and immune cells including the innate and adaptive cells
associated with cancer. The output from such screens will be important
in understanding the inflammatory contributions of bacterial strains’
oncogenic potential.
3.2. Genotoxicity
Beyond eliciting inflammation, many bacteria have evolved mech­
anisms to damage DNA to confer a survival advantage over other mi­
croorganisms [168,169]. These mechanisms are important for bacterial
ecology and evolution, with evidence that such behavior can prevent
competing strains from invading a niche or inhibit the growth of coex­
isting strains [168,170]. However, these bacterial defense strategies can
lead to host chromosomal instability - a hallmark of carcinogenesis. For
instance, E. coli (as well as other Enterobacteriaceae) expressing the
genomic island polyketide synthase (pks+) produces the genotoxin col­
ibactin that is believed to alkylate adenine residues in DNA, causing
DNA damage [171,172]. The detection of pks+ E. coli in human CRCs,
along with the ability of these strains to potentiate tumorigenesis in
animal models of disease, generated great interest in colibactin as a
molecule of interest in colorectal carcinogenesis [173–176]. Cytolethal
distending toxin (CDT) produced by several ε- and γ-proteobacteria has
also been shown to produce DNA double-strand breaks (DSBs) in
mammalian cells and promote colorectal tumorigenesis in mice [177,
178]. Accumulating data also support the role for enterotoxigenic Bac­
teroides fragilis (ETBF), Enterococcus faecalis, and Peptostreptococcus
anaerobius in animal and human tumorigenesis via ROS-mediated
damage of host DNA [179–182]. At high levels, ROS can lead to DNA
damage and mutations.
The characterization and measurement of genotoxicity may be
extremely valuable in predicting the pathogenicity of bacterial strains,
specifically those associated with increased cancer risk. Many of the
traditional DNA damage test methods, such as the bacterial reverse
mutation (Ames) test, are laborious, time-consuming and harbor a va­
riety of other methodological issues including low sample throughput
[183,184]. However, a number of sensitive, high-throughput genotox­
icity platforms have been recently developed for rapid measurement of
DNA damage (single- and double-strand breaks). Most notably, the
extent of DNA damage in intestinal epithelial cells has been quantified in
vitro and in vivo using phosphorylated histone H2AX (γH2AX) as a
surrogate marker of DNA damage. Immunofluorescence (IF) staining of
rat intestinal epithelial cells (IEC-6) infected with the pks+ harboring
strain of E. coli NC101 revealed γH2AX in ~30% of cells compared with
γH2AX in < 5% in cells infected with isogenic pks-deficient NC101
[175]. Similar results have been reported in germ-free IL10-/- mice and
transparent zebrafish larva using immunohistochemistry (IHC) and
flow-cytometry based detection assays of γH2AX [185]. Smaller-scale IF
screens using an antibody against the DNA lesion 8-oxo-7,8-dihy­
dro-2′ -deoxyguanosine (8-oxoG), a more general marker of oxidative
DNA damage, have also been used to demonstrate the effects of microbe
induced DNA damage in IL10-/- inflammatory CRC models [186].
The mechanism of CDT-induced DNA damage was defined using
similar, although low-throughput, genotoxic screens including IF using
antibodies against a well-defined DSB markers 53BP [187] and the
comet assay [178], which is another reliable method for measuring DNA
strand breaks. Bacterial lysates from wild-type and CDT-deficient
Campylobacter jejuni strains were used to treat the human colon cancer
cell line HT-29, IEC-6 cell line, and culture enteroids for 24 h. In all three
experimental models, bacterial lysates from wild-type C. jejuni promoted
DNA damage, while this response was strongly attenuated in cells
7
Seminars in Immunology 66 (2023) 101735
exposed to lysates from CDT-deficient C. jejuni [178].
While insofar these genotoxic assays of cancer-associated microbes
described above remain limited to a handful of bacterial strains, it re­
mains possible to scale up these assays to evaluate and predict the
genotoxic (and potentially, carcinogenic) potential of additional strains.
In an excellent recent example of such a scale-up, Cao et al., screened the
genotoxicity of more than 100 phylogenetically diverse human gut
commensal bacterial strains through co-incubation of each strain with
plasmid DNA followed by gel electrophoresis [188]. They identified
several new genotoxic strains, a subset of which drove increased tumor
formation in susceptible mice, and the genotoxic metabolite produced
by one of their top hits – a strain of Morganella morganii.
3.3. Proliferation
Several cancer-associated bacteria were shown to alter signaling
pathways involved in cell stemness and growth. The Wnt/β-catenin
pathway is a pleiotropic signaling pathway commonly mutated in many
cancers. This pathway regulates embryo development, cell proliferation
and differentiation, and apoptosis [189]. Unsurprisingly, pathogenic
bacteria have evolved strategies to hijack these processes to establish a
replicative niche [190]. Bacteria such as Helicobacter pylori, F. nucleatum,
ETBF, Salmonella typhi, Mycobacterium tuberculosis, and C. difficile secrete
virulence factors that alter the activity of the Wnt/β-catenin pathway
[97,189]. For instance, oncogenic strains of H. pylori inject an exogenous
cancer-promoting protein, CagA, intermittently into gastric epithelial
cells leading to activation of the Wnt/β-catenin pathway and subsequent
upregulation of genes involved in proliferation, survival, migration, and
angiogenesis [97,151,191–193]. On the other hand, F. nucleatum, a
member of the oral microbiota that is associated with human colorectal
adenomas, uses its FadA adhesin to bind and invade epithelial and
endothelial cells [157,194–196]. FadA binds host E-cadherin, activating
the Wnt/β-catenin pathway and stimulating the growth of cancer cells
[157]. ETBF, enriched in some human CRCs, secretes a 20-kDa metal­
loprotease toxin, BFT [197–199]. This toxin cleaves host E-cadherin,
leading to the stimulation of β-catenin-dependent cell proliferation
[198,200]. Finally, S. typhi, overabundant in hepatobiliary cancers,
activate the β-catenin pathway through its pathogenic product, AvrA
[201–203]. AvrA alters ubiquitination and acetylation of target proteins
to modulate epithelial proliferation and apoptosis [203,204].
