TIMI 2235 No.

of Pages 20

Trends in
Microbiology
Review

The interaction between autophagy,

Helicobacter pylori, and gut microbiota
in gastric carcinogenesis
Ali Nabavi-Rad, 1 Abbas Yadegar , 1,* Amir Sadeghi, 2 Hamid Asadzadeh Aghdaei, 3
Mohammad Reza Zali, 2 Daniel J. Klionsky , 4,5,* and Yoshio Yamaoka 6,7,8,*

Chronic infection with Helicobacter pylori is the primary risk factor for the Highlights
development of gastric cancer. Hindering our ability to comprehend the precise Autophagy orchestrates cell homeosta-
role of autophagy during H. pylori infection is the complexity of context- sis and cellular stress signaling, and im-
plements a dichotomous function in
dependent autophagy signaling pathways. Recent and ongoing progress in
different stages of cancer development.
understanding H. pylori virulence allows new frontiers of research for the
crosstalk between autophagy and H. pylori. Novel approaches toward discover- The stress signals in the tumor microen-
ing autophagy signaling networks have further revealed their critical influence on vironment differentially regulate the
autophagy pathway, subsequently mod-
the structure of gut microbiota and the metabolome. Here we intend to present a
ulating tumor immunity, progression, and
holistic view of the perplexing role of autophagy in H. pylori pathogenesis and metastasis.
carcinogenesis. We also discuss the intermediate role of autophagy in
H. pylori-mediated modification of gut inflammatory responses and microbiota Autophagy manipulation represents a
prominent strategy for intracellular
structure. Helicobacter pylori replication and timely
release of oncoproteins.

Autophagy signaling network widely

An overview of autophagy contributes to gut homeostasis persis-
Three primary types of autophagy (see Glossary) have been described based on different mech- tence, gut ecology architecture, and anti-
microbial protection.
anisms underlying cargo sequestration. Direct lysosomal uptake and degradation of cytoplasmic
components is referred to as microautophagy, while selective unfolding of individual cargo pro- H. pylori-induced dysbiosis of gut micro-
teins that are subsequently translocated into lysosomes via the action of chaperone complexes biota and metabolic profiles can provide
an infrastructure for gastric cancer devel-
is distinguished as chaperone-mediated autophagy. Finally, the main autophagic response
opment and progression.
maintaining eukaryotic cell homeostasis, macroautophagy (autophagy hereafter), extensively
contributes to the sequestration of damaged or otherwise dispensable proteins, aggregates, Although not confirmed, evidence sug-
droplets, and organelles [1]. Considering the involvement of several autophagic components in gests the potential for H. pylori to modify
the gut microbiota through autophagy
a variety of intracellular signaling networks and intercellular interactions, the detailed molecular
modulation.
mechanism of autophagy is illustrated in Figure 1.
1
Foodborne and Waterborne Diseases
Research Center, Research Institute for
Following intracellular stress and/or nutrient or energy deprivation, the initiation of basal or Gastroenterology and Liver Diseases,
adaptive autophagy is a keystone of preserving cellular homeostasis [2]. However, a Shahid Beheshti University of Medical
disrupted autophagy response can cause the aggregation of impaired cellular components, Sciences, Tehran, Iran
2
Gastroenterology and Liver Diseases
which results in cellular senescence or autophagic cell death. Typically, autophagy shuts Research Center, Research Institute for
off apoptosis, and pro-apoptotic caspase activation interferes with the autophagy flux. In Gastroenterology and Liver Diseases,
particular conditions, however, autophagy may excessively degrade the cytoplasm and facilitate Shahid Beheshti University of Medical
Sciences, Tehran, Iran
apoptosis or necrosis, bringing about autophagic cell death [3]. The intricate interplay between 3
Basic and Molecular Epidemiology of
autophagic cell death, apoptosis, and necrosis determines the pathological conditions of Gastrointestinal Disorders Research
several diseases [4]. Among the wide spectrum of cellular functions regulated by autophagy, Center, Research Institute for Gastroen-
terology and Liver Diseases, Shahid
metabolic responses may critically contribute to the orchestration of autophagic cell death and Beheshti University of Medical Sciences,
apoptosis. Tehran, Iran

Trends in Microbiology, Month 2023, Vol. xx, No. xx https://doi.org/10.1016/j.tim.2023.04.001 1

© 2023 Elsevier Ltd. All rights reserved.
 Trends in Microbiology

Nucleus
Plasma membrane

Cytoplasm
Recycling Mitochondrion h
endosome ug
Ro
E R
Membrane
sources

ATG9-containing

ATG9
vesicle NRBF2
PIK3C3 PIK3R4 PtdIns3K
Golgi Omegasome
COPII vesicle Ptd ATG14 AMBRA1 BECN1 complex I
ATG2 I
e

ns
ATG9 hor
gop

3P
Cargo Pha

1
VE
sequestration ULK1
on P

Pt

ZFY
ATG9 nsi complex

dIns3P
a
Exp Phagophore
ZFYVE1

nucleation RPTOR

I2
WIP
ATG13 RB1CC1
ATG101
Expansion
I2
IP

Initiation
W

Acidic
Autophagosome– hydrolases AMPK
Sealing
lysosome fusion
Lysosome MTOR
RAB7 DEPTOR MLST8
HOPS RPTOR
ATG4
AKT1S1
Autolysosome

MTORC1
Autophagosome SNARE-like
proteins

Degradation

LC3 UBL system

LC3 LC3 LC3-I LC3
PE
ATG3 ATG4
ATG7

ATG16L1 ATG10 ATG5

ATG5 ATG5
ATG16L1 ATG12 UBL system
ATG12 ATG12 ATG12

Trends in Microbiology

Figure 1. The molecular mechanism of autophagy flux in the host cell. Upon suppression of mechanistic target of rapamycin kinase complex 1 (MTORC1) or
stimulation of AMP-activated protein kinase (AMPK), the autophagy process initiates following the activation of the ULK1 complex. Following ULK1 activation,
membrane nucleation requires phosphatidylinositol-3-phosphate (PtdIns3P), which is produced by the lipid kinase function of the PtdIns3K-CI at the phagophore (the
initial sequestering compartment). PtdIns3K-CI subunits include ATG14, PIK3C3, BECN1, PIK3R4/VPS15/p150 (phosphoinositide-3-kinase regulatory subunit 4),
AMBRA1 (autophagy and beclin 1 regulator 1), and NRBF2 (nuclear receptor binding factor 2). Various membranous organelles such as the mitochondria, plasma
membrane, and Golgi complex donate membrane, contributing to phagophore expansion. Phagophore expansion proceeds as ATG12 conjugates to ATG5 and the
noncovalent interaction of the ATG12–ATG5 complex with ATG16L1 forms the ATG12–ATG5-ATG16L1 ternary complex. The recruitment of WIPI2 (WD repeat
domain, phosphoinositide interacting 2) and ZFYVE1/DFCP1 (zinc finger FYVE-type containing 1) by PtdIns3P promotes the conjugation of LC3 to PE
(phosphatidylethanolamine). LC3 mediates selective autophagy by interacting with cargo and further facilitates phagophore expansion and sealing. ATG4 removes LC3
from the autophagosome outer membrane and then autophagosome–lysosome fusion is mediated by HOPS, small GTPase-family proteins such as RAB7, and
soluble N-ethylmaleimide-sensitive fusion attachment protein receptors/SNAREs. The reformation of lysosomes from autolysosomes facilitates the next cycle of
autolysosome formation.