Proliferation is an important part of cancer development and pro­
gression and so, screening bacterial strains for their ability to alter
mammalian cell proliferation could identify potentially oncogenic mi­
crobes. For instance, CRC-associated ETBF has been shown to stimulate
cell proliferation through the secretion of BFT in multiple cancerous
intestinal epithelial cell lines using the thymidine incorporation assay
[200]. Furthermore, immunohistochemistry evaluation of β-catenin
abundance in the colon of mice colonized with Citrobacter rodentium
revealed colonocyte hyperproliferation [205]. C. rodentium has been
shown to promote adenoma formation in ApcMin/+ mice [206]. Finally,
BrdU and XTT proliferation assays have been used to show proliferation
and migration of pancreatic cancer cells upon F. nucleatum invasion
[207]. Many of these proliferation assays can be modified to scale up the
screening of pro-proliferative microbes associated with various diseases.
4. Challenges and opportunities
Performing functional screens at the scale of hundreds to thousands
of microbial strains provides both advantages and disadvantages over
more common experiments on individual strains or small groups of
strains. To practically move beyond one-hundred different strains
assayed individually requires a break from individual gnotobiotic mice
and a transition to either ex vivo assays on mice previously colonized
with sets of strains or in vitro assays. A key feature of these large-scale
platforms will then be the relevance of the implemented assay for in
vivo and human biology. Furthermore, working with hundreds of
T. Plitt and J.J. Faith
microbial strains is inherently more error prone than working with small
numbers of strains. During such screens, it is not reasonable to assume
that each strain will grow every time the assay is run and that there will
not be a contamination of any wells if hundreds of strains are being
grown in multi-well plates in close proximity. Therefore, it is critical that
the purity of each strain is assayed with each screen to eliminate any
wells that are not the expected strain or that did not grow. Furthermore,
we need improved methods for replicating and distributing screening
libraries of bacterial strains so that assays can be repeated across labs
and different screens can be performed on the same sets of strains
allowing for a growing knowledgebase of these strains.
Other challenges that lie ahead for large-scale functional screens of
microbiome strains include how to build on prior knowledge rather than
have each study be an island. Our accumulation of knowledge from the
scientific community functional efforts have traditionally been focused
across individual bacterial strains, model organisms (e.g., mouse), or
human. Heavily used human functional resources like TCGA [208] and
ImmGen [209] have an advantage that they target a single or limited
number of mammalian hosts, whereas in the context the human
microbiome, each new screen could include hundreds of new strains that
have never been studied before. At the same time, there is high overlap
at the species and other taxonomic levels and even in the genetic se­
quences available for more strains used in high-throughput screens.
Therefore, envisioning systems that organize the results from different
high-throughput screens into a shared community database is a daunt­
ing but not impossible task – one that requires some ingenuity to
maximize the utility across studies but with a potential payback of
increased data reutilization, a growing knowledge of the probability of
different functions as a function of taxonomic or genetic context, and
community organization. Other key aspects of functional screens
include the scale of the screen and the generalizability of results. With
dozens of organisms one can cover multiple common species, with
hundreds of organisms more rare species will be observed as well as
multiple strains of common species, and with thousands of organisms in
a screen multiple strains can be tested for each species. As more genomes
become available for each bacterial species, pangenomics will also
provide a strategy to select strains and determine the number of strains
necessary to cover a specified fraction of a species’ pangenomic space.
With the majority of high-throughput functional screens of over 100
microbial strains emerging as publications or preprints only in the past
year, it is likely that we will see this trend expanding in the coming
years. As a community, we must balance the utility of each lab using
different screening sets of strains versus shared strains across screens
versus some combination of the two. Regardless, these larger screens
that often include multiple strains per species for common species pro­
vide a wealth of information across studies even when different strains
are used because the higher-level trends observed across taxonomic
groups are easily transferable to other strains. The key to success for
these new types of experiments will be to balance the value of the
screening assays, in vivo validations, and human interventions to ulti­
mately determine the utility of new discoveries.
Acknowledgements
This work was supported by National Institutes of Health grants (nos.
NIDDK DK112978, NIDDK DK124133, and NIDDK DK123749). We
thank Graham Britton for critical comments on the manuscript and
Philip Ahern for helpful tweets on T cell specificity.
References
[1] F.R. Blattner, et al., The complete genome sequence of Escherichia coli K-12,
Science 277 (1997) 1453–1462.
[2] T. Hayashi, et al., Complete genome sequence of enterohemorrhagic Escherichia
coli O157:H7 and genomic comparison with a laboratory strain K-12, DNA Res 8
(2001) 11–22.
8
Seminars in Immunology 66 (2023) 101735
[3] T. Baba, et al., Construction of Escherichia coli K-12 in-frame, single-gene
knockout mutants: the Keio collection, Mol. Syst. Biol. 2 (2006) (2006) 0008.
[4] J.J. Faith, et al., Large-scale mapping and validation of Escherichia coli
transcriptional regulation from a compendium of expression profiles, PLoS Biol. 5
(2007), e8.
[5] S. Kasif, R.J. Roberts, We need to keep a reproducible trace of facts, predictions,
and hypotheses from gene to function in the era of big data, PLoS Biol. 18 (2020),
e3000999.
[6] R.J. Roberts, et al., COMBREX: a project to accelerate the functional annotation
of prokaryotic genomes, Nucleic Acids Res 39 (2011) D11–D14.
[7] D.J. Lane, et al., Rapid determination of 16S ribosomal RNA sequences for
phylogenetic analyses, Proc. Natl. Acad. Sci. USA 82 (1985) 6955–6959.
[8] C.R. Woese, G.E. Fox, Phylogenetic structure of the prokaryotic domain: the
primary kingdoms, Proc. Natl. Acad. Sci. USA 74 (1977) 5088–5090.
[9] H.P. Browne, et al., Culturing of ‘unculturable’ human microbiota reveals novel
taxa and extensive sporulation, Nature 533 (2016) 543–546.
[10] M. Derrien, E.E. Vaughan, C.M. Plugge, W.M. de Vos, Akkermansia muciniphila
gen. nov., sp. nov., a human intestinal mucin-degrading bacterium, Int J. Syst.
Evol. Microbiol 54 (2004) 1469–1476.
[11] A.L. Goodman, et al., Extensive personal human gut microbiota culture
collections characterized and manipulated in gnotobiotic mice, Proc. Natl. Acad.
Sci. USA 108 (2011) 6252–6257.
[12] E.E. Hansen, et al., Pan-genome of the dominant human gut-associated archaeon,
Methanobrevibacter smithii, studied in twins, Proc. Natl. Acad. Sci. USA 108
(Suppl 1) (2011) 4599–4606.
[13] J.-C. Lagier, et al., Microbial culturomics: paradigm shift in the human gut
microbiome study, Clin. Microbiol Infect. 18 (2012) 1185–1193.
[14] F.E. Rey, et al., Metabolic niche of a prominent sulfate-reducing human gut
bacterium, Proc. Natl. Acad. Sci. USA 110 (2013) 13582–13587.
[15] G.J. Britton, et al., Defined microbiota transplant restores Th17/RORγt(+)
regulatory T cell balance in mice colonized with inflammatory bowel disease
microbiotas, Proc. Natl. Acad. Sci. USA 117 (2020) 21536–21545.
[16] S. Brugiroux, et al., Genome-guided design of a defined mouse microbiota that
confers colonization resistance against Salmonella enterica serovar Typhimurium,
Nat. Microbiol 2 (2016) 16215.
[17] A.G. Cheng, et al., Design, construction, and in vivo augmentation of a complex
gut microbiome, Cell 185 (3617–3636) (2022), e19.
[18] C.S. Eun, et al., Induction of bacterial antigen-specific colitis by a simplified
human microbiota consortium in gnotobiotic interleukin-10-/- mice, Infect.
Immun. 82 (2014) 2239–2246.
[19] J.J. Faith, et al., Creating and characterizing communities of human gut microbes
in gnotobiotic mice, ISME J. 4 (2010) 1094–1098.
[20] S.C. Forster, et al., A human gut bacterial genome and culture collection for
improved metagenomic analyses, Nat. Biotechnol. 37 (2019) 186–192.
[21] M. Poyet, et al., A library of human gut bacterial isolates paired with longitudinal
multiomics data enables mechanistic microbiome research, Nat. Med 25 (2019)
1442–1452.
[22] K.A. Romano, E.I. Vivas, D. Amador-Noguez, F.E. Rey, Intestinal microbiota
composition modulates choline bioavailability from diet and accumulation of the
proatherogenic metabolite trimethylamine-N-oxide, mBio 6 (2015), e02481.
[23] M.P. Spindler, et al., Human gut microbiota stimulate defined innate immune
responses that vary from phylum to strain, 00408–5, Cell Host Microbe
S1931–3128 (22) (2022), https://doi.org/10.1016/j.chom.2022.08.009.
[24] C. Yang, et al., Immunoglobulin A antibody composition is sculpted to bind the
self gut microbiome, Sci. Immunol. 7 (2022), eabg3208.
[25] J. Qin, et al., A human gut microbial gene catalogue established by metagenomic
sequencing, Nature 464 (2010) 59–65.
[26] P.M. Smith, et al., The microbial metabolites, short-chain fatty acids, regulate
colonic Treg cell homeostasis, Science 341 (2013) 569–573.
[27] K. Atarashi, et al., Treg induction by a rationally selected mixture of Clostridia
strains from the human microbiota, Nature 500 (2013) 232–236.
[28] I.I. Ivanov, et al., Induction of intestinal Th17 cells by segmented filamentous
bacteria, Cell 139 (2009) 485–498.
[29] C.G. Buffie, et al., Precision microbiome reconstitution restores bile acid
mediated resistance to Clostridium difficile, Nature 517 (2015) 205–208.
[30] C. Campbell, et al., Bacterial metabolism of bile acids promotes generation of
peripheral regulatory T cells, Nature 581 (2020) 475–479.
[31] S. Hang, et al., Bile acid metabolites control T(H)17 and T(reg) cell
differentiation, Nature 576 (2019) 143–148.
[32] L.N. Lucas, et al., Dominant Bacterial Phyla from the Human Gut Show
Widespread Ability To Transform and Conjugate Bile Acids, mSystems e0080521
(2021), https://doi.org/10.1128/mSystems.00805-21.
[33] D. Paik, et al., Human gut bacteria produce Τ(Н)17-modulating bile acid
metabolites, Nature 603 (2022) 907–912.
[34] Structure, function and diversity of the healthy human microbiome, Nature 486
(2012) 207–214.
[35] P.B. Eckburg, et al., Diversity of the human intestinal microbial flora, Science 308
(2005) 1635–1638.
[36] D. Gevers, et al., The treatment-naive microbiome in new-onset Crohn’s disease,
Cell Host Microbe 15 (2014) 382–392.
[37] J.M. Shapiro, et al., Immunoglobulin a targets a unique subset of the microbiota
in inflammatory bowel disease, Cell Host Microbe 29 (83–93) (2021), e3.
[38] E.A. Franzosa, et al., Gut microbiome structure and metabolic activity in
inflammatory bowel disease, Nat. Microbiol 4 (2019) 293–305.
[39] V. Gopalakrishnan, et al., Gut microbiome modulates response to anti-PD-1
immunotherapy in melanoma patients, Science 359 (2018) 97–103.
T. Plitt and J.J. Faith
[40] B. Routy, et al., Gut microbiome influences efficacy of PD-1-based
immunotherapy against epithelial tumors, Science 359 (2018) 91–97.
[41] F.Y. Shaikh, et al., A uniform computational approach improved on existing
pipelines to reveal microbiome biomarkers of nonresponse to immune checkpoint
inhibitors, Clin. Cancer Res 27 (2021) 2571–2583.
[42] B. Yilmaz, et al., Microbial network disturbances in relapsing refractory Crohn’s
disease, Nat. Med 25 (2019) 323–336.
[43] V. Aggarwala, et al., Precise quantification of bacterial strains after fecal
microbiota transplantation delineates long-term engraftment and explains
outcomes, Nat. Microbiol 6 (2021) 1309–1318.
[44] M.R. Olm, et al., inStrain profiles population microdiversity from metagenomic
data and sensitively detects shared microbial strains, Nat. Biotechnol. 39 (2021)
727–736.
[45] D.T. Truong, A. Tett, E. Pasolli, C. Huttenhower, N. Segata, Microbial strain-level
population structure and genetic diversity from metagenomes, Genome Res 27
(2017) 626–638.
[46] S. Zhao, C.L. Dai, E.D. Evans, Z. Lu, E.J. Alm, Tracking strains predicts personal
microbiomes and reveals recent adaptive evolution, 2020.09.14, bioRxiv (2020),
296970, https://doi.org/10.1101/2020.09.14.296970.
[47] W. Zheng, et al., High-throughput, single-microbe genomics with strain
resolution, applied to a human gut microbiome, Science 376 (2022) eabm1483.