2 Trends in Microbiology, Month 2023, Vol. xx, No. xx

Trends in Microbiology

4
Autophagy and gut homeostasis Life Sciences Institute, University of
Michigan, Ann Arbor, MI 48109, USA
Autophagy and the gut epithelial barrier 5
Department of Molecular, Cellular and
The integrity of the gut epithelium has a major influence on gut homeostasis and the host’s Developmental Biology, University of
inflammatory response. Dysfunctional autophagy can impair tight junction proteins and damage Michigan, Ann Arbor, MI 48109, USA
6
Department of Environmental and Pre-
epithelial barrier integrity (Box 1), which would be vulnerable to bleeding and perforation, leading ventive Medicine, Oita University Faculty
to the development of primary gastrointestinal diseases [5]. Therefore, autophagy modulation can of Medicine, Oita, Japan
7
influence the integrity of the gut epithelium. As such, high concentrations of the proinflammatory Department of Medicine, Gastroenterol-
ogy and Hepatology Section, Baylor Col-
cytokine TNF/TNF-α (tumor necrosis factor) stimulate CLDN2 (claudin 2) expression in colonic lege of Medicine, Houston, TX, USA
8
Caco-2 monolayers and induce intestinal permeability partly by inhibiting autophagy [6]. Autophagy Research Center for Global and Local
is negatively associated with non-essential amino acid deprivation, which can lower transepithelial Infectious Diseases, Oita University,
Oita, Japan
resistance and downregulate the production of CLDN1 and TJP1/ZO-1 (tight junction protein 1),
tight junction proteins in intestinal porcine epithelial cells [7]. As H. pylori exotoxin-dependent
mitochondrial perturbations interrupt cellular metabolism and alter cellular amino acid homeostasis
*Correspondence:
[8], the effect of amino acid homeostasis disruption on the production of tight junction proteins a.yadegar@sbmu.ac.ir (A. Yadegar),
could be investigated in H. pylori-infected gastric epithelial cells. Moreover, BECN1 (beclin 1) klionsky@umich.edu (D.J. Klionsky),
promotes OCLN (occludin) endocytosis in colonic Caco-2 cells, thereby reducing the extracellular and
yyamaoka@oita-u.ac.jp (Y. Yamaoka).
presence of OCLN tight junctions in an autophagy-independent manner. However, an enhanced
autophagy response suppresses the constitutive activity of BECN1 and ultimately preserves intesti-
nal homeostasis [9]. Furthermore, autophagy signaling plays a major role in regulating the production
of adherens junction proteins (CTNNB1/β-catenin and CDH1/E-cadherin) and the degradation of
gap junctions [10]. While several studies have deepened our understanding of autophagy in intestinal
stem cells, Paneth cells, and goblet cells, much more remains to be elucidated regarding other
epithelial cell populations. Additionally, the underpinning mechanism of cytokine-oriented autophagy
dysregulation and the ultimate epithelial cell apoptosis is yet to be fully explained.

Box 1. Autophagy and gut immune homeostasis

Autophagy-mediated modulation of the host immunity occurs through maintaining immune cell homeostasis and regulat-
ing cellular activity (Figure I). Cytosolic receptors for pathogen-associated molecular patterns (PAMPs) and DAMPs, NOD
(nucleotide binding oligomerization domain containing)-like receptors (NLRs) include inflammasome subunits that
contribute to autophagy, pyroptosis, and cytokine expression. Key members of the NLR family, NOD1 and NOD2, signal
by the adaptor kinase RIPK2/RIP2 (receptor interacting serine/threonine kinase 2) to activate NFKB and MAPK
(mitogen-activated protein kinase). Additionally, NOD1 and NOD2 accelerate MAP1LC3/LC3 recruitment to phagosomes
and stimulate xenophagy [94]. H. pylori peptidoglycan (PG) can induce gastric inflammation through NOD1 activation [95].
Likewise, outer membrane vesicles (OMVs) of H. pylori can activate the NOD1-RIPK2 axis to induce autophagy and
promote the production of IL8 (interleukin 8) and CXCL1 (C-X-C motif chemokine ligand 1). Both the inflammatory and
autophagy responses require autophagosome formation as ATG5 and LC3 knockdown or knockout epithelial cells
demonstrate lower response rates [64]. Conversely, autophagy can regulate NLR activation by eliminating endogenous
NLR activators as well as NLR-forming components [96].

Whereas NLRs sense cytosolic components, cell surface TLR (toll like receptor) signals the extracellular presence of
PAMPs through MYD88 (MYD88 innate immune signal transduction adaptor), TICAM1/TRIF (TIR domain containing adap-
tor molecule 1), MAPK/ERK, and/or MAPK/JNK. TLR2 promotes the host’s innate immunity by activating MAPK and
inducing autophagy through MAPK/ERK and MAPK/JNK [97,98]. Upon lipopolysaccharide (LPS) exposure, TLR4 interac-
tion with TRAF6 accelerates lysine 63 (K63) ubiquitination of BECN1 to facilitate the formation of the class III phos-
phatidylinositol 3-kinase complex I (PtdIns3K-CI) [99]. TLR4 further takes part in xenophagy via K63 ubiquitination of
TBK1 (TANK binding kinase 1) following TRIF and TRAF3 activation to increase LC3 recruitment of Gram-negative bacteria
[100]. TLR7 stimulates mitophagy by engaging with BNIP3 (BCL2 interacting protein 3) and BECN1 and might elevate
autophagic programmed cell death through MAPK/p38 or MAP2K/MEK-MAPK/ERK signaling pathways [101].

Autophagy regulatory proteins are involved in various critical aspects of innate and adaptive immunity. Autophagy is
involved in mast cell degranulation [102], monocyte differentiation [103], eosinophil differentiation and infiltration [104],
neutrophil proliferation and differentiation [105,106], and natural killer (NK) cell development [107]. In the context of the
adaptive immune response, autophagy regulates dendritic cell presentation of endogenous antigens and cross-presentation
of exogenous antigens [108], differentiation of T helper 1 (Th1), Th2, and Th9 cells [109,110], memory T cell response [111],
B cell maturation [112], and secondary immune responses in memory B cells [113].

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 Trends in Microbiology

Autophagy and the immune response

Autophagy signaling can converge with the immune response to establish intricate signaling
networks that orchestrate immune defense strategies (Box 1). Throughout all immune cell types
and subsets, autophagy is critically involved in cellular maturation, activity, and homeostasis. Addi-
tionally, autophagy contributes to other strategies in host defense such as the engulfment and

Tauro-conjugated
bile acids

Commensals
M. tuberculosis

Lipoprotein
M. bovis
Bifidobacterium
Mucus layer CAMP Butyrate TMAO TMA E. coli
probiotic strains
Gut epithelial
TLR2
cells

Endosome LC3 Gap junction

ATG16L1
MAPK
ssRNA SQSTM1
CDH1 CTNNB1
AMPK ATG12 ATG16L1

CDKN1B ATG5
TLR7 NLRP3

CLDN1 TJP1
Autophagy Autophagy Autophagy
BNIP3
Autophagy ROS Autophagy
BECN1

Lamina propria
TLR4
LPS

Imature cDC
Apoptosis
TRAF6
ATG3
MAP1LC3-II
Maturation
BECN1
ATG16L1 ATG5

Autophagy ATG3
Neutrophil
BECN1
Macrophage

MTOR
ATG7 ATG5
PI3K
T cell Memory T cell
HIF1A
Mature DC
Antigen presentation
TLR4
ATG16L1 ATG7
LPS BECN1 ATG5
ROS
IL15

TRAF3
MTOR
HEX/E-hexosaminidase
TBK1
ATG7
Autophagy

Bioenergetic
LC3 LC3
Histamine metabolism
Mast cell Degranulation
NK cell

ATG3

Blood vessel
MTOR

ULK1
AMPK
Eosinophil
ATG7
Memory NK cell
ATG5
Monocyte

Trends in Microbiology

(See figure legend at the bottom of the next page.)

4 Trends in Microbiology, Month 2023, Vol. xx, No. xx

Trends in Microbiology

degradation of intracellular pathogens (known as xenophagy), antigen presentation/cross-

presentation, and the modulation of inflammatory responses [11]. However, the reasons for the
difference in the sensitivity of immune cells to the loss of autophagy remain unanswered.