[48] J.J. Faith, et al., Strain population structure varies widely across bacterial species
and predicts strain colonization in unrelated individuals, 2020.10.17, bioRxiv
(2020), 343640, https://doi.org/10.1101/2020.10.17.343640.
[49] A. Conwill, et al., Anatomy promotes neutral coexistence of strains in the human
skin microbiome, Cell Host Microbe 30 (171–182) (2022), e7.
[50] T.D. Lieberman, Detecting bacterial adaptation within individual microbiomes,
Philos. Trans. R. Soc. Lond. B Biol. Sci. 377 (2022) 20210243.
[51] B. Yilmaz, et al., Long-term evolution and short-term adaptation of microbiota
strains and sub-strains in mice, Cell Host Microbe 29 (650–663) (2021), e9.
[52] S. Zhao, et al., Adaptive evolution within gut microbiomes of healthy people, Cell
Host Microbe 25 (656–667) (2019), e8.
[53] J.S. Johnson, et al., Evaluation of 16S rRNA gene sequencing for species and
strain-level microbiome analysis, Nat. Commun. 10 (2019) 5029.
[54] R.C. Edgar, Updating the 97% identity threshold for 16S ribosomal RNA OTUs,
Bioinformatics 34 (2018) 2371–2375.
[55] A. Chen-Liaw, et al., Gut microbiota bacterial strain richness is species specific
and limits therapeutic engraftment, 2022.11.01, bioRxiv (2022), 514782,
https://doi.org/10.1101/2022.11.01.514782.
[56] J.J. Faith, et al., The long-term stability of the human gut microbiota, Science 341
(2013) 1237439.
[57] S. Paramsothy, et al., Specific Bacteria and Metabolites Associated With Response
to Fecal Microbiota Transplantation in Patients With Ulcerative Colitis,
Gastroenterology 156 (1440–1454) (2019), e2.
[58] E.N. Baruch, et al., Fecal microbiota transplant promotes response in
immunotherapy-refractory melanoma patients, Science 371 (2021) 602–609.
[59] D. Davar, et al., Fecal microbiota transplant overcomes resistance to anti-PD-1
therapy in melanoma patients, Science 371 (2021) 595–602.
[60] M. Schirmer, A. Garner, H. Vlamakis, R.J. Xavier, Microbial genes and pathways
in inflammatory bowel disease, Nat. Rev. Microbiol 17 (2019) 497–511.
[61] S. Tomkovich, et al., Human colon mucosal biofilms from healthy or colon cancer
hosts are carcinogenic, J. Clin. Invest 129 (2019) 1699–1712.
[62] F. Colombo, et al., Gut microbiota composition in colorectal cancer patients is
genetically regulated, Sci. Rep. 12 (2022) 11424.
[63] S. Gupta, et al., Cutaneous surgical wounds have distinct microbiomes from intact
skin, Microbiol Spectr. e0330022 (2022), https://doi.org/10.1128/
spectrum.03300-22.
[64] J.P. Hugot, et al., Association of NOD2 leucine-rich repeat variants with
susceptibility to Crohn’s disease, Nature 411 (2001) 599–603.
[65] Y. Ogura, et al., A frameshift mutation in NOD2 associated with susceptibility to
Crohn’s disease, Nature 411 (2001) 603–606.
[66] E. Fleming, et al., Cultivation of common bacterial species and strains from
human skin, oral, and gut microbiota, BMC Microbiol 21 (2021) 278.
[67] X. He, et al., Cultivation of a human-associated TM7 phylotype reveals a reduced
genome and epibiotic parasitic lifestyle, Proc. Natl. Acad. Sci. USA 112 (2015)
244–249.
[68] B. Bor, et al., Insights obtained by culturing saccharibacteria with their bacterial
hosts, J. Dent. Res 99 (2020) 685–694.
[69] K. Nagashima, et al., Mapping the T cell repertoire to a complex gut bacterial
community, 2022.05.04, bioRxiv (2022), 490632, https://doi.org/10.1101/
2022.05.04.490632.
[70] E. van Nood, et al., Duodenal infusion of donor feces for recurrent Clostridium
difficile, N. Engl. J. Med 368 (2013) 407–415.
[71] M. Dsouza, et al., Colonization of the live biotherapeutic product VE303 and
modulation of the microbiota and metabolites in healthy volunteers, Cell Host
Microbe 30 (583–598) (2022), e8.
[72] E.O. Petrof, et al., Stool substitute transplant therapy for the eradication of
Clostridium difficile infection: ‘RePOOPulating’ the gut, Microbiome 1 (2013) 3.
[73] D. Kao, et al., The effect of a microbial ecosystem therapeutic (MET-2) on
recurrent Clostridioides difficile infection: a phase 1, open-label, single-group
trial, Lancet Gastroenterol. Hepatol. 6 (2021) 282–291.
[74] F. Cold, C.K. Svensson, A.M. Petersen, L.H. Hansen, M. Helms, Long-Term Safety
Following Faecal Microbiota Transplantation as a Treatment for Recurrent
Clostridioides difficile Infection Compared with Patients Treated with a Fixed
Bacterial Mixture: Results from a Retrospective Cohort Study, Cells 11 (2022).
9
Seminars in Immunology 66 (2023) 101735
[75] G.J. Britton, J.J. Faith, Causative Microbes in Host-Microbiome Interactions,
Annu Rev. Microbiol 75 (2021) 223–242.
[76] M.B. Geuking, et al., Intestinal bacterial colonization induces mutualistic
regulatory T cell responses, Immunity 34 (2011) 794–806.
[77] H.C. Rath, et al., Normal luminal bacteria, especially Bacteroides species, mediate
chronic colitis, gastritis, and arthritis in HLA-B27/human beta2 microglobulin
transgenic rats, J. Clin. Invest 98 (1996) 945–953.
[78] S. Naik, et al., Commensal-dendritic-cell interaction specifies a unique protective
skin immune signature, Nature 520 (2015) 104–108.
[79] K. Atarashi, et al., Ectopic colonization of oral bacteria in the intestine drives T
(H)1 cell induction and inflammation, Science 358 (2017) 359–365.
[80] M. Viladomiu, et al., IgA-coated E. coli enriched in Crohn’s disease
spondyloarthritis promote T(H)17-dependent inflammation, Sci. Transl. Med 9
(2017).
[81] J.J. Faith, P.P. Ahern, V.K. Ridaura, J. Cheng, J.I. Gordon, Identifying gut
microbe-host phenotype relationships using combinatorial communities in
gnotobiotic mice, Sci. Transl. Med 6 (2014) 220ra11.
[82] N. Geva-Zatorsky, et al., Mining the Human Gut Microbiota for
Immunomodulatory Organisms, Cell 168 (928–943) (2017), e11.
[83] S. Paramsothy, et al., Multidonor intensive faecal microbiota transplantation for
active ulcerative colitis: a randomised placebo-controlled trial, Lancet 389 (2017)
1218–1228.
[84] M.P. Spindler, I. Mogno, P. Suri, G.J. Britton, J.J. Faith, Species-specific CD4+ T
cells Enable Prediction of Mucosal Immune Phenotypes from Microbiota
Composition, 2022.08.13, bioRxiv (2022), 503851, https://doi.org/10.1101/
2022.08.13.503851.
[85] D.J. Morrison, T. Preston, Formation of short chain fatty acids by the gut
microbiota and their impact on human metabolism, Gut Microbes 7 (2016)
189–200.
[86] M. Zimmermann, M. Zimmermann-Kogadeeva, R. Wegmann, A.L. Goodman,
Mapping human microbiome drug metabolism by gut bacteria and their genes,
Nature 570 (2019) 462–467.
[87] N. Koppel, V. Maini Rekdal, E.P. Balskus, Chemical transformation of xenobiotics
by the human gut microbiota, Science 356 (2017).
[88] L.J. Cohen, et al., Unraveling function and diversity of bacterial lectins in the
human microbiome, Nat. Commun. 13 (2022) 3101.
[89] D.A. Colosimo, et al., Mapping Interactions of Microbial Metabolites with Human
G-Protein-Coupled Receptors, Cell Host Microbe 26 (273–282) (2019), e7.
[90] Y. Asano, et al., Critical role of gut microbiota in the production of biologically
active, free catecholamines in the gut lumen of mice, Am. J. Physiol. Gastrointest.
Liver Physiol. 303 (2012) G1288–G1295.
[91] J.M. Yano, et al., Indigenous bacteria from the gut microbiota regulate host
serotonin biosynthesis, Cell 161 (2015) 264–276.
[92] D.R. Donohoe, et al., A gnotobiotic mouse model demonstrates that dietary fiber
protects against colorectal tumorigenesis in a microbiota- and butyrate-
dependent manner, Cancer Disco 4 (2014) 1387–1397.
[93] F. Hosseinkhani, et al., The contribution of gut bacterial metabolites in the human
immune signaling pathway of non-communicable diseases, Gut Microbes 13
(2021) 1–22.
[94] N. Tennoune, et al., Bacterial ClpB heat-shock protein, an antigen-mimetic of the
anorexigenic peptide α-MSH, at the origin of eating disorders, Transl. Psychiatry
4 (2014), e458.
[95] J. Breton, et al., Gut Commensal E. coli Proteins Activate Host Satiety Pathways
following Nutrient-Induced Bacterial Growth, Cell Metab. 23 (2016) 324–334.
[96] Y. Lai, et al., High-coverage metabolomics uncovers microbiota-driven
biochemical landscape of interorgan transport and gut-brain communication in
mice, Nat. Commun. 12 (2021) 6000.
[97] W.S. Garrett, Cancer and the microbiota, Science 348 (2015) 80–86.
[98] S.J.D. O’Keefe, Diet, microorganisms and their metabolites, and colon cancer,
Nat. Rev. Gastroenterol. Hepatol. 13 (2016) 691–706.
[99] P. Liu, et al., The role of short-chain fatty acids in intestinal barrier function,
inflammation, oxidative stress, and colonic carcinogenesis, Pharm. Res 165
(2021), 105420.
[100] K.M. Maslowski, et al., Regulation of inflammatory responses by gut microbiota
and chemoattractant receptor GPR43, Nature 461 (2009) 1282–1286.
[101] N. Arpaia, et al., Metabolites produced by commensal bacteria promote
peripheral regulatory T-cell generation, Nature 504 (2013) 451–455.
[102] Y. Furusawa, et al., Commensal microbe-derived butyrate induces the
differentiation of colonic regulatory T cells, Nature 504 (2013) 446–450.
[103] P.V. Chang, L. Hao, S. Offermanns, R. Medzhitov, The microbial metabolite
butyrate regulates intestinal macrophage function via histone deacetylase
inhibition, Proc. Natl. Acad. Sci. USA 111 (2014) 2247–2252.
[104] K.Y.C. Fung, L. Cosgrove, T. Lockett, R. Head, D.L. Topping, A review of the
potential mechanisms for the lowering of colorectal oncogenesis by butyrate, Br.
J. Nutr. 108 (2012) 820–831.
[105] Y. Cheng, Z. Ling, L. Li, The Intestinal Microbiota and Colorectal Cancer, Front
Immunol. 11 (2020), 615056.
[106] Y. Tang, Y. Chen, H. Jiang, G.T. Robbins, D. Nie, G-protein-coupled receptor for
short-chain fatty acids suppresses colon cancer, Int J. Cancer 128 (2011)
847–856.
[107] A.J. Brown, et al., The Orphan G protein-coupled receptors GPR41 and GPR43 are
activated by propionate and other short chain carboxylic acids, J. Biol. Chem. 278
(2003) 11312–11319.
[108] V. Ganapathy, M. Thangaraju, P.D. Prasad, P.M. Martin, N. Singh, Transporters
and receptors for short-chain fatty acids as the molecular link between colonic
bacteria and the host, Curr. Opin. Pharm. 13 (2013) 869–874.
T. Plitt and J.J. Faith
[109] N. Singh, et al., Activation of Gpr109a, receptor for niacin and the commensal
metabolite butyrate, suppresses colonic inflammation and carcinogenesis,
Immunity 40 (2014) 128–139.
[110] L.B. Bindels, et al., Gut microbiota-derived propionate reduces cancer cell
proliferation in the liver, Br. J. Cancer 107 (2012) 1337–1344.
[111] A. Belcheva, et al., Gut microbial metabolism drives transformation of MSH2-
deficient colon epithelial cells, Cell 158 (2014) 288–299.
[112] R.E. Ley, P.J. Turnbaugh, S. Klein, J.I. Gordon, Microbial ecology: human gut
microbes associated with obesity, Nature 444 (2006) 1022–1023.