The extracellular presence of danger/damage-associated molecular patterns (DAMPs) that

include nucleic acid, ATP, and HMGB1 (high mobility group box 1) can potentially regulate
autophagy flux and inflammatory response [12]. H. pylori-induced expression of HMGB1 triggers
gastric inflammation through NFKB/NF-κB activation [13]. As the extracellular release of HMGB1
can be detected by AGER/RAGE (advanced glycation end-product specific receptor) and sup-
press the MTOR (mechanistic target of rapamycin kinase) complex, the primary negative
regulator of autophagy, H. pylori infection may stimulate autophagy through the excessive accu-
mulation of HMGB1 as well as the increased rate of cellular apoptosis and necrosis [14,15].
Reactive oxygen species (ROS), a key mediator of H. pylori pathogenesis, promote the
cytosolic translocation of HMGB1 wherein it prevents BECN1 suppression by BCL2 (BCL2
apoptosis regulator) and ultimately induces an autophagic response [16]. The inhibition of
calpain-mediated cleavage of BECN1 and ATG5 (autophagy related 5) represents another
autophagy-inducing mechanism for cytosolic HMGB1 [17].

Given the particular significance of autophagy in the immune response, many conceptual and tech-
nical questions remain to be answered. How can we explain the mechanistic basis of the disparate
impact of autophagy deletions on different immune cells or subsets? Particularly, what source of
metabolites regulates the autophagic signaling pathways in each immune cell? One major obstacle
hindering the resolution of these knowledge gaps is the complexity of detecting autophagosome
contents during autophagy flux. Thus, the development of novel techniques that would be able
to do so is of great importance in addressing several pending issues in the field.

Autophagy and the gut microbiota

The overwhelming majority of the human microbiota reside within the gastrointestinal tract, collec-
tively referred to as the gut microbiota. These microorganisms are fundamentally important for
various host physiological functions. Therefore, preserving the inherent composition of the gut
microbiota is of great significance, and an aberrant profile of the gut microbiota (known as
dysbiosis) may lead to various gastrointestinal and extragastrointestinal disorders [18]. Dysfunc-
tional autophagy is associated with gut dysbiosis, which might also be the case for tumor micro-
biota and its influence on the immune landscape of the tumor microenvironment (TME).

ATG7 deficiency of intestinal epithelial cells in mouse models was demonstrated to cause intes-
tinal dysbiosis characterized by increased abundance of Firmicutes Gram-positive bacteria and a

Figure I. Autophagy and gut immune homeostasis. The gut microbiota, particularly probiotic strains, can induce
autophagy, whereas pathogenic bacteria mainly suppress autophagy flux. Autophagy activation preserves the integrity of
the epithelial barrier by stimulating the production of tight junctions while suppressing gap junctions. Autophagy-mediated
secretion of CAMP and inhibition of reactive oxygen species (ROS) production and NLRP3 inflammasome activation
reduce the risk of inflammation. Conversely, phosphoinositide 3-kinase/PI3K and the mechanistic target of rapamycin
kinase complex 1 (MTORC1)–HIF1A/HIF-1α (hypoxia inducible factor 1 subunit alpha) axis engage with ROS production
by neutrophils. ATG7 contributes to the degranulation of mast cells. The MTOR complex is involved in eosinophil
differentiation and infiltration. MTOR also activates bioenergetic metabolism via interleukin (IL)15 signaling in natural killer
(NK) cells, whereas ATG3 is required for memory NK cell formation. The activation of AMPK, ULK1, ATG5, and ATG7
directs monocytes away from apoptosis and toward differentiation. ATG3, ATG16L1, and MAP1LC3-II regulate dendritic
cell (DC) maturation, whereas ATG5, ATG7, ATG16L1, and BECN1 coordinate antigen presentation and cross-
presentation. Furthermore, lipopolysaccharide (LPS)-mediated stimulation of Toll-like receptor 4 (TLR4) in macrophages
can trigger different autophagy signaling pathways. Abbreviations: CAMP, cathelicidin antimicrobial peptide; MAPK,
mitogen-activated protein kinase; TMA, trimethylamine; TMAO, trimethylamine N-oxide.

Trends in Microbiology, Month 2023, Vol. xx, No. xx 5

 Trends in Microbiology

reduced proportion of Pseudomonadota/Proteobacteria in mice stool samples [19]. Colonic Glossary

epithelial cell-specific atg7 conditional knockout mice further exhibit higher fecal DNA loads, as Apoptosis: a type of regulated cell
well as enriched bacterial populations compared to the control group [20]. Similar outcomes death that involves the action of
caspases.
are seen with an increased number of proinflammatory bacteria due to autophagy failure in the Autophagic cell death: autophagy-
intestine of atg5 knockout mice [21] and in a mouse model for the ATG16L1T300A polymorphism, mediated induction of apoptosis or the
which contributes to the development of inflammatory bowel disease [22]. activation of a distinct autophagy-
dependent mechanism that leads to cell
death.
Pathogenic bacteria are considered targets of xenophagy, through which autophagy prevents Autophagy: processes involved in the
bacterial infection and directly influences gut dysbiosis. Intracellular restriction of bacterial replica- lysosomal degradation and recycling of
tion is mainly executed by Atg8-family protein recognition of receptors that target bacterial com- cytoplasmic components.
Autophagy-related genes: these
ponents or ubiquitin on the bacterial surface, leading to sequestration by a phagophore [23].
genes encode the components of the
Furthermore, Paneth cell secretion of lysozyme is a key antimicrobial defense occurring via secre- macroautophagy machinery. The
tory autophagy; therefore, autophagy deficiency can ultimately disrupt the defensive mechanism acronym ‘ATG’ indicates 'autophagy
of Paneth cells and promote intestinal inflammation [24,25]. related', but some components such as
BECN1 are also considered to be part of
this group.
In the context of autophagy gut microbiota interaction, efforts have been made to develop BECN1 (beclin 1): a component of the
autophagy-regulating therapeutics. Notably, some derivatives of dietary products such as vitamin class III PtdIns3K that generates
D modulate autophagy and modify the gut microbial structure, including H. pylori decolonization phosphatidylinositol-3-phosphate; this
lipid is required for autophagy.
[26]. However, one issue concerns the huge intraindividual variation of the gut microbiota profile cag pathogenicity island (cag PAI): a
and how this affects H. pylori virulence. Accordingly, the development of individually tailored ther- 40-kb genomic DNA segment that
apeutics appears mandatory. encodes the T4SS; these genes are the
most well-recognized virulence
determinants of H. pylori.
H. pylori infection Cytokine: a small protein secreted by
The infection-driven nature of most gastric cancers has encouraged researchers to seek for potential different types of cells including immune
carcinogenic signaling networks affecting cancer development and progression [27]. The high and endothelial cells that interact with
cell surface receptors to trigger various
pathogenicity of H. pylori along with its genetic diversity and the presence of various virulence factors
responses within the target cells.
have challenged our understanding of the definite mechanisms underpinning H. pylori-induced Dysbiosis: aberrant compositional and
gastric carcinogenesis. Chronic inflammation, mucosal damage, genetic and epigenetic alteration, functional modification of the microbiota
and modification in gene expression are the foremost characteristics of H. pylori infection resulting that disturbs the indigenous microbial
ecosystem.
in gastric tumorigenesis [28]. H. pylori pathogenesis mainly relies on the detrimental effect of virulence
Inflammasome: a cytoplasmic
factors VacA (vacuolating cytotoxin A) and CagA (cytotoxin-associated gene A). Intracellular VacA ex- multiprotein complex that detects
erts several cellular effects including cell vacuolation to increase the longevity of infection, as well as pathogenic microorganisms and danger
mitochondrial stress and perturbation, and apoptosis [29]. The injection of CagA oncoprotein into signals in the host cells.
Mitophagy: a type of selective
the host cell by a type IV secretion system (T4SS), encoded by cag pathogenicity island
autophagy that degrades dysfunctional
(cag PAI), impairs cellular proliferation and the apoptotic process. However, a noteworthy character- or damaged mitochondria.
istic of H. pylori infection lies in its capacity for immune escape. H. pylori-induced alteration in the MTOR (mechanistic target of
expression of autophagy-related genes and manipulation of autophagy flux and apoptotic process rapamycin kinase): MTOR regulates
cell growth and is the primary negative
are of great significance for its survival, replication, and pathogenesis. Autophagy flux is disrupted regulator of macroautophagy.
and utilized by H. pylori to flee from host immunity and antibiotic exposure [30]. Moreover, H. pylori Phagophore: the initial sequestering
modulation of autophagy and subsequent alteration of cellular apoptosis contributes to the etiology compartment of macroautophagy; the
of gastritis, peptic ulcers, and neoplasia. The interplay between autophagy and apoptosis in the phagophore surrounds cargo and
matures into a double-membrane
acute and chronic stages of H. pylori infection is yet to be elucidated. In the next section, we autophagosome.
meticulously discuss the cross-talk between autophagy, H. pylori, and its virulence factors. Pyroptosis: a lytic type of cell death
triggered by inflammasome stimulation
Autophagy and H. pylori infection and consequent inflammatory caspase
activation.
H. pylori is attached to the plasma membrane during the early exposure of macrophages, den- Reactive oxygen species (ROS):
dritic cells, and gastric epithelial cells, whereas at 12 h of post-infection in vitro, H. pylori bacteria chemical byproducts derived from oxygen
are mainly internalized into the cytoplasm, residing within autophagosomes; this localization al- including peroxides, superoxide, and
hydroxy radical; these highly reactive
lows the bacteria to avoid acidic conditions, and resist bactericidal treatments, and impairs the
species can damage cellular components.
cellular immune response [31,32]. Thus, researchers have envisioned the development of novel