[113] T.M. Loo, et al., Gut Microbiota Promotes Obesity-Associated Liver Cancer
through PGE(2)-Mediated Suppression of Antitumor Immunity, Cancer Disco 7
(2017) 522–538.
[114] P. Marzullo, et al., Spot-light on microbiota in obesity and cancer, Int J. Obes.
(Lond. ) 45 (2021) 2291–2299.
[115] J. Park, T.S. Morley, M. Kim, D.J. Clegg, P.E. Scherer, Obesity and cancer–
mechanisms underlying tumour progression and recurrence, Nat. Rev.
Endocrinol. 10 (2014) 455–465.
[116] R.W. O’Rourke, Obesity and cancer: at the crossroads of cellular metabolism and
proliferation, Surg. Obes. Relat. Dis. 10 (2014) 1208–1219.
[117] G. Brandi, et al., Microbiota, NASH, HCC and the potential role of probiotics,
Carcinogenesis 38 (2017) 231–240.
[118] M.F. Gregor, G.S. Hotamisligil, Inflammatory mechanisms in obesity, Annu Rev.
Immunol. 29 (2011) 415–445.
[119] R. Mirzaei, et al., Role of microbiota-derived short-chain fatty acids in cancer
development and prevention, Biomed. Pharm. 139 (2021), 111619.
[120] S. Yoshimoto, et al., Obesity-induced gut microbial metabolite promotes liver
cancer through senescence secretome, Nature 499 (2013) 97–101.
[121] V. Matson, C.S. Chervin, T.F. Gajewski, Cancer and the Microbiome-Influence of
the Commensal Microbiota on Cancer, Immune Responses, and Immunotherapy,
Gastroenterology 160 (2021) 600–613.
[122] C. Ma, et al., Gut microbiome-mediated bile acid metabolism regulates liver
cancer via NKT cells, Science 360 (2018).
[123] S. Han, et al., A metabolomics pipeline for the mechanistic interrogation of the
gut microbiome, Nature 595 (2021) 415–420.
[124] M. Wang, et al., Strain dropouts reveal interactions that govern the metabolic
output of the gut microbiome, 2022.07.25, bioRxiv (2022), 501461, https://doi.
org/10.1101/2022.07.25.501461.
[125] A.C. Tolonen, et al., Synthetic glycans control gut microbiome structure and
mitigate colitis in mice, Nat. Commun. 13 (2022) 1244.
[126] M.L. Patnode, et al., Strain-level functional variation in the human gut microbiota
based on bacterial binding to artificial food particles, Cell Host Microbe 29
(664–673) (2021), e5.
[127] N.A. Pudlo, et al., Phenotypic and Genomic Diversification in Complex
Carbohydrate-Degrading Human Gut Bacteria, mSystems 7 (2022), e0094721.
[128] L.C.-H. Yu, Microbiota dysbiosis and barrier dysfunction in inflammatory bowel
disease and colorectal cancers: exploring a common ground hypothesis,
J. Biomed. Sci. 25 (2018) 79.
[129] M.A. Fischbach, Microbiome: focus on causation and mechanism, Cell 174 (2018)
785–790.
[130] M.C. Andrews, et al., Gut microbiota signatures are associated with toxicity to
combined CTLA-4 and PD-1 blockade, Nat. Med 27 (2021) 1432–1441.
[131] T.K. Pedersen, et al., The CD4(+) T cell response to a commensal-derived epitope
transitions from a tolerant to an inflammatory state in Crohn’s disease, Immunity
S1074–7613 (22) (2022) 00410–00411, https://doi.org/10.1016/j.
immuni.2022.08.016.
[132] M.E. Perez-Muñoz, P. Joglekar, Y.-J. Shen, K.Y. Chang, D.A. Peterson,
Identification and Phylogeny of the First T Cell Epitope Identified from a Human
Gut Bacteroides Species, PLoS One 10 (2015), e0144382.
[133] Y. Yang, et al., Focused specificity of intestinal TH17 cells towards commensal
bacterial antigens, Nature 510 (2014) 152–156.
[134] M. Xu, et al., c-MAF-dependent regulatory T cells mediate immunological
tolerance to a gut pathobiont, Nature 554 (2018) 373–377.
[135] E. Sefik, et al., MUCOSAL IMMUNOLOGY. Individual intestinal symbionts induce
a distinct population of RORγ+ regulatory T cells, Science 349 (2015) 993–997.
[136] P.J.P.L. Teixeira, et al., Specific modulation of the root immune system by a
community of commensal bacteria, Proc. Natl. Acad. Sci. USA 118 (2021).
[137] Gardnerella vaginalis alters cervicovaginal epithelial cell function through
microbe-specific immune responses. vol. 10, 2022.
[138] T. Rollenske, et al., Parallelism of intestinal secretory IgA shapes functional
microbial fitness, Nature 598 (2021) 657–661.
[139] C. Yang, et al., Fecal IgA levels are determined by strain-level differences in
bacteroides ovatus and are modifiable by gut microbiota manipulation, Cell Host
Microbe 27 (467–475) (2020), e6.
[140] D.A. Peterson, N.P. McNulty, J.L. Guruge, J.I. Gordon, IgA response to symbiotic
bacteria as a mediator of gut homeostasis, Cell Host Microbe 2 (2007) 328–339.
[141] S. Hapfelmeier, et al., Reversible microbial colonization of germ-free mice reveals
the dynamics of IgA immune responses, Science 328 (2010) 1705–1709.
[142] N.W. Palm, et al., Immunoglobulin A coating identifies colitogenic bacteria in
inflammatory bowel disease, Cell 158 (2014) 1000–1010.
[143] K.L. Alexander, et al., Human Microbiota Flagellins Drive Adaptive Immune
Responses in Crohn’s Disease, Gastroenterology 161 (522–535) (2021), e6.
[144] J.J. Bunker, A. Bendelac, IgA Responses to Microbiota, Immunity 49 (2018)
211–224.
[145] M.Y. Zeng, et al., Gut Microbiota-Induced Immunoglobulin G Controls Systemic
Infection by Symbiotic Bacteria and Pathogens, Immunity 44 (2016) 647–658.
10
Seminars in Immunology 66 (2023) 101735
[146] M.A. Koch, et al., Maternal IgG and IgA Antibodies Dampen Mucosal T Helper,
Cell Responses Early Life. Cell 165 (2016) 827–841.