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drug-delivery systems that target intracellular pathogens, using immune-cell-derived exosomes Tumor microenvironment (TME): the
dynamic surrounding microenvironment
and nanotechnology. of tumor cells that is spatially and
temporally modified in response to
Acute H. pylori infection predominantly induces autophagosome formation while at the same time environmental factors and antitumor
interfering with autophagy flux to establish intracellular niches for bacterial replication [33] therapeutics.
Type IV secretion system (T4SS): a
(Figure 2). The nutrient requirement of H. pylori can further explain autophagy stimulation as bacterial protein complex located in the
this pathogen shifts certain metabolic pathways within the host cell. The upregulation of the tricar- plasma membrane that transports
boxylic acid (TCA) cycle and amino acid metabolism are reported as the in vitro characteristic of protein and DNA into the extracellular
AGS cells 6 h post-infection, which can regulate the MTOR complex 1 (MTORC1) signaling path- space.
Xenophagy: a selective form of
way in the gastric epithelium and immune cells [34,35]. macroautophagy involving the
elimination of intracellular microbes.
The H. pylori type 1 capJ (capsular polysaccharide biosynthesis protein J) gene encodes
cholesterol-α-glucosyltransferase (CGT) that transforms cellular cholesterol into cholesteryl

H. pylori

Mucus NH4+
layer
Urease NH4+
Integrin VacA Plasma
NH4+ OMV CGT
membrane

ILK Vacuolation

GSH ER stress O-glycans MIR99B

LRP1
PG
Mitochondrial
perturbation
RHOA

RAC1 ROS EIF2S1 Lysosomal

CASP7
damage
PARP
MTORC1
NOD1 RIPK2 LGALS8
MAPK

Autophagy AKT ATF4 STAT3

Apoptosis Autophagosome–
lysosome fusion
Cytoplasm

GJA1

MDM2 TRIB3

CagA

MTOR TP53 Autophagy

Trends in Microbiology

Figure 2. Autophagy and acute infection with Helicobacter pylori. H. pylori suppresses ILK (integrin linked kinase) to induce autophagy and apoptosis in
the host cell. Urease-mediated accumulation of ammonia inhibits vacuolating cytotoxin A (VacA) degradation and augments VacA-induced apoptosis. The interaction of VacA
with LRP1 stimulates reactive oxygen species (ROS) production, endoplasmic reticulum (ER) stress, apoptosis, and autophagy, which leads to cytotoxin-associated gene A (CagA)
degradation. Similarly, cholesterol-α-glucosyltransferase (CGT) and outer-membrane vesicle (OMV)-derived peptidoglycan (PG) induce autophagy; meanwhile, CGT prevents
autophagosome–lysosome fusion. H. pylori-mediated mitochondrial perturbation, lysosomal damage, and MIR99B upregulation further trigger an autophagy response.

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glucosides to stimulate autophagy, while interfering with lysosome–autophagosome fusion in mac-

rophage cell line J774A.1 [36]. Cholesteryl 6′-O-acyl-α-D-glucoside augments the H. pylori-induced
autophagy response by inhibiting autophagosome degradation and suppressing lysosome biogen-
esis [37]. Autophagy induction may facilitate H. pylori-induced gastric carcinogenesis. For example,
excessive production of ROS contributes to the stimulation of the NFE2L2/Nrf2–HMOX1/HO-1
axis, which is considered a mechanism underlying H. pylori-associated gastric carcinogenesis via
autophagy induction [38]. The progression of gastric cancer, however, mainly relies on the persis-
tence of H. pylori infection, which is characterized by the suppression of the autophagy response
[39] (Figure 3). Through the NOD1–RIPK2–MAPK/ERK–FOXO4 pathway, prolonged exposure of
gastric epithelial cells to H. pylori lysate promotes cellular proliferation, inhibits autophagy and apo-
ptosis, and suppresses the production of CCL20, CCL28, and CXCL2 [40]. Furthermore, the infec-
tion of gastric epithelial cell lines and gastric mucosal tissue of patients with H. pylori is accompanied
by MIR30B (microRNA 30b) upregulation and consequent suppression of ATG12 and BECN1 [41].
MIR30D was further described in AGS and GES-1 cells as the regulator of core autophagy proteins
ATG2B, ATG5, ATG12, BECN1, and BNIP3L (BCL2 interacting protein 3 like), through which
H. pylori suppresses the autophagy pathway in the gastric epithelium [42]. However, MIR99B over-
expression in H. pylori-infected gastric cancer tissues substantially induces autophagy and inhibits
cellular proliferation via regulating the MTOR signaling pathway [43]. Likewise, H. pylori-induced
overexpression of MIF (macrophage migration inhibitory factor) in the serum and gastric epithelium
is associated with gastric cancer and intestinal metaplasia, respectively. Yet, MIF probably contrib-
utes to the H. pylori induction of autophagy as the MIF concentration is correlated with autophagy
markers LC3A, LC3B, and ATG5 [44].

The convergence of bacteria with autophagy illustrates the double-edged nature of this process.
That is, autophagy can be used to eliminate bacteria via xenophagy, but H. pylori promotes au-
tophagy – while blocking complete autophagic flux – to establish a replicative niche. Later on,
however, H. pylori may suppress autophagy, which disrupts cellular homeostasis and promotes
carcinogenesis. As clinical manifestations attributed to chronic H. pylori infection, gastric tissue
samples predominantly present autophagy suppression, whereas in vitro experiments or the ex-
posure of organoids to H. pylori within several hours mainly present autophagy induction. Owing
to this type of complexity, H. pylori interaction with the autophagy response requires further elu-
cidation. To simplify the multivariate structure of this interaction, several studies have evaluated
the specific effect of H. pylori virulence factors on the autophagy flux, as further described in
the following text (Table 1).

VacA
To provide the nutrients required for H. pylori colonization and render intracellular niches for
H. pylori replication, VacA inhibits MTORC1 and induces autophagy through mitochondria pertur-
bation and subsequent depletion of cellular amino acids [8]. The exposure of the SGC7901
human gastric cancer cell line to VacA leads to increased production levels of ROS, BECN1,
ATG7, and PIK3C3/VPS34 (phosphatidylinositol 3-kinase catalytic subunit type 3) [45]. VacA is
sufficient for autophagy formation and its channel-forming activity significantly influences
autophagy initiation [46]. Disruption of cellular osmotic properties and stimulation of V-ATPase
activity describes the potential mechanism through which VacA induces endolysosomal LC3
lipidation and noncanonical autophagy in MCF10A human breast epithelial cells [47], which
should also be investigated in gastric epithelial cells. VacA further prevents endolysosomal
trafficking by targeting the lysosomal calcium channel MCOLN1/TRPML1 and disrupting CTSD
(cathepsin D) delivery to lysosomes in the gastric epithelium of mouse models [48]. Sustained ex-
posure of AGS and mouse gastric cells to VacA triggers the formation of autophagosomes defi-
cient in CTSD and consequently results in the accumulation of ROS and the SQSTM1 autophagy

8 Trends in Microbiology, Month 2023, Vol. xx, No. xx

Trends in Microbiology

receptor [49]. VacA is normally degraded in the acidified lysosome; importantly, accumulation of
VacA can lead to apoptosis presented in different gastric epithelial cell lines [50].

CagA
CagA injection into the epithelial cells by the T4SS leads to genomic instability, tumor-promoting
inflammation, and sustained proliferation of tumor cells [51]. Furthermore, T4SS-dependent
translocation of H. pylori DNA into epithelial cells activates STING1 (stimulator of interferon

H. pylori MIF

Macrophage

Mucus
layer
HpGGT CagA Hp0175
VacA Plasma
MET membrane

LRP1
ATF4 NOD1 RIPK2
MIR30D MIR30B
ICD Lysosome

DDIT3 BECN1 MAP3K7

ATG12

Ca 2+
Cytoplasm

MCOLN1 ATG5 MAPK/ERK

BNIP3L ATG2B

CTSD

FOXO4
Endolysosomal
MCOLN1 inhibition
trafficking
Autophagy BCL6
MTORC1
BNIP3

ATG12
CagA TRIP12 CDKN2A
accumulation

Apoptosis
MIR543 SIRT1

CAPZA1
ATG5
LAMP1 MIF
LC3

Nucleus

Trends in Microbiology

Figure 3. Autophagy and chronic infection with Helicobacter pylori. Sustained exposure of the host cell to H. pylori mainly results in autophagy suppression. In the
chronic stage of H. pylori infection, vacuolating cytotoxin A (VacA) and secretory protein HpGGT disrupt endolysosomal trafficking, leading to CagA accumulation and
autophagy inhibition. H. pylori-mediated activation of NOD1 and expression of MIR30B and MIR30D further prevent autophagy flux. However, secretory protein
Hp0175 and H. pylori-induced secretion of MIF from macrophages might induce the autophagy process in the host cell. Abbreviations: MET, MET proto-oncogene,
receptor tyrosine kinase; MIF, macrophage migration inhibitory factor.

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10

Table 1. H. pylori-mediated mechanisms of autophagy regulation

Modulatory H. pylori Experimental model Mediators Molecular mechanism Outcome Refs
Trends in Microbiology, Month 2023, Vol. xx, No. xx

agent strain/isolate
H. pylori (104 ATCC 700392 J774A.1 macrophage cells ↑BECN1, LC3-II, Cholesterol modulation ↑Autophagy, bacterial [36]
CFU/ml) and SQSTM1, EEA1, survival, lipid raft formation
recombinant ATG12 ↓Lysosome formation,
CGT (10 μg/ml) ↓LAMP1 autophagosome–lysosome
fusion
H. pylori (104–6 ATCC 700392 AGS gastric cancer cells, C57BL/6 ↑LC3-II - ↑Autophagy, H. pylori [37]
CFU/ml) and male mice intercellular survival
CAG (20 μM) ↓Lysosome formation,
autophagosome
degradation
H. pylori (200 ATCC 700392, AGS gastric cancer cells ↑LC3-II, Lysosomal damage, O-glycan and LGALS8 ↑Autophagy [114]
and 400 MOI) NCTC 11637 CALCOCO2/NDP52 accumulation
H. pylori (100 ATCC 700392 GES-1 gastric epithelial cells, clinical ↑LC3-II, PAK1, ILK suppression, F-actin depolymerization ↑Autophagy [115]
MOI) specimens, SPF grade BALB/c male ROCK1, ROCK2,
mice LIMK1
↓RAC1, RHOA, CFL
(cofilin)
H. pylori (100 ATCC 43504 AGS gastric cancer cells ↑LC3-II STAT3 phosphorylation on Ser727 ↑Autophagy, mitochondrial [116]
MOI) damage
H. pylori (100 ATCC 43504, SS1 AGS gastric cancer cells, female ↑LC3-II, ROS production ↑Autophagy [38]
MOI) C57BL/6J mice NFE2L2/Nrf2, ↓Apoptosis
HMOX1/HO-1
H. pylori (6 × ATCC 43504 MKN-45 gastric cancer cells, GES-1 ↑SQSTM1 Targeting ↑Proliferation [40]
106 CFU/ml) or gastric epithelial cells, Mongolian ↓LC3-II, CASP3 NOD1-RIPK2-MAPK/ERK-FOXO4 pathway, ↓Autophagy, apoptosis,
H. pylori lysate gerbils (caspase 3), SLPI CCL20, CCL28, and CXCL2 decreased in GES-1 cell migration
(1.5–2 μg/ml) GES-1 cells but increased in MKN-45 cells
H. pylori (100 ATCC 700392 AGS and HGC-27 gastric cancer ↑SQSTM1 MIR30B upregulation, ATG12 and BECN1 ↑H. pylori intercellular [41]
MOI) cells, chronic gastritis clinical ↓LC3-II suppression survival
specimens ↓Autophagy
H. pylori ATCC 700392 AGS gastric cancer cells, GES-1 ↓LC3-II, ATG2B, MIR30D upregulation ↑H. pylori intercellular [42]
gastric epithelial cells ATG5, ATG12, survival
BECN1, BNIP3L ↓Autophagy

Trends in Microbiology
H. pylori (100 ATCC 700392 BGC-823 gastric cancer cells ↑LC3-II MIR99B upregulation, MTOR suppression ↑Autophagy [43]
MOI) ↓Proliferation, H. pylori
intercellular survival
H. pylori – Gastric dysplasia and cancer clinical ↑LC3A, LC3B, MIF upregulation ↑Autophagy, risk of cancer [44]
specimens ATG5 development
H. pylori (100 ATCC 49503, ATCC HEK293T, AGS, NCI-N87 and ↑LC3-II MTORC1 suppression, ULK1 activation ↑Autophagy, mitochondrial [8]
MOI), VacA 700392 AZ-521 cell lines, C57BL/6J mice ↓p-RPS6KB/S6K damage
(35–250 nM) ↓Cell energy and nutrients
H. pylori (100 ATCC 43629 SGC-7901 gastric cancer cells ↑LC3-II, BECN1, ROS production ↑Autophagy [45]
MOI), H. pylori ATG7, PIK3C3
supernatant ↓SQSTM1
(1:25)
 Trends in Microbiology
VacA (10 μM) – MCF10A human breast epithelial ↑LC3-II V-ATPase-dependency of LC3 lipidation ↑Autophagy [47]
cells
H. pylori (50 ATCC 49503, J166, AGS gastric cancer cells, gastric ↑LC3-II MCOLN1 inhibition, lysosomal calcium ↑H. pylori intercellular [48]
MOI) SS1 organoid, C57/Bl6 mice ↓CTSD dysregulation survival
↓Endolysosomal trafficking
H. pylori ATCC 49503 AGS gastric cancer cells, murine ↑SQSTM1, LC3-II ROS production ↑Autophagy [49]
primary gastric cells ↓CTSD ↓Endolysosomal trafficking,
H. pylori intercellular
survival
H. pylori (100 cagA−/vacA s1m1 AGS and AZ-521 gastric cancer ↑LC3-II, EIF2S1 ER stress, EIF2S1 phosphorylation, DDIT3 ↑Autophagy, autophagic [117]
MOI), purified and cagA−/vacA cells, C57BL/6 mice and TRIB3 upregulation cell death
VacA (100 s1m2 H. pylori
ng/ml) clinical isolates
Purified VacA ATCC 49503 AGS and AZ-521 gastric cancer cells ↑LC3-II, CASP7 LRP1 stimulation, PARP activation ↑Autophagy, apoptosis [118]
(100 nM)
Wild-type or ATCC 49503 AZ-521 gastric cancer cells, clinical ↑LC3-II, CASP9 GJA1/Cx43 accumulation in autophagic ↑Autophagy, apoptosis [50]
heat-- specimens ↓GSH vesicles, RAC1-GTP formation, MAPK/ERK
inactivated activation
VacA (120 nM)
H. pylori (50 ATCC 700392 (VacA AGS gastric cancer cells ↑LC3-II LRP1 stimulation, ROS production, MDM2 ↑Autophagy, CagA [54]
MOI) s1m1) ↓GSH, SQSTM1 activation, AKT activation degradation
Purified VacA ATCC 49503 AGS gastric cancer cells ↑SQSTM1 Suppression of CagA proteasome ↑CagA accumulation [55]
(35 nM) ↓LC3-II degradation ↓Autophagy
H. pylori ATCC 43504 GES-1 gastric epithelial cells, ↑SQSTM1, γH2AX, S-phase arrest, CagA-dependent DNA ↑DNA damage, DNA [56]
C57BL/6 mice, Mongolian gerbils TP53 damage damage response
↓RAD51 ↓Autophagy
H. pylori (100 B128, 7.13 AGS and SNU-1 gastric cancer cells, ↑TRIP12 CDKN2A/p14ARF ubiquitination and ↑CagA-dependent TRIP12 [57]
MOI) clinical specimens ↓LC3-II, BECN1, degradation induction
ARF ↓Autophagy
H. pylori (50 ATCC 700392 AGS gastric cancer cells, Mongolian ↑CAPZA1 LRP1 stimulation, LRP1-ICD nuclear ↑CagA accumulation [119]
MOI/109 gerbils, gastric adenocarcinoma clini- ↓LAMP1 translocation, CAPZA1 linkage to LRP1-ICD ↓Autolysosome formation
CFU/ml) cal specimens
Trends in Microbiology, Month 2023, Vol. xx, No. xx