[147] R. Ahmad, M.F. Sorrell, S.K. Batra, P. Dhawan, A.B. Singh, Gut permeability and
mucosal inflammation: bad, good or context dependent, Mucosal Immunol. 10
(2017) 307–317.
[148] L.R. Lopez, J.-H. Ahn, T. Alves, J.C. Arthur, Microenvironmental Factors that
Shape Bacterial Metabolites in Inflammatory Bowel Disease, Front Cell Infect.
Microbiol 12 (2022), 934619.
[149] L. Robrahn, L. Jiao, T. Cramer, Barrier integrity and chronic inflammation
mediated by HIF-1 impact on intestinal tumorigenesis, Cancer Lett. 490 (2020)
186–192.
[150] A. Piechota-Polanczyk, J. Fichna, Review article: the role of oxidative stress in
pathogenesis and treatment of inflammatory bowel diseases, Naunyn Schmiede
Arch. Pharm. 387 (2014) 605–620.
[151] M.T. Abreu, R.M.J. Peek, Gastrointestinal malignancy and the microbiome,
Gastroenterology 146 (1534–1546) (2014), e3.
[152] R.F. Schwabe, C. Jobin, The microbiome and cancer, Nat. Rev. Cancer 13 (2013)
800–812.
[153] J.A. DiDonato, F. Mercurio, M. Karin, NF-κB and the link between inflammation
and cancer, Immunol. Rev. 246 (2012) 379–400.
[154] G. Zhang, R. Chen, J.D. Rudney, Streptococcus cristatus modulates the
Fusobacterium nucleatum-induced epithelial interleukin-8 response through the
nuclear factor-kappa B pathway, J. Periodontal Res 46 (2011) 558–567.
[155] M.R. Milward, et al., Differential activation of NF-kappaB and gene expression in
oral epithelial cells by periodontal pathogens, Clin. Exp. Immunol. 148 (2007)
307–324.
[156] E. Allen-Vercoe, J. Strauss, K. Chadee, Fusobacterium nucleatum: an emerging gut
pathogen? Gut Microbes 2 (2011) 294–298.
[157] M.R. Rubinstein, et al., Fusobacterium nucleatum promotes colorectal
carcinogenesis by modulating E-cadherin/β-catenin signaling via its FadA
adhesin, Cell Host Microbe 14 (2013) 195–206.
[158] S.M.N. Udden, et al., NOD2 Suppresses Colorectal Tumorigenesis via
Downregulation of the TLR Pathways, Cell Rep. 19 (2017) 2756–2770.
[159] M.H. Zaki, et al., The NOD-like receptor NLRP12 attenuates colon inflammation
and tumorigenesis, Cancer Cell 20 (2011) 649–660.
[160] I.C. Allen, et al., The NLRP3 inflammasome functions as a negative regulator of
tumorigenesis during colitis-associated cancer, J. Exp. Med 207 (2010)
1045–1056.
[161] K.-M. Lin, et al., IRAK-1 bypasses priming and directly links TLRs to rapid NLRP3
inflammasome activation, Proc. Natl. Acad. Sci. USA 111 (2014) 775–780.
[162] S.I. Grivennikov, et al., Adenoma-linked barrier defects and microbial products
drive IL-23/IL-17-mediated tumour growth, Nature 491 (2012) 254–258.
[163] S. Wu, et al., A human colonic commensal promotes colon tumorigenesis via
activation of T helper type 17 T cell responses, Nat. Med 15 (2009) 1016–1022.
[164] E. Elinav, et al., Inflammation-induced cancer: crosstalk between tumours,
immune cells and microorganisms, Nat. Rev. Cancer 13 (2013) 759–771.
[165] T. Irrazábal, A. Belcheva, S.E. Girardin, A. Martin, D.J. Philpott, The multifaceted
role of the intestinal microbiota in colon cancer, Mol. Cell 54 (2014) 309–320.
[166] A.E. Overacre-Delgoffe, et al., Microbiota-specific T follicular helper cells drive
tertiary lymphoid structures and anti-tumor immunity against colorectal cancer,
Immunity 54 (2812–2824) (2021), e4.
[167] N. Li, S.I. Grivennikov, M. Karin, The unholy trinity: inflammation, cytokines, and
STAT3 shape the cancer microenvironment, Cancer Cell 19 (2011) 429–431.
[168] R. Niehus, N.M. Oliveira, A. Li, A.G. Fletcher, K.R. Foster, The evolution of
strategy in bacterial warfare via the regulation of bacteriocins and antibiotics,
Elife 10 (2021).
[169] L.-S. Ma, A. Hachani, J.-S. Lin, A. Filloux, E.-M. Lai, Agrobacterium tumefaciens
deploys a superfamily of type VI secretion DNase effectors as weapons for
interbacterial competition in planta, Cell Host Microbe 16 (2014) 94–104.
[170] J.E. Silpe, J.W.H. Wong, S.V. Owen, M. Baym, E.P. Balskus, The bacterial toxin
colibactin triggers prophage induction, Nature 603 (2022) 315–320.
[171] J.-P. Nougayrède, et al., Escherichia coli induces DNA double-strand breaks in
eukaryotic cells, Science 313 (2006) 848–851.
[172] J. Putze, et al., Genetic structure and distribution of the colibactin genomic island
among members of the family Enterobacteriaceae, Infect. Immun. 77 (2009)
4696–4703.
[173] E. Buc, et al., High prevalence of mucosa-associated E. coli producing
cyclomodulin and genotoxin in colon cancer, PLoS One 8 (2013), e56964.
[174] M. Prorok-Hamon, et al., Colonic mucosa-associated diffusely adherent afaC+
Escherichia coli expressing lpfA and pks are increased in inflammatory bowel
disease and colon cancer, Gut 63 (2014) 761–770.
[175] J.C. Arthur, et al., Intestinal inflammation targets cancer-inducing activity of the
microbiota, Science 338 (2012) 120–123.
[176] J.C. Arthur, et al., Microbial genomic analysis reveals the essential role of
inflammation in bacteria-induced colorectal cancer, Nat. Commun. 5 (2014)
4724.
[177] L. Guerra, R. Guidi, T. Frisan, Do bacterial genotoxins contribute to chronic
inflammation, genomic instability and tumor progression? FEBS J. 278 (2011)
4577–4588.
[178] Z. He, et al., Campylobacter jejuni promotes colorectal tumorigenesis through the
action of cytolethal distending toxin, Gut 68 (2019) 289–300.