H. pylori (10, ATCC 43504, AGS gastric cancer cells, dyspeptic ↑SQSTM1 MET/c-Met-AKT activation CagA suppression of [82]
50, 100, 200 cagA−/vacAs1m2 clinical specimens ↓LC3-II, LAMP1 autophagosome formation
MOI) and and starvation-induced
cagA+/vacAs1m2 autophagy
clinical isolates
H. pylori (100 ATCC 700392, SNU1, AGS, MGC-803, and MKN1 ↑SQSTM1, CagA-dependent MIR543 upregulation, ↑EMT, cell proliferation, [58]
MOI/108 CFU) ATCC 49503, ATCC gastric cancer cells, C57BL/6 mice, CDH2/N-cadherin, SIRT1 suppression metastasis, invasion
51932 gastric cancer clinical specimens SNAI/Snail ↓Autophagy
↓LC3-II
H. pylori (100 ATCC 700392, AGS, BGC-823, SGC-7901 and ↑SQSTM1, LC3-II SIRT1 suppression ↑Autophagosome [120]
MOI) ATCC 43504 GES-1 cell lines, superficial gastritis, ↓RUNX3 formation
atrophic gastritis and dysplasia ↓Autophagy
clinical specimens
(continued on next page)
11
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Table 1. (continued)
Trends in Microbiology, Month 2023, Vol. xx, No. xx

Modulatory H. pylori Experimental model Mediators Molecular mechanism Outcome Refs

agent strain/isolate
H. pylori (100 ATCC 43504 AGS, GES-1, and HGC-27 cell lines, ↑p-MTOR, p-AKT, CagA-dependent MTORC1 activation ↑Inflammation [59]
MOI) C57BL/6 mice, clinical specimens p-RPS6KB/S6K, ↓Bacterial colonization
DEPTOR burden
H. pylori (50, ATCC 700392 AGS gastric cancer cells ↑LC3-II, ULK1, UPR activation ↑Autophagy [60]
100 MOI), ATG5, BECN1, ↓Apoptosis
HP0175 EIF2AK3/PERK,
(1 μg/ml) ATF4, DDIT3/CHOP
↓CASP3
H. pylori (100 ATCC 700392 AGS gastric cancer cells, GES-1 ↓CTSD Lysosomal damage ↑H. pylori intercellular [61]
MOI), H. pylori gastric epithelial cells survival
supernatant ↓Autophagy
(1:25)
Purified OMV 251 HeLa and AGS cell lines, primary ↑LC3-II, ATG5 NOD1 stimulation, RIPK2 activation ↑Autophagy [64]
(50 mg/ml) epithelial cells
H. pylori (10 B128, 7.13 AGS and HEK 293 cell lines, ↑LC3-II pgdA-mediated peptidoglycan ↑Autophagy [65]
MOI) Mongolian gerbils deacetylation

Trends in Microbiology
Trends in Microbiology

response cGAMP interactor 1). Recruitment of autophagy-inducing effectors following the

detection of cytosolic DNA fragments is an innate immune response by STING1 complexes.
However, it has been recently demonstrated that H. pylori suppresses STING1 signaling while
the H. pylori-induced autophagic response remains intact [52].

As an innate immune mechanism, autophagy can degrade the CagA protein preventing its
proinflammatory and precancerous activity. The autophagy mediator LRP1 prevents CagA accu-
mulation in patients with noninvasive differentiated-type gastric cancer; consequently, LRP1 mu-
tation significantly contributes to the progression of gastric cancer through CagA-mediated
signaling pathways [53]. Notably, the VacA m1 allelic type can activate autophagy in gastric ep-
ithelial cells, resulting in consequent CagA degradation [54]. However, upon sustained exposure,
as noted previously, VacA perturbs endolysosomal trafficking, which leads to the formation of au-
tophagosomes deficient in CTSD. Thus, in addition to promoting the intracellular replication of
H. pylori, VacA facilitates CagA accumulation during chronic infection with H. pylori [55].

CagA can contribute to carcinogenesis in several other ways, as follows: (i) gradual suppression
of autophagy by CagA in the chronic stage of H. pylori infection leads to the accumulation of
SQSTM1, which reduces RAD51 expression. This can cause the aggravation of the DNA damage
response owing to the induction of double-strand breaks and dysfunctional DNA repair, which
can ultimately induce intestinal metaplasia and gastric cancer [56]. (ii) CagA promotes the produc-
tion of the TRIP12 protein that induces the ubiquitination and degradation of the tumor suppres-
sor CDKN2A/p14ARF. The deficiency of CDKN2A can compromise the oncogenic stress
response and further inhibit autophagy in a TP53-independent manner [57]. (iii) CagA promotes
MIR543 expression to target SIRT1 (sirtuin 1) and thereafter inhibit autophagy flux [58]. (iv) In a
CagA-dependent manner, H. pylori promotes MTORC1 activation, which inhibits autophagy
and upregulates the expression of pro-inflammatory cytokines IL1B, IL6, TNF, CCL7,
and CXCL16, as well as antimicrobial peptide CAMP/LL37 to reduce the gastric bacterial
burden [59].

Secretory proteins
H. pylori Hp0175 (peptidyl-prolyl cis-trans isomerase) contributes to H. pylori stimulation of
autophagy through upregulating the production of ULK1 (unc-51-like autophagy activating
kinase 1), ATG5, BECN1, and MAP1LC3B independently of VacA vacuolating activity. The
exposure of AGS cells to Hp0175 results in high expression levels of ATF4 (activating
transcription factor 4) and DDIT3/CHOP (DNA damage inducible transcript 3); as transcription
factors, ATF4 regulates MAP1LC3B, DDIT3, ULK1, and ATG5, whereas DDIT3 regulates ATG5
expression. In gastric epithelial cells, Hp0175 can also induce the conversion of LC3-I to LC3-II
through the unfolded protein response (UPR) activation of EIF2AK3/PERK [60].

H. pylori HpGGT (γ-glutamyl transpeptidase) is involved in the pathogenesis of almost all H. pylori
strains and is required for H. pylori internalization into AGS and GES-1 gastric epithelial cells. In
AGS cells, but not in GES-1 cells, HpGGT inhibits the late stages of autophagy influx by reducing
the activity of CTSB (cathepsin B) in the lysosome and inducing lysosomal permeabilization;
however, lysosome acidification remains intact [61]. Therefore, HpGGT inhibition of autophagy
and induction of oxidative stress and genome instability renders a favorable environment for the
development of gastric adenocarcinoma.

Outer membrane vesicles

The bilayered lipid membrane nanostructures of outer membrane vesicles (OMVs) contain a wide
spectrum of bacterial biological contents including peptidoglycan (PG), lipopolysaccharide (LPS),

Trends in Microbiology, Month 2023, Vol. xx, No. xx 13

 Trends in Microbiology

outer membrane proteins, periplasmic proteins, enzymes, DNA, and toxins [62]. The introduction
of H. pylori PG fragments to gastric epithelial cells through OMVs or the T4SS can induce a
proinflammatory response through NOD1 receptor stimulation and NFKB signaling activation
[63]. Using LC3 or ATG5 siRNA-transfected AGS cells, an in vitro study elucidated the significant
involvement of autophagic components in the H. pylori OMV-induced inflammatory response
through NOD1. NOD1 migration following the detection of PG-containing OMVs is independent
of NFKB activation, yet intracellular migration of NOD1 is regulated by RIPK2. Thus, OMV-
delivered PG can signal through the NOD1-RIPK2 axis to induce autophagosome formation
and stimulate a proinflammatory response [64]. Mutation in the H. pylori PG deacetylase PgdA
reduces H. pylori intracellular survival, attenuates the autophagy response through the PG-
NOD1 axis, and increases lysosome formation independent of autophagy pathways [65].

Taken together, many molecular mechanisms in the interaction of H. pylori and its virulence fac-
tors with autophagy core proteins remain to be elucidated. For instance, several aspects of the
interplay between the MTOR signaling network, VacA, and host cells’ energy metabolism require
further in-depth investigation. Furthermore, the fate of CagA translocation by OMVs into the host
cell during acute H. pylori infection remains to be addressed. However, the main issue is the
discrimination of acute and chronic stages of H. pylori infection. Despite the resolution of key
autophagy signaling pathways during H. pylori infection, the precise role of autophagy in gastric
cancer differentiation and metastasis is contentious. The conceptual fog around the functional
influence of autophagy on H. pylori carcinogenesis is retained by a lack of adequate
mechanism-oriented clinical studies.

Autophagy and gastric carcinogenesis

From the acute to chronic stages of infection, H. pylori can contribute in different ways to promote
the risk of cancer progression (Figure 4). Acute infection with H. pylori is mainly accompanied by
autophagy stimulation and the coexpression of the gastric cancer stem cell marker CD44,
which is associated with the mesenchymal phenotype and metastatic properties of epithelial–
mesenchymal transition (EMT) [66]. However, autophagy inhibition in the chronic stages of
H. pylori infection may establish the major signaling network contributing to gastric carcinogenesis.
For example, the accumulation of SQSTM1 in gastric epithelial cells following autophagy disruption
can alter NFKB regulation [49], and, as noted in the section on CagA in the preceding text, reduce
RAD51 expression, induce double-strand breaks, and prevent DNA repair [56]. H. pylori can further
induce genomic instability by oxidative stress and ROS aggregation as a consequence of autophagy
impairment [67]. Aggravation of the DNA damage response and genomic instability can stimulate
EMT unequivocally. Conversely, H. pylori suppression of MIR1298-5p and autophagy promotes
gastric cancer cell proliferation and motility through targeting the MAP2K6-MAPK/p38 axis in
human and mouse gastric tissues [68]. Methylation silencing of the MAP1LC3A variant 1
(MAP1LC3Av1) by H. pylori impairs the autophagy response and induces the survival, migration,
and invasion of the normal gastric epithelium in rats [69]. More precisely, CagA-induced expression
of MIR543 in human gastric cancer tissue was shown by in vitro experiments to suppress SIRT1 and
autophagy, leading to EMT, cell migration, and invasion [58]. Moreover, ATG16L1T300A polymor-
phism is considered a risk factor for the development of gastric cancer [70]. Regulation of
H. pylori-induced ER stress and proinflammatory responses may explain the underlying
mechanisms associating the genetic polymorphism with the development of premalignant lesions
of gastric cancer [71]. Notwithstanding the suppressing role of autophagy in gastric carcinogenesis,
autophagy is required for tumor angiogenesis in human gastric cancer SGC7901 cells [72].

As noted previously, a major complexity concerns the context-dependent dual role of autophagy
in gastric carcinogenesis – autophagy can inhibit and promote tumor formation depending on

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Trends in Microbiology

(A) (B)
Acute H. pylori infection
Gastric
epithelium

Neutrophil

Autophagy
DC T cell Blood
Macrophage vessel

Chronic gastritis
CD44 expression Angiogenesis

EMT

Apoptosis

Proliferation, metastasis and invasion

(C)
Chronic H. pylori infection
Collagen

Dysplasia

Autophagy
MIR1298-5p

DNA repair ROS SQSTM1 MAP2K6

Tumor cells Necrosis

DNA damage NFKB MAPK/p38

EMT Inflammation Proliferation, metastasis and invasion

Proliferation, metastasis and invasion

Carcinoma

Trends in Microbiology

Figure 4. Helicobacter pylori-induced gastric cancer through autophagy modulation. (A) Epithelial–mesenchymal transition (EMT) progression from chronic
gastritis to dysplasia and carcinoma. (B) Acute H. pylori infection induces autophagy that can promote angiogenesis and the expression of the CD44 tumor marker.
(C) Chronic H. pylori infection mainly suppresses autophagy, resulting in DNA damage, inflammation, and EMT progression.

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 Trends in Microbiology

the stage of development and specific conditions. Autophagy is essential for angiogenesis and
tumor cells’ energy metabolism, for which autophagy inhibitors (e.g., chloroquine) are being de-
veloped as antitumor therapeutics [73]. Yet, H. pylori suppresses autophagy in the last stages
of infection, which promotes gastric carcinogenesis. The mechanistic role of H. pylori-induced
autophagy in EMT and the development of cancer stem cell features should be considered for
anticancer therapeutics.

Autophagy as the mediator of H. pylori–gut microbiota interaction

The clinical implications of H. pylori infection include the alteration of gastric, intestinal, and fecal
microbial composition. Modification of mucosal immunity, alteration of host cell signaling, disrup-
tion of the gastric epithelial cell architecture, and modulation of gastric acidity are considered the
predominant mechanisms shifting the structure of the gut microbiota [74]. We now discuss the
potential role of autophagy signaling in coordinating H. pylori–gut microbiota interaction.

Gut metabolic homeostasis

The gut microbiota modulates host physiology and pathophysiology through the production of a di-
verse array of metabolites and byproducts. The absorption of these metabolites contributes to host
metabolic reactions as signaling molecules [75]. Short-chain fatty acids (SCFAs), key bacterial me-
tabolites, not only contribute to the promotion of local immune responses but also to the modifica-
tion of intracellular signaling pathways. In colon cancer cells, propionate and butyrate stimulate
autophagy to modulate apoptosis through the suppression of MTOR signaling [76]. Butyrate is fur-
ther suggested to trigger autophagy by inducing endoplasmic reticulum (ER) stress in HCT-116 and
HT-29 colorectal cancer cells [77]. Through the induction of MTOR-dependent autophagy and
ROS-mediated apoptosis, butyrate prevents bladder cancer cell metastasis [78]. Controversially,
the utilization of butyrate as the primary energy source in colonocytes prevents the development
of starvation conditions and dampens the autophagy flux [79]. Notwithstanding the mechanisms
underlying the potential effect of SCFAs on the autophagy response of gastric epithelial cells, alter-
ation in the composition of gastric microbiota, particularly SCFA-producing bacteria, may affect cel-
lular autophagy flux and H. pylori-induced pathogenesis and carcinogenesis. Shifts in the
concentration of other microbial metabolites and byproducts that regulate energy metabolism
such as branched-chain amino acids might influence the host cell autophagy and consequently
H. pylori carcinogenesis [34]. Microbiome-based therapeutics, from dietary changes to the admin-
istration of defined microbial consortia, are developing exciting opportunities to more precisely
modify the gut microbial and metabolic profile. Recent and ongoing advances in the knowledge
about host–microbiota interactions are unveiling novel frontiers of research for modulating the
host immune response and fighting against pathophysiological conditions [80].