[179] H. Tsoi, et al., Peptostreptococcus anaerobius Induces Intracellular Cholesterol
Biosynthesis in Colon Cells to Induce Proliferation and Causes Dysplasia in Mice,
Gastroenterology 152 (1419–1433) (2017), e5.
T. Plitt and J.J. Faith
[180] X. Wang, et al., Enterococcus faecalis induces aneuploidy and tetraploidy in
colonic epithelial cells through a bystander effect, Cancer Res 68 (2008)
9909–9917.
[181] C.M. Dejea, et al., Patients with familial adenomatous polyposis harbor colonic
biofilms containing tumorigenic bacteria, Science 359 (2018) 592–597.
[182] L. Chung, et al., Bacteroides fragilis toxin coordinates a pro-carcinogenic
inflammatory cascade via targeting of colonic epithelial cells, Cell Host Microbe
23 (203–214) (2018), e5.
[183] B.C. Nelson, et al., Emerging metrology for high-throughput nanomaterial
genotoxicology, Mutagenesis 32 (2017) 215–232.
[184] S. Dubbert, B. Klinkert, M. Schimiczek, T.M. Wassenaar, R. Bünau, von. No
Genotoxicity Is Detectable for Escherichia coli Strain Nissle 1917 by Standard In
Vitro and In Vivo Tests. Eur, J. Microbiol Immunol. (Bp) 10 (2020) 11–19.
[185] J.J. Mousa, et al., MATE transport of the E. coli-derived genotoxin colibactin, Nat.
Microbiol 1 (2016) 15009.
[186] T. Irrazabal, et al., Limiting oxidative DNA damage reduces microbe-induced
colitis-associated colorectal cancer, Nat. Commun. 11 (2020) 1802.
[187] V. Graillot, et al., Genotoxicity of Cytolethal Distending Toxin (CDT) on Isogenic
Human Colorectal Cell Lines: Potential Promoting Effects for Colorectal
Carcinogenesis, Front Cell Infect. Microbiol 6 (2016) 34.
[188] Cao, Y. et al. Commensal microbiota from patients with inflammatory bowel
disease produce genotoxic metabolites. Science 378, eabm3233 (2022).
[189] O. Silva-García, J.J. Valdez-Alarcón, V.M. Baizabal-Aguirre, Wnt/β-Catenin
Signaling as a Molecular Target by Pathogenic Bacteria, Front Immunol. 10
(2019) 2135.
[190] M.R. Rogan, L.L. Patterson, J.Y. Wang, J.W. McBride, Bacterial Manipulation of
Wnt Signaling: A Host-Pathogen Tug-of-Wnt, Front Immunol. 10 (2019) 2390.
[191] S. Alzahrani, et al., Effect of Helicobacter pylori on gastric epithelial cells, World
J. Gastroenterol. 20 (2014) 12767–12780.
[192] M. Hatakeyama, Structure and function of Helicobacter pylori CagA, the first-
identified bacterial protein involved in human cancer, Proc. Jpn Acad. Ser. B
Phys. Biol. Sci. 93 (2017) 196–219.
[193] L.F. Jiménez-Soto, R. Haas, The CagA toxin of Helicobacter pylori: abundant
production but relatively low amount translocated, Sci. Rep. 6 (2016) 23227.
[194] A.D. Kostic, et al., Fusobacterium nucleatum potentiates intestinal tumorigenesis
and modulates the tumor-immune microenvironment, Cell Host Microbe 14
(2013) 207–215.
11
Seminars in Immunology 66 (2023) 101735
[195] A.D. Kostic, et al., Genomic analysis identifies association of Fusobacterium with
colorectal carcinoma, Genome Res 22 (2012) 292–298.
[196] M. Castellarin, et al., Fusobacterium nucleatum infection is prevalent in human
colorectal carcinoma, Genome Res 22 (2012) 299–306.
[197] C.L. Enterotoxigenic Sears, Bacteroides fragilis: a rogue among symbiotes, Clin.
Microbiol Rev. 22, 349–369, Table Contents (2009).
[198] S. Wu, K.-J. Rhee, M. Zhang, A. Franco, C.L. Sears, Bacteroides fragilis toxin
stimulates intestinal epithelial cell shedding and gamma-secretase-dependent E-
cadherin cleavage, J. Cell Sci. 120 (2007) 1944–1952.
[199] A. Boleij, et al., The Bacteroides fragilis toxin gene is prevalent in the colon
mucosa of colorectal cancer patients, Clin. Infect. Dis. 60 (2015) 208–215.
[200] S. Wu, P.J. Morin, D. Maouyo, C.L. Sears, Bacteroides fragilis enterotoxin induces
c-Myc expression and cellular proliferation, Gastroenterology 124 (2003)
392–400.
[201] U. Dutta, P.K. Garg, R. Kumar, R.K. Tandon, Typhoid carriers among patients with
gallstones are at increased risk for carcinoma of the gallbladder, Am. J.
Gastroenterol. 95 (2000) 784–787.
[202] E.C. Lazcano-Ponce, et al., Epidemiology and molecular pathology of gallbladder
cancer, CA Cancer J. Clin. 51 (2001) 349–364.
[203] F. de la Gala Sánchez, J.J. Jorge Gómez, P. García Méndez, M. González Estecha,
[Ferritin and iron deficiency anemias], Med Interna 6 (1989) 133–136.
[204] R. Lu, et al., Presence of Salmonella AvrA in colorectal tumor and its precursor
lesions in mouse intestine and human specimens, Oncotarget 8 (2017)
55104–55115.
[205] J.H. Sellin, S. Umar, J. Xiao, A.P. Morris, Increased beta-catenin expression and
nuclear translocation accompany cellular hyperproliferation in vivo, Cancer Res
61 (2001) 2899–2906.
[206] J.V. Newman, T. Kosaka, B.J. Sheppard, J.G. Fox, D.B. Schauer, Bacterial
infection promotes colon tumorigenesis in Apc(Min/+) mice, J. Infect. Dis. 184
(2001) 227–230.
[207] B. Udayasuryan, et al., Fusobacterium nucleatum induces proliferation and
migration in pancreatic cancer cells through host autocrine and paracrine
signaling, Sci. Signal 15 (2022).
[208] G.F. Gao, et al., Before and After: Comparison of Legacy and Harmonized TCGA
Genomic Data Commons’ Data, Cell Syst. 9 (24–34) (2019), e10.
[209] ImmGen at 15. Nat Immunol 21, 700–703, 2020.