Host inflammatory response

H. pylori infection can induce local inflammation, which may develop into systemic inflammation
and establish low-grade and chronic inflammation. An inflammatory environment within the gut
is conducive to compositional and functional alterations of the microbial community often by
blooms of opportunistic pathogens and depletion of commensal bacteria [81]. In vivo ATG5
knockdown and in vitro LC3 or ATG5 siRNA transfection confirm that H. pylori-induced activation
of NFKB through the NOD1 receptor and subsequent production of inflammatory cytokines, par-
ticularly CXCL2 and IL8, depends on autophagic pathways [64]. CagA-dependent activation of
MTORC1 and inhibition of autophagy increase the expression and secretion of proinflammatory
cytokines IL1B, IL6, TNF, CCL7, and CXCL16. Furthermore, the overexpression of antimicrobial
peptide CAMP/LL37 reduces the colonization burden of gastric niches [59]. Transfection of AGS
cells with siRNAs for ATG5 or ATG12 stimulates proinflammatory cytokine production through
NFKB activation in a CagA-dependent manner [82]. A cytokine storm can further disrupt the

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Trends in Microbiology

integrity of epithelial tight junctions, perturb epithelial barriers, and aggravate gut dysbiosis during
inflammation [83]. Thus, as a key mediator, autophagy contributes to H. pylori-induced cytokine
production, inflammation, and subsequent gastric dysbiosis.

H. pylori inhibition of autophagy impairs the trafficking of major histocompatibility complex class II
(MHC-II) components from the cytoplasm to the plasma membrane and prevents the surface ex-
pression of CD80 and CD86 in murine bone marrow-derived dendritic cells (BMDCs) [32]. The in-
tact activity of dendritic cells is required by the immune system to establish tolerance against
commensal microbiota and to develop an adaptive immune response against pathogenic micro-
organisms [84]. Dysregulation in the cross-presentation of exogenous antigens to CD4+ T cells
and disruption of indigenous immune tolerance might be considered other mechanisms through
which H. pylori modifies the composition of gastric microbiota utilizing autophagy flux.

A defective autophagy response following ER stress in intestinal epithelial cells is characterized by

an elevated abundance of IgA-producing plasma cells [85]. IgA mainly blocks the translocation of
bacteria from the lamina propria to the bloodstream, prevents conjugative plasmid transfer,
restricts pathogen overgrowth, and accelerates the survival of commensal microorganisms. IgA
binding to gut-colonizing bacteria can vastly modify the composition of the gut microbiota [86].
Thus, H. pylori modification of autophagy may influence the host microbiota structure via
adjusting the concentration of IgA antibody in the gastric environment.

To our knowledge the intermediate role of autophagy in H. pylori–gut microbiota crosstalk has not
been previously discussed; this paves the way for novel study design considerations. In this regard,
we propose multi-omics approaches to further explore the dynamics and interactions of the gut mi-
crobiota with autophagy core proteins and regulatory factors during H. pylori infection. Subse-
quently, we suggest utilizing artificial intelligence-based technologies and techniques to describe
the infrastructure of the intriguing triangle between H. pylori, autophagy, and gut microbiota.

Knowledge gaps and golden opportunities

Over the past decades, progress in biotechnology, development of myriad therapeutics, and
improvement in clinical infrastructure have provided us with heightened efficacy in preventing
and treating gastric malignancies. However, due to the high risk of developing metastatic
gastric adenocarcinoma from localized gastric cancer, therapeutic choices in the clinic are
still quite limited. The complex systemic nature of gastric carcinogenesis hinders the
understanding of some basic notions regarding the progression of H. pylori-induced gastric
cancer [87].

One major bottleneck in the study of gastric carcinogenesis is the lack of a competent biological
model to thoroughly mimic the carcinogenic cascades. The emergence of cancer organoids has
provided a comprehensive tool for studying cancer development, progression, and the TME.
Moreover, the potential of organ-on-a-chip technology holds great promise in facilitating cancer
organoid sustained coculture with the gut microbiota to better anticipate human clinical re-
sponses [88]. The extended coculture of cancer tissues with living microbiota not only can predict
the human drug pharmacokinetics but also measure the potential capacity of microbiome-based
therapeutics in modulating gastric malignancies. However, this technology is in its infancy and
primarily requires the standardization of different platforms for reproducibility and robustness in
distinct conditions [89].

Recent studies not only focus on modifying the gut microbiota structure but also seek to modu-
late the tumor microbiota to barricade tumor growth and metastasis while improving tumor cell

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 Trends in Microbiology

sensitivity to cancer therapy and immunotherapy [90]. In patients with cancer, gut microbiota Outstanding questions
signatures can predict the toxicity and efficacy of immunotherapy, such as the immune Given the changes in the gut
checkpoint blockade-promoting effect of microbiome-derived inosine [91]. A recent study microbiota composition during gastric
cancer development, could these
demonstrated the translocation of particular gut bacteria into secondary lymphoid organs and
structural or functional alterations
subcutaneous melanoma tumors upon immune checkpoint blockade therapy [92]. This, in drive suppression rather than
principle, presents a fundamental mechanism through which gut microbiota can modify the promotion of autophagy flux?
TME. However, to establish a predictive signature, sophisticated metagenomics approaches
What are the impacts of autophagy-
should be developed and integrated with microbiome multi-omics data.
mediated regulation of host cell nutri-
ent and energy status on the gut mi-
Concluding remarks and future perspectives crobial profile during H. pylori
There is an urgent need for the investigation of more specific mechanisms underlying gastric car- infection?

cinogenesis. H. pylori modulation of autophagy signaling may directly contribute to gastric Can H. pylori infection of gastric
dysbiosis, inflammation, tumorigenesis, and metastasis. While the biphasic activity of autophagy epithelial cells interfere with secretory
determines the turnover of cytoplasmic organelles, apoptosis profoundly influences the entire autophagy? Will the aberrant
cell. The complex ambiguous interconnection between autophagy and apoptosis is one factor secretory autophagy compromise the
innate gastric antimicrobial defense
that determines the fate of gastric tumor cells [93]. Further studies are required to explain the dis- against H. pylori?
crepancies in the protective and suppressive role of autophagy in H. pylori-induced gastric cancer
progression, probably based on its dichotomous function in different stages of cancer develop- What are the effects of OMV-mediated
translocation of CagA on the host cell
ment (see Outstanding questions). Broad clinical trials should focus on how to preserve the del-
autophagy signaling during H. pylori in-
icate equilibrium between the dual roles of autophagy. Moreover, mechanism-oriented in vitro fection?
and in vivo studies should clarify the shifts in autophagy signaling as H. pylori diminishes toward
advanced stages of gastric cancer. How does the complex
interconnection between autophagy
and apoptosis affect the turnover of
Acknowledgments H. pylori-infected cells and the devel-
This work was funded by the Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of opment of cancer stem cells?
Medical Sciences, Tehran, Iran (Project No. RIGLD 1128, IR.SBMU.RIGLD.REC.1399.046), NIH grant GM131919 and
Grants-in Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of What other polymorphisms in
Japan (18KK0266, 19H03473, 21H00346, and 22H02871). autophagy-related genes contribute
to H. pylori pathogenesis and carcino-
genesis? Do germline genetic variants
Declaration of interests influencing autophagy flux also modu-
There are no interests to declare. late H. pylori-induced carcinogenic
pathways?
